**Meet the editor**

Isin Akyar graduated from School of Medicine, Cukurova University, Adana, Turkey in 1989. She finished her Medical Microbiology Specialty training in Gazi University, Ankara, Turkey in 1999. She joined the Acibadem Labmed Clinical Laboratories in Istanbul as a Specialist of Microbiology in 2004. She became Coordinator of Microbiology in 2007. Since 2004 she has had several Quali-

ty Control trainings. She works in the first accredited laboratory according to ISO 15189 for clinical laboratories in Turkey. In 2008 she joined the Department of Medical Microbiology at Acibadem University in Istanbul. In 2011 she was promoted to serve as an Assistant Professor. Her special interests are laboratory quality control, molecular microbiology, parasitology and proteomics studies. Currently she is working as both Microbiology Coordinator and Assistant Professor. She has been an Associate Editor for the Journal of Acibadem University Science of Health since 2009.

Contents

**Preface IX**

**Sennosides 3**

Shoyama

**Regulation 83**

**Section 2 Quality Control in Food Science 109**

**Fingerprinting 111**

**Section 1 Quality Control of Herbal Medicine 1**

Chapter 1 **Quality Control of Rheum and Cassia Species by Immunological Methods Using Monoclonal Antibodies Against**

Takuhiro Uto, Nguyen Huu Tung, Hiroyuki Tanaka and Yukihiro

**Spectrophotometric Determination of Phenolic Compounds**

Elba Lúcia Cavalcanti de Amorim, Valérium Thijan Nobre de Almeida de Castro, Joabe Gomes de Melo, Allan Jonathan Chernichiarro Corrêa and Tadeu José da Silva Peixoto Sobrinho

Marcelo Gonzaga de Freitas Araújo and Taís Maria Bauab

Sunday Ameh, Florence Tarfa, Magaji Garba and Karniyus Gamaniel

Chapter 5 **Application of ISO 9001 Industrial Standard to Herbal Drug**

Chapter 6 **QA: Fraud Control for Foods and Other Biomaterials by Product**

Edoardo Capuano and Saskia M. van Ruth

Osamu Morinaga and Yukihiro Shoyama

**Purification on Quality Control 29**

Chapter 3 **Standard Operating Procedures (SOP) for the**

**Contained in Plant Samples 47**

Chapter 4 **Microbial Quality of Medicinal Plant Materials 67**

Chapter 2 **Applications of Anti-natural Compound Immunoaffinity**

## Contents



**Section 6 Quality Control in Cosmetics 335**

**Section 7 Sops: What Are They Good For? 365**

Bruna Galdorfini Chiari, Maria Gabriela José de Almeida, Marcos

Contents **VII**

Antonio Corrêa and Vera Lucia Borges Isaac

**Section 8 Quality Control in Clinical Laboratory Medicine 393**

Chapter 18 **Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome 395** Stella Lai, Renate Marquis-Nicholson, Chuan-Ching Lan, Jennifer M. Love, Elaine Doherty, Jonathan R. Skinner and Donald R. Love

Chapter 19 **Quality Assurance in Antimicrobial Susceptibility Testing 413**

Chapter 20 **The Investigation of Gene Regulation and Variation in Human**

**Hybridisation of Paraffin-Embedded Pathology Specimens in a**

Lisa Duffy, Liangtao Zhang, Donald R. Love and Alice M. George

F. G. Ravagnani, D. M. Saidemberg, A. L. C. Faria, S. B. Sartor, D. N.

Chapter 21 **Quality Control Considerations for Fluorescence In Situ**

**Diagnostic Laboratory Environment 469**

Chapter 22 **Quality Control of Biomarkers: From the Samples to Data**

**Cancers and Other Diseases 435** Shihori Tanabe and Sun Ha Jee

Chapter 17 **Standard Operating Procedures (What Are They**

Chapter 16 **Cosmetics' Quality Control 337**

**Good For ?) 367**

Onur Karatuna

**Interpretation 491**

Oliveira and R. R. Catharino

Isin Akyar


#### **Section 6 Quality Control in Cosmetics 335**

Chapter 7 **Principle of Meat Aroma Flavors and Future Prospect 145**

Chapter 9 **Microbial Quality Concerns for Biopharmaceuticals 195** Farzaneh Lotfipour and Somayeh Hallaj-Nezhadi

Chapter 10 **New Approachs in Drug Quality Control: Matrices and**

**Section 4 Quality Control in Radiology and Clinical Imaging 243**

Chapter 14 **Unified Procedures for Quality Controls in Analogue and**

Chapter 15 **The Quality Management of The R&D in High Energy Physics**

**A Medical Physicist's Perspective 245**

Chapter 12 **Quality Assurance in Diagnostic Medical Exposures in Ghana -**

Chapter 11 **Quality Control of Formulated Medicines 227**

da Cunha and Radojko Jaćimović

Chapter 13 **Quality by Design and Risk Assessment for**

**Digital Mammography 293**

**Section 5 Quality Control in Energy 317**

Xuemin Zhu and Sen Qian

**Detector 319**

Christian W. Huck

**VI** Contents

**Chemometrics 215**

Stephen Inkoom

Chen

**Section 3 Quality Control in Pharmaceutics 193**

Hoa Van Ba, Inho Hwang, Dawoon Jeong and Amna Touseef

Sigrid Mennickent, M. de Diego, B. Schulz, M. Vega and C. G. Godoy

Alexandre S. Leal, Maria Ângela de B. C. Menezes, Ilza Dalmázio, Fernanda P. Sepe, Tatiana C. B. Gomes, Amalia S. Santana, Luzia H.

**Radiopharmaceutical Manufacturing and Clinical Imaging 255** Kung-Tien Liu, Jian-Hua Zhao, Lee-Chung Men and Chien-Hsin

Barbara Testagrossa, Giuseppe Acri, Federica Causa, Raffaele Novario, Maria Giulia Tripepi and Giuseppe Vermiglio

Chapter 8 **Novel Analytical Tools for Quality Control in Food Science 177**

Chapter 16 **Cosmetics' Quality Control 337** Bruna Galdorfini Chiari, Maria Gabriela José de Almeida, Marcos Antonio Corrêa and Vera Lucia Borges Isaac

#### **Section 7 Sops: What Are They Good For? 365**


Preface

techniques.

Quality control has an emerging importance in every field of life. Quality control is a process that is used to guarantee a certain level of quality in a product or service. It might include whatever actions a business deems necessary to provide for the control and verification of certain characteristics of a product or service. Most often, it involves thoroughly examining and testing the quality of products or the results of services. The basic goal of this process is to ensure that the products or services that are provided meet specific requirements and characteristics, such as being dependable, satisfactory, safe and fiscally sound. There are some standards which guarantee quality control. In those standards you've got to document everything and track it. You should write what you do, do what you write. Groups that engage in quality control typically have a team of workers who focus on testing a certain number of products or observing services being done. The goal of the quality control team is to identify products or services that do not meet a company's specified standards of quality. If a problem is identified, the job of a quality control team or professional might involve stopping production or service until the problem has been corrected. Depending on the particular service or product as well as the type of problem identified, production or services might not cease entirely. There should be well

With the improvement of technology everyday we meet new and complicated devices and methods in different fields. Quality control should be performed in all of those new

In this book "Latest Research Into Quality Control" our aim was to collect information about

organized procedures and management for ensuring quality control.

quality control in many different fields such as:

Quality Control in Clinical Laboratory Medicine

Quality Control in Radiology and Clinical Imaging

Quality Control in general: SOPs

Quality Control of Herbal Medicine Quality Control in Food Science Quality Control in Pharmaceutics

Quality Control in Energy Quality Control in Cosmetics

## Preface

Quality control has an emerging importance in every field of life. Quality control is a process that is used to guarantee a certain level of quality in a product or service. It might include whatever actions a business deems necessary to provide for the control and verification of certain characteristics of a product or service. Most often, it involves thoroughly examining and testing the quality of products or the results of services. The basic goal of this process is to ensure that the products or services that are provided meet specific requirements and characteristics, such as being dependable, satisfactory, safe and fiscally sound. There are some standards which guarantee quality control. In those standards you've got to document everything and track it. You should write what you do, do what you write. Groups that engage in quality control typically have a team of workers who focus on testing a certain number of products or observing services being done. The goal of the quality control team is to identify products or services that do not meet a company's specified standards of quality. If a problem is identified, the job of a quality control team or professional might involve stopping production or service until the problem has been corrected. Depending on the particular service or product as well as the type of problem identified, production or services might not cease entirely. There should be well organized procedures and management for ensuring quality control.

With the improvement of technology everyday we meet new and complicated devices and methods in different fields. Quality control should be performed in all of those new techniques.

In this book "Latest Research Into Quality Control" our aim was to collect information about quality control in many different fields such as:

Quality Control in general: SOPs Quality Control in Clinical Laboratory Medicine Quality Control of Herbal Medicine Quality Control in Food Science Quality Control in Pharmaceutics Quality Control in Radiology and Clinical Imaging Quality Control in Energy Quality Control in Cosmetics

#### XIV Preface

The aim of this book is to share useful and practical knowledge about quality control in several fields with the people who want to improve their knowledge.

> **Dr Isin Akyar** Acibadem University, School of Medicine, Department of Medical Microbiology, Istanbul, Turkey

**Section 1**

**Quality Control of Herbal Medicine**

**Quality Control of Herbal Medicine**

The aim of this book is to share useful and practical knowledge about quality control in

**Dr Isin Akyar**

Istanbul, Turkey

Acibadem University, School of Medicine, Department of Medical Microbiology,

several fields with the people who want to improve their knowledge.

X Preface

**Chapter 1**

**Quality Control of** *Rheum* **and** *Cassia* **Species by**

**Against Sennosides**

http://dx.doi.org/10.5772/51272

**1. Introduction**

this laboratry.

Osamu Morinaga and Yukihiro Shoyama

Additional information is available at the end of the chapter

**Immunological Methods Using Monoclonal Antibodies**

Recently, medical usage of Japanese traditional medicine has been expanded by reaching ag‐ ing society and increasing various chronic diseases. Therefore, the demand of crude drugs prescribed for Japanese traditional medicine has been increased. However, over 90% of crude drugs are imported in our country, and those over 70% are supplied by the collection of wild species. It is well known that the natural resources bring the difficulty of quality con‐ trol depending on collection season, cultivation place, a variety of species and so on. The other problem, shortage of crude drug comes up. For these general environment, micropro‐ pagation and clonal propagation systems using tissue and cell culture were investigated in

Sennoside A (SA) and B (SB) have the strong catharsis activity and contained in rhubarb and senna (Figure 1) [1]. The concentration of sennosides in rhubarb and senna is variously dependent on the genetic heterogeneity of species, differences in soil condition and climate influence. Sennosides are metabolized by intestinal bacteria to rheinanthrone which acts in the intestines as a direct purgatives [2, 3] and functions as similar to a natural prodrug (Figure 2). Despite the rising availability of a number of synthetic cathartics, sennoside- containing prescriptions are still among the most widely used today, and their importance is increasing. Rhubarb, the rhizome and root of *Rheum* spp. (Polygonaceae), is an important drug in tradi‐ tional Japanese herbal medicine as well as in western medicine since ancient times. It was already recorded in *Chinese Materia Medica* 2000 years ago. It is used in many traditional Jap‐ anese herbal medicines prescribed with other herbal medicines for the syndrome of stasis of blood, as an anti-inflammatory, sedative agent and as a stomachic. Furthermore, it is widely

> © 2012 Morinaga and Shoyama; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Morinaga and Shoyama; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

## **Quality Control of** *Rheum* **and** *Cassia* **Species by Immunological Methods Using Monoclonal Antibodies Against Sennosides**

Osamu Morinaga and Yukihiro Shoyama

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51272

#### **1. Introduction**

Recently, medical usage of Japanese traditional medicine has been expanded by reaching ag‐ ing society and increasing various chronic diseases. Therefore, the demand of crude drugs prescribed for Japanese traditional medicine has been increased. However, over 90% of crude drugs are imported in our country, and those over 70% are supplied by the collection of wild species. It is well known that the natural resources bring the difficulty of quality con‐ trol depending on collection season, cultivation place, a variety of species and so on. The other problem, shortage of crude drug comes up. For these general environment, micropro‐ pagation and clonal propagation systems using tissue and cell culture were investigated in this laboratry.

Sennoside A (SA) and B (SB) have the strong catharsis activity and contained in rhubarb and senna (Figure 1) [1]. The concentration of sennosides in rhubarb and senna is variously dependent on the genetic heterogeneity of species, differences in soil condition and climate influence. Sennosides are metabolized by intestinal bacteria to rheinanthrone which acts in the intestines as a direct purgatives [2, 3] and functions as similar to a natural prodrug (Figure 2). Despite the rising availability of a number of synthetic cathartics, sennoside- containing prescriptions are still among the most widely used today, and their importance is increasing.

Rhubarb, the rhizome and root of *Rheum* spp. (Polygonaceae), is an important drug in tradi‐ tional Japanese herbal medicine as well as in western medicine since ancient times. It was already recorded in *Chinese Materia Medica* 2000 years ago. It is used in many traditional Jap‐ anese herbal medicines prescribed with other herbal medicines for the syndrome of stasis of blood, as an anti-inflammatory, sedative agent and as a stomachic. Furthermore, it is widely

© 2012 Morinaga and Shoyama; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Morinaga and Shoyama; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

used as cathartics in Japan. The main purgative principles of rhubarb have proved to be sen‐ nosides [1], identical with those isolated from senna leaves, and rheinosides, which were al‐ so isolated as purgatives of rhubarb, together with various kinds of phenolics, like tannins, stilbenes, naphthalenes and lindleyin. The quality of rhubarb is severely regulated by Japa‐ nese Pharmacopeia as rhubarb contains SA of over 0.25% dry weight in root [4].

in *Cassia acutifolia* Delile and *Cassia angustifolia* Vahl. *C. angustifolia* listed in Japanese Phar‐ macopeia, and the quality is severely regulated as senna contains total sennosides (SA and

Quality Control of *Rheum* and *Cassia* Species by Immunological Methods...

http://dx.doi.org/10.5772/51272

5

In the breeding research on the plant, a lot of stages are required as follows : dedifferentia‐ tion, extension of mutation by the mutagen, redifferentiation, analysis of the redifferentiated plant, mass propagation of the higher yielding plant and transplanting to soil. Therefore, it is very important to study a large number of plant samples in the phytochemical field and a small sample size *in vitro* for the breeding of *Rheum* and *Cassia* species yielding high concen‐ tration of sennosides. Many analytical approaches have been investigated for the determina‐ tion of sennosides in plant extracts. Among these methods, the use of high-performance liquid chromatography (HPLC) appears most frequently and widely today. However, when the assay of very low concentration of sennosides in the regenerated plantlets is needed, the

Recently, the immunological assay method is widely developed for the purpose of analysis for a small amount of constituent. In general immunological methodologies in particular en‐ zyme-linked immunosorbent assay (ELISA) have promoted the development of higher sen‐

On the one hand, monoclonal antibodies (MAbs) have many potential uses in addition to immunological methods in plant sciences. MAbs are superior to polyclonal antibodies (PAbs) in the antigenic specificity and stability. Therefore, immunoassay using MAbs against pharmacologically active compound having small molecular weight has become an important tool for the studies on receptor binding analysis, enzyme assay and quantitative and/or qualitative analytical techniques in plants owing to its specific affinity, and possesses an extremely high possibility in the phytochemical analysis. Up to now, immunological ap‐ proach for assaying quantities of sennosides in *C. angustifolia* using PAb against SB has been investigated by Atzorn *et al* [5]. However, since no success with MAbs against SA and SB

**3.** Establishments of a new eastern blotting, double staining and immunohistochemical

**2. Production of MAb against SA, its characterization and use for ELISA**

In the immunologically analytical methodology, there are two measuring methods using the antiserum (polyclonal antibody ; PAb) and MAb in general. PAb is a heterogeneous mixture of antibody molecules arising from a variety of constantly evolving B lymphocytes. There‐ fore, PAb can often show high affinity because different antibody populations react with the

SB) of over 1.0% dry weight in leaf [4].

HPLC method is not appropriate and efficient.

staining using anti-SA and SB MAbs.

has been reported, objectives of this work are shown as following.

**1.** Production of MAb against SA, its characterization and use for ELISA.

**2.** Production of MAbs against SB, their characterization and use for ELISA.

sitive assay system.

**2.1. Preface**


**Figure 1.** Structures of sennosides

**Figure 2.** Metabolic pathways of sennosides by intestinal bacteria.

Senna, the leaf and pod of *Cassia* spp. (Leguminosae), is one of the most popular in herbal remedies and in health food industry. It has been widely used in cathartics for the relief of constipation prescribed with other health teas and dieter's teas in Japan, and often used as natural dietary supplements for enhancement of bloodflow and metabolism in USA, Europe and Australia. These pharmaceutical properties are due to sennosides, which are contained in *Cassia acutifolia* Delile and *Cassia angustifolia* Vahl. *C. angustifolia* listed in Japanese Phar‐ macopeia, and the quality is severely regulated as senna contains total sennosides (SA and SB) of over 1.0% dry weight in leaf [4].

In the breeding research on the plant, a lot of stages are required as follows : dedifferentia‐ tion, extension of mutation by the mutagen, redifferentiation, analysis of the redifferentiated plant, mass propagation of the higher yielding plant and transplanting to soil. Therefore, it is very important to study a large number of plant samples in the phytochemical field and a small sample size *in vitro* for the breeding of *Rheum* and *Cassia* species yielding high concen‐ tration of sennosides. Many analytical approaches have been investigated for the determina‐ tion of sennosides in plant extracts. Among these methods, the use of high-performance liquid chromatography (HPLC) appears most frequently and widely today. However, when the assay of very low concentration of sennosides in the regenerated plantlets is needed, the HPLC method is not appropriate and efficient.

Recently, the immunological assay method is widely developed for the purpose of analysis for a small amount of constituent. In general immunological methodologies in particular en‐ zyme-linked immunosorbent assay (ELISA) have promoted the development of higher sen‐ sitive assay system.

On the one hand, monoclonal antibodies (MAbs) have many potential uses in addition to immunological methods in plant sciences. MAbs are superior to polyclonal antibodies (PAbs) in the antigenic specificity and stability. Therefore, immunoassay using MAbs against pharmacologically active compound having small molecular weight has become an important tool for the studies on receptor binding analysis, enzyme assay and quantitative and/or qualitative analytical techniques in plants owing to its specific affinity, and possesses an extremely high possibility in the phytochemical analysis. Up to now, immunological ap‐ proach for assaying quantities of sennosides in *C. angustifolia* using PAb against SB has been investigated by Atzorn *et al* [5]. However, since no success with MAbs against SA and SB has been reported, objectives of this work are shown as following.


#### **2. Production of MAb against SA, its characterization and use for ELISA**

#### **2.1. Preface**

used as cathartics in Japan. The main purgative principles of rhubarb have proved to be sen‐ nosides [1], identical with those isolated from senna leaves, and rheinosides, which were al‐ so isolated as purgatives of rhubarb, together with various kinds of phenolics, like tannins, stilbenes, naphthalenes and lindleyin. The quality of rhubarb is severely regulated by Japa‐

nese Pharmacopeia as rhubarb contains SA of over 0.25% dry weight in root [4].

**Figure 1.** Structures of sennosides

4 Latest Research into Quality Control

**Figure 2.** Metabolic pathways of sennosides by intestinal bacteria.

Senna, the leaf and pod of *Cassia* spp. (Leguminosae), is one of the most popular in herbal remedies and in health food industry. It has been widely used in cathartics for the relief of constipation prescribed with other health teas and dieter's teas in Japan, and often used as natural dietary supplements for enhancement of bloodflow and metabolism in USA, Europe and Australia. These pharmaceutical properties are due to sennosides, which are contained

In the immunologically analytical methodology, there are two measuring methods using the antiserum (polyclonal antibody ; PAb) and MAb in general. PAb is a heterogeneous mixture of antibody molecules arising from a variety of constantly evolving B lymphocytes. There‐ fore, PAb can often show high affinity because different antibody populations react with the variety of epitopes that characterize the antigen. On the other hand, there are some prob‐ lems of PAb that the extensive cross-reactivity occurs between the antibody and the multiple antigens which have the same antigenic determinant, and it is impossible to supply for iden‐ tical antibody permanently. In the meantime, MAb is produced from a single B lymphocyte and can react with one antigenic determinant of the specific antigen. Besides MAb has iden‐ tical specificity and affinity. There are some advantages that the complete purity of the im‐ munized antigen is not required and the hybridoma cells can be preserved as freeze stock, and it is possible to get MAb depending on necessary respond.

*2.2.2. Extraction of various rhubarb samples*

*2.2.3. Synthesis of antigen conjugates*

thesized in the same manner.

jugate was mixed with a 103

*2.2.5. Competitive ELISA for SA*

490 nm.

lowing.

the ELISA.

Dried samples (30 mg) of various rhubarb roots were powdered, and then extracted five times with MeOH containing 0.1% (w/v) NH4OH (0.5 mL) with sonication, filtered using a Cosmonice Filter W (0.45 μm Filter Unit, Nacalai Tesque Inc., Kyoto, Japan), and the combined extracts were diluted with 10 mM NaHCO3 to prepare a solution suitable for

To SA (6 mg) dissolved in 1 mL of tetrahydrofuran-20 mM phosphate buffer of pH 5.5 (7:3), 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of EDC was added. Then, 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of BSA was added, with stirring at room temperature for 14 hr. The reaction mixture was dialyzed five times against H2O, and then lyophilized to give 5.8 mg of SA conjugate (SA-BSA). SA-HSA conjugate was also syn‐

*2.2.4. Determination of hapten density in SA-carrier protein conjugate by matrix-assisted laser*

The hapten number in the SA-carrier protein conjugate was determined by MALDI-TOF mass spectrometry as previously described [15]. A small amount (1-10 pmol) of antigen con‐

taining 0.15% trifluoroacetic acid (TFA). The mixture was subjected to a JEOL Mass Spectrometers (JMS) time-of-flight (TOF) mass monitor (model Voyager Elite, PerSeptive Bi‐ osystems Inc., Framingham, MA, USA) and irradiated with a N2 laser (337 nm, 150 ns pulse). The ions formed by each pulse were accelerated by a 20 kV potential into a 2.0 m evacuated

SA-HSA (five molecules of SA per molecule of HSA) (100 μL, 1 μg/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate then treated with 300 μL S-PBS for 1 hr to reduce non-specific adsorption. Fifty μL of various concentra‐ tions of SA or samples dissolved in 10 mM NaHCO3 solution were incubated with 50 μL of MAb solution (0.218 μg/mL) for 1 hr. The plate was washed three times with T-PBS, and then incubated with 100 μL of a 1:1000 dilution of POD-labeled anti-mouse IgG for 1 hr. Af‐ ter washing the plate three times with T-PBS, 100 μL of substrate solution [0.1 M citrate buf‐ fer (pH 4) containing 0.003% H2O2 and 0.3 mg/mL of ABTS] was added to each well and incubated for 15 min. The absorbance was measured by a micro plate reader at 405 nm and

The cross-reactivities (CR) of sennosides and related compounds were determined as fol‐

*μg* / mL of compound under investigation yielding *<sup>A</sup>* / *<sup>A</sup>*<sup>0</sup> <sup>=</sup> <sup>50</sup>*%* ×100

CR(*%*) <sup>=</sup> *μg* / mL of SA yielding *<sup>A</sup>* / *<sup>A</sup>*<sup>0</sup> =50*%*


Quality Control of *Rheum* and *Cassia* Species by Immunological Methods...

http://dx.doi.org/10.5772/51272

7

*desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry*

tube and detected using a compatible computer as previously reported [15].

There are several formats for ELISA like direct ELISA, competitive ELISA, sandwich ELI‐ SA and competitive ELISA according to the immune complexes formed during manipula‐ tion. Analysis of low molecular weight compound by immunoassay is still limited to competitive format.

Quality control of the Japanese herbal medicine is necessary because it is believed that ap‐ proximately 70% of these crude drugs prescribed are collected from natural resource. Fur‐ thermore, since MAbs become necessary for the assay of concentrations of active constituents in our on-going plant biotechnological projects, we have already produced MAbs against natural compounds such as forskolin [6], solamargine [7], opium alkaloids [8], marihuana compounds [9], glycyrrhizin [10], crocin [11], ginsenoside Rb1 [12] and Rg1 [13], and developed individual competitive ELISAs. An immunological approach for assaying quantities of sennosides using a PAbs has been investigated by Atzorn *et al*.[5]. However, since no result of MAb related to sennosides has been reported yet, anti-SA MAb was pro‐ duced as described [14].

#### **2.2. Experimental**

#### *2.2.1. Chemicals and immunochemicals*

SA was purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). 1-Ethyl-3-(3'-dime‐ thylaminopropyl)-carbodiimide HCl (EDC) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). BSA and HSA were provided by Pierce (Rockford, IL, USA). Peroxidase-labeled antimouse IgG was provided by Organon Teknika Cappel Products (West Chester, PA, USA). Enriched RPMI1640-Dulbecco's-Ham's F12 (eRDF) medium and RD-1 additives (containing 9 μg/mL insulin, 20 μg/mL transferrin, 20 μM ethanolamine, 25 μM sodium selenite) were purchased from Kyokuto Pharmaceutical Industrial Co., Ltd. (Tokyo, Japan). Hypoxanthineaminopterin-thymidine (HAT) additives were obtained from Sigma Chemical Company (St. Louis, MO, USA). Fetal calf serum (FCS) was purchased from Cambrex Corporation (Wal‐ kersville, MA, USA). All other chemicals were standard commercial products of analytical grade. Samples of various rhubarb roots were purchased from the Tochimototenkaido Cor‐ poration (Osaka, Japan).

#### *2.2.2. Extraction of various rhubarb samples*

variety of epitopes that characterize the antigen. On the other hand, there are some prob‐ lems of PAb that the extensive cross-reactivity occurs between the antibody and the multiple antigens which have the same antigenic determinant, and it is impossible to supply for iden‐ tical antibody permanently. In the meantime, MAb is produced from a single B lymphocyte and can react with one antigenic determinant of the specific antigen. Besides MAb has iden‐ tical specificity and affinity. There are some advantages that the complete purity of the im‐ munized antigen is not required and the hybridoma cells can be preserved as freeze stock,

There are several formats for ELISA like direct ELISA, competitive ELISA, sandwich ELI‐ SA and competitive ELISA according to the immune complexes formed during manipula‐ tion. Analysis of low molecular weight compound by immunoassay is still limited to

Quality control of the Japanese herbal medicine is necessary because it is believed that ap‐ proximately 70% of these crude drugs prescribed are collected from natural resource. Fur‐ thermore, since MAbs become necessary for the assay of concentrations of active constituents in our on-going plant biotechnological projects, we have already produced MAbs against natural compounds such as forskolin [6], solamargine [7], opium alkaloids [8], marihuana compounds [9], glycyrrhizin [10], crocin [11], ginsenoside Rb1 [12] and Rg1 [13], and developed individual competitive ELISAs. An immunological approach for assaying quantities of sennosides using a PAbs has been investigated by Atzorn *et al*.[5]. However, since no result of MAb related to sennosides has been reported yet, anti-SA MAb was pro‐

SA was purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). 1-Ethyl-3-(3'-dime‐ thylaminopropyl)-carbodiimide HCl (EDC) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). BSA and HSA were provided by Pierce (Rockford, IL, USA). Peroxidase-labeled antimouse IgG was provided by Organon Teknika Cappel Products (West Chester, PA, USA). Enriched RPMI1640-Dulbecco's-Ham's F12 (eRDF) medium and RD-1 additives (containing 9 μg/mL insulin, 20 μg/mL transferrin, 20 μM ethanolamine, 25 μM sodium selenite) were purchased from Kyokuto Pharmaceutical Industrial Co., Ltd. (Tokyo, Japan). Hypoxanthineaminopterin-thymidine (HAT) additives were obtained from Sigma Chemical Company (St. Louis, MO, USA). Fetal calf serum (FCS) was purchased from Cambrex Corporation (Wal‐ kersville, MA, USA). All other chemicals were standard commercial products of analytical grade. Samples of various rhubarb roots were purchased from the Tochimototenkaido Cor‐

and it is possible to get MAb depending on necessary respond.

competitive format.

6 Latest Research into Quality Control

duced as described [14].

poration (Osaka, Japan).

*2.2.1. Chemicals and immunochemicals*

**2.2. Experimental**

Dried samples (30 mg) of various rhubarb roots were powdered, and then extracted five times with MeOH containing 0.1% (w/v) NH4OH (0.5 mL) with sonication, filtered using a Cosmonice Filter W (0.45 μm Filter Unit, Nacalai Tesque Inc., Kyoto, Japan), and the combined extracts were diluted with 10 mM NaHCO3 to prepare a solution suitable for the ELISA.

#### *2.2.3. Synthesis of antigen conjugates*

To SA (6 mg) dissolved in 1 mL of tetrahydrofuran-20 mM phosphate buffer of pH 5.5 (7:3), 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of EDC was added. Then, 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of BSA was added, with stirring at room temperature for 14 hr. The reaction mixture was dialyzed five times against H2O, and then lyophilized to give 5.8 mg of SA conjugate (SA-BSA). SA-HSA conjugate was also syn‐ thesized in the same manner.

#### *2.2.4. Determination of hapten density in SA-carrier protein conjugate by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry*

The hapten number in the SA-carrier protein conjugate was determined by MALDI-TOF mass spectrometry as previously described [15]. A small amount (1-10 pmol) of antigen con‐ jugate was mixed with a 103 -fold molar excess of sinapinic acid in an aqueous solution con‐ taining 0.15% trifluoroacetic acid (TFA). The mixture was subjected to a JEOL Mass Spectrometers (JMS) time-of-flight (TOF) mass monitor (model Voyager Elite, PerSeptive Bi‐ osystems Inc., Framingham, MA, USA) and irradiated with a N2 laser (337 nm, 150 ns pulse). The ions formed by each pulse were accelerated by a 20 kV potential into a 2.0 m evacuated tube and detected using a compatible computer as previously reported [15].

#### *2.2.5. Competitive ELISA for SA*

SA-HSA (five molecules of SA per molecule of HSA) (100 μL, 1 μg/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate then treated with 300 μL S-PBS for 1 hr to reduce non-specific adsorption. Fifty μL of various concentra‐ tions of SA or samples dissolved in 10 mM NaHCO3 solution were incubated with 50 μL of MAb solution (0.218 μg/mL) for 1 hr. The plate was washed three times with T-PBS, and then incubated with 100 μL of a 1:1000 dilution of POD-labeled anti-mouse IgG for 1 hr. Af‐ ter washing the plate three times with T-PBS, 100 μL of substrate solution [0.1 M citrate buf‐ fer (pH 4) containing 0.003% H2O2 and 0.3 mg/mL of ABTS] was added to each well and incubated for 15 min. The absorbance was measured by a micro plate reader at 405 nm and 490 nm.

The cross-reactivities (CR) of sennosides and related compounds were determined as fol‐ lowing.

$$\text{CR} \text{(\%)} = \frac{\mu \text{g/mL} \quad \text{of SA yielding } A/A\_0 \text{=50\%}}{\mu \text{g/mL} \quad \text{of compound under investigation} \quad \text{yielding } A/A\_0 \text{=50\%}} \times 100$$

where *A* is the absorbance in the presence of the test compound and *A* <sup>0</sup> is the absorbance in the absence of the test compound.

#### **2.3. Results and discussion**

#### *2.3.1. Direct determination of SA-carrier protein conjugate by MALDI-TOF mass spectrometry*

In general, the low molecular weight compounds (hapten) like plant secondary metabolite have no immunogenicity. Therefore, it should be conjugated with some high molecular compound like protein resulting in immunogenic. The specificity of immunoassay method is dependent on the site of linkage between hapten and carrier protein moiety, and enumer‐ ation of hapten in immunogen conjugate. SA-BSA and SA-HSA conjugates were synthesized as immunogen and the immobilization antigen for ELISA, respectively. Figure 3 shows the typical synthetic pathway of SA-BSA conjugate. The commonly used methods to link car‐ boxyl group and amino group in a hapten or carrier involve activation by carbodiimides, isobutylchloroformate or carbonyldiimidazole. Carbodiimides react with carboxyl groups to form an unstable *O*-acetylisourea intermediate, which reacts with amines to form amide bonds. EDC can be used commonly as a carbodiimide. In this case, carrier protein combined directly to antigen as indicated in Figure 3.

**Figure 4.** Direct determination of SA-BSA by MALDI-TOF MS. [M+H]+, [M+2H]2+ are single and double protonated

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After the cell fusion and HAT selection, hybridoma producing MAb reactive to SA was ob‐ tained, and classfied into IgG1 which had *k* light chains. Refined MAb was confirmed to be IgG compared to the MALDI-TOF MS measurement. The molecular weight of MAb was 151,396 calculated [16]. The reactivity of IgG type MAb 6G8 was tested for varying the anti‐ body concentration and for performing a dilution curve in direct ELISA. The antibody con‐ centration of 0.218 μg/mL showed the absorbance at 0.8 in direct ELISA, therefore it was

The free MAb 6G8, following incubation with competing antigen, was bound to the poly‐ styrene microtitre plates precoated with SA-HSA. Under these conditions, the full measur‐

SA is a unique anthraquinone having individual double of carboxylic acid-, hydroxyl-, car‐ bonyl- and *O*-glucosyl-groups at C-3, C-1, C-9 and C-8 positions in a molecule, respective‐ ly. Moreover, SA possessed a *threo*- configuration between C-10 and C-10' positions as indicated in Figure 6. Therefore, a MAb should detect all these functions, and also the stereochemical recognition is needed for this complicated compound. Since the newly established ELISA against SA is expected to be applied for phytochemical investigations involving crude plant extracts, the assay specificity was checked by determining the cross-reactivities of MAb with various related compounds. The cross-reactivities of the MAb was examined by competi‐

ing range of the assay extended from 20 to 200 ng/mL as indicated in Figure 5.

molecules of SA-BSA, respectively.

selected for the competitive ELISA.

tive ELISA.

*2.3.3. Assay sensitivity and assay specificity*

*2.3.2. Production and characteristic of MAb against SA*

**Figure 3.** Typical synthetic pathway of SA-BSA. Carboxyl group of SA was activated by EDC and subsequently com‐ bined to amino residues of lysine and/or arginine on the protein to form amide bond.

Figure 4 shows the MALDI-TOF mass spectrum of the antigen, SA-BSA conjugate. A broad peak coinciding with the conjugate of SA and BSA appeared from *m/z* 68,500 to 73,500 cen‐ tering at around *m/z* 70,600. Using experimental results and a molecular weight of 66,433 for BSA, the calculated values of SA component (MW 862) are 4,218 resulting in the range of two to eight molecules of SA (five on average) conjugated with BSA. In general eight to twenty five molecules of hapten conjugated with carrier protein in the conjugate were suffi‐ cient for immunization. Therefore, the hapten number was estimated to be sufficient for im‐ munization because an antigen conjugate having a similar hapten number was sufficient for immunization in a previous study [10]. The number of SA contained in the SA-HSA conju‐ gate was also determined to be around five molecules by its spectrum.

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**Figure 4.** Direct determination of SA-BSA by MALDI-TOF MS. [M+H]+, [M+2H]2+ are single and double protonated molecules of SA-BSA, respectively.

#### *2.3.2. Production and characteristic of MAb against SA*

where *A* is the absorbance in the presence of the test compound and *A* <sup>0</sup> is the absorbance in

*2.3.1. Direct determination of SA-carrier protein conjugate by MALDI-TOF mass spectrometry*

In general, the low molecular weight compounds (hapten) like plant secondary metabolite have no immunogenicity. Therefore, it should be conjugated with some high molecular compound like protein resulting in immunogenic. The specificity of immunoassay method is dependent on the site of linkage between hapten and carrier protein moiety, and enumer‐ ation of hapten in immunogen conjugate. SA-BSA and SA-HSA conjugates were synthesized as immunogen and the immobilization antigen for ELISA, respectively. Figure 3 shows the typical synthetic pathway of SA-BSA conjugate. The commonly used methods to link car‐ boxyl group and amino group in a hapten or carrier involve activation by carbodiimides, isobutylchloroformate or carbonyldiimidazole. Carbodiimides react with carboxyl groups to form an unstable *O*-acetylisourea intermediate, which reacts with amines to form amide bonds. EDC can be used commonly as a carbodiimide. In this case, carrier protein combined

**Figure 3.** Typical synthetic pathway of SA-BSA. Carboxyl group of SA was activated by EDC and subsequently com‐

Figure 4 shows the MALDI-TOF mass spectrum of the antigen, SA-BSA conjugate. A broad peak coinciding with the conjugate of SA and BSA appeared from *m/z* 68,500 to 73,500 cen‐ tering at around *m/z* 70,600. Using experimental results and a molecular weight of 66,433 for BSA, the calculated values of SA component (MW 862) are 4,218 resulting in the range of two to eight molecules of SA (five on average) conjugated with BSA. In general eight to twenty five molecules of hapten conjugated with carrier protein in the conjugate were suffi‐ cient for immunization. Therefore, the hapten number was estimated to be sufficient for im‐ munization because an antigen conjugate having a similar hapten number was sufficient for immunization in a previous study [10]. The number of SA contained in the SA-HSA conju‐

bined to amino residues of lysine and/or arginine on the protein to form amide bond.

gate was also determined to be around five molecules by its spectrum.

the absence of the test compound.

directly to antigen as indicated in Figure 3.

**2.3. Results and discussion**

8 Latest Research into Quality Control

After the cell fusion and HAT selection, hybridoma producing MAb reactive to SA was ob‐ tained, and classfied into IgG1 which had *k* light chains. Refined MAb was confirmed to be IgG compared to the MALDI-TOF MS measurement. The molecular weight of MAb was 151,396 calculated [16]. The reactivity of IgG type MAb 6G8 was tested for varying the anti‐ body concentration and for performing a dilution curve in direct ELISA. The antibody con‐ centration of 0.218 μg/mL showed the absorbance at 0.8 in direct ELISA, therefore it was selected for the competitive ELISA.

#### *2.3.3. Assay sensitivity and assay specificity*

The free MAb 6G8, following incubation with competing antigen, was bound to the poly‐ styrene microtitre plates precoated with SA-HSA. Under these conditions, the full measur‐ ing range of the assay extended from 20 to 200 ng/mL as indicated in Figure 5.

SA is a unique anthraquinone having individual double of carboxylic acid-, hydroxyl-, car‐ bonyl- and *O*-glucosyl-groups at C-3, C-1, C-9 and C-8 positions in a molecule, respective‐ ly. Moreover, SA possessed a *threo*- configuration between C-10 and C-10' positions as indicated in Figure 6. Therefore, a MAb should detect all these functions, and also the stereochemical recognition is needed for this complicated compound. Since the newly established ELISA against SA is expected to be applied for phytochemical investigations involving crude plant extracts, the assay specificity was checked by determining the cross-reactivities of MAb with various related compounds. The cross-reactivities of the MAb was examined by competi‐ tive ELISA.

have appreciable cross-reactivities. From these results it is suggested that a basal structure of rhein and sugar moiety caused immunization. In addition the most important property of MAb 6G8 is an ability of stereochemical recognition because the differences of structure be‐ tween SA and SB are only the stereochemical configuration at the C-10 and C-10' positions. Therefore, it is suggested that *threo*-configurational structure of bisanthrone is indispensable

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as an immunodominant molecule for reactivity of MAb 6G8.

sennoside A 100 sennoside B 0.28 rhein 0.35 emodin < 0.04 aloe-emodin < 0.04 barbaloin < 0.04 1,4-dihydroxy-anthraquinone < 0.04

rhaponticin < 0.04

gallic acid < 0.04 vanillic acid < 0.04 caffeic acid < 0.04 homogentisic acid < 0.04

**Table 1.** Cross-reactivities (%) of MAb-6G8 against sennosides and other compounds.

*2.3.4. Correlation of results of SA determination in crude extracts of rhubarb roots between HPLC*

The ELISA was utilized to measure the concentrations of SA in various rhubarb (Table 2). Oshio and Kawamura determined sennoside concentrations in various crude rhubarbs by HPLC [17]. More recently Seto *et al.* reported the comparative concentrations of sennosides determined by HPLC in various commercial rhubarbs [18]. They required a lager sample size compared to the newly established ELISA due to some pretreatments because the crude materials contained several kinds of phenolics such as tannins, stilbens, naphthalen deriva‐

Table 2 shows the SA concentrations in various rhubarbs. Shinshu Daio bred by crossing *R. palmatum* and *R. coreanum* in order to increase the concentration of SA in Japan, contained the highest SA; 13.69±0.69 μg/mg dry wt. Ga-wo which was estimated to be high grade, con‐

**Anthraquinone and anthrone**

**Stilbene**

**Phenol carboxylic acid**

*and ELISA using MAb 6G8*

tives and lindleyin as previously indicated.

**Compound Cross-reactivities (%)**

**Figure 5.** Calibration curve for SA.

**Figure 6.** Chemical structures of SA, SB and its structurally related compounds.

Table 1 indicates the cross-reactivities of anti-SA MAb against related anthraquinone, an‐ throne and phenol carboxylic acid. MAb 6G8 cross-reacted with rhein and SB weakly; 0.28 and 0.35%, respectively. However, the other related anthraquinone and anthrone did not have appreciable cross-reactivities. From these results it is suggested that a basal structure of rhein and sugar moiety caused immunization. In addition the most important property of MAb 6G8 is an ability of stereochemical recognition because the differences of structure be‐ tween SA and SB are only the stereochemical configuration at the C-10 and C-10' positions. Therefore, it is suggested that *threo*-configurational structure of bisanthrone is indispensable as an immunodominant molecule for reactivity of MAb 6G8.


**Table 1.** Cross-reactivities (%) of MAb-6G8 against sennosides and other compounds.

**Figure 5.** Calibration curve for SA.

10 Latest Research into Quality Control

**Figure 6.** Chemical structures of SA, SB and its structurally related compounds.

Table 1 indicates the cross-reactivities of anti-SA MAb against related anthraquinone, an‐ throne and phenol carboxylic acid. MAb 6G8 cross-reacted with rhein and SB weakly; 0.28 and 0.35%, respectively. However, the other related anthraquinone and anthrone did not

#### *2.3.4. Correlation of results of SA determination in crude extracts of rhubarb roots between HPLC and ELISA using MAb 6G8*

The ELISA was utilized to measure the concentrations of SA in various rhubarb (Table 2). Oshio and Kawamura determined sennoside concentrations in various crude rhubarbs by HPLC [17]. More recently Seto *et al.* reported the comparative concentrations of sennosides determined by HPLC in various commercial rhubarbs [18]. They required a lager sample size compared to the newly established ELISA due to some pretreatments because the crude materials contained several kinds of phenolics such as tannins, stilbens, naphthalen deriva‐ tives and lindleyin as previously indicated.

Table 2 shows the SA concentrations in various rhubarbs. Shinshu Daio bred by crossing *R. palmatum* and *R. coreanum* in order to increase the concentration of SA in Japan, contained the highest SA; 13.69±0.69 μg/mg dry wt. Ga-wo which was estimated to be high grade, con‐ tained 6.62±0.42 μg/mg dry wt. The other three species showed almost the same concentra‐ tions of SA, around 3.3 μg/mg dry wt. These results are in good agreement with the previous reports [18]. The correlation between results from ELISA and HPLC is reasonable except for Kinmon Daio. The concentration analyzed by HPLC was very low compared to the others. The reason is still obscure although individual peaks separated by HPLC were analyzed by ELISA.

*3.2.2. Sample preparation*

*3.2.3. Synthesisi of antigen conjugates*

mass spectrometry as previously described [15].

sized in the same manner.

*3.2.5. Competitive ELISA for SB*

**3.3. Results and discussion**

*spectrometry*

Dried samples (30 mg) of various rhubarb roots, *Cassia* plant leaves, traditional Japanese prescriptions and dietary supplements were powdered, and then extracted five times with MeOH containing 0.1% (w/v) NH4OH (0.5 mL) with sonication, filtered using a Cosmonice Filter W (0.45 μm Filter Unit, Nacalai Tesque Inc., Kyoto, Japan), and the combined extracts

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To SB (6 mg) dissolved in 1 mL of tetrahydrofuran-20 mM phosphate buffer of pH 5.5 (7:3), 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of EDC was added. Then, 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of BSA was added, with stirring at room temperature for 14 hr. The reaction mixture was dialyzed five times against H2O, and then lyophilized to give 5.5 mg of SB-BSA conjugate. SB-HSA conjugate was also synthe‐

*3.2.4. Determination of hapten density in SB-carrier protein conjugate by MALDI-TOF mass*

The hapten number in the SB-carrier protein conjugate was determined by MALDI-TOF

SB-HSA (four molecules of SB per molecule of HSA) (100 μL, 1 μg/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate then treated with 300 μL S-PBS for 1 hr to reduce non-specific adsorption. Fifty μL of various concentra‐ tions of SB or samples dissolved in 10 mM NaHCO3 solution were incubated with 50 μL of MAb solution (0.121 μg/mL) for 1 hr. The plate was washed three times with T-PBS, and then incubated with 100 μL of a 1:1000 dilution of POD-labeled anti-mouse IgG for 1 hr. After washing the plate three times with T-PBS, 100 μL of substrate solution [0.1 M citrate buffer (pH 4) containing 0.003% H2O2 and 0.3 mg/mL of ABTS] was added to each well and incubat‐ ed for 15 min. The absorbance was measured by a micro plate reader at 405 nm and 490 nm.

*3.3.1. Direct determination of SB-carrier protein conjugate by MALDI-TOF mass spectrometry*

It is well known that hapten number in an antigen conjugate is important for immunization against low molecular weight compounds. Figure 7 shows the MALDI-TOF mass spectrum of the antigen, SB-BSA conjugate. A broad peak coinciding with the conjugate of SB and BSA appeared from *m/z* 67,300 to 70,700 centering at around *m/z* 68,900. Using experimental results and a molecular weight of 66,433 for BSA, the calculated values of SB component

were diluted with 10 mM NaHCO3 to prepare a solution suitable for the ELISA.


**Table 2.** SA concentrations in various rhubarb samples. Data are the means of triplicate assays.

### **3. Production of MAbs against SB, their characterization and use for ELISA**

#### **3.1. Preface**

SB is a very important natural bioactive component of rhubarb and senna as well as SA. To‐ tal sennoside (SA and SB) concentrations are important, when rhubarb and senna are used as a raw material of medical supply and traditional Japanese herbal medicine for the purga‐ tive effect.

A number of methods for the quantification of SB have been published, most of which have been performed by HPLC [17]. Immunological approaches for assaying quantities of senno‐ sides and SA using PAb and MAb have been investigated by Atzorn *et al*. [5] and by us [14], respectively. However, no success with MAb against SB has been reported. In here, produc‐ tion of anti-SB MAb and the competitive ELISA using anti-SA and SB MAbs for the direct determination of SA and SB in various samples are described [19].

#### **3.2. Experimental**

#### *3.2.1. Plant materials*

Samples of various rhubarb roots were purchased from the Tochimototenkaido Corporation (Osaka, Japan). Samples of leaves of *Cassia* plants were collected in Thailand. Traditional Japanese prescriptions were procured from Tsumura & Co. (Tokyo, Japan). Dietary supple‐ ments (health teas and dieter's teas) were purchased from drug and department stores.

#### *3.2.2. Sample preparation*

tained 6.62±0.42 μg/mg dry wt. The other three species showed almost the same concentra‐ tions of SA, around 3.3 μg/mg dry wt. These results are in good agreement with the previous reports [18]. The correlation between results from ELISA and HPLC is reasonable except for Kinmon Daio. The concentration analyzed by HPLC was very low compared to the others. The reason is still obscure although individual peaks separated by HPLC were

**ELISA HPLC**

**Sample Concentration (μg/mg dry wt. powder)**

Shinshu Daio 13.69±0.69 12.28±0.41 Ga-wo 6.62±0.42 6.93±0.02 Kinmon Daio 3.34±0.02 0.85±0.04 Itto-Ga-wo (powder) 3.27±0.20 3.69±0.32 Itto-Ga-wo (refuse) 3.43±0.16 3.69±0.28

**Table 2.** SA concentrations in various rhubarb samples. Data are the means of triplicate assays.

determination of SA and SB in various samples are described [19].

**3. Production of MAbs against SB, their characterization and use for**

SB is a very important natural bioactive component of rhubarb and senna as well as SA. To‐ tal sennoside (SA and SB) concentrations are important, when rhubarb and senna are used as a raw material of medical supply and traditional Japanese herbal medicine for the purga‐

A number of methods for the quantification of SB have been published, most of which have been performed by HPLC [17]. Immunological approaches for assaying quantities of senno‐ sides and SA using PAb and MAb have been investigated by Atzorn *et al*. [5] and by us [14], respectively. However, no success with MAb against SB has been reported. In here, produc‐ tion of anti-SB MAb and the competitive ELISA using anti-SA and SB MAbs for the direct

Samples of various rhubarb roots were purchased from the Tochimototenkaido Corporation (Osaka, Japan). Samples of leaves of *Cassia* plants were collected in Thailand. Traditional Japanese prescriptions were procured from Tsumura & Co. (Tokyo, Japan). Dietary supple‐ ments (health teas and dieter's teas) were purchased from drug and department stores.

analyzed by ELISA.

12 Latest Research into Quality Control

**ELISA**

**3.1. Preface**

tive effect.

**3.2. Experimental**

*3.2.1. Plant materials*

Dried samples (30 mg) of various rhubarb roots, *Cassia* plant leaves, traditional Japanese prescriptions and dietary supplements were powdered, and then extracted five times with MeOH containing 0.1% (w/v) NH4OH (0.5 mL) with sonication, filtered using a Cosmonice Filter W (0.45 μm Filter Unit, Nacalai Tesque Inc., Kyoto, Japan), and the combined extracts were diluted with 10 mM NaHCO3 to prepare a solution suitable for the ELISA.

#### *3.2.3. Synthesisi of antigen conjugates*

To SB (6 mg) dissolved in 1 mL of tetrahydrofuran-20 mM phosphate buffer of pH 5.5 (7:3), 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of EDC was added. Then, 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of BSA was added, with stirring at room temperature for 14 hr. The reaction mixture was dialyzed five times against H2O, and then lyophilized to give 5.5 mg of SB-BSA conjugate. SB-HSA conjugate was also synthe‐ sized in the same manner.

#### *3.2.4. Determination of hapten density in SB-carrier protein conjugate by MALDI-TOF mass spectrometry*

The hapten number in the SB-carrier protein conjugate was determined by MALDI-TOF mass spectrometry as previously described [15].

#### *3.2.5. Competitive ELISA for SB*

SB-HSA (four molecules of SB per molecule of HSA) (100 μL, 1 μg/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate then treated with 300 μL S-PBS for 1 hr to reduce non-specific adsorption. Fifty μL of various concentra‐ tions of SB or samples dissolved in 10 mM NaHCO3 solution were incubated with 50 μL of MAb solution (0.121 μg/mL) for 1 hr. The plate was washed three times with T-PBS, and then incubated with 100 μL of a 1:1000 dilution of POD-labeled anti-mouse IgG for 1 hr. After washing the plate three times with T-PBS, 100 μL of substrate solution [0.1 M citrate buffer (pH 4) containing 0.003% H2O2 and 0.3 mg/mL of ABTS] was added to each well and incubat‐ ed for 15 min. The absorbance was measured by a micro plate reader at 405 nm and 490 nm.

#### **3.3. Results and discussion**

#### *3.3.1. Direct determination of SB-carrier protein conjugate by MALDI-TOF mass spectrometry*

It is well known that hapten number in an antigen conjugate is important for immunization against low molecular weight compounds. Figure 7 shows the MALDI-TOF mass spectrum of the antigen, SB-BSA conjugate. A broad peak coinciding with the conjugate of SB and BSA appeared from *m/z* 67,300 to 70,700 centering at around *m/z* 68,900. Using experimental results and a molecular weight of 66,433 for BSA, the calculated values of SB component (MW 862) are 2,500 resulting in the range of one to five molecules of SB (three on average) conjugated with BSA. This conjugate, although having a relatively low hapten number, proved sufficiently immunogenic in agreement with our previous results [10]. The number of SB contained in the SB-HSA conjugate was also determined to be around four molecules by its spectrum.

**Figure 8.** Calibration curve for SB.

investigate stereochemical recognition precisely.

SB is a unique anthraquinone having individual double-carboxylic acid-, hydroxyl-, carbon‐ yl- and *O*-glucosyl-groups at C-3, C-1, C-9 and C-8 positions in the molecule, respectively. Moreover, SB possesses an *erythro*-configuration between C-10 and C-10' positions. There‐ fore, MAbs should distinguish all these functional groups, and also recognize the stereo‐ chemistry of this complicated compound. Since the newly established ELISA against SB is expected to be used for phytochemical investigations involving crude plant extracts, the as‐ say specificity was checked by determining the cross-reactivities of the MAbs with various related compounds. The cross-reactivities of MAbs were examined by the competitive ELI‐ SA. Table 3 indicates the cross-reactivities of anti-SB MAbs against related anthraquinone, anthrone and phenol carboxylic acid. MAb 7H12 has weak cross-reactivities with SA (2.45%) and rhein (0.012%). However, the other related anthraquinone and anthrone did not have appreciable cross-reactivities. From these results it is suggested that the epitope consists of a basal structure of rhein and sugar moiety. In addition the most important property of MAb 7H12 is its ability to distinguish between SB and SA, which differ only in the stereochemical configuration at the C-10 and C-10' positions. Therefore, it is suggested that *erythro*-configu‐ rational structure of bisanthrone is indispensable as an immunodominant molecule for the reactivity of MAb 7H12. So the ELISA using a MAb 7H12 possesses apparently high sensi‐ tivity and specificity for SB. Because we have also prepared an anti-SA MAb having a weak cross-reactivity with SB (0.28%) as already discussed, these two MAbs make it possible to

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**Figure 7.** Direct determination of SB-BSA by MALDI-TOF MS.

#### *3.3.2. Production and characteristics of Mabs against SB*

The immunized BALB/c mice yielded splenocytes which were fused with P3-X63-Ag8-653 myeloma cells by the routinely established procedure in this laboratory [6]. Hybridoma pro‐ ducing MAbs reactive to SB were obtained, and classified as IgG1 (5G6, 7H12) and IgG2b (5C7) which had *k* light chains. The reactivity of IgG type MAb 7H12 was tested by varying the antibody concentration and by performing a dilution curve in direct ELISA. The anti‐ body concentration (0.121 μg/mL) at which the absorbance was about 1.0 in direct ELISA was selected for competitive ELISA.

#### *3.3.3. Assay sensitivity and assay specificity*

The free MAb 7H12 following competition was bound to the polystyrene microtitre plates precoated with SB-HSA. Under these conditions, the full measuring range of the assay ex‐ tends from 0.5 ng/mL to 15 ng/mL as indicated in Figure 8 and the ELISA using a MAb 7H12 is more sensitive than those using MAb 5C7 and 5G6.

**Figure 8.** Calibration curve for SB.

(MW 862) are 2,500 resulting in the range of one to five molecules of SB (three on average) conjugated with BSA. This conjugate, although having a relatively low hapten number, proved sufficiently immunogenic in agreement with our previous results [10]. The number of SB contained in the SB-HSA conjugate was also determined to be around four molecules

The immunized BALB/c mice yielded splenocytes which were fused with P3-X63-Ag8-653 myeloma cells by the routinely established procedure in this laboratory [6]. Hybridoma pro‐ ducing MAbs reactive to SB were obtained, and classified as IgG1 (5G6, 7H12) and IgG2b (5C7) which had *k* light chains. The reactivity of IgG type MAb 7H12 was tested by varying the antibody concentration and by performing a dilution curve in direct ELISA. The anti‐ body concentration (0.121 μg/mL) at which the absorbance was about 1.0 in direct ELISA

The free MAb 7H12 following competition was bound to the polystyrene microtitre plates precoated with SB-HSA. Under these conditions, the full measuring range of the assay ex‐ tends from 0.5 ng/mL to 15 ng/mL as indicated in Figure 8 and the ELISA using a MAb 7H12

by its spectrum.

14 Latest Research into Quality Control

**Figure 7.** Direct determination of SB-BSA by MALDI-TOF MS.

was selected for competitive ELISA.

*3.3.3. Assay sensitivity and assay specificity*

is more sensitive than those using MAb 5C7 and 5G6.

*3.3.2. Production and characteristics of Mabs against SB*

SB is a unique anthraquinone having individual double-carboxylic acid-, hydroxyl-, carbon‐ yl- and *O*-glucosyl-groups at C-3, C-1, C-9 and C-8 positions in the molecule, respectively. Moreover, SB possesses an *erythro*-configuration between C-10 and C-10' positions. There‐ fore, MAbs should distinguish all these functional groups, and also recognize the stereo‐ chemistry of this complicated compound. Since the newly established ELISA against SB is expected to be used for phytochemical investigations involving crude plant extracts, the as‐ say specificity was checked by determining the cross-reactivities of the MAbs with various related compounds. The cross-reactivities of MAbs were examined by the competitive ELI‐ SA. Table 3 indicates the cross-reactivities of anti-SB MAbs against related anthraquinone, anthrone and phenol carboxylic acid. MAb 7H12 has weak cross-reactivities with SA (2.45%) and rhein (0.012%). However, the other related anthraquinone and anthrone did not have appreciable cross-reactivities. From these results it is suggested that the epitope consists of a basal structure of rhein and sugar moiety. In addition the most important property of MAb 7H12 is its ability to distinguish between SB and SA, which differ only in the stereochemical configuration at the C-10 and C-10' positions. Therefore, it is suggested that *erythro*-configu‐ rational structure of bisanthrone is indispensable as an immunodominant molecule for the reactivity of MAb 7H12. So the ELISA using a MAb 7H12 possesses apparently high sensi‐ tivity and specificity for SB. Because we have also prepared an anti-SA MAb having a weak cross-reactivity with SB (0.28%) as already discussed, these two MAbs make it possible to investigate stereochemical recognition precisely.


*3.3.5. Determination of concentrations of SA and SB in various Cassia species*

**Sample Concentration (μg/mg dry wt. powder)**

*Cassia angustifolia* 4.56±0.25 5.10±0.15 9.66±0.40 *C. alata* 1.19±0.12 1.16±0.15 2.35±0.27 *C. bakeriana* 0.40±0.03 0.44±0.02 0.84±0.05 *C. fistula* (A) 1.14±0.08 0.75±0.08 1.89±0.16 *C. fistula* (B) 2.04±0.32 1.52±0.12 3.56±0.44 *C. fistula* (C) 1.90±0.16 2.05±0.24 3.95±0.40 *C. fistula* (D) 0.10±0.01 0.13±0.00 0.23±0.01 *C. mimosoides* (1.30±0.24)×10-2 (1.88±0.29)×10-4 (1.32±0.24)×10-2 *C. floribunda* (2.78±0.11)×10-3 (1.04±0.03)×10-4 (2.88±0.11)×10-3 *C. surattensis* (1.15±0.18)×10-2 (2.44±0.17)×10-4 (1.17±0.18)×10-2 *C. tora* (2.13±0.21)×10-3 (3.64±0.21)×10-5 (2.17±0.23)×10-3 *C. siamea* (4.45±0.14)×10-3 (1.87±0.13)×10-3 (6.32±0.27)×10-3

**Table 5.** Total sennoside concentrations in leaves of various *Cassia* species. Data are the means of triplicate assays.

Thin-layer chromatography (TLC) is most widely used for detection, separation and moni‐ toring of small molecular compounds like sennosides. If the direct TLC immunostaining with MAb can be done, this procedure must be contributive to the development of structur‐ al analysis of small molecular compounds. However, this procedure cannot be used for the direct detection of small molecular compounds on a TLC plate because the silica gel is sloughed off from the plate and the compounds on the plate are easily washed out without fixing during treatment. If the compounds are transferred from the TLC plate to a plastic

**4. Establishments of a new eastern blotting, double staining and immunohistochemical staining using anti-SA and SB MAbs**

powder of SB, respectively.

**4.1. Preface**

The concentrations of SA and SB in leaves of various *Cassia* species were determined by ELISA using anti-SA and SB MAbs (Table 5). The results indicate that *C. angustifolia* con‐ tains 4.56±0.25 μg/mg dry wt. powder of SA and 5.10±0.15 μg/mg dry wt. powder of SB indicating higher amounts of SA and SB compared to the other species. *C. alata* contains 1.19±0.12 μg/mg dry wt. powder of SA and 1.16±0.15 μg/mg dry wt. powder of SB. *C. fistula* (A)~(D) contain 0.10-2.04 μg/mg dry wt. powder of SA and 0.13-2.05 μg/mg dry wt.

**Sennoside A Sennoside B Total sennosides**

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**Table 3.** Cross-reactivities of anti-SB MAbs against various compounds.

#### *3.3.4. Correlation of results of SB determination in crude extracts of rhubarb roots between HPLC and ELISA using MAb 7H12*

The concentrations of SB in various rhubarb samples were determined by ELISA (Table 4). Shinshu Daio, bred by crossing *R. palmatum* and *R. coreanum* in order to increase the level of SB concentration in Japan, contained the highest SB level of 6.01±0.18 μg/mg dry wt. Ga-wo, estimated to be high grade in the traditional Japanese medicine, contained SB level of 3.14±0.27 μg/mg dry wt. These results are in good agreement with previous reports [18]. The correlation between results from ELISA and HPLC is also good.


**Table 4.** SB concentrations in various rhubarb samples. Data are the means of triplicate assays.

#### *3.3.5. Determination of concentrations of SA and SB in various Cassia species*

The concentrations of SA and SB in leaves of various *Cassia* species were determined by ELISA using anti-SA and SB MAbs (Table 5). The results indicate that *C. angustifolia* con‐ tains 4.56±0.25 μg/mg dry wt. powder of SA and 5.10±0.15 μg/mg dry wt. powder of SB indicating higher amounts of SA and SB compared to the other species. *C. alata* contains 1.19±0.12 μg/mg dry wt. powder of SA and 1.16±0.15 μg/mg dry wt. powder of SB. *C. fistula* (A)~(D) contain 0.10-2.04 μg/mg dry wt. powder of SA and 0.13-2.05 μg/mg dry wt. powder of SB, respectively.


**Table 5.** Total sennoside concentrations in leaves of various *Cassia* species. Data are the means of triplicate assays.

### **4. Establishments of a new eastern blotting, double staining and immunohistochemical staining using anti-SA and SB MAbs**

#### **4.1. Preface**

**Compound**

16 Latest Research into Quality Control

**Stilbene**

**Phenol carboxylic acid**

*and ELISA using MAb 7H12*

**Anthraquinone and anthrone**

**Cross-reactivities (%)**

**7H12 5G6 5C7**

sennoside B 100 100 100 sennoside A 2.45 2.30 8.53 rhein 0.012 0.030 0.007 emodin < 0.004 < 0.023 < 0.006 aloe-emodin < 0.040 < 0.023 < 0.006 barbaloin < 0.004 < 0.023 < 0.006 1,4-dihydroxy-anthraquinone < 0.004 < 0.023 < 0.006

rhaponticin < 0.004 < 0.023 < 0.006

gallic acid < 0.004 < 0.023 < 0.006 vanillic acid < 0.004 < 0.023 < 0.006 caffeic acid < 0.004 < 0.023 < 0.006 homogentisic acid < 0.004 < 0.023 < 0.006

*3.3.4. Correlation of results of SB determination in crude extracts of rhubarb roots between HPLC*

The concentrations of SB in various rhubarb samples were determined by ELISA (Table 4). Shinshu Daio, bred by crossing *R. palmatum* and *R. coreanum* in order to increase the level of SB concentration in Japan, contained the highest SB level of 6.01±0.18 μg/mg dry wt. Ga-wo, estimated to be high grade in the traditional Japanese medicine, contained SB level of 3.14±0.27 μg/mg dry wt. These results are in good agreement with previous reports [18]. The

**ELISA HPLC**

**Table 3.** Cross-reactivities of anti-SB MAbs against various compounds.

correlation between results from ELISA and HPLC is also good.

**Sample Concentration (μg/mg dry wt. powder)**

Shinshu Daio 6.01±0.18 6.15±0.59 Ga-wo 3.14±0.27 3.80±0.16 Kinmon Daio 0.35±0.01 0.38±0.02 Itto-Ga-wo (powder) 1.44±0.12 1.52±0.18 Itto-Ga-wo (refuse) 1.42±0.07 1.40±0.11

**Table 4.** SB concentrations in various rhubarb samples. Data are the means of triplicate assays.

Thin-layer chromatography (TLC) is most widely used for detection, separation and moni‐ toring of small molecular compounds like sennosides. If the direct TLC immunostaining with MAb can be done, this procedure must be contributive to the development of structur‐ al analysis of small molecular compounds. However, this procedure cannot be used for the direct detection of small molecular compounds on a TLC plate because the silica gel is sloughed off from the plate and the compounds on the plate are easily washed out without fixing during treatment. If the compounds are transferred from the TLC plate to a plastic membrane with hydrophobic properties and immobilized on the membrane, these difficul‐ ties can be solved. Therefore, I examined the transfer of sennosides from a TLC plate to a plastic membrane. Towbin *et al*. first reported the transfer of glycosphingolipids using nitro‐ cellulose membranes [20]. However, since its transfer efficiency was poor and reproducible results were not obtained, I tested various plastic membranes and transfer conditions result‐ ing in a polyvinylidene difluoride (PVDF) membrane to be the best [21]. The membrane is very stable against heating and various organic solvents in addition to retaining sennosides with high efficiency. I named this new method as eastern blotting (EB), because theoretically same methodology compared to previous EB except the way of sennoside-BSA conjugation for fixing sennosides on the membrane [22]. I communicate here the EB procedure for sen‐ nosides and its application for analytical survey of sennosides [23].

#### **4.2. Experimental**

#### *4.2.1. Chemicals and immunochemicals*

Polyvinylidene difluoride (PVDF) membranes (Immobilon-N) were purchased from Milli‐ pore Corporation (Bedford, MA, USA). Glass microfiber filter sheets (GF/A) were purchased from Whatman International Ltd. (Maidstone, England). All other chemicals were standard commercial products of analytical grade.

#### *4.2.2. EB and Double staining*

Sennosides were applied to a TLC plate and developed with 1-propanol-ethyl acetate-wateracetic acid (40:40:30:1, by volume). The developed TLC plate was dried and then sprayed with a blotting solution mixture of isopropanol-methanol-water (1:4:16, by volume). It was placed on a stainless steel plate and then covered with a PVDF membrane sheet. After cov‐ ering with a glass microfiber filter sheet, the whole assembly was pressed evenly for 70 s with a 120 ˚C hot plate as previously described with some modifications [24, 25]. The PVDF membrane was separated from the TLC plate and dried.

**Figure 9.** Eastern blotting protocol.

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**Figure 10.** Double staining protocol.

The blotted PVDF membrane was dipped in 20 mM carbonate buffer solution (pH 9.6) con‐ taining BSA (1%) and EDC (20 mg/mL), and stirred at room temperature for 14 hr. After washing the PVDF membrane twice with T-PBS for 5 min and then treated with S-PBS for 3 hr to reduce non-specific adsorption. The PVDF membrane was washed with T-PBS twice for 5 min, and then immersed in anti-SA MAb (6G8) and stirred at room temperature for 3 hr. After washing the PVDF membrane twice with T-PBS for 5 min, a 1:1000 dilution of POD-labeled goat anti-mouse IgG in PBS cotaining 0.2% of gelatin (G-PBS) was added and stirred at room temperature for 1 hr. The PVDF membrane was washed twice with T-PBS and water, then exposed to 1 mg/mL 4-chloro-1-naphtol-0.03% H2O2 in PBS solution which was freshly prepared before use for 10 min at room temperature. The protocol of the EB technique is shown in Figure 9.

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**Figure 9.** Eastern blotting protocol.

membrane with hydrophobic properties and immobilized on the membrane, these difficul‐ ties can be solved. Therefore, I examined the transfer of sennosides from a TLC plate to a plastic membrane. Towbin *et al*. first reported the transfer of glycosphingolipids using nitro‐ cellulose membranes [20]. However, since its transfer efficiency was poor and reproducible results were not obtained, I tested various plastic membranes and transfer conditions result‐ ing in a polyvinylidene difluoride (PVDF) membrane to be the best [21]. The membrane is very stable against heating and various organic solvents in addition to retaining sennosides with high efficiency. I named this new method as eastern blotting (EB), because theoretically same methodology compared to previous EB except the way of sennoside-BSA conjugation for fixing sennosides on the membrane [22]. I communicate here the EB procedure for sen‐

Polyvinylidene difluoride (PVDF) membranes (Immobilon-N) were purchased from Milli‐ pore Corporation (Bedford, MA, USA). Glass microfiber filter sheets (GF/A) were purchased from Whatman International Ltd. (Maidstone, England). All other chemicals were standard

Sennosides were applied to a TLC plate and developed with 1-propanol-ethyl acetate-wateracetic acid (40:40:30:1, by volume). The developed TLC plate was dried and then sprayed with a blotting solution mixture of isopropanol-methanol-water (1:4:16, by volume). It was placed on a stainless steel plate and then covered with a PVDF membrane sheet. After cov‐ ering with a glass microfiber filter sheet, the whole assembly was pressed evenly for 70 s with a 120 ˚C hot plate as previously described with some modifications [24, 25]. The PVDF

The blotted PVDF membrane was dipped in 20 mM carbonate buffer solution (pH 9.6) con‐ taining BSA (1%) and EDC (20 mg/mL), and stirred at room temperature for 14 hr. After washing the PVDF membrane twice with T-PBS for 5 min and then treated with S-PBS for 3 hr to reduce non-specific adsorption. The PVDF membrane was washed with T-PBS twice for 5 min, and then immersed in anti-SA MAb (6G8) and stirred at room temperature for 3 hr. After washing the PVDF membrane twice with T-PBS for 5 min, a 1:1000 dilution of POD-labeled goat anti-mouse IgG in PBS cotaining 0.2% of gelatin (G-PBS) was added and stirred at room temperature for 1 hr. The PVDF membrane was washed twice with T-PBS and water, then exposed to 1 mg/mL 4-chloro-1-naphtol-0.03% H2O2 in PBS solution which was freshly prepared before use for 10 min at room temperature. The protocol of the EB

nosides and its application for analytical survey of sennosides [23].

**4.2. Experimental**

18 Latest Research into Quality Control

*4.2.1. Chemicals and immunochemicals*

commercial products of analytical grade.

membrane was separated from the TLC plate and dried.

*4.2.2. EB and Double staining*

technique is shown in Figure 9.

**Figure 10.** Double staining protocol.

For successive staining by anti-SB MAb (7H12), the PVDF membrane stained by anti-SA MAb was treated in the same way as anti-SA MAb (6G8) except that it was exposed to 2 mg/10 mL 3-amino-9-ethylcarbazole-0.03% H2O2 in acetate buffer (0.05 M, pH 5.0) containing 0.5 mL of *N,N*-dimethyl formamide. The protocol of double staining is shown in Figure 10.

#### *4.2.3. EB for immunohistochemical staining of SA*

A piece of PVDF membrane was placed on a glass microfiber filter sheet. A sliced fresh rhubarb root was placed on the PVDF membrane, and they were pressed together evenly for 1 hr. The blotted PVDF membrane was stained using the same procedure described for the EB method.

#### **4.3. Results and discussion**

#### *4.3.1. EB of SA using anti-SA MAb*

Previously we established a new immunostaining method named as eastern blotting for sev‐ eral glycosides like solasodine glycosides [21], ginsenosides [26, 27] and glycyrrhizin [22, 28] by using individual MAbs. In this methodology we separated the sugar moiety in a mole‐ cule into two functions, the epitope part and fixation ability part on a membrane after blot‐ ted to a PVDF membrane from a TLC plate, since small molecular compounds can not be fixed on the membrane. Although I followed the previous methodology for SA, unfortunate‐ ly staining was not succeeded. Therefore, a new blotting method onto a PVDF membrane from the developed TLC plate is required. SA was transferred to the PVDF membrane by the same way as previously described, and treated with EDC solution followed by the addi‐ tion of BSA as indicated in Figure 9. This reaction enhanced the fixation of SA via SA-BSA conjugate on the PVDF membrane and the pathway was indicated diagrammatically in Fig‐ ure 11. When the blotted PVDF membrane was incubated in the absence of EDC, it was es‐ sentially free of immunostaining (data not shown).

**Figure 11.** Schematic diagram illustrating the eastern blotting of SA onto the PVDF membrane and the detection us‐

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**Figure 12.** Eastern blotting of sennosides and related compounds stained by anti-SA MAb (A). B shows a TLC plate stained by 10% H2SO4. Lanes 1, 2, 3, 4, 5, 6 and 7 indicate rhaponticin, barbaloin, aloe-emodin, emodin, rhein, SB and

ing anti-SA MAb.

SA (3 μg), respectively.

Figure 12 shows the EB of sennosides and other structurally related compounds using anti-SA MAb (A) and the H2SO4 staining (B). The EB indicated only limited staining of SA as shown in Figure 12A, lane 7. Moreover, the EB method was considerably more sensitive than that of H2SO4 staining. Since anti-SA MAb cross-reacts against SB and rhein as 0.28 and 0.35%, respectively, they can be stained very weakly by anti-SA MAb, as described in the previous section. Previously Fukuda *et al.* succeeded the EB of ginsenoside Rb1 by using an‐ ti-ginsenoside Rb1 MAb resulting in staining together with ginsenoside Rc, Rd, Re and Rg1 [26, 27]. The difference between the newly established EB and the previous methodology is combine system of sugar moiety to PVDF membrane. The sugar moiety in ginsenosides was oxidatively cleavaged to release aldehyde groups which were conjugated with a protein to fix on a PVDF membrane. Since it was evident that a part of sugar moiety in ginsenoside Rb1 was immunized, the cleavage of sugar moiety by NaIO4 expanded its cross-reactivity against other ginsenosides resulting in possibility of staining for ginsenoside Rc, Rd, Re and Rg1, though their cross reactivities are weak. On the other hand, the newly established EB in here does not hinder around sugar moiety in SA. Therefore, strength of staining for SA, SB and rhein was proportional to their cross-reactivities as described in ELISA.

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For successive staining by anti-SB MAb (7H12), the PVDF membrane stained by anti-SA MAb was treated in the same way as anti-SA MAb (6G8) except that it was exposed to 2 mg/10 mL 3-amino-9-ethylcarbazole-0.03% H2O2 in acetate buffer (0.05 M, pH 5.0) containing 0.5 mL of

A piece of PVDF membrane was placed on a glass microfiber filter sheet. A sliced fresh rhubarb root was placed on the PVDF membrane, and they were pressed together evenly for 1 hr. The blotted PVDF membrane was stained using the same procedure described for the EB method.

Previously we established a new immunostaining method named as eastern blotting for sev‐ eral glycosides like solasodine glycosides [21], ginsenosides [26, 27] and glycyrrhizin [22, 28] by using individual MAbs. In this methodology we separated the sugar moiety in a mole‐ cule into two functions, the epitope part and fixation ability part on a membrane after blot‐ ted to a PVDF membrane from a TLC plate, since small molecular compounds can not be fixed on the membrane. Although I followed the previous methodology for SA, unfortunate‐ ly staining was not succeeded. Therefore, a new blotting method onto a PVDF membrane from the developed TLC plate is required. SA was transferred to the PVDF membrane by the same way as previously described, and treated with EDC solution followed by the addi‐ tion of BSA as indicated in Figure 9. This reaction enhanced the fixation of SA via SA-BSA conjugate on the PVDF membrane and the pathway was indicated diagrammatically in Fig‐ ure 11. When the blotted PVDF membrane was incubated in the absence of EDC, it was es‐

Figure 12 shows the EB of sennosides and other structurally related compounds using anti-SA MAb (A) and the H2SO4 staining (B). The EB indicated only limited staining of SA as shown in Figure 12A, lane 7. Moreover, the EB method was considerably more sensitive than that of H2SO4 staining. Since anti-SA MAb cross-reacts against SB and rhein as 0.28 and 0.35%, respectively, they can be stained very weakly by anti-SA MAb, as described in the previous section. Previously Fukuda *et al.* succeeded the EB of ginsenoside Rb1 by using an‐ ti-ginsenoside Rb1 MAb resulting in staining together with ginsenoside Rc, Rd, Re and Rg1 [26, 27]. The difference between the newly established EB and the previous methodology is combine system of sugar moiety to PVDF membrane. The sugar moiety in ginsenosides was oxidatively cleavaged to release aldehyde groups which were conjugated with a protein to fix on a PVDF membrane. Since it was evident that a part of sugar moiety in ginsenoside Rb1 was immunized, the cleavage of sugar moiety by NaIO4 expanded its cross-reactivity against other ginsenosides resulting in possibility of staining for ginsenoside Rc, Rd, Re and Rg1, though their cross reactivities are weak. On the other hand, the newly established EB in here does not hinder around sugar moiety in SA. Therefore, strength of staining for SA, SB

and rhein was proportional to their cross-reactivities as described in ELISA.

*N,N*-dimethyl formamide. The protocol of double staining is shown in Figure 10.

*4.2.3. EB for immunohistochemical staining of SA*

sentially free of immunostaining (data not shown).

**4.3. Results and discussion**

20 Latest Research into Quality Control

*4.3.1. EB of SA using anti-SA MAb*

**Figure 11.** Schematic diagram illustrating the eastern blotting of SA onto the PVDF membrane and the detection us‐ ing anti-SA MAb.

**Figure 12.** Eastern blotting of sennosides and related compounds stained by anti-SA MAb (A). B shows a TLC plate stained by 10% H2SO4. Lanes 1, 2, 3, 4, 5, 6 and 7 indicate rhaponticin, barbaloin, aloe-emodin, emodin, rhein, SB and SA (3 μg), respectively.

#### *4.3.2. Double staining of sennosides using anti-SA and SB MAbs*

Previously, I used 4-chloro-1-naphthol for staining of SB. However, since it could not func‐ tion well for SB, the combination of 4-chloro-1-naphthol and 3-amino-9-ethylcarbazole was selected to improve double staining of sennosides as indicated in Figure 10. SA and SB were stained clearly by the purple and red color, respectively (Figure 13). From this result both antibodies can distinguish stereochemical configurations, *threo* and *erythro* between C-10 and C-10' positions in a molecule on PVDF membrane stained as double coloring, respectively.

ration from its red color. The *R*f value clearly showed that band 2 includes one sugar moiety having a HOOC-CO group. From these results I supposed that band 2 is sennoside F (SF) that has *erythro*-configuration as indicated previously [1]. The double staining by EB indi‐ cates that *C. angustifolia*, *C. alata*, *C. bakeriana* and *C. fistula* contain a higher concentration of sennosides compared to the other species. This result has a good agreement with that of ELI‐ SA. The limit of detection by the double staining method was confirmed to be 48 μg/mL of

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**Figure 14.** Double staining of SA and SB in various *Cassia* species (A). B shows a result of H2SO4 staining. Lefthand lane

As an other application of the EB method, the immunohistochemical staining of SA in rhu‐ barb root, was investigated. A sliced fresh rhubarb root was placed on the PVDF membrane, and they were pressed together evenly for 1 hr. The blotted PVDF membrane was stained using the same procedure described for the EB method. Figure 15II illustrates the immuno‐ histochemical staining of SA in fresh Hokkai Daio root. The phloem and cambium contained a higher concentration of SA compared to other tissues, pith and bud. To confirm this result, I analyzed these tissues individually by ELISA and HPLC. The concentrations of SA were determined by ELISA to determine 64.4±4.5, 48.1±8.2, 15.0±1.6 and 1.8±0.3 ng/mg fresh wt. in phloem, cambium, pith and bud, respectively. This result was a good agreement with those of HPLC resulting in 58.4±2.6, 49.0±3.9 and 13.3±0.5 ng/mg fresh wt. in phloem, cambium

indicates SA (4 μg)and SB (3 μg). Lanes 1~12 indicate various *Cassia* species (3 μL).

*4.3.4. Validation of EB for immunohistochemical staining of SA*

both SA and SB.

and pith, respectively.

**Figure 13.** Double staining of sennosides using eastern blotting technique (A). B shows a result of H2SO4 staining. Red and purple colors were stained by anti-SB and SA MAb, respectively.

#### *4.3.3. Detection of SA and SB in various Cassia species using double staining with a new EB technique*

The crude extracts of various *Cassia* species were analyzed by the newly developed double staining system and TLC stained with H2SO4 as shown in Figure 14. Although H2SO4 stain‐ ing (Figure 14B) detected many spots including probably sugars and different types of an‐ thraquinone glycosides in various *Cassia* species, double staining (Figure 14A) detected clearly SA and SB, and very weakly other sennosides except appearance of chlorophylls around top. Band 1 indicated a purple color that means a *threo-*configuration between C-10 and C-10' positions detected by EB using anti-SA MAb as shown in Figure 14A. Moreover, its *R*f value indicated that band 1 has one sugar moiety and a CH2OH group instead of COOH group in a molecule. I surveyed the previous papers regarding sennosides in senna [1]. Judging from these evidences, I suggested that band 1 is sennoside C (SC) having *threo*configuration as indicated previously [1]. Band 2 was easily suggested to be *erythro*-configu‐ ration from its red color. The *R*f value clearly showed that band 2 includes one sugar moiety having a HOOC-CO group. From these results I supposed that band 2 is sennoside F (SF) that has *erythro*-configuration as indicated previously [1]. The double staining by EB indi‐ cates that *C. angustifolia*, *C. alata*, *C. bakeriana* and *C. fistula* contain a higher concentration of sennosides compared to the other species. This result has a good agreement with that of ELI‐ SA. The limit of detection by the double staining method was confirmed to be 48 μg/mL of both SA and SB.

**Figure 14.** Double staining of SA and SB in various *Cassia* species (A). B shows a result of H2SO4 staining. Lefthand lane indicates SA (4 μg)and SB (3 μg). Lanes 1~12 indicate various *Cassia* species (3 μL).

#### *4.3.4. Validation of EB for immunohistochemical staining of SA*

*4.3.2. Double staining of sennosides using anti-SA and SB MAbs*

22 Latest Research into Quality Control

and purple colors were stained by anti-SB and SA MAb, respectively.

*technique*

Previously, I used 4-chloro-1-naphthol for staining of SB. However, since it could not func‐ tion well for SB, the combination of 4-chloro-1-naphthol and 3-amino-9-ethylcarbazole was selected to improve double staining of sennosides as indicated in Figure 10. SA and SB were stained clearly by the purple and red color, respectively (Figure 13). From this result both antibodies can distinguish stereochemical configurations, *threo* and *erythro* between C-10 and C-10' positions in a molecule on PVDF membrane stained as double coloring, respectively.

**Figure 13.** Double staining of sennosides using eastern blotting technique (A). B shows a result of H2SO4 staining. Red

The crude extracts of various *Cassia* species were analyzed by the newly developed double staining system and TLC stained with H2SO4 as shown in Figure 14. Although H2SO4 stain‐ ing (Figure 14B) detected many spots including probably sugars and different types of an‐ thraquinone glycosides in various *Cassia* species, double staining (Figure 14A) detected clearly SA and SB, and very weakly other sennosides except appearance of chlorophylls around top. Band 1 indicated a purple color that means a *threo-*configuration between C-10 and C-10' positions detected by EB using anti-SA MAb as shown in Figure 14A. Moreover, its *R*f value indicated that band 1 has one sugar moiety and a CH2OH group instead of COOH group in a molecule. I surveyed the previous papers regarding sennosides in senna [1]. Judging from these evidences, I suggested that band 1 is sennoside C (SC) having *threo*configuration as indicated previously [1]. Band 2 was easily suggested to be *erythro*-configu‐

*4.3.3. Detection of SA and SB in various Cassia species using double staining with a new EB*

As an other application of the EB method, the immunohistochemical staining of SA in rhu‐ barb root, was investigated. A sliced fresh rhubarb root was placed on the PVDF membrane, and they were pressed together evenly for 1 hr. The blotted PVDF membrane was stained using the same procedure described for the EB method. Figure 15II illustrates the immuno‐ histochemical staining of SA in fresh Hokkai Daio root. The phloem and cambium contained a higher concentration of SA compared to other tissues, pith and bud. To confirm this result, I analyzed these tissues individually by ELISA and HPLC. The concentrations of SA were determined by ELISA to determine 64.4±4.5, 48.1±8.2, 15.0±1.6 and 1.8±0.3 ng/mg fresh wt. in phloem, cambium, pith and bud, respectively. This result was a good agreement with those of HPLC resulting in 58.4±2.6, 49.0±3.9 and 13.3±0.5 ng/mg fresh wt. in phloem, cambium and pith, respectively.

ces of sennosides and quickly determine their structures. Furthermore, EB also can be used for the survey of distribution of SA and/or SB in the *Rheum* specimen by immunohistochem‐

Quality Control of *Rheum* and *Cassia* Species by Immunological Methods...

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25

We thank Dr. Hiroyuki Tanaka (Faculty of Pharmaceutical Sciences, Kyushu University) for useful suggestions in this work. This research was supported in part by Japan Science and Technology Agency, Grant-in-Aid from the Ministry of Education, Culture, Sports, Science

Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International

[1] Oshio, H., Naruse, Y., & Tsukui, M. (1978). Quantilative analysis of the purgative components of rhubarb and senna. *Chemical Pharmaceutical Bulletin*, 26, 2458-2464. [2] Yang, L., Akao, T., Kobashi, K., & Hattori, M. (1996). A sennoside-hydrolyzing β-glu‐ cosidase from Bifidobacterium sp.strain SEN is inducible. *Biological Pharmaceutical*

[3] Yang, L., Akao, T., Kobashi, K., & Hattori, M. (1996). Purification and characteriza‐ tion of a novel sennoside-hydrolyzing β-glucosidase from Bifidobacterium sp. strain SEN, a human intestinal anaerobe. *Biological Pharmaceutical Bulletin*, 19, 705-709.

[5] Atzorn, R., Weiler, E. W., & Zenk, M. H. (1981). Formation and distribution of senno‐ sides in Cassia angustifolia, as determined by a sensitive and specific radioimmuno‐

[6] Sakata, R., Shoyama, Y., & Murakami, H. (1994). Production of monoclonal antibod‐ ies and enzyme immunoassay for typical adenylate cyclase activator, forskolin. *Cyto‐*

[4] Japanese Pharmacopoeia (2011). The Japanese Pharmacopoeia, 16. th ed.; Jiho

and Technology of Japan, the research grant from Takeda Science Foundation.

ical staining.

**Acknowledgements**

**Author details**

University, Sasebo, Japan

*Bulletin*, 19, 701-704.

assay. *Planta Medica*, 41, 1-14.

*technology*, 16, 101-108.

**References**

Osamu Morinaga and Yukihiro Shoyama\*

\*Address all correspondence to: shoyama@niu.ac.jp

**Figure 15.** Immunohistochemical staining of SA using anti-SA MAb in rhubarb root. I, cross section of Hokkai Daio root; II, direct eastern blotting on PVDF membrane of a cross section of Hokkai Daio root. A, Phloem; B, Cambium; C, Pith; D, Bud, respectively.

#### **5. Conclusion**

The recent developments of molecular biosciences and their biotechnological applications have opened up many new avenues of pharmaceutical areas. MAbs have many potential uses in addition to immunological methods to plant sciences. Therefore, immunoassay sys‐ tem using MAbs against pharmacologically active natural products having low molecular weight have become an important tool for the studies on receptor binding analysis, enzyme assay, and quantitative and/or qualitative analytical techniques in plants owing to their spe‐ cific affinity.

In order to analyze the stereochemical isomers, SA and SB in plants, medicaments, prescrip‐ tions, health foods and patients'sera, I have produced MAbs against them. These MAbs have the most important ability to distinguish between SA and SB, which differ only in the stereochemical configuration at the C-10 and C-10' positions, respectively. Moreover, they have no detectable cross-reaction with the other related anthraquinone and anthrone.

Analytical systems of SA and SB by competitive ELISA using anti-SA and SB MAbs were established. These ELISA systems are capable of measuring SA and SB in complex matrics without any pretreatments. Furthermore, these ELISA methods are approximately 2,000 times for SA and 10,000 times for SB more sensitive than that of HPLC method.

The newly developed EB methodology can be theoretically expanded for all compounds having carboxylic acid such as phenol carboxylic acids, glucuronides, furthermore com‐ pounds having only a carboxylic group in a molecule. A new double staining with EB method for sennosides using anti-SA and SB MAbs was established. SA and SB were stained pur‐ ple and red color, respectively. This system visualized sennosides on a PVDF membrane. In fact, SA and SB in the crude extracts of various *Cassia* species were distinguished by their coloring and *R*f values. Moreover, it could make it possible to survey the natural resour‐ ces of sennosides and quickly determine their structures. Furthermore, EB also can be used for the survey of distribution of SA and/or SB in the *Rheum* specimen by immunohistochem‐ ical staining.

#### **Acknowledgements**

We thank Dr. Hiroyuki Tanaka (Faculty of Pharmaceutical Sciences, Kyushu University) for useful suggestions in this work. This research was supported in part by Japan Science and Technology Agency, Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the research grant from Takeda Science Foundation.

#### **Author details**

**Figure 15.** Immunohistochemical staining of SA using anti-SA MAb in rhubarb root. I, cross section of Hokkai Daio root; II, direct eastern blotting on PVDF membrane of a cross section of Hokkai Daio root. A, Phloem; B, Cambium; C,

The recent developments of molecular biosciences and their biotechnological applications have opened up many new avenues of pharmaceutical areas. MAbs have many potential uses in addition to immunological methods to plant sciences. Therefore, immunoassay sys‐ tem using MAbs against pharmacologically active natural products having low molecular weight have become an important tool for the studies on receptor binding analysis, enzyme assay, and quantitative and/or qualitative analytical techniques in plants owing to their spe‐

In order to analyze the stereochemical isomers, SA and SB in plants, medicaments, prescrip‐ tions, health foods and patients'sera, I have produced MAbs against them. These MAbs have the most important ability to distinguish between SA and SB, which differ only in the stereochemical configuration at the C-10 and C-10' positions, respectively. Moreover, they

Analytical systems of SA and SB by competitive ELISA using anti-SA and SB MAbs were established. These ELISA systems are capable of measuring SA and SB in complex matrics without any pretreatments. Furthermore, these ELISA methods are approximately 2,000

The newly developed EB methodology can be theoretically expanded for all compounds having carboxylic acid such as phenol carboxylic acids, glucuronides, furthermore com‐ pounds having only a carboxylic group in a molecule. A new double staining with EB method for sennosides using anti-SA and SB MAbs was established. SA and SB were stained pur‐ ple and red color, respectively. This system visualized sennosides on a PVDF membrane. In fact, SA and SB in the crude extracts of various *Cassia* species were distinguished by their coloring and *R*f values. Moreover, it could make it possible to survey the natural resour‐

have no detectable cross-reaction with the other related anthraquinone and anthrone.

times for SA and 10,000 times for SB more sensitive than that of HPLC method.

Pith; D, Bud, respectively.

24 Latest Research into Quality Control

**5. Conclusion**

cific affinity.

Osamu Morinaga and Yukihiro Shoyama\*

\*Address all correspondence to: shoyama@niu.ac.jp

Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Japan

#### **References**


[7] Ishiyama, M., Shoyama, Y., Murakami, H., & Shinohara, H. (1995). Production of monoclonal antibodies and development of an ELISA for solamargine. *Cytotechnolo‐ gy*, 18, 153-158.

[20] Towbin, H., Schoenenberger, C., Ball, R., Braun, D. G., & Rosenfelder, G. (1984). Gly‐ cosphingolipid-blotting: an immunological detection procedure after separation by

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[21] Tanaka, H., Putalun, W., Tsuzaki, C., & Shoyama, Y. (1997). A simple determination of steroidal alkaloid glycosides by thin-layer chromatography immunostaining using

[22] Shan, S. J., Tanaka, H., & Shoyama, Y. (2001). Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid. *Analytical Chemistry*, 73, 5784-5790.

[23] Morinaga, O., Uto, T., Sakamoto, S., Putalun, W., Lhieochaiphant, S., Tanaka, H., & Shoyama, Y. (2009). Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-sennoside B monoclonal antibodies.

[24] Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. *Proceedings of the National Academy of Sciences of the United States of America*, 76, 4350-4.

[25] Taki, K., Kasama, T., Handa, S., & Ishikawa, D. (1994). A simple and quantitative pu‐ rification of glycosphingolipids and phospholipids by thin-layer chromatography

[26] Fukuda, N., Tanaka, H., & Shoyama, Y. (1999). Western blotting for ginseng sapo‐ nins, ginsenosides using anti-ginsenoside Rb1 monoclonal antibody. *Biological Phar‐*

[27] Fukuda, N., Tanaka, H., & Shoyama, Y. (2001). Double staining of ginsenosides by Western blotting using anti-ginsenoside Rb1 and Rg1 monoclonal antibodies. *Biologi‐*

[28] Shan, S. J., Tanaka, H., & Shoyama, Y. (1999). Western blotting method for the immu‐ nostaining detection of glucuronides of glycyrrhetic acid using anti-glycyrrhizin

monoclonal antibody. *Biological Pharmaceutical Bulletin*, 22, 221-223.

thin layer chromatography. *Journal of Immunological Methods*, 72, 471-479.

monoclonal antibody against solamargine. *FEBS Letters*, 404, 279-82.

*Phytochemical Analysis*, 20, 154-158.

blotting. *Analytical Biochemistry*, 223, 232-8.

*cal Pharmaceutical Bulletin*, 24, 1157-1160.

*maceutical Bulletin*, 22, 219-20.


[20] Towbin, H., Schoenenberger, C., Ball, R., Braun, D. G., & Rosenfelder, G. (1984). Gly‐ cosphingolipid-blotting: an immunological detection procedure after separation by thin layer chromatography. *Journal of Immunological Methods*, 72, 471-479.

[7] Ishiyama, M., Shoyama, Y., Murakami, H., & Shinohara, H. (1995). Production of monoclonal antibodies and development of an ELISA for solamargine. *Cytotechnolo‐*

[8] Shoyama, Y., Fukada, T., & Murakami, H. (1995). Production of monoclonal antibod‐

[9] Tanaka, H., Goto, Y., & Shoyama, Y. (1996). Monoclonal antibody based enzyme im‐ munoassay for marihuana (cannabinoid) compounds. *Journal of Immunoassay*, 17,

[10] Tanaka, H., & Shoyama, Y. (1998). Formation of a monoclonal antibody against gly‐ cyrrhizin and development of an ELISA. *Biological Pharmaceutical Bulletin*, 21,

[11] Xuan, L., Tanaka, H., Xu, Y., & Shoyama, Y. (1999). Preparation of monoclonal anti‐

[12] Tanaka, H., Fukuda, N., & Shoyama, Y. (1999). Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization. *Cyto‐*

[13] Fukuda, N., Tanaka, H., & Shoyama, Y. (2000). Formation of monoclonal antibody against a major ginseng component, ginsenoside Rg1 and its characterization. Mono‐

[14] Morinaga, O., Tanaka, H., & Shoyama, Y. (2000). Production of monoclonal antibody against a major purgative component, sennoside A, its characterization and ELISA.

[15] Shoyama, Y., Sakata, R., Isobe, R., & Murakami, H. (1993). Direct determination of forskolin-bovine serum albumin conjugate by matrix-assisted laser desorption ioni‐

[16] Langone, J. J. (1980). A simple procedure to use whole serum as a source of either

[17] Oshio, H., & Kawamura, N. (1985). Determination of the laxative compounds in rhu‐ barb by high performance liquid chromatography. *Shoyakugaku Zasshi*, 39, 131-38.

[18] Seto, T., Yasuda, I., Hamano, T., Takano, I., Kiyono, S., Nishijima, M., & Akiyama, K. (1996). Determination method of sennoside A, sennoside B, rhein and rhein 8-gluco‐ side in kampo or crude drug preparations and the comparison of these components

[19] Morinaga, O., Nakajima, S., Tanaka, H., & Shoyama, Y. (2001). Production of mono‐ clonal antibodies against a major purgative component, sennoside B, their characteri‐

body against crocin and its characterization. *Cytotechnology*, 29, 65-70.

clonal antibody for a ginseng saponin. *Cytotechnology*, 34, 197-204.

zation mass spectrometry. *Organic Mass Spectrometry*, 28, 987-988.

IgG- or IgM-specific antibody. *Journal of Immunol Methods*, 32, 51-58.

in processed rhubarb. *Natural Medicines*, 50, 138-144.

zation and use in ELISA. *Analyst*, 126, 1372-1376.

ies and ELISA for thebaine and codeine. *Cytotechnology*, 19, 55-61.

*gy*, 18, 153-158.

26 Latest Research into Quality Control

321-342.

1391-1393.

*technology*, 29, 115-120.

*Analyst*, 125, 1109-1113.


**Chapter 2**

**Applications of Anti-natural Compound**

Takuhiro Uto, Nguyen Huu Tung, Hiroyuki Tanaka

Additional information is available at the end of the chapter

and Yukihiro Shoyama

http://dx.doi.org/10.5772/45955

**1. Introduction**

and long analysis time.

**Immunoaffinity Purification on Quality Control**

Worldwide demand of herbal medicines has increased in recent years owing to rising inter‐ est in the health benefits. Among with this, the quality control of plant extracts and plantderived medicines is growing in importance to ensure their efficacy and safety. Effective quality control of the traditional Chinese medicines (TCM) and plant crude extracts requires the rapid and sensitive methods for separation and quantification of bioactive compounds. Various methods have been employed for the separation and quantification of certain con‐ stituents in medicinal plants or herbal medicines. However, the current methods in use are not necessarily optimal approaches. For example, separation and quantification of glycyrrhi‐ zin (GC), the main active constituent in licorice (*Glycyrrhiza* spp.), have been used gas chro‐ matography, high performance liquid chromatography (HPLC) and micellar trokinetic chromatography and so on [1,2]. Commercial purification of GC typically progressed through several steps, including crystallization, column chromatography, and liquid parti‐ tioning. These current methods are not sufficiently approaches because of insufficient sensi‐ tivity and reproducibility, large consumption of organic solvent for extraction and analysis,

Immunoassay systems using monoclonal antibody (MAb) against drugs and small molecu‐ lar weight bioactive compounds have become an important tool for studies on receptor binding assays, enzyme assays, and quantitative and qualitative analytical techniques both *in vivo* and *in vitro* studies. Although immunoaffinity purification against higher molecule analyte such as peptides and proteins are widely used in the research and commercial ways, there are too few cases of immunoaffinity purification targeting a small molecule com‐ pound such as natural compounds. Our laboratory has prepared many kinds of MAbs against

> © 2012 Uto et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Uto et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

## **Applications of Anti-natural Compound Immunoaffinity Purification on Quality Control**

Takuhiro Uto, Nguyen Huu Tung, Hiroyuki Tanaka and Yukihiro Shoyama

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45955

#### **1. Introduction**

Worldwide demand of herbal medicines has increased in recent years owing to rising inter‐ est in the health benefits. Among with this, the quality control of plant extracts and plantderived medicines is growing in importance to ensure their efficacy and safety. Effective quality control of the traditional Chinese medicines (TCM) and plant crude extracts requires the rapid and sensitive methods for separation and quantification of bioactive compounds. Various methods have been employed for the separation and quantification of certain con‐ stituents in medicinal plants or herbal medicines. However, the current methods in use are not necessarily optimal approaches. For example, separation and quantification of glycyrrhi‐ zin (GC), the main active constituent in licorice (*Glycyrrhiza* spp.), have been used gas chro‐ matography, high performance liquid chromatography (HPLC) and micellar trokinetic chromatography and so on [1,2]. Commercial purification of GC typically progressed through several steps, including crystallization, column chromatography, and liquid parti‐ tioning. These current methods are not sufficiently approaches because of insufficient sensi‐ tivity and reproducibility, large consumption of organic solvent for extraction and analysis, and long analysis time.

Immunoassay systems using monoclonal antibody (MAb) against drugs and small molecu‐ lar weight bioactive compounds have become an important tool for studies on receptor binding assays, enzyme assays, and quantitative and qualitative analytical techniques both *in vivo* and *in vitro* studies. Although immunoaffinity purification against higher molecule analyte such as peptides and proteins are widely used in the research and commercial ways, there are too few cases of immunoaffinity purification targeting a small molecule com‐ pound such as natural compounds. Our laboratory has prepared many kinds of MAbs against

© 2012 Uto et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Uto et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

naturally occurring bioactive compounds such as terpenoids [3-5], alkaloids [6,7], saponins [8-12], and phenolics [13-16], and developed several applications. One of the applications by using MAbs is immunoaffinity column conjugated with anti-natural compound-specific MAbs and work by specifically binding and removing the target compounds. We have been estab‐ lishing several affinity columns against a kind of terpenoid, forskolin [17], solasodine glyco‐ sides [18], ginsenosides Rb1 [19], and GC [20]. Application of an immunoaffinity column to isolate and concentrate a natural compound may decrease the amount of solvent consump‐ tion and the number of purification steps, shorten analysis time, and simplify sample analy‐ sis compared to traditional cleanup techniques.

In order to develop efficient quality control of ginseng, we have prepared anti-G-Rb1 MAb, set up of enzyme-linked immunosorbent assay (ELISA), and a new immunostaining meth‐ od named Eastern blotting [8,32]. Furthermore, we established an immunoaffinity column against G-Rb1 and its application for one-step isolation from crude extract of ginseng root [19, 32]. Herein we describe the preparation of anti-G-Rb1 immunoaffinity column and it appli‐

Applications of Anti-natural Compound Immunoaffinity Purification on Quality Control

http://dx.doi.org/10.5772/45955

31

*2.1.1. Analytical methodology for determination of hapten number in antigen, hapten-carrier protein*

The first step for the MAb production is the synthesis of a hapten-carrier protein conjugate. Bovine serum albumin (BSA) conjugated with G-Rb1 was produced for the preparation of specific MAb in mouse [8]. There had been no direct and appropriate methods for the deter‐ mination of haptens conjugated carrier proteins without differential UV analysis, radiochem‐ ical or chemical methods. Therefore, immunization by the injection of hapten-carrier protein conjugate was unreliable. Wengatez *et al.* determined the hapten density of immuno-conju‐ gates by matrix-assisted UV laser desorption/ionization (MALDI) mass spectrometry [33]. We also reported the direct analytical method of hapten and carrier protein conjugates by a MALDI tof mass spectrometry using internal standard [3-16]. Figure 1 shows the MALDI tof mass spectra of G-Rb1-BSA conjugate. A broad peak coinciding with the conjugate of G-Rb1 and BSA appeared from *m/z* 70,000 to 90,000 centering at around *m/z* 79,469. Using experimental results and a molecular weight of 66,433 for BSA, the calculated values of G-Rb1component (MW1,109) are from 3,327 to 23,289 resulting in the range of 3 to 21 (12 in average) mole‐ cules of G-Rb1 conjugated with BSA [8]. This method is suitable for characterization of conju‐

gates between small molecule natural compound and carrier protein conjugates.

**Figure 1.** Direct detection of G-Rb1-BSA conjugate by MALDI tof mass spectrometry. [M+H]+, [M+2H]2+ are single and

double protonated molecules of G-Rb1-BSA, respectively.

cations for identification and concentration of G-Rb1.

*conjugate*

**2.1. Preparation of MAb and immunoaffinity column against G-Rb1**

In this chapter, we focus on the immunoaffinity purification to separate and concentrate the target bioactive compounds from the crude extract. Our approaches effectively succeeded one-step purification of target compounds by MAb-conjugated immunoaffinity column, which leads to high-sensitivity detection and isolation of target compounds. In addition, the immunoaffinity column can prepare the knockout (KO) extract which contains all compo‐ nents except an antigen molecule, and KO extract will be useful for the pharmacological investigation to reveal the real effects of bioactive compound in the crude extract.The infor‐ mation in this chapter may provide new insight into quality control of plant-derived medicines.

### **2. Preparation of anti-ginsenoside Rb1 immunoaffinity column and its application**

Ginseng, the root of *Panax ginseng*, has been an important component in traditional medi‐ cines for more than 1000 years in Eastern Asia. It is now one of the most extensively used alternative medicines all over the world and appears in the pharmacopoeias of several coun‐ tries. The biological and pharmacological activities of ginseng have been reported to have antiaging, anti-cancer, anti-inflammation, anti-diabetics, anti-stress, maintenance of homeostasis, and to affect on central nervous system and immune function [21]. The bioactive compo‐ nents responsible for ginseng actions are ginsenosides, which are triterpenes saponins that possess a dammarane skeleton with sugar moieties [22]. Up to now more than 60 kinds of ginsenosides have been isolated from *Panax* genus [23]. It is well-known that the concentra‐ tions of ginsenosides vary in the ginseng root or the root extracts depending on the method of extraction, subsequent treatment, or even the season of its collection [24,25]. Due to the importance of ginseng, a number of researches has been carried out to develop the methods for the identification, quantification and quality control of ginsenosides in raw plants materi‐ als, extracts and commercial products. Currently, analytical and preparative HPLC are com‐ monly used to quantify and purify the individual ginsenosides from ginseng [26]. However, isolation of ginsenosides by HPLC requires the repeated purification steps, including cumber‐ some handling and lengthy analysis times, and may result in the decrease of the final yield.Thus, the developed approaches are required for quality control of ginseng in the field of TCM.

Ginsenoside Rb1 (G-Rb1) is one of the main ginsenosides responsible for many pharmaceut‐ ical actions of ginseng [27]. G-Rb1 has various biological activities, including facilitating acquisition and retrieval of memory [28], scavenging free radicals [29], inhibition of calci‐ um over-influx into neurons [30], and preserving the structural integrity of the neurons [31]. In order to develop efficient quality control of ginseng, we have prepared anti-G-Rb1 MAb, set up of enzyme-linked immunosorbent assay (ELISA), and a new immunostaining meth‐ od named Eastern blotting [8,32]. Furthermore, we established an immunoaffinity column against G-Rb1 and its application for one-step isolation from crude extract of ginseng root [19, 32]. Herein we describe the preparation of anti-G-Rb1 immunoaffinity column and it appli‐ cations for identification and concentration of G-Rb1.

#### **2.1. Preparation of MAb and immunoaffinity column against G-Rb1**

naturally occurring bioactive compounds such as terpenoids [3-5], alkaloids [6,7], saponins [8-12], and phenolics [13-16], and developed several applications. One of the applications by using MAbs is immunoaffinity column conjugated with anti-natural compound-specific MAbs and work by specifically binding and removing the target compounds. We have been estab‐ lishing several affinity columns against a kind of terpenoid, forskolin [17], solasodine glyco‐ sides [18], ginsenosides Rb1 [19], and GC [20]. Application of an immunoaffinity column to isolate and concentrate a natural compound may decrease the amount of solvent consump‐ tion and the number of purification steps, shorten analysis time, and simplify sample analy‐

In this chapter, we focus on the immunoaffinity purification to separate and concentrate the target bioactive compounds from the crude extract. Our approaches effectively succeeded one-step purification of target compounds by MAb-conjugated immunoaffinity column, which leads to high-sensitivity detection and isolation of target compounds. In addition, the immunoaffinity column can prepare the knockout (KO) extract which contains all compo‐ nents except an antigen molecule, and KO extract will be useful for the pharmacological investigation to reveal the real effects of bioactive compound in the crude extract.The infor‐ mation in this chapter may provide new insight into quality control of plant-derived medicines.

**2. Preparation of anti-ginsenoside Rb1 immunoaffinity column and its**

Ginseng, the root of *Panax ginseng*, has been an important component in traditional medi‐ cines for more than 1000 years in Eastern Asia. It is now one of the most extensively used alternative medicines all over the world and appears in the pharmacopoeias of several coun‐ tries. The biological and pharmacological activities of ginseng have been reported to have antiaging, anti-cancer, anti-inflammation, anti-diabetics, anti-stress, maintenance of homeostasis, and to affect on central nervous system and immune function [21]. The bioactive compo‐ nents responsible for ginseng actions are ginsenosides, which are triterpenes saponins that possess a dammarane skeleton with sugar moieties [22]. Up to now more than 60 kinds of ginsenosides have been isolated from *Panax* genus [23]. It is well-known that the concentra‐ tions of ginsenosides vary in the ginseng root or the root extracts depending on the method of extraction, subsequent treatment, or even the season of its collection [24,25]. Due to the importance of ginseng, a number of researches has been carried out to develop the methods for the identification, quantification and quality control of ginsenosides in raw plants materi‐ als, extracts and commercial products. Currently, analytical and preparative HPLC are com‐ monly used to quantify and purify the individual ginsenosides from ginseng [26]. However, isolation of ginsenosides by HPLC requires the repeated purification steps, including cumber‐ some handling and lengthy analysis times, and may result in the decrease of the final yield.Thus, the developed approaches are required for quality control of ginseng in the field of TCM.

Ginsenoside Rb1 (G-Rb1) is one of the main ginsenosides responsible for many pharmaceut‐ ical actions of ginseng [27]. G-Rb1 has various biological activities, including facilitating acquisition and retrieval of memory [28], scavenging free radicals [29], inhibition of calci‐ um over-influx into neurons [30], and preserving the structural integrity of the neurons [31].

sis compared to traditional cleanup techniques.

30 Latest Research into Quality Control

**application**

#### *2.1.1. Analytical methodology for determination of hapten number in antigen, hapten-carrier protein conjugate*

The first step for the MAb production is the synthesis of a hapten-carrier protein conjugate. Bovine serum albumin (BSA) conjugated with G-Rb1 was produced for the preparation of specific MAb in mouse [8]. There had been no direct and appropriate methods for the deter‐ mination of haptens conjugated carrier proteins without differential UV analysis, radiochem‐ ical or chemical methods. Therefore, immunization by the injection of hapten-carrier protein conjugate was unreliable. Wengatez *et al.* determined the hapten density of immuno-conju‐ gates by matrix-assisted UV laser desorption/ionization (MALDI) mass spectrometry [33]. We also reported the direct analytical method of hapten and carrier protein conjugates by a MALDI tof mass spectrometry using internal standard [3-16]. Figure 1 shows the MALDI tof mass spectra of G-Rb1-BSA conjugate. A broad peak coinciding with the conjugate of G-Rb1 and BSA appeared from *m/z* 70,000 to 90,000 centering at around *m/z* 79,469. Using experimental results and a molecular weight of 66,433 for BSA, the calculated values of G-Rb1component (MW1,109) are from 3,327 to 23,289 resulting in the range of 3 to 21 (12 in average) mole‐ cules of G-Rb1 conjugated with BSA [8]. This method is suitable for characterization of conju‐ gates between small molecule natural compound and carrier protein conjugates.

**Figure 1.** Direct detection of G-Rb1-BSA conjugate by MALDI tof mass spectrometry. [M+H]+, [M+2H]2+ are single and double protonated molecules of G-Rb1-BSA, respectively.

#### *2.1.2. Preparation of anti-G-Rb1 MAb and ELISA as an assay system*

A hybridoma-producing MAb reactive to G-Rb1 was obtained by general procedure and classified into IgG2b which had κ light chains [8]. The reactivity of IgG type MAb, 9G7 was tested by varying antibody concentration and by performing a dilution curve. The antibody concentration was selected for competitive ELISA. The free MAb following competition is bound to polystyrene microtiter plates precoated with G-Rb1-human serum albumin (HSA). Under these conditions, the full measurement range of the assay extends from 20 to 400 ng/mL. The cross-reactivity against G-Rc and G-Rd, which possess a diglucose moiety at‐ tached to the C-3 hydroxy group, were weak compared with G-Rb1 (0.024 and 0.020 %, re‐ spectively). G-Re and G-Rg1 showed no cross-reactivity (less than 0.005 %). It is evident that the MAb reacted only with a small number of structurally related G-Rb1 molecules, and very weakly and did not react with other steroidal compounds.

**2.2. Purification of G-Rb1 by immunoaffinity column**

from a crude extract of *Coleus forslohlii* root [17].

*immunoaffinity column*

by ELISA. a

presence the test compounds.

*2.2.1. One-step purification of G-Rb1 from crude extract of P. ginseng roots by anti-G-Rb1*

A crude extract (3.8 mg) of *P. ginseng* roots was loaded onto anti-G-Rb1 immunoaffinity col‐ umn. The column was washed with the washing buffer (fractions 1-20), and then eluted with elution buffer (fractions 21-40). As shown in Figure 3, the fractions 1-8 contained the overloaded G-Rb1, which determined by ELISA. The other ginsenosides such as G-Rg1, Rc, Re and Rd were also detected in these fractions by Eastern blotting procedure. After wash‐ ing, a sharp peak was observed around fractions 21-24 of elution buffer, which contained G-Rb1. However, these eluted fractions were still contaminated by a small amount of malonyl-G-Rb1 as detected by Eastern blotting. The malonyl-G-Rb1 has almost the same crossreactivity with G-Rb1 [32]. Therefore, the eluted fractions were treated with a mild alkaline solution (0.1 % KOH in MeOH) at room temperature to give pure G-Rb1 [19]. Overcharged G-Rb1 in washing solution (fractions 1-8) was repeatedly loaded and finally isolated in pure form. The anti-G-Rb1 MAb was stable during all procedures, and the immunoaffinity col‐ umn showed almost no decrease in capacity (20 μg of G-Rb1/ml gel) after repeated use more than 10 times under same conditions, as reported for a one-step purification of forskolin

Applications of Anti-natural Compound Immunoaffinity Purification on Quality Control

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33

**Figure 3.** Elution profile of *P. ginseng* crude extract separated by anti-G-Rb1 immunoaffinity column. The concentra‐ tion of G-Rb1 in each fraction was monitoring by ELISA using anti-G-Rb1 MAb. Individual fraction (2 mL) were assayed

This methodology is effective for the rapid and simple purification of G-Rb1 and may open up a wide field of comparable studies with other families of saponins for which an accepta‐ ble method for one-step separation has not yet been developed. Furthermore, to separate the

Inhibition = (A0-A)/A0 ;A0 is the absorbance in the absence of the test compounds. A is the absence in the

#### *2.1.3. Preparation of anti-G-Rb1 immunoaffinity column and appropriate buffer systems for separation of G-Rb1*

The purified IgG (10 mg) was treated by NaIO4 to give dialdehyde group in sugar moiety which was coupled to Affi-Gel Hz hydrazide gel resulting in a hydrozone-type immunoaf‐ finity gel [32]. The immunoaffinity gel was packed into plastic mini-column (Figure 2). Due to examine the optimal conditions of adsorption and elution, 400 μg of G-Rb1 was dissolved in phosphate buffered saline (PBS) and loaded on anti-G-Rb1 affinity column. After washing with washing buffer (20 mM PB containing 0.5 M NaCl), various buffer solutions for elution were loaded on the column, and then the recovery efficiency was determined by ELISA. The G-Rb1 concentration was somewhat increased by eluting with a 20 mM phosphate buffer containing 0.5 M KSCN and 10 % MeOH. When the 20 mM phosphate buffer was changed to 100 mM AcOH buffer (pH 4), the elution ability reached the optimal level. Although 20 % MeOH could enhance the elution of G-Rb1, higher MeOH concentration of over 20 % was ineffective. Thus, 100 mM AcOH buffer containing 0.5 M KSCN and 20 % MeOH could be used as an elution buffer in the immunoaffinity chromatography.

**Figure 2.** Preparation of anti-G-Rb1 immunoaffinity column

#### **2.2. Purification of G-Rb1 by immunoaffinity column**

*2.1.2. Preparation of anti-G-Rb1 MAb and ELISA as an assay system*

weakly and did not react with other steroidal compounds.

used as an elution buffer in the immunoaffinity chromatography.

**Figure 2.** Preparation of anti-G-Rb1 immunoaffinity column

*separation of G-Rb1*

32 Latest Research into Quality Control

A hybridoma-producing MAb reactive to G-Rb1 was obtained by general procedure and classified into IgG2b which had κ light chains [8]. The reactivity of IgG type MAb, 9G7 was tested by varying antibody concentration and by performing a dilution curve. The antibody concentration was selected for competitive ELISA. The free MAb following competition is bound to polystyrene microtiter plates precoated with G-Rb1-human serum albumin (HSA). Under these conditions, the full measurement range of the assay extends from 20 to 400 ng/mL. The cross-reactivity against G-Rc and G-Rd, which possess a diglucose moiety at‐ tached to the C-3 hydroxy group, were weak compared with G-Rb1 (0.024 and 0.020 %, re‐ spectively). G-Re and G-Rg1 showed no cross-reactivity (less than 0.005 %). It is evident that the MAb reacted only with a small number of structurally related G-Rb1 molecules, and very

*2.1.3. Preparation of anti-G-Rb1 immunoaffinity column and appropriate buffer systems for*

The purified IgG (10 mg) was treated by NaIO4 to give dialdehyde group in sugar moiety which was coupled to Affi-Gel Hz hydrazide gel resulting in a hydrozone-type immunoaf‐ finity gel [32]. The immunoaffinity gel was packed into plastic mini-column (Figure 2). Due to examine the optimal conditions of adsorption and elution, 400 μg of G-Rb1 was dissolved in phosphate buffered saline (PBS) and loaded on anti-G-Rb1 affinity column. After washing with washing buffer (20 mM PB containing 0.5 M NaCl), various buffer solutions for elution were loaded on the column, and then the recovery efficiency was determined by ELISA. The G-Rb1 concentration was somewhat increased by eluting with a 20 mM phosphate buffer containing 0.5 M KSCN and 10 % MeOH. When the 20 mM phosphate buffer was changed to 100 mM AcOH buffer (pH 4), the elution ability reached the optimal level. Although 20 % MeOH could enhance the elution of G-Rb1, higher MeOH concentration of over 20 % was ineffective. Thus, 100 mM AcOH buffer containing 0.5 M KSCN and 20 % MeOH could be

#### *2.2.1. One-step purification of G-Rb1 from crude extract of P. ginseng roots by anti-G-Rb1 immunoaffinity column*

A crude extract (3.8 mg) of *P. ginseng* roots was loaded onto anti-G-Rb1 immunoaffinity col‐ umn. The column was washed with the washing buffer (fractions 1-20), and then eluted with elution buffer (fractions 21-40). As shown in Figure 3, the fractions 1-8 contained the overloaded G-Rb1, which determined by ELISA. The other ginsenosides such as G-Rg1, Rc, Re and Rd were also detected in these fractions by Eastern blotting procedure. After wash‐ ing, a sharp peak was observed around fractions 21-24 of elution buffer, which contained G-Rb1. However, these eluted fractions were still contaminated by a small amount of malonyl-G-Rb1 as detected by Eastern blotting. The malonyl-G-Rb1 has almost the same crossreactivity with G-Rb1 [32]. Therefore, the eluted fractions were treated with a mild alkaline solution (0.1 % KOH in MeOH) at room temperature to give pure G-Rb1 [19]. Overcharged G-Rb1 in washing solution (fractions 1-8) was repeatedly loaded and finally isolated in pure form. The anti-G-Rb1 MAb was stable during all procedures, and the immunoaffinity col‐ umn showed almost no decrease in capacity (20 μg of G-Rb1/ml gel) after repeated use more than 10 times under same conditions, as reported for a one-step purification of forskolin from a crude extract of *Coleus forslohlii* root [17].

**Figure 3.** Elution profile of *P. ginseng* crude extract separated by anti-G-Rb1 immunoaffinity column. The concentra‐ tion of G-Rb1 in each fraction was monitoring by ELISA using anti-G-Rb1 MAb. Individual fraction (2 mL) were assayed by ELISA. a Inhibition = (A0-A)/A0 ;A0 is the absorbance in the absence of the test compounds. A is the absence in the presence the test compounds.

This methodology is effective for the rapid and simple purification of G-Rb1 and may open up a wide field of comparable studies with other families of saponins for which an accepta‐ ble method for one-step separation has not yet been developed. Furthermore, to separate the total ginseng saponins, a wide cross-reactive MAb against ginsenoside, like anti G-Re MAb which showed wide cross-reactivity, could be designed [34]. A combination of immunoaf‐ finity column, Eastern blotting and ELISA could be used to survey low concentrations of ginsenoside Rb1 of plant origin and/or in experimental animals and human. In fact we have succeeded in the isolation of G-Rb1 from a different plant, *Kalopanax pictus* Nakai, which was not known previously to contain ginsenosides, using this combination of methods [35].

ed fraction by the washing buffer, and showed many spots, indicating chikusetsusaponins, similar to the original extract of *P. japonicus*. After washing, the column was eluted by elu‐ tion buffer (fraction 5), and then one spot was detected. As shown in Figure 4B, Eastern blot‐ ting indicated two different spots in washing fraction (Compound 1) and eluted fraction (Compound 2). These compounds bound with anti-G-Rb1 MAb have a dammarane saponin

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**Figure 4.** Purification and determination of ginsenosides of *P. japonicus* by the anti-G-Rb1 immunoaffinity column. TLC (A) and Eastern blotting (B) profiles of the separated fractions from *P. japonicus* crude extract by the anti-G-Rb1 immu‐ noaffinity column. Lane S indicates the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane 1-4 and

**Figure 5.** Chemical structures of G-Rb1 and chikusetsusaponins purified from *P. japonicus* by the anti-G-Rb1 immunoaf‐

Compound 1 has three sugar moieties in a molecule because that the *R* <sup>F</sup> value closes to that of G-Rd, indicating that this compound is chikusetsusaponin III (Figure 5). Finally, we iden‐ tified that this compound as chikusetsusaponin III in a direct comparison with authentic sample [32]. Another unknown spot, compound 2 appeared in fraction 5 of the eluted frac‐ tion. Thus, compound 2 has a similar molecular structure and high cross-reactivity with G-Rb1, and seems to be related ginseng saponin having protopanaxadiol as an aglycone. Moreover, compound 2 might have the same sugar fragments and possess five sugar moiety compared with G-Rb1, as indicated by their *R* F value. From these evidences compound 2

having protopanaxadiol as a framework.

Lane 5 were the washing fractions and the eluted fraction, respectively.

finity column.

#### *2.2.2. Isolation and determination of unknown compounds related to G-Rb1 by anti-G-Rb1 immunoaffinity column*

Several species of ginseng are known to exist and contain different amount and kinds of gin‐ senosides. *P. japonicus* is distributed in Japan and China and it is morphologically different from the other *Panax* species. Yahara *et al.* indicated that G-Rbl was not detected in *P. japoni‐ cus*, and isolated oleanane-type saponins called chikusetsusaponins and determined their structures [36]. Morita *et al*. reported the varieties of saponins in *P. japonicus* by chemical analysis. These results suggested that the concentration of G-Rb1 might be trace level in *P. japonicas* [37]. We previously analyzed the G-Rb1 concentration in several ginseng roots by ELISA using anti-G-Rb1 MAb and HPLC after pre-treatment under mildly alkaline condition [32]. As shown in Table 1, G-Rb1 concentrationsof *P. ginseng*, *P. notoginseng* and *P. quinquefo‐ lius* were correlated between ELISA and HPLC. However, the G-Rb1 of *P. japonicus* was higher concentrations compared with HPLC and previous reports [37]. This data suggest that anti-G-Rb1 MAb using ELISA has the cross-reactivity with some unknown compounds contained in *P. japonicus*.



To clarify the unknown compounds bound to anti-G-Rb1 MAb, the crude extract of *P. japoni‐ cus* was concentrated by immunoaffinity column using anti-G-Rb1 MAb. The crude root ex‐ tract was loaded on the column and washed with the washing Buffer, followed by the elution buffer as already indicated. Figure 4 shows the H2SO4 staining (A) and the Eastern blotting (B) profiles of the washing fractions 1-4 and eluted fraction 5. Fraction 1 is first elut‐ ed fraction by the washing buffer, and showed many spots, indicating chikusetsusaponins, similar to the original extract of *P. japonicus*. After washing, the column was eluted by elu‐ tion buffer (fraction 5), and then one spot was detected. As shown in Figure 4B, Eastern blot‐ ting indicated two different spots in washing fraction (Compound 1) and eluted fraction (Compound 2). These compounds bound with anti-G-Rb1 MAb have a dammarane saponin having protopanaxadiol as a framework.

total ginseng saponins, a wide cross-reactive MAb against ginsenoside, like anti G-Re MAb which showed wide cross-reactivity, could be designed [34]. A combination of immunoaf‐ finity column, Eastern blotting and ELISA could be used to survey low concentrations of ginsenoside Rb1 of plant origin and/or in experimental animals and human. In fact we have succeeded in the isolation of G-Rb1 from a different plant, *Kalopanax pictus* Nakai, which was not known previously to contain ginsenosides, using this combination of methods [35].

Several species of ginseng are known to exist and contain different amount and kinds of gin‐ senosides. *P. japonicus* is distributed in Japan and China and it is morphologically different from the other *Panax* species. Yahara *et al.* indicated that G-Rbl was not detected in *P. japoni‐ cus*, and isolated oleanane-type saponins called chikusetsusaponins and determined their structures [36]. Morita *et al*. reported the varieties of saponins in *P. japonicus* by chemical analysis. These results suggested that the concentration of G-Rb1 might be trace level in *P. japonicas* [37]. We previously analyzed the G-Rb1 concentration in several ginseng roots by ELISA using anti-G-Rb1 MAb and HPLC after pre-treatment under mildly alkaline condition [32]. As shown in Table 1, G-Rb1 concentrationsof *P. ginseng*, *P. notoginseng* and *P. quinquefo‐ lius* were correlated between ELISA and HPLC. However, the G-Rb1 of *P. japonicus* was higher concentrations compared with HPLC and previous reports [37]. This data suggest that anti-G-Rb1 MAb using ELISA has the cross-reactivity with some unknown compounds

> **Sample ELISA HPLC** *P. ginseng* **White ginseng 5.49 ±0.75 4.96 ± 0.05**

> *P. notoginseng* **47.08 ± 3.34 42.39 ± 1.39** *P. quinquefolium* **48.51 ± 1.79 47.96 ± 1.04** *P. japonicus* **1.37 ± 0.34 0.63 ± 0.06**

To clarify the unknown compounds bound to anti-G-Rb1 MAb, the crude extract of *P. japoni‐ cus* was concentrated by immunoaffinity column using anti-G-Rb1 MAb. The crude root ex‐ tract was loaded on the column and washed with the washing Buffer, followed by the elution buffer as already indicated. Figure 4 shows the H2SO4 staining (A) and the Eastern blotting (B) profiles of the washing fractions 1-4 and eluted fraction 5. Fraction 1 is first elut‐

**Red ginseng 3.57 ± 0.62 3.93 ± 0.34 Fibrous ginseng 64.44 ± 3.64 69.75± 1.45**

**G-Rb1 concentration (µg/mg dry weight powder)**

*2.2.2. Isolation and determination of unknown compounds related to G-Rb1 by anti-G-Rb1*

*immunoaffinity column*

34 Latest Research into Quality Control

contained in *P. japonicus*.

**Table 1.** G-Rb1 concentration in various ginseng samples

**Figure 4.** Purification and determination of ginsenosides of *P. japonicus* by the anti-G-Rb1 immunoaffinity column. TLC (A) and Eastern blotting (B) profiles of the separated fractions from *P. japonicus* crude extract by the anti-G-Rb1 immu‐ noaffinity column. Lane S indicates the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane 1-4 and Lane 5 were the washing fractions and the eluted fraction, respectively.

**Figure 5.** Chemical structures of G-Rb1 and chikusetsusaponins purified from *P. japonicus* by the anti-G-Rb1 immunoaf‐ finity column.

Compound 1 has three sugar moieties in a molecule because that the *R* <sup>F</sup> value closes to that of G-Rd, indicating that this compound is chikusetsusaponin III (Figure 5). Finally, we iden‐ tified that this compound as chikusetsusaponin III in a direct comparison with authentic sample [32]. Another unknown spot, compound 2 appeared in fraction 5 of the eluted frac‐ tion. Thus, compound 2 has a similar molecular structure and high cross-reactivity with G-Rb1, and seems to be related ginseng saponin having protopanaxadiol as an aglycone. Moreover, compound 2 might have the same sugar fragments and possess five sugar moiety compared with G-Rb1, as indicated by their *R* F value. From these evidences compound 2 might be chikusetsusaponin III-20-*O*-gentiobiose, chikusetsusaponin VI (Figure 5), which has 5 sugars in a molecule in good agreement with the *R* F value previously reported [38] and we confirmed that compound 2 is chikusetsusaponin VI by the direct comparison with authentic sample.

**3. Glycyrrhizin-knockout extract and its application for** *in vitro* **assay**

application for functional analysis of GC in licorice crude extract.

tive ELISA and Eastern blotting method using anti-GC MAb [11,49].

unbound fraction "GC-knockout (GC-KO) extract" [50].

**characterization of GC-KO extract**

**3.1. Preparation of GC-KO extract by anti-GC immunoaffinity column and the**

Our previous study demonstrated the preparation of anti-GC MAb [11]. The cross-reactivi‐ ties of the anti-GC MAb against glycyrrhetic acid-3-*O*-glucuronide and glycyrrhetic acid were 0.585 % and 1.865 %, respectively. The other related compounds (deoxycholic acid, ur‐ solic acid, and oleanolic acid) were all less than 0.005 %. Moreover, we established competi‐

The immunoaffinity column against GC was prepared by coupling the purified 60 mg of the anti-GC MAb to 25 ml of an Affi-Gel Hz gel [11]. To eliminate GC from licorice extract, 12 mg of licorice crude extract (GC content: 1275.0 μg) in loading buffer (5 % MeOH) was ap‐ plied on the anti-GC MAb immunoaffinity column, and then the loading buffer was contin‐ uously circulated through the column to enhance the binding efficiency. After overnight circulation at 4 °C, the unbound fraction was separated. The column was washed with washing buffer (5 % MeOH) and then eluted with elution buffer (20 mM phosphate buffer containing 30 % MeOH). After separation, each fraction was deionized and the solvent was lyophilized. Figure 7 showed the recovery ratio of GC checked by ELISA. In the unbound fraction, 3.50 μg of GC (0.27% of the applied GC) was detected. On the other hand, 1269.26 μg of GC (99.55% of the applied GC) was obtained in the bound fraction. These data indicate that the anti-GC column could eliminate 99.55 % of the loading GC. Thus, we named this

Licorice (*Glycyrrhiza* spp.) is also important crude drug used in over 70 % of the TCMs and Japanese Kampo medicines. It is prescribed with other herbal medicines as a demulcent in the treatment of sore throats, an expectorant for coughs and bronchial catarrh, an antitus‐ sive, a taste-modifying agent for relieving pain, an anti-inflammatory agent for anti-allergic reactions, rheumatism and arthritis, a prophylactic for liver disease and tuberculosis and adrenocorticoid insufficiency [41-43]. Accumulated evidence indicated that GC, a main sap‐ onin component of licorice, is one of the biologically active compounds. It has been reported that GC exhibits numerous pharmacological effects such as anti-inflammation, anti-ulcer, anti-tumor, anti-allergy, and hepatoprotective activities [44,45]. Clinically, GC has been used to treat patients with chronic hepatitis [46,47]. Although GC is supposed to be a major active principle in licorice crude extract, a number of studies by HPLC profiles suggested that lico‐ rice has many other bioactive components, including flavonoids, isoflavonoids and chal‐ cones [43,48]. Biological studies showed that various flavonoid glycosides and their aglycones of licorice exhibit anti-inflammatory, anti-oxidative, anti-microbial, superoxide scavenging, and anti-carcinogenic activities [43,48]. In order to confirm the role of GC in TCM, we previously purified GC from TCM using an immunoaffinity column conjugated with anti-GC MAb [20]. In this section, we describe the preparation of GC-KO extract and its

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These data suggested that the anti-G-Rb1 immunoaffinity column could isolate some un‐ known structurally resemble compounds having cross-reactivity against anti-G-Rb1MAb. Therefore, this purification system will be applied to survey new compounds related to target compound of MAb. In our previous studies, we demonstrated the immunoaffinity purification against all solasodine glycosides from crude extract by one-step purification. In this case, all solasodine glycoside have almost same cross-reactivity against anti-solamar‐ gine MAb [12].

#### *2.2.3. Preparation of G-Rb1 knockout extract by anti-G-Rb1 immunoaffinity column*

The capacity of this anti-G-Rb1immunoaffinity column is 20 μg of G-Rb1/ml gel [32]. By load‐ ing the samples not to exceed the binding capacity against G-Rb1, this immunoaffinity col‐ umn becomes possible to remove all G-Rb1 from crude ginseng extract. Figure 6 showed H2SO4 staining of TLC of the purification steps by the immunoaffinity column. Lane 1 and 2 were spotted the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane A, B, and C were the crude extract, the washing fraction, and the eluted fraction, respectively. In the crude extract (lane A), all spots of ginsenosides were clearly detected. On the other hand, the washing fraction (lane B) contained all of the ginsenosides in the crude extract except G-Rb1. Furthermore, the spot of G-Rb1 was detected in the eluted fractions (lane C). These data strongly indicated that G-Rb1 molecule in the ginseng extract can be eliminated by an anti-G-Rb1 immunoaffinity column and the washing fractions was knockout only by the antigen molecule, G-Rb1. Thus, we named the washing fractions a knockout (KO) extract [39,40]. This KO extract may be useful for the determination of real pharmacologically active princi‐ ple in the TCMs.

**Figure 6.** Preparation of G-Rb1-KO extract from *P. ginseng* crude extract using anti-G-Rb1 immunoaffinity column. Lane S1 and S2 indicate the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1 and G-Re). Lane A, B, and C were the crude extract, the washing fraction, and the eluted fraction, respectively.

### **3. Glycyrrhizin-knockout extract and its application for** *in vitro* **assay**

might be chikusetsusaponin III-20-*O*-gentiobiose, chikusetsusaponin VI (Figure 5), which has 5 sugars in a molecule in good agreement with the *R* F value previously reported [38] and we confirmed that compound 2 is chikusetsusaponin VI by the direct comparison with

These data suggested that the anti-G-Rb1 immunoaffinity column could isolate some un‐ known structurally resemble compounds having cross-reactivity against anti-G-Rb1MAb. Therefore, this purification system will be applied to survey new compounds related to target compound of MAb. In our previous studies, we demonstrated the immunoaffinity purification against all solasodine glycosides from crude extract by one-step purification. In this case, all solasodine glycoside have almost same cross-reactivity against anti-solamar‐

The capacity of this anti-G-Rb1immunoaffinity column is 20 μg of G-Rb1/ml gel [32]. By load‐ ing the samples not to exceed the binding capacity against G-Rb1, this immunoaffinity col‐ umn becomes possible to remove all G-Rb1 from crude ginseng extract. Figure 6 showed H2SO4 staining of TLC of the purification steps by the immunoaffinity column. Lane 1 and 2 were spotted the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane A, B, and C were the crude extract, the washing fraction, and the eluted fraction, respectively. In the crude extract (lane A), all spots of ginsenosides were clearly detected. On the other hand, the washing fraction (lane B) contained all of the ginsenosides in the crude extract except G-Rb1. Furthermore, the spot of G-Rb1 was detected in the eluted fractions (lane C). These data strongly indicated that G-Rb1 molecule in the ginseng extract can be eliminated by an anti-G-Rb1 immunoaffinity column and the washing fractions was knockout only by the antigen molecule, G-Rb1. Thus, we named the washing fractions a knockout (KO) extract [39,40]. This KO extract may be useful for the determination of real pharmacologically active princi‐

**Figure 6.** Preparation of G-Rb1-KO extract from *P. ginseng* crude extract using anti-G-Rb1 immunoaffinity column. Lane S1 and S2 indicate the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1 and G-Re). Lane A, B, and C were the

crude extract, the washing fraction, and the eluted fraction, respectively.

*2.2.3. Preparation of G-Rb1 knockout extract by anti-G-Rb1 immunoaffinity column*

authentic sample.

36 Latest Research into Quality Control

gine MAb [12].

ple in the TCMs.

Licorice (*Glycyrrhiza* spp.) is also important crude drug used in over 70 % of the TCMs and Japanese Kampo medicines. It is prescribed with other herbal medicines as a demulcent in the treatment of sore throats, an expectorant for coughs and bronchial catarrh, an antitus‐ sive, a taste-modifying agent for relieving pain, an anti-inflammatory agent for anti-allergic reactions, rheumatism and arthritis, a prophylactic for liver disease and tuberculosis and adrenocorticoid insufficiency [41-43]. Accumulated evidence indicated that GC, a main sap‐ onin component of licorice, is one of the biologically active compounds. It has been reported that GC exhibits numerous pharmacological effects such as anti-inflammation, anti-ulcer, anti-tumor, anti-allergy, and hepatoprotective activities [44,45]. Clinically, GC has been used to treat patients with chronic hepatitis [46,47]. Although GC is supposed to be a major active principle in licorice crude extract, a number of studies by HPLC profiles suggested that lico‐ rice has many other bioactive components, including flavonoids, isoflavonoids and chal‐ cones [43,48]. Biological studies showed that various flavonoid glycosides and their aglycones of licorice exhibit anti-inflammatory, anti-oxidative, anti-microbial, superoxide scavenging, and anti-carcinogenic activities [43,48]. In order to confirm the role of GC in TCM, we previously purified GC from TCM using an immunoaffinity column conjugated with anti-GC MAb [20]. In this section, we describe the preparation of GC-KO extract and its application for functional analysis of GC in licorice crude extract.

#### **3.1. Preparation of GC-KO extract by anti-GC immunoaffinity column and the characterization of GC-KO extract**

Our previous study demonstrated the preparation of anti-GC MAb [11]. The cross-reactivi‐ ties of the anti-GC MAb against glycyrrhetic acid-3-*O*-glucuronide and glycyrrhetic acid were 0.585 % and 1.865 %, respectively. The other related compounds (deoxycholic acid, ur‐ solic acid, and oleanolic acid) were all less than 0.005 %. Moreover, we established competi‐ tive ELISA and Eastern blotting method using anti-GC MAb [11,49].

The immunoaffinity column against GC was prepared by coupling the purified 60 mg of the anti-GC MAb to 25 ml of an Affi-Gel Hz gel [11]. To eliminate GC from licorice extract, 12 mg of licorice crude extract (GC content: 1275.0 μg) in loading buffer (5 % MeOH) was ap‐ plied on the anti-GC MAb immunoaffinity column, and then the loading buffer was contin‐ uously circulated through the column to enhance the binding efficiency. After overnight circulation at 4 °C, the unbound fraction was separated. The column was washed with washing buffer (5 % MeOH) and then eluted with elution buffer (20 mM phosphate buffer containing 30 % MeOH). After separation, each fraction was deionized and the solvent was lyophilized. Figure 7 showed the recovery ratio of GC checked by ELISA. In the unbound fraction, 3.50 μg of GC (0.27% of the applied GC) was detected. On the other hand, 1269.26 μg of GC (99.55% of the applied GC) was obtained in the bound fraction. These data indicate that the anti-GC column could eliminate 99.55 % of the loading GC. Thus, we named this unbound fraction "GC-knockout (GC-KO) extract" [50].

**3.2.** *in vitro* **Assay by GC-KO extract prepared by anti-GC immunoaffinity column**

expression may be useful strategy to treat a variety of inflammatory diseases.

ly suppressed [50].

Nitric oxide (NO), synthesized by NO synthase (NOS) from L-arginine, is an important reg‐ ulatory/modulatory mediator for several physiological processes [51]. However, during in‐ flammatory process, a large amount of NO is produced by inducible NOS (iNOS) stimulated by bacterial lipopolysaccharide (LPS) and inflammatory cytokines participates in the patho‐ genesis of inflammatory diseases [52]. Overproduced NO synthesized by iNOS triggers a variety of inflammatory diseases including sepsis, psoriasis, arthritis, multiple sclerosis, and systemic lupus *erythematosus* [53]*.* Therefore, inhibiting NO production by blocking iNOS

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In LPS-treated mouse RAW264 macrophages, licorice extract inhibited NO production and iNOS expression. At 100 μg/mL of licorice extract, iNOS protein and mRNA were complete‐

**Figure 9.** Effect of GC-KO extract and the combination of GC-KO extract and GC on NO production (A) and iNOS pro‐ tein expression (B) in LPS-treated RAW264 cells.Each bar indicates the mean S.D. of four individual experiments.

We next examined the inhibitory effect of GC alone, GC-KO extract and the combined treat‐ ment with GC and GC-KO extract on NO production [50]. Since 100 μg of licorice extract contains 10.6 ±0.618 μg of GC,the cells were pre-treated with licorice extract (100 μg/ml), GC-KO extract (89.4 μg/ml), or the combination of GC-KO extract (89.4 μg/ml) and GC (10.6 μg/ml). Figure9A indicated that the treatment of licorice extract led to a marked suppression of NO production as compared to LPS treatment [inhibition ratio (IR) 57.7% ]. Interestingly, the inhibitory effect of GC-KO extract was lower (IR 17.8% ) compared with licorice extract although GC alone could not block NO production as indicated above. On the other hand, the combined treatment with GC-KO extract and GC significantly improved the inhibitory ability (IR 33.5% ). To determine whether the combinational effect of GC-KO extract and GC was related to iNOS expression, we performed Western blotting. As shown in Figure 9B, the treatment of GC-KO extract diminished the inhibitory ability of LE on iNOS expression, and addition of GC to GC-KO extract could improve it. These data suggest that GC alone cannot

\**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001 indicate significant differences from the LPS alone

**Figure 7.** Preparation of GC-KO extract from licorice extract by anti-GC immunoaffinity column.

To further characterize GC-KO extract, the TLC analysis and Eastern blotting were per‐ formed [50]. As shown in Figure 8A, several spots including GC were detected in lico‐ rice extract (Lane B). However, the spot of GC was completely disappeared in GC-KO extract, although all other spots were clearly detected (lane C). Eastern blotting by anti-GC MAb indicated that GC was detected in licorice extract (Figure 8B, lane B), but the spot of GC was disappeared in GC-KO extract (Figure 8B, lane C). Therefore, these data suggest that GC was specifically eliminated from licorice extract by anti-GC MAb immu‐ noaffinity column.

**Figure 8.** TLC profiles (A) and Eastern blotting by anti-GC MAb (B). Lane A, B, and C indicate GC, licorice extract, and GC-KO extract, respectively.

#### **3.2.** *in vitro* **Assay by GC-KO extract prepared by anti-GC immunoaffinity column**

Nitric oxide (NO), synthesized by NO synthase (NOS) from L-arginine, is an important reg‐ ulatory/modulatory mediator for several physiological processes [51]. However, during in‐ flammatory process, a large amount of NO is produced by inducible NOS (iNOS) stimulated by bacterial lipopolysaccharide (LPS) and inflammatory cytokines participates in the patho‐ genesis of inflammatory diseases [52]. Overproduced NO synthesized by iNOS triggers a variety of inflammatory diseases including sepsis, psoriasis, arthritis, multiple sclerosis, and systemic lupus *erythematosus* [53]*.* Therefore, inhibiting NO production by blocking iNOS expression may be useful strategy to treat a variety of inflammatory diseases.

In LPS-treated mouse RAW264 macrophages, licorice extract inhibited NO production and iNOS expression. At 100 μg/mL of licorice extract, iNOS protein and mRNA were complete‐ ly suppressed [50].

**Figure 7.** Preparation of GC-KO extract from licorice extract by anti-GC immunoaffinity column.

noaffinity column.

38 Latest Research into Quality Control

GC-KO extract, respectively.

To further characterize GC-KO extract, the TLC analysis and Eastern blotting were per‐ formed [50]. As shown in Figure 8A, several spots including GC were detected in lico‐ rice extract (Lane B). However, the spot of GC was completely disappeared in GC-KO extract, although all other spots were clearly detected (lane C). Eastern blotting by anti-GC MAb indicated that GC was detected in licorice extract (Figure 8B, lane B), but the spot of GC was disappeared in GC-KO extract (Figure 8B, lane C). Therefore, these data suggest that GC was specifically eliminated from licorice extract by anti-GC MAb immu‐

**Figure 8.** TLC profiles (A) and Eastern blotting by anti-GC MAb (B). Lane A, B, and C indicate GC, licorice extract, and

**Figure 9.** Effect of GC-KO extract and the combination of GC-KO extract and GC on NO production (A) and iNOS pro‐ tein expression (B) in LPS-treated RAW264 cells.Each bar indicates the mean S.D. of four individual experiments. \**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001 indicate significant differences from the LPS alone

We next examined the inhibitory effect of GC alone, GC-KO extract and the combined treat‐ ment with GC and GC-KO extract on NO production [50]. Since 100 μg of licorice extract contains 10.6 ±0.618 μg of GC,the cells were pre-treated with licorice extract (100 μg/ml), GC-KO extract (89.4 μg/ml), or the combination of GC-KO extract (89.4 μg/ml) and GC (10.6 μg/ml). Figure9A indicated that the treatment of licorice extract led to a marked suppression of NO production as compared to LPS treatment [inhibition ratio (IR) 57.7% ]. Interestingly, the inhibitory effect of GC-KO extract was lower (IR 17.8% ) compared with licorice extract although GC alone could not block NO production as indicated above. On the other hand, the combined treatment with GC-KO extract and GC significantly improved the inhibitory ability (IR 33.5% ). To determine whether the combinational effect of GC-KO extract and GC was related to iNOS expression, we performed Western blotting. As shown in Figure 9B, the treatment of GC-KO extract diminished the inhibitory ability of LE on iNOS expression, and addition of GC to GC-KO extract could improve it. These data suggest that GC alone cannot suppress iNOS expression, but combinational inhibition of iNOS expression may occur when GC coexists with the other constituents contained in licorice extract. The *in vitro* and in *vivo* analysis by using KO extract prepared by immunoaffinity column is a useful approach for determination of potential function of natural compound on *in vitro* and in *vivo* assays.

**References**

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[2] Tan, T. W., Huo, Q., & Ling, Q. (2002). Purification of glycyrrhizin from glycyrrhiza uralensis fisch with ethanol/phosphate aqueous two phase system. *Biochem Lett*,

[3] Sakata, R., Shoyama, Y., & Murakami, H. (1994). Production of monoclonal antibod‐ ies and enzyme immunoassay for typical adenylate cyclase activator, Forskolin. *Cyto‐*

[4] Xuan, L., Tanaka, H., Xu, Y., & Shoyama, Y. (1999). Preparation of monoclonal anti‐

[5] Lu, Z., Morinaga, O., Tanaka, H., & Shoyama, Y. (2003). A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and tradi‐

[6] Shoyama, Y., Fukada, T., & Murakami, H. (1996). Production of monoclonal antibod‐

[7] Kim, J. S., Tanaka, H., & Shoyama, Y. (2004). Immunoquantitative analysis for ber‐ berine and its related compounds using monoclonal antibodies in herbal medicines.

[8] Tanaka, H., Fukuda, N., & Shoyama, Y. (1999). Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization. Cyto‐

[9] Fukuda, N., Tanaka, H., & Shoyama, Y. (2000). Formation of monoclonal antibody against a major ginseng component, ginsenoside Rg1 and its characterization Mono‐

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#### **4. Conclusion**

In this chapter, we introduce the unique strategy of one-step purification of target com‐ pounds from crude extract by anti-natural compound specific MAb-conjugated immunoaf‐ finity column. The immunoaffinity column conjugated with anti-G-Rb1 MAb could purify the G-Rb1 from *P. ginseng* extract, and the washing fraction contained all compounds ex‐ pect only G-Rb1, which was named G-Rb1-KO extract. By the use of the cross-reactivity of MAb, the anti-G-Rb1 immunoaffinity column can identify new unknown compounds relat‐ ed to target compound of MAb and determine their structures. Furthermore, our data suggest that the combination of the immunoaffinity column and ELISA by using MAb provided a reliable and high sensitivity analysis for target compound in various TCMs and crude extract. We also demonstrated the in vitro assay by using GC-KO extract prepared by anti-GC immunoaffinity column from licorice extract. The KO extract may be able to support the pharmacological investigation for finding out a really active compound in a TCM and crude drug.

#### **Acknowledgements**

This work was funded by the Asahi Beer Science Promoting Foundation and Takeda Science Foundation. The research in this paper was also supported in part by Sasakawa Scientific Research Grant from Japan Science Society and "Science and Technology Research Partner‐ ship for Sustainable Development (SATREPS)" supported by the Japan Science and Technol‐ ogy Agency (JST) and the Japan International Cooperation Agency (JICA).

#### **Author details**

Takuhiro Uto1 , Nguyen Huu Tung1 , Hiroyuki Tanaka2 and Yukihiro Shoyama1\*

\*Address all correspondence to: shoyama@niu.ac.jp

1 Faculty of Pharmaceutical Sciences, Nagasaki International University,, Japan

2 Faculty of Pharmaceutical Sciences, Kyushu University,, Japan

#### **References**

suppress iNOS expression, but combinational inhibition of iNOS expression may occur when GC coexists with the other constituents contained in licorice extract. The *in vitro* and in *vivo* analysis by using KO extract prepared by immunoaffinity column is a useful approach for determination of potential function of natural compound on *in vitro* and in *vivo* assays.

In this chapter, we introduce the unique strategy of one-step purification of target com‐ pounds from crude extract by anti-natural compound specific MAb-conjugated immunoaf‐ finity column. The immunoaffinity column conjugated with anti-G-Rb1 MAb could purify the G-Rb1 from *P. ginseng* extract, and the washing fraction contained all compounds ex‐ pect only G-Rb1, which was named G-Rb1-KO extract. By the use of the cross-reactivity of MAb, the anti-G-Rb1 immunoaffinity column can identify new unknown compounds relat‐ ed to target compound of MAb and determine their structures. Furthermore, our data suggest that the combination of the immunoaffinity column and ELISA by using MAb provided a reliable and high sensitivity analysis for target compound in various TCMs and crude extract. We also demonstrated the in vitro assay by using GC-KO extract prepared by anti-GC immunoaffinity column from licorice extract. The KO extract may be able to support the pharmacological investigation for finding out a really active compound in a

This work was funded by the Asahi Beer Science Promoting Foundation and Takeda Science Foundation. The research in this paper was also supported in part by Sasakawa Scientific Research Grant from Japan Science Society and "Science and Technology Research Partner‐ ship for Sustainable Development (SATREPS)" supported by the Japan Science and Technol‐

, Hiroyuki Tanaka2

1 Faculty of Pharmaceutical Sciences, Nagasaki International University,, Japan

and Yukihiro Shoyama1\*

ogy Agency (JST) and the Japan International Cooperation Agency (JICA).

, Nguyen Huu Tung1

\*Address all correspondence to: shoyama@niu.ac.jp

2 Faculty of Pharmaceutical Sciences, Kyushu University,, Japan

**4. Conclusion**

40 Latest Research into Quality Control

TCM and crude drug.

**Acknowledgements**

**Author details**

Takuhiro Uto1


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**Chapter 3**

**Standard Operating Procedures (SOP) for the**

**Compounds Contained in Plant Samples**

Elba Lúcia Cavalcanti de Amorim,

Joabe Gomes de Melo,

http://dx.doi.org/10.5772/51686

standardisation of the final product.

ologies for such determinations.

**1. Introduction**

Valérium Thijan Nobre de Almeida de Castro,

Allan Jonathan Chernichiarro Corrêa and

Additional information is available at the end of the chapter

Tadeu José da Silva Peixoto Sobrinho

**Spectrophotometric Determination of Phenolic**

The quality control of raw materials and products from plants is one of the topics most discussed by universities and health surveillance agencies. One of the primary tools used to ensure the reliability of production processes is the use of Standard Operating Procedures (SOPs). SOPs sequentially describe the steps of a particular methodology so that it can be reproduced by different analysts, which minimises variations in their implementation and improves the

Several techniques, such as high performance liquid chromatography, gas chromatography and mass spectrometry, can be used in SOPs to control the quality of plant phenolic com‐ pounds [1,2]. However, these compounds have a characteristic spectrum produced by the double bonds in the aromatic rings and substituent positions that facilitates their identifica‐ tion and the development of spectrometric analytical techniques is easily accomplished. In this sense, spectrophotometric methods are more practical, reproducible and inexpensive than other techniques and are therefore favoured for the development of analytical method‐

In addition to producing compounds such as carbohydrates, lipids, proteins and nucleic acids directly involved in their essential growth functions, plants have an arsenal of enzymes capable

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Cavalcanti de Amorim et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Cavalcanti de Amorim et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

## **Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained in Plant Samples**

Elba Lúcia Cavalcanti de Amorim, Valérium Thijan Nobre de Almeida de Castro, Joabe Gomes de Melo, Allan Jonathan Chernichiarro Corrêa and Tadeu José da Silva Peixoto Sobrinho

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51686

**1. Introduction**

The quality control of raw materials and products from plants is one of the topics most discussed by universities and health surveillance agencies. One of the primary tools used to ensure the reliability of production processes is the use of Standard Operating Procedures (SOPs). SOPs sequentially describe the steps of a particular methodology so that it can be reproduced by different analysts, which minimises variations in their implementation and improves the standardisation of the final product.

Several techniques, such as high performance liquid chromatography, gas chromatography and mass spectrometry, can be used in SOPs to control the quality of plant phenolic com‐ pounds [1,2]. However, these compounds have a characteristic spectrum produced by the double bonds in the aromatic rings and substituent positions that facilitates their identifica‐ tion and the development of spectrometric analytical techniques is easily accomplished. In this sense, spectrophotometric methods are more practical, reproducible and inexpensive than other techniques and are therefore favoured for the development of analytical method‐ ologies for such determinations.

In addition to producing compounds such as carbohydrates, lipids, proteins and nucleic acids directly involved in their essential growth functions, plants have an arsenal of enzymes capable

© 2012 Cavalcanti de Amorim et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Cavalcanti de Amorim et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of producing, processing and accumulating several other substances not necessarily related to the maintenance of their life [3]. All of these reactions can be defined as secondary metab‐ olism, the products of which provide advantages for both survival and species perpetuation in the plant's ecosystem [4]. However, this protection has a cost for the plant because meta‐ bolic resources that could increase its biomass are used to produce these compounds. In addition to protection, secondary metabolites perform important ecological functions such as inhibiting the germination and growth of other plants, attracting both pollinators and seeddispersing animals and providing chemical defences against microorganisms [5].

Condensed tannins, also known as proanthocyanidins (Figure 2), can contain dozens of units of flavan-3-ols (catechin) or flavan-3,4-diols (leucoanthocyanidins). These units have a complex structure and are resistant to hydrolysis; however, they can be soluble in aqueous

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

49

http://dx.doi.org/10.5772/51686

Hydrolysable and condensed tannins may occur in the same plant simultaneously. Howev‐ er, the hydrolysable tannins are characteristic of Magnoliopsida herbaceous and woody plants and are restricted to certain taxonomic families. Ellagitannins have been used as taxonomic markers, particularly for Hemamelidaceae, Dilenidaceaa and Rosaceae. Condensed tannins have been identified in all plant groups, including Gymnosperms and Pteridophytes [10]. These secondary metabolites were initially identified by their astringent taste and capacity to bind proteins, which allows for the precipitation and formation of complexes with colla‐ gen skin fibres to increase their resistance to water and heat. Chemically speaking, hydro‐ phobic interactions and hydrogen bonds between the phenolic groups in tannins and some macromolecules explain these features. However, the stability of the formed complexes only results after the formation of covalent bonds via the oxidation of tannins by quinones [11].

Since antiquity, plants containing tannins have been used medicinally as anti-inflamma‐ tory, antimicrobial, antitumor and antiviral agents and to treat both wound sand burns [7]. Tannins are also used to manufacture beverages and process animal skin into leath‐ er. Some researchers have shown that tannins protect plants against attack by herbi‐

Although the use of tannin in the tanning industry has become restricted, interest in study‐ ing the ingestion of foods containing tannins to prevent diseases such as atherosclerosis or certain types of cancer has increased because of various epidemiological studies. Some stud‐ ies report that the complexation of tannins with proteins gives them an important role in controlling bacteria, fungi and insects [13-15]. Other studies examined the inhibitory action of the enzyme reverse transcriptase [16] and the anticarcinogenic activity associated with

organic solvents because of their structure [7].

**Figure 2.** Basic structures of catechin and proanthocyanidins (condensed tannin).

vores and pathogens [12].

Phenolic compounds, which have one or more hydroxyl groups linked to an aromatic ring, stand out from other classes of plant secondary metabolites because they are widely distrib‐ uted and have various ecological functions that are scientifically proven to have numerous pharmacological activities and are well represented by tannins, flavonoids and coumarins.

#### **1.1. Tannins**

Tannins are water soluble phenolic compounds with a molecular weight between 500 and 3000 Daltons and may be chemically classified into two groups: hydrolysable tannins and condensed tannins [6,7]. Hydrolysable tannins are connected by ester-carboxyl linkages, which undergo hydrolysis under acidic and basic conditions [8]. Figure 1 presents an exam‐ ple of hydrolysable tannin (gallotannin), connected through a polyol (usually β-D-glucose) with the hydroxyl group esterified by gallic acid. Polyphenols connected with ellagic acid are called ellagitannins [9].

**Figure 1.** Structure of gallic acid, ellagic acid and 1,3,6-tri-O-galloyl-β-D-glucose, a hydrolysable tannin.

Condensed tannins, also known as proanthocyanidins (Figure 2), can contain dozens of units of flavan-3-ols (catechin) or flavan-3,4-diols (leucoanthocyanidins). These units have a complex structure and are resistant to hydrolysis; however, they can be soluble in aqueous organic solvents because of their structure [7].

**Figure 2.** Basic structures of catechin and proanthocyanidins (condensed tannin).

of producing, processing and accumulating several other substances not necessarily related to the maintenance of their life [3]. All of these reactions can be defined as secondary metab‐ olism, the products of which provide advantages for both survival and species perpetuation in the plant's ecosystem [4]. However, this protection has a cost for the plant because meta‐ bolic resources that could increase its biomass are used to produce these compounds. In addition to protection, secondary metabolites perform important ecological functions such as inhibiting the germination and growth of other plants, attracting both pollinators and seed-

Phenolic compounds, which have one or more hydroxyl groups linked to an aromatic ring, stand out from other classes of plant secondary metabolites because they are widely distrib‐ uted and have various ecological functions that are scientifically proven to have numerous pharmacological activities and are well represented by tannins, flavonoids and coumarins.

Tannins are water soluble phenolic compounds with a molecular weight between 500 and 3000 Daltons and may be chemically classified into two groups: hydrolysable tannins and condensed tannins [6,7]. Hydrolysable tannins are connected by ester-carboxyl linkages, which undergo hydrolysis under acidic and basic conditions [8]. Figure 1 presents an exam‐ ple of hydrolysable tannin (gallotannin), connected through a polyol (usually β-D-glucose) with the hydroxyl group esterified by gallic acid. Polyphenols connected with ellagic acid

dispersing animals and providing chemical defences against microorganisms [5].

**Figure 1.** Structure of gallic acid, ellagic acid and 1,3,6-tri-O-galloyl-β-D-glucose, a hydrolysable tannin.

**1.1. Tannins**

48 Latest Research into Quality Control

are called ellagitannins [9].

Hydrolysable and condensed tannins may occur in the same plant simultaneously. Howev‐ er, the hydrolysable tannins are characteristic of Magnoliopsida herbaceous and woody plants and are restricted to certain taxonomic families. Ellagitannins have been used as taxonomic markers, particularly for Hemamelidaceae, Dilenidaceaa and Rosaceae. Condensed tannins have been identified in all plant groups, including Gymnosperms and Pteridophytes [10].

These secondary metabolites were initially identified by their astringent taste and capacity to bind proteins, which allows for the precipitation and formation of complexes with colla‐ gen skin fibres to increase their resistance to water and heat. Chemically speaking, hydro‐ phobic interactions and hydrogen bonds between the phenolic groups in tannins and some macromolecules explain these features. However, the stability of the formed complexes only results after the formation of covalent bonds via the oxidation of tannins by quinones [11].

Since antiquity, plants containing tannins have been used medicinally as anti-inflamma‐ tory, antimicrobial, antitumor and antiviral agents and to treat both wound sand burns [7]. Tannins are also used to manufacture beverages and process animal skin into leath‐ er. Some researchers have shown that tannins protect plants against attack by herbi‐ vores and pathogens [12].

Although the use of tannin in the tanning industry has become restricted, interest in study‐ ing the ingestion of foods containing tannins to prevent diseases such as atherosclerosis or certain types of cancer has increased because of various epidemiological studies. Some stud‐ ies report that the complexation of tannins with proteins gives them an important role in controlling bacteria, fungi and insects [13-15]. Other studies examined the inhibitory action of the enzyme reverse transcriptase [16] and the anticarcinogenic activity associated with green tea and diets rich in fruits containing tannins [17]. It is generally believed that the pharmacological activity of tannins occurs via their complexation with metal ions, antioxi‐ dant activity or the ability to complex with macromolecules.

medicine as a hypoglycemic, diuretic and antihypertensive agent [31,32]. Several plantbased products are sold in open markets, pharmacies and natural product stores [33]. Though not officially recommended, herbal products from the leaves of pata-de-vaca (*Bauhi‐ nia* L.) are popularly used for therapeutic purposes. This plant was used in our study as a

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

51

http://dx.doi.org/10.5772/51686

Coumarins are lactones of *O*-hydroxy-cinnamic acid derived from trans-cinnamic acid via oxidation-reduction and isomerisation to produce 1,2-benzopyrone. Coumarins are divided into simple coumarins, furanocoumarins, pyranocoumarins, dimeric coumarins and chro‐ mones (Figure 4) [34]. The difference between these classes is the position of the radical in the coumarin lactone ring, which varies between C-6, C-7 and C-8. Chromones represent a group of natural isomers to coumarin that may be linearly and angularly prenylated at C-6

Coumarins are used as antioxidants, anti-HIV drugs, antispasmodics, spasmolytics, hypoli‐ pidemics, hypotensives and vasodilating agents [34]; however, they are also used in food flavouring, perfumes, tobacco and cosmetic products [35]. It is estimated that the daily hu‐

Coumarins are a class of secondary metabolites widely distributed throughout the Plantae kingdom and found in Fungi and Bacteria as well [37]. In plants, coumarins are found fre‐ quently in the families of Apiaceae, Rutaceae and Asteraceae, and less frequently in the fam‐

man exposure to coumarins from cosmetics and perfumes is 0.04 mg/kg/day [36].

ilies of Fabaceae, Oleaceae, Moraceae and Thymeleaceae [38].

and C-8, respectively, in furanochromones and pyranochromones, respectively [34].

reference species for flavonoids.

**Figure 4.** Basic structure of coumarins and chromones.

**1.3. Coumarins**

*Maytenus ilicifolia* (Schrad.) Planch. (Celastraceae), popularly known as "espinheira-santa", is a species native to Brazil traditionally used to treat digestive disorders and the literature cites tannins as compounds that act to protect the stomach by helping to treat ulcers and gastritis [18-20]. In addition to these activities, *M. ilicifolia* has other pharmacological appli‐ cations, such as anticancer, antimicrobial and antioxidant activity and treatment of the cen‐ tral nervous system [21]. For these reasons, *M. ilicifolia* was chosen as a reference species in this study.

#### **1.2. Flavonoids**

Flavonoids comprise a group of natural substances with great structural diversity and there are currently more than nine thousand known flavonoids that do not occur in humans but can be found in various plant parts such as the leaves, fruits, bark, roots, stems and flowers [22,23]. Flavonoids (Figure 3) are composed of a simple skeleton containing two phenol rings connected by a propionic chain; where ring A is the acetate derivative and both ring B and the three-carbon bridge are derived via a shikimate pathway, which may be associated with carbohydrates (heterosides), un associated (aglycones) or polymerised further (antho‐ cyanins) [24].

**Figure 3.** Basic structure of flavonoids.

This class of metabolites has several biological functions, such as defence against both herbi‐ vores and pathogens, the perpetuation of the species by attracting seed dispersing animals, protection from ultraviolet rays and allelopathy [24,25]. Flavonoids also possess important pharmacological properties, such as antioxidant, antiinflammatory, anti-thrombogenic, anti‐ microbial, anticancer, antidiabetic and hypocholesterolemic activities [23,26].

Studies show that flavonoids are chemical markers responsible for various pharmacological activities performed by the genus *Bauhinia* [27-29]. *Bauhinia forficata* Link (Fabaceae), popu‐ larly known as "pata-de-vaca", is a tree native to Brazil that prevails in the phytogeographi‐ cal area of Mata Atlântica [30] and can also be found in Cerrado. Pata-de-vaca is used in folk medicine as a hypoglycemic, diuretic and antihypertensive agent [31,32]. Several plantbased products are sold in open markets, pharmacies and natural product stores [33]. Though not officially recommended, herbal products from the leaves of pata-de-vaca (*Bauhi‐ nia* L.) are popularly used for therapeutic purposes. This plant was used in our study as a reference species for flavonoids.

#### **1.3. Coumarins**

green tea and diets rich in fruits containing tannins [17]. It is generally believed that the pharmacological activity of tannins occurs via their complexation with metal ions, antioxi‐

*Maytenus ilicifolia* (Schrad.) Planch. (Celastraceae), popularly known as "espinheira-santa", is a species native to Brazil traditionally used to treat digestive disorders and the literature cites tannins as compounds that act to protect the stomach by helping to treat ulcers and gastritis [18-20]. In addition to these activities, *M. ilicifolia* has other pharmacological appli‐ cations, such as anticancer, antimicrobial and antioxidant activity and treatment of the cen‐ tral nervous system [21]. For these reasons, *M. ilicifolia* was chosen as a reference species in

Flavonoids comprise a group of natural substances with great structural diversity and there are currently more than nine thousand known flavonoids that do not occur in humans but can be found in various plant parts such as the leaves, fruits, bark, roots, stems and flowers [22,23]. Flavonoids (Figure 3) are composed of a simple skeleton containing two phenol rings connected by a propionic chain; where ring A is the acetate derivative and both ring B and the three-carbon bridge are derived via a shikimate pathway, which may be associated with carbohydrates (heterosides), un associated (aglycones) or polymerised further (antho‐

This class of metabolites has several biological functions, such as defence against both herbi‐ vores and pathogens, the perpetuation of the species by attracting seed dispersing animals, protection from ultraviolet rays and allelopathy [24,25]. Flavonoids also possess important pharmacological properties, such as antioxidant, antiinflammatory, anti-thrombogenic, anti‐

Studies show that flavonoids are chemical markers responsible for various pharmacological activities performed by the genus *Bauhinia* [27-29]. *Bauhinia forficata* Link (Fabaceae), popu‐ larly known as "pata-de-vaca", is a tree native to Brazil that prevails in the phytogeographi‐ cal area of Mata Atlântica [30] and can also be found in Cerrado. Pata-de-vaca is used in folk

microbial, anticancer, antidiabetic and hypocholesterolemic activities [23,26].

dant activity or the ability to complex with macromolecules.

this study.

**1.2. Flavonoids**

50 Latest Research into Quality Control

cyanins) [24].

**Figure 3.** Basic structure of flavonoids.

Coumarins are lactones of *O*-hydroxy-cinnamic acid derived from trans-cinnamic acid via oxidation-reduction and isomerisation to produce 1,2-benzopyrone. Coumarins are divided into simple coumarins, furanocoumarins, pyranocoumarins, dimeric coumarins and chro‐ mones (Figure 4) [34]. The difference between these classes is the position of the radical in the coumarin lactone ring, which varies between C-6, C-7 and C-8. Chromones represent a group of natural isomers to coumarin that may be linearly and angularly prenylated at C-6 and C-8, respectively, in furanochromones and pyranochromones, respectively [34].

**Figure 4.** Basic structure of coumarins and chromones.

Coumarins are used as antioxidants, anti-HIV drugs, antispasmodics, spasmolytics, hypoli‐ pidemics, hypotensives and vasodilating agents [34]; however, they are also used in food flavouring, perfumes, tobacco and cosmetic products [35]. It is estimated that the daily hu‐ man exposure to coumarins from cosmetics and perfumes is 0.04 mg/kg/day [36].

Coumarins are a class of secondary metabolites widely distributed throughout the Plantae kingdom and found in Fungi and Bacteria as well [37]. In plants, coumarins are found fre‐ quently in the families of Apiaceae, Rutaceae and Asteraceae, and less frequently in the fam‐ ilies of Fabaceae, Oleaceae, Moraceae and Thymeleaceae [38].

*Mikania glomerata* Spreng. (Asteraceae), popularly known in Brazil as "guaco", has existed in the Brazilian Pharmacopoeia since 1929 and is used as an expectorant to treat respiratory problems. Most herbal products are marketed as some form of syrup; however, they are produced from fluid extracts and tinctures [39,40]. Chemical studies of this species show coumarins to be a major component that can be used as a chemical marker for the quality control of products based on guaco [41-43].

**3.1. Standard operating procedure for the quantification of tannins**

calculated as the difference between the total and waste phenol content.

or more molecules of flavan-3-ol or flavan-3,4-diol [3] [7].

**Reagents:**

distilled water.

tion of 1 mg/mL.

tannic acid concentration will be 1-5 μg/mL.

**Description:** Tannins are phenolic compounds with the ability to bind macromolecules, es‐ pecially proteins. This class of compounds has traditionally been divided into two groups: hydrolysable and condensed tannins. Hydrolysable tannins are characterised by a central polyol, usually β-D-glucose, containing hydroxy groups esterified with gallic acid and ella‐ gic acid. Condensed tannins are oligomers or polymers formed by the condensation of two

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

53

http://dx.doi.org/10.5772/51686

**Principle of the method:** The phenolic compounds in the sample are oxidised using the Folin-Ciocalteu reagent. This reagent is a mixture of phosphotungstic and phosphomolybdic acids that are reduced by the oxidation of phenolic compounds in a mixture of tungsten and molybdenum oxides. The blue colour produced by the oxides has a maximum absorption at 760 nm and is proportional to the total phenolic concentration [47]. The tannin content is

(1) Tannic acid (0.1 mg/mL, w/v): Dissolve 10 mg of tannic acid in 100 ml of distilled water.

(2) Folin-Ciocalteu reagent (10%, v/v): Dilute 5 ml of Folin-Ciocalteu reagent with 45 mL of

(3) Sodium carbonate Na2CO3 (7.5%, w/v): Dissolve 7.5 g of Na2CO3 in 100 ml of distilled

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter pa‐ per into a 50 mL volumetric flask and the volume should be completed using the same sol‐ vent. The final extract concentration will be 10 mg/mL. If the products are liquid, they must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentra‐

**Calibration curve:** The calibration curve must be prepared using 100-500 μL aliquots of the tannic acid solution, 500 μL of the Folin-Ciocalteu solution and 1 mL of the sodium carbo‐ nate solution. The final volume should be adjusted to 10 mL with distilled water. The final

**Measurement procedure:** To quantify the total phenol concentration (that is, all of the phe‐ nols present in the sample), 500 μL of the extract must be transferred to a test tube. Next, 500 μL of the Folin-Ciocalteu solution, 1 mL of the sodium carbonate solution and 8 ml of distil‐ led water are added. The samples remain at room temperature for 30 minutes. The spectro‐ photometer should be adjusted to a wavelength of 760 nm and the equipment must be rinsed with distilled water. To quantify the phenol waste, (i.e., the phenols present in the sample except for the proteins precipitated with the tannin) 500 mg of casein is weighed and

water. If necessary, solubilising the solution on a heating plate and magnetic stirrer.

(4) Methanol (80%, v/v): Dilute 800 ml of methanol with 200 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

Coumarins have a characteristic UV spectrum due to the nature and position of their sub‐ stituents, which facilitates both their identification and the development of analytical spec‐ trophotometric techniques [11]. For these reasons, *M. glomerata* was chosen as the reference species to quantify the coumarin level.

#### **2. Problem statement**

Despite extensive literature presenting various analytical methods, the development of an SOP is often difficult for three reasons: 1) the work does not detail the difficulties and adjust‐ ments required to implement the methodology, 2) the steps are not clearly presented for re‐ production and 3) the limits of interpretation are not discussed. One criterion recommended by the National Sanitary Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) in Brazil for the standardisation of herbal drugs is the active compound content or chemical class, which is the total concentration of tannins for products based on *M. ilicifolia* (espinheira-santa) [44], flavonoids for *B. forficata* (pata-de-vaca) [45,46] and coumarins for *M. glomerata* (guaco) [44].

Thus, this paper presents research protocols adopted by our research group to study the lev‐ els of tannins, flavonoids and coumarins from plant extracts and the experimental applica‐ tion of these SOPs to analyse products sold in markets (pharmacies and natural product stores) as phytomedicines1 or plant drugs2 with high commercial value.

#### **3. Standard operating procedures (POP)**

The following SOPs describe the chemical classes to be analysed and the chemical basis of the methods. They provide a detailed list of all the reagents required for the preparation and describe the experimental procedure to be followed. Finally, there is a list of references used in the development of the SOP.

<sup>1 &</sup>quot;All medicine is obtained using solely active raw vegetables. It is characterised by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of its quality. Its efficacy and safety are validated through ethnopharmacological surveys of use, documentation, technical and scientific publications or clinical trial phase 3"[44].

<sup>2 &</sup>quot;Medicinal plant or their parts, after collection processes, stabilisation and drying and can be full, erasures, crushed or powdered" [44].

#### **3.1. Standard operating procedure for the quantification of tannins**

**Description:** Tannins are phenolic compounds with the ability to bind macromolecules, es‐ pecially proteins. This class of compounds has traditionally been divided into two groups: hydrolysable and condensed tannins. Hydrolysable tannins are characterised by a central polyol, usually β-D-glucose, containing hydroxy groups esterified with gallic acid and ella‐ gic acid. Condensed tannins are oligomers or polymers formed by the condensation of two or more molecules of flavan-3-ol or flavan-3,4-diol [3] [7].

**Principle of the method:** The phenolic compounds in the sample are oxidised using the Folin-Ciocalteu reagent. This reagent is a mixture of phosphotungstic and phosphomolybdic acids that are reduced by the oxidation of phenolic compounds in a mixture of tungsten and molybdenum oxides. The blue colour produced by the oxides has a maximum absorption at 760 nm and is proportional to the total phenolic concentration [47]. The tannin content is calculated as the difference between the total and waste phenol content.

#### **Reagents:**

*Mikania glomerata* Spreng. (Asteraceae), popularly known in Brazil as "guaco", has existed in the Brazilian Pharmacopoeia since 1929 and is used as an expectorant to treat respiratory problems. Most herbal products are marketed as some form of syrup; however, they are produced from fluid extracts and tinctures [39,40]. Chemical studies of this species show coumarins to be a major component that can be used as a chemical marker for the quality

Coumarins have a characteristic UV spectrum due to the nature and position of their sub‐ stituents, which facilitates both their identification and the development of analytical spec‐ trophotometric techniques [11]. For these reasons, *M. glomerata* was chosen as the reference

Despite extensive literature presenting various analytical methods, the development of an SOP is often difficult for three reasons: 1) the work does not detail the difficulties and adjust‐ ments required to implement the methodology, 2) the steps are not clearly presented for re‐ production and 3) the limits of interpretation are not discussed. One criterion recommended by the National Sanitary Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) in Brazil for the standardisation of herbal drugs is the active compound content or chemical class, which is the total concentration of tannins for products based on *M. ilicifolia* (espinheira-santa) [44], flavonoids for *B. forficata* (pata-de-vaca) [45,46] and coumarins for *M.*

Thus, this paper presents research protocols adopted by our research group to study the lev‐ els of tannins, flavonoids and coumarins from plant extracts and the experimental applica‐ tion of these SOPs to analyse products sold in markets (pharmacies and natural product

The following SOPs describe the chemical classes to be analysed and the chemical basis of the methods. They provide a detailed list of all the reagents required for the preparation and describe the experimental procedure to be followed. Finally, there is a list of references used

1 "All medicine is obtained using solely active raw vegetables. It is characterised by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of its quality. Its efficacy and safety are validated through ethnopharmacological surveys of use, documentation, technical and scientific publications or clinical trial

2 "Medicinal plant or their parts, after collection processes, stabilisation and drying and can be full, erasures, crushed

with high commercial value.

or plant drugs2

**3. Standard operating procedures (POP)**

control of products based on guaco [41-43].

species to quantify the coumarin level.

**2. Problem statement**

52 Latest Research into Quality Control

*glomerata* (guaco) [44].

stores) as phytomedicines1

in the development of the SOP.

phase 3"[44].

or powdered" [44].

(1) Tannic acid (0.1 mg/mL, w/v): Dissolve 10 mg of tannic acid in 100 ml of distilled water.

(2) Folin-Ciocalteu reagent (10%, v/v): Dilute 5 ml of Folin-Ciocalteu reagent with 45 mL of distilled water.

(3) Sodium carbonate Na2CO3 (7.5%, w/v): Dissolve 7.5 g of Na2CO3 in 100 ml of distilled water. If necessary, solubilising the solution on a heating plate and magnetic stirrer.

(4) Methanol (80%, v/v): Dilute 800 ml of methanol with 200 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter pa‐ per into a 50 mL volumetric flask and the volume should be completed using the same sol‐ vent. The final extract concentration will be 10 mg/mL. If the products are liquid, they must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentra‐ tion of 1 mg/mL.

**Calibration curve:** The calibration curve must be prepared using 100-500 μL aliquots of the tannic acid solution, 500 μL of the Folin-Ciocalteu solution and 1 mL of the sodium carbo‐ nate solution. The final volume should be adjusted to 10 mL with distilled water. The final tannic acid concentration will be 1-5 μg/mL.

**Measurement procedure:** To quantify the total phenol concentration (that is, all of the phe‐ nols present in the sample), 500 μL of the extract must be transferred to a test tube. Next, 500 μL of the Folin-Ciocalteu solution, 1 mL of the sodium carbonate solution and 8 ml of distil‐ led water are added. The samples remain at room temperature for 30 minutes. The spectro‐ photometer should be adjusted to a wavelength of 760 nm and the equipment must be rinsed with distilled water. To quantify the phenol waste, (i.e., the phenols present in the sample except for the proteins precipitated with the tannin) 500 mg of casein is weighed and transferred into a 25 ml Erlenmeyer flask before adding 5 ml of the extract and 5 ml of water distilled. After two hours (time required for the complexing of the tannins to the total pro‐ tein), the extracts are filtered into a 10 mL volumetric flasks and its volume is adjusted with distilled water. The phenols are considered to be equal to the residue from the total phenol. These assays are performed at least in triplicate and the total phenolic content is expressed as milligrams of tannic acid equivalents per gram of sample or extract (mg TAE/g).

**Calibration curve:** The calibration curve must be prepared using 100-1000 μL aliquots of the rutin solution, 500 μL of the acetic acid solution, 2 mL of the pyridine solution and 1 ml of the reagent aluminium chloride solution. The final volume should be adjusted to 10 mL with

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

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http://dx.doi.org/10.5772/51686

**Measurement procedure:** To quantify the flavonoids, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 500 μL of the acetic acid solution, 2 mL of the pyridine solution, 1 ml of the reagent aluminium chloride solution and 6 ml of 80% methanol will be added. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a wavelength of 420 nm and the equipment must be rinsed with distilled water. The test shall be performed at least in triplicate and the flavonoid content is expressed as

Peixoto Sobrinho T. J. S, Silva C. H. T. P., Nascimento J. E., Monteiro J. M., Albuquerque U. P., Amorim E. L. C. Validação de metodologia espectrofotométrica para quantificação dos flavonóides de *Bauhinia cheilantha* (Bongard) Steudel. Brazilian Journal of Pharmaceutical

Zuanazzi, J. A. S.; Montanha, J. A. Flavonóides. In: Simões C. M. O., Schenkel E. P., Gos‐ manm, G, Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao med‐ icamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.577-614 [24].

**Description:** Coumarins are lactones of *O*-hydroxy-cinnamic acid and are metabolites of phenylalanine. 1,2-benzopyrone is the simplest coumarin representative, others being furo‐

**Principle of the method:** The Borntrager reaction is based on the solubility of free coumarin derivatives in polar organic solvents and the solubility of their soluble alkali phenolates. Coumarin absorbs at 280 nm; however, ionisation of phenolic hydroxyls in the molecule by alkaline hydroxide causes a bathochromic deviation to 320 nm, which is proportional to the

(1) 1,2-benzopyrone (1 mg/mL, w/v): Dissolve 10 mg of coumarin in 10 ml of distilled water.

(3) Hydrochloric acid solution, HCl (0.1 M, v/v): Dilute 10 ml of concentrated hydrochloric

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter paper

(2) Lead acetate (5%, w/v): Dissolve 2,5 g of lead acetate in 50 ml of distilled water.

(4) Methanol (80%, v/v): Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

80% methanol. The final rutin concentration will be 1-10 μg/mL.

milligrams of rutin equivalents per gram of sample or extract (mg RE/g).

**3.3. Standard operating procedure for the quantification of coumarins**

coumarins, pyranocoumarins, dimeric coumarins and chromones [34].

**References:**

Sciences 2008; 44 (4) 683-689 [48].

coumarin concentration [40].

acid with 1000 ml of distilled water.

**Reagents:**

#### **References:**

Amorim E. L. C, Nascimento J. E., Monteiro J. M., Peixoto Sobrinho T. J. S, Araújo T. A. S., Albuquerque U. P. A simple and accurate procedure for the determination of tannin and fla‐ vonoid levels and some applications in ethnobotany and ethnopharmacology. Functional Ecosystems and Communities 2008; 2(1) 88-94 [47].

Santos S. C., Mello J. C. P. Taninos. In: Simões C. M. O., Schenkel E. P., Gosmanm, G., Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004. p.615-656 [3].

#### **3.2. Standard operating procedure for the quantification of flavonoids**

**Description:** Flavonoids are the most important and diverse phenolic compounds. Most fla‐ vonoids have 15 carbon atoms and consist of two phenol rings connected by a chain of three carbons to form a tricyclic compound [24].

**Principle of the method:** The aluminium ion (Al3+) is reacted with the flavonoids in the sam‐ ple to form the stable flavonoid-Al3+complex, which has a yellow colour and intensity pro‐ portional to the flavonoid concentration. This reaction causes a bathochromic shift and intensification in the absorption, which can be measured without influence from other phe‐ nolic compounds present in the sample [48].

#### **Reagents:**

(1) Rutin (0.1 mg/mL, w/v): Dissolve 10 mg of rutin in 100 ml of methanol.

(2) Acetic acid solution (60%, v/v): Dilute 30 ml of acetic acid with 20 ml of methanol.

(3) Pyridine Solution (20%, v/v): Dilute 40 ml of pyridine with 160 ml of methanol.

(4) Aluminium chloride solution AlCl3 (5%, w/v): Dissolve 5 g AlCl3 in 100 mL of methanol. If necessary, complete dissolution via magnetic stirring.

(5) Methanol (80%, v/v). Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol on a hot plate for 30 minutes. The extract should be filtered through filter paper into a 50 mL volumetric flask and the volume should be adjusted with the same solvent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol to obtain a final concentration of 1 mg/mL.

**Calibration curve:** The calibration curve must be prepared using 100-1000 μL aliquots of the rutin solution, 500 μL of the acetic acid solution, 2 mL of the pyridine solution and 1 ml of the reagent aluminium chloride solution. The final volume should be adjusted to 10 mL with 80% methanol. The final rutin concentration will be 1-10 μg/mL.

**Measurement procedure:** To quantify the flavonoids, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 500 μL of the acetic acid solution, 2 mL of the pyridine solution, 1 ml of the reagent aluminium chloride solution and 6 ml of 80% methanol will be added. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a wavelength of 420 nm and the equipment must be rinsed with distilled water. The test shall be performed at least in triplicate and the flavonoid content is expressed as milligrams of rutin equivalents per gram of sample or extract (mg RE/g).

#### **References:**

transferred into a 25 ml Erlenmeyer flask before adding 5 ml of the extract and 5 ml of water distilled. After two hours (time required for the complexing of the tannins to the total pro‐ tein), the extracts are filtered into a 10 mL volumetric flasks and its volume is adjusted with distilled water. The phenols are considered to be equal to the residue from the total phenol. These assays are performed at least in triplicate and the total phenolic content is expressed

Amorim E. L. C, Nascimento J. E., Monteiro J. M., Peixoto Sobrinho T. J. S, Araújo T. A. S., Albuquerque U. P. A simple and accurate procedure for the determination of tannin and fla‐ vonoid levels and some applications in ethnobotany and ethnopharmacology. Functional

Santos S. C., Mello J. C. P. Taninos. In: Simões C. M. O., Schenkel E. P., Gosmanm, G., Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao medicamento. Porto

**Description:** Flavonoids are the most important and diverse phenolic compounds. Most fla‐ vonoids have 15 carbon atoms and consist of two phenol rings connected by a chain of three

**Principle of the method:** The aluminium ion (Al3+) is reacted with the flavonoids in the sam‐ ple to form the stable flavonoid-Al3+complex, which has a yellow colour and intensity pro‐ portional to the flavonoid concentration. This reaction causes a bathochromic shift and intensification in the absorption, which can be measured without influence from other phe‐

as milligrams of tannic acid equivalents per gram of sample or extract (mg TAE/g).

Alegre: Universidade Federal do Rio Grande do Sul; 2004. p.615-656 [3].

**3.2. Standard operating procedure for the quantification of flavonoids**

(1) Rutin (0.1 mg/mL, w/v): Dissolve 10 mg of rutin in 100 ml of methanol.

(2) Acetic acid solution (60%, v/v): Dilute 30 ml of acetic acid with 20 ml of methanol.

(4) Aluminium chloride solution AlCl3 (5%, w/v): Dissolve 5 g AlCl3 in 100 mL of methanol.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol on a hot plate for 30 minutes. The extract should be filtered through filter paper into a 50 mL volumetric flask and the volume should be adjusted with the same solvent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol to obtain a final concentration of 1 mg/mL.

(3) Pyridine Solution (20%, v/v): Dilute 40 ml of pyridine with 160 ml of methanol.

(5) Methanol (80%, v/v). Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

Ecosystems and Communities 2008; 2(1) 88-94 [47].

carbons to form a tricyclic compound [24].

nolic compounds present in the sample [48].

If necessary, complete dissolution via magnetic stirring.

**References:**

54 Latest Research into Quality Control

**Reagents:**

Peixoto Sobrinho T. J. S, Silva C. H. T. P., Nascimento J. E., Monteiro J. M., Albuquerque U. P., Amorim E. L. C. Validação de metodologia espectrofotométrica para quantificação dos flavonóides de *Bauhinia cheilantha* (Bongard) Steudel. Brazilian Journal of Pharmaceutical Sciences 2008; 44 (4) 683-689 [48].

Zuanazzi, J. A. S.; Montanha, J. A. Flavonóides. In: Simões C. M. O., Schenkel E. P., Gos‐ manm, G, Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao med‐ icamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.577-614 [24].

#### **3.3. Standard operating procedure for the quantification of coumarins**

**Description:** Coumarins are lactones of *O*-hydroxy-cinnamic acid and are metabolites of phenylalanine. 1,2-benzopyrone is the simplest coumarin representative, others being furo‐ coumarins, pyranocoumarins, dimeric coumarins and chromones [34].

**Principle of the method:** The Borntrager reaction is based on the solubility of free coumarin derivatives in polar organic solvents and the solubility of their soluble alkali phenolates. Coumarin absorbs at 280 nm; however, ionisation of phenolic hydroxyls in the molecule by alkaline hydroxide causes a bathochromic deviation to 320 nm, which is proportional to the coumarin concentration [40].

#### **Reagents:**

(1) 1,2-benzopyrone (1 mg/mL, w/v): Dissolve 10 mg of coumarin in 10 ml of distilled water.

(2) Lead acetate (5%, w/v): Dissolve 2,5 g of lead acetate in 50 ml of distilled water.

(3) Hydrochloric acid solution, HCl (0.1 M, v/v): Dilute 10 ml of concentrated hydrochloric acid with 1000 ml of distilled water.

(4) Methanol (80%, v/v): Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter paper into a 50 mL volumetric flask and the final volume should be adjusted with the same sol‐ vent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentration of 1 mg/mL.

Analysis of the active component concentrations were conducted as listed in the described

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

To quantify the active components, calibration curves with increasing concentrations pro‐ portional to their absorbance were constructed. A correlation equation was obtained from these curves (generally linear) of the type *y = ax + b*, where *y* corresponds to the absorbance of the sample and *x* the concentration. To convert the absorbance values (nm) to sample concentration (μg/mL), it is necessary to place the sample absorbance into the equation as y.

tional results between the analyte concentration and the device response. The interval be‐ tween the lowest and highest scalar values, which is also called the linearity range, should be determined with both precision and accuracy. The correlation coefficient must be equal to

The calibration curve constructed for tannic acid and tannins and used to quantify *M. ilicifo‐ lia* demonstrated a correlation equation of y = 0.067x + 0.01 and a correlation coefficient of R2

**Figure 5.** Calibration curve constructed using tannic acid concentrations of 1-5 μg/mL at 760 nm used to quantify the

A calibration curve was constructed from rutin to quantify flavonoids in products from *B. forficata* and yielded the correlation equation y = 0.022 + 0.0039 x and correlation coefficient

) shows the ability of the method to provide directly propor‐

in Plant Samples

57

http://dx.doi.org/10.5772/51686

standard operating protocols (SOPs) and the results are presented below.

**4.1. Calibration curves**

The correlation coefficient (R2

tannin content of *Maytenus ilicifolia* (Schrad.) Planch.

or greater than 0.98.

= 0.996 (Figure 5).

R2

= 0.991 (Figure 6).

**Calibration curve:** The calibration curve must be prepared using 50-500 μL aliquots of the coumarin solution, 2 ml of distilled water and 500 μL of the lead acetate solution. The sam‐ ple should be shaken and the final volume should be adjusted to 10 mL with distilled water before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric acid solution. The final concentration of rutin will be 1-10 μg/mL.

**Measurement procedure:** To quantify the coumarins, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 2 ml of distilled water and 500 μL of lead acetate solution will be added. The sample is shaken and then 7 ml of distilled water are added before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric acid solution. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a 320 nm wavelength and the equipment must be rinsed with distilled water. The test should be performed in at least triplicate and the total coumarin content is expressed as milligrams of coumarin equivalents per gram of the sample extract (mg CE/g).

#### **References:**

Kuster R. A. M., Rocha L. A. M. A. Cumarinas, coronas e cantinas. In: Simões CMO, Schen‐ kel EP, Gosmanm, G, Mello JCP, Mentz LA, Petrovick PR. (ed.) Farmacognosia: da plan‐ ta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p. 537-556 [34].

Osório O. K., Martins J. L. S. Determinação de cumarina em extrato fluido e tintura de guaco por espectrofotometria derivada de primeira ordem. Brazilian Journal of Pharmaceutical Sci‐ ences 2004; 40 (4) 481-486 [40].

#### **4. Results**

Analysis of the active component levels in raw plant materials and phytomedicines is essen‐ tial for the safety and efficacy of pharmaceutical products [49]. The quantification of active compounds in herbals is still only incidentally performed due to the presence of active phy‐ tocomplexes plants and their extracts [50], which complicates their analysis. Through this framework, the use of standardised extracts focusing on specific groups of active compo‐ nents ensures the chemical homogeneity of the product, which improves product quality [51]. The compounds selected for this quality adjustment process should be the same as the assets in the product [52].

In this way, five products containing *M. ilicifolia* (all plant drugs), four containing *B. forficata* (all plant drugs) and five containing *M. glomerata* (three plant drugs and two fluid extract) sold in pharmacies and health food stores in Recife/PE, Northeast Brazil were obtained. Analysis of the active component concentrations were conducted as listed in the described standard operating protocols (SOPs) and the results are presented below.

#### **4.1. Calibration curves**

into a 50 mL volumetric flask and the final volume should be adjusted with the same sol‐ vent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentration of 1 mg/mL.

**Calibration curve:** The calibration curve must be prepared using 50-500 μL aliquots of the coumarin solution, 2 ml of distilled water and 500 μL of the lead acetate solution. The sam‐ ple should be shaken and the final volume should be adjusted to 10 mL with distilled water before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric

**Measurement procedure:** To quantify the coumarins, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 2 ml of distilled water and 500 μL of lead acetate solution will be added. The sample is shaken and then 7 ml of distilled water are added before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric acid solution. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a 320 nm wavelength and the equipment must be rinsed with distilled water. The test should be performed in at least triplicate and the total coumarin content is expressed as

Kuster R. A. M., Rocha L. A. M. A. Cumarinas, coronas e cantinas. In: Simões CMO, Schen‐ kel EP, Gosmanm, G, Mello JCP, Mentz LA, Petrovick PR. (ed.) Farmacognosia: da plan‐ ta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.

Osório O. K., Martins J. L. S. Determinação de cumarina em extrato fluido e tintura de guaco por espectrofotometria derivada de primeira ordem. Brazilian Journal of Pharmaceutical Sci‐

Analysis of the active component levels in raw plant materials and phytomedicines is essen‐ tial for the safety and efficacy of pharmaceutical products [49]. The quantification of active compounds in herbals is still only incidentally performed due to the presence of active phy‐ tocomplexes plants and their extracts [50], which complicates their analysis. Through this framework, the use of standardised extracts focusing on specific groups of active compo‐ nents ensures the chemical homogeneity of the product, which improves product quality [51]. The compounds selected for this quality adjustment process should be the same as the

In this way, five products containing *M. ilicifolia* (all plant drugs), four containing *B. forficata* (all plant drugs) and five containing *M. glomerata* (three plant drugs and two fluid extract) sold in pharmacies and health food stores in Recife/PE, Northeast Brazil were obtained.

milligrams of coumarin equivalents per gram of the sample extract (mg CE/g).

acid solution. The final concentration of rutin will be 1-10 μg/mL.

**References:**

56 Latest Research into Quality Control

537-556 [34].

**4. Results**

ences 2004; 40 (4) 481-486 [40].

assets in the product [52].

To quantify the active components, calibration curves with increasing concentrations pro‐ portional to their absorbance were constructed. A correlation equation was obtained from these curves (generally linear) of the type *y = ax + b*, where *y* corresponds to the absorbance of the sample and *x* the concentration. To convert the absorbance values (nm) to sample concentration (μg/mL), it is necessary to place the sample absorbance into the equation as y. The correlation coefficient (R2 ) shows the ability of the method to provide directly propor‐ tional results between the analyte concentration and the device response. The interval be‐ tween the lowest and highest scalar values, which is also called the linearity range, should be determined with both precision and accuracy. The correlation coefficient must be equal to or greater than 0.98.

The calibration curve constructed for tannic acid and tannins and used to quantify *M. ilicifo‐ lia* demonstrated a correlation equation of y = 0.067x + 0.01 and a correlation coefficient of R2 = 0.996 (Figure 5).

**Figure 5.** Calibration curve constructed using tannic acid concentrations of 1-5 μg/mL at 760 nm used to quantify the tannin content of *Maytenus ilicifolia* (Schrad.) Planch.

A calibration curve was constructed from rutin to quantify flavonoids in products from *B. forficata* and yielded the correlation equation y = 0.022 + 0.0039 x and correlation coefficient R2 = 0.991 (Figure 6).

**4.2. Content of active principles**

ins of *M. glomerata* are shown in Table 1.

*Maytenus ilicifolia* Tannins

*Bauhinia forficata* Flavonoids

*Mikania glomerata* Coumarins

Recife/PE, Northeast of Brazil.

statistically different (n = 6, p<0.05).

cient of variation (CV = 37.61%).

The results of quality control of tannins of *M. Ilicifolia*, flavonoids of *B. forficata* and coumar‐

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

**Specie / Marker Sample Concentration ± SD (mg/g) CV (%)**

**Table 1.** Results of quality control of tannins, flavonoids and coumarins contained in products based on *Maytenus ilicifolia (*Schrad.) Planch., *Bauhinia forficata* Link and *Mikania glomerata* Spreng. respectively, sold in pharmacies in

Values are mean ± standard deviation. Values followed by the same letter in column are not

Analysis of variance (ANOVA) is one way to indicate significant differences (p<0.01) for the drugs of *M. ilicifolia*, which is made from five plants. Samples Mi1 (12.57 ± 2.15 mg TAE/g) and Mi5 (11.81 ± 1.0 mg TAE/g) both had higher concentrations of tannins and were not sig‐ nificantly different, whereas at least three of the other samples showed tannins (Figure 8). Comparing the average tannin concentration from different samples showed a low coeffi‐

Analysis of four products containing *B. forficata* showed a significant difference (p<0.01). The sample Bf4 presented the highest flavonoid concentration (65.98 ± 3.62 mg RE/g), whereas samples Bf1 and Bf2 had the lowest concentrations (4.89 ± 0.11 and 7.27 ± 0.39 mg RE/g, re‐ spectively) with a content approximately 13 times lower. These results indicate that there is

no standardisation regarding the flavonoid concentrations in products (Figure 9).

MI1 12.57 ± 2.15a 17.12% MI2 4.04 ± 0.23b 5.75% MI3 5.61 ± 0.55bc 9.76% MI4 7.72 ± 0.84c 10.84% MI5 11.81 ± 1.00a 8.44%

in Plant Samples

59

http://dx.doi.org/10.5772/51686

BF1 4.89 ± 0.11a 2.33% BF2 7.27 ± 0.39a 5.41% BF3 50.38 ± 5.36b 10.64% BF4 65.98 ± 3.62c 5.49%

MG1 3.06 ± 0.20a 6.67% MG2 5.17 ± 0.59b 11.40% MG3 6.80 ± 0.24c 3.46% MG4 1.63 ± 0.20d 12.50% MG5 4.49 ± 0.20b 4.55%

**Figure 6.** Calibration curve constructed from rutin for concentrations of 1-10 μg/mL at 420 nm used to quantify the flavonoids content in *Bauhinia forficata* Link.

The correlation equation and coefficient obtained from the calibration curve used to analyse coumarins in products containing *M. glomerata* were y = 0.049 x + 0.031 and R2 = 0.994 (Figure 7), respectively.

**Figure 7.** The calibration curve constructed using 1,2-benzopyrone in concentrations of 1-12 μg/mL using 320 nm ex‐ citation to quantify the coumarin content in *Mikania glomerata* Spreng.

#### **4.2. Content of active principles**

**Figure 6.** Calibration curve constructed from rutin for concentrations of 1-10 μg/mL at 420 nm used to quantify the

The correlation equation and coefficient obtained from the calibration curve used to analyse

**Figure 7.** The calibration curve constructed using 1,2-benzopyrone in concentrations of 1-12 μg/mL using 320 nm ex‐

citation to quantify the coumarin content in *Mikania glomerata* Spreng.

= 0.994 (Figure

coumarins in products containing *M. glomerata* were y = 0.049 x + 0.031 and R2

flavonoids content in *Bauhinia forficata* Link.

58 Latest Research into Quality Control

7), respectively.


The results of quality control of tannins of *M. Ilicifolia*, flavonoids of *B. forficata* and coumar‐ ins of *M. glomerata* are shown in Table 1.

**Table 1.** Results of quality control of tannins, flavonoids and coumarins contained in products based on *Maytenus ilicifolia (*Schrad.) Planch., *Bauhinia forficata* Link and *Mikania glomerata* Spreng. respectively, sold in pharmacies in Recife/PE, Northeast of Brazil.

Values are mean ± standard deviation. Values followed by the same letter in column are not statistically different (n = 6, p<0.05).

Analysis of variance (ANOVA) is one way to indicate significant differences (p<0.01) for the drugs of *M. ilicifolia*, which is made from five plants. Samples Mi1 (12.57 ± 2.15 mg TAE/g) and Mi5 (11.81 ± 1.0 mg TAE/g) both had higher concentrations of tannins and were not sig‐ nificantly different, whereas at least three of the other samples showed tannins (Figure 8). Comparing the average tannin concentration from different samples showed a low coeffi‐ cient of variation (CV = 37.61%).

Analysis of four products containing *B. forficata* showed a significant difference (p<0.01). The sample Bf4 presented the highest flavonoid concentration (65.98 ± 3.62 mg RE/g), whereas samples Bf1 and Bf2 had the lowest concentrations (4.89 ± 0.11 and 7.27 ± 0.39 mg RE/g, re‐ spectively) with a content approximately 13 times lower. These results indicate that there is no standardisation regarding the flavonoid concentrations in products (Figure 9).

sample Mg4 had the lowest level (1.63 ± 0.20 mg CE/g), with an approximately four times

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

61

http://dx.doi.org/10.5772/51686

**Figure 10.** Concentration of coumarins (mg CE/g) contained in products from *Mikania glomerata* Spreng. sold in Re‐

The quantitative analysis of raw vegetables and phytomedicines is a fundamental quality control process that leads to security, stability, consistency and effectiveness in the produced phytomedicines [49]. It is important to emphasise the need for standardisation in analysing herbal medicines to determine the concentration of their active components in raw vegetable

This chapter provides easily reproducible standard operating procedures (SOPs) for the quality control of raw materials and herbal plants to ensure a minimal standard of quality in products sold. The implementation of these SOPs allows for the analysis of samples sold in establishments in Recife/PE and reveals an inconsistency in the concentration of tannins, fla‐

The low level of these metabolites may alter their effectiveness and more rigorous quality control and standardisation of these products is required to prevent compromising their

lower coumarin concentration.

cife/PE, Brazil.

**5. Conclusion**

therapeutic activity.

materials as well as for species identification.

vonoids and coumarins within these products.

**Figure 8.** Concentration of tannin (mg TAE/g) contained in products from *Maytenus ilicifolia* (Schrad.) Planch. Sold in Recife/PE, Brazil.

**Figure 9.** Concentration of flavonoids (mg RE/g) contained within *Bauhinia forficata* link. sold in Recife/PE, Brazil.

Of the five products from *M. glomerata*, three were plant drugs and two were fluid extracts. The plant drugs were extracted with ethanol as recommended by the Brazilian Pharmaco‐ poeia 4th Edition to remove discrepancies from the results [53]. Both the extracts and liquids were evaporated to dryness. The one way ANOVA showed significant differences between the products (p<0.01) and sample Mg3 had the highest level (6.80 ± 0.24 mg CE/g), whereas sample Mg4 had the lowest level (1.63 ± 0.20 mg CE/g), with an approximately four times lower coumarin concentration.

**Figure 10.** Concentration of coumarins (mg CE/g) contained in products from *Mikania glomerata* Spreng. sold in Re‐ cife/PE, Brazil.

The quantitative analysis of raw vegetables and phytomedicines is a fundamental quality control process that leads to security, stability, consistency and effectiveness in the produced phytomedicines [49]. It is important to emphasise the need for standardisation in analysing herbal medicines to determine the concentration of their active components in raw vegetable materials as well as for species identification.

#### **5. Conclusion**

**Figure 8.** Concentration of tannin (mg TAE/g) contained in products from *Maytenus ilicifolia* (Schrad.) Planch. Sold in

**Figure 9.** Concentration of flavonoids (mg RE/g) contained within *Bauhinia forficata* link. sold in Recife/PE, Brazil.

Of the five products from *M. glomerata*, three were plant drugs and two were fluid extracts. The plant drugs were extracted with ethanol as recommended by the Brazilian Pharmaco‐ poeia 4th Edition to remove discrepancies from the results [53]. Both the extracts and liquids were evaporated to dryness. The one way ANOVA showed significant differences between the products (p<0.01) and sample Mg3 had the highest level (6.80 ± 0.24 mg CE/g), whereas

Recife/PE, Brazil.

60 Latest Research into Quality Control

This chapter provides easily reproducible standard operating procedures (SOPs) for the quality control of raw materials and herbal plants to ensure a minimal standard of quality in products sold. The implementation of these SOPs allows for the analysis of samples sold in establishments in Recife/PE and reveals an inconsistency in the concentration of tannins, fla‐ vonoids and coumarins within these products.

The low level of these metabolites may alter their effectiveness and more rigorous quality control and standardisation of these products is required to prevent compromising their therapeutic activity.

#### **Author details**

Elba Lúcia Cavalcanti de Amorim1\*, Valérium Thijan Nobre de Almeida de Castro1 , Joabe Gomes de Melo2 , Allan Jonathan Chernichiarro Corrêa1 and Tadeu José da Silva Peixoto Sobrinho3

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Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

63

http://dx.doi.org/10.5772/51686

[10] Heil, M., Baumann, B., Andary, C., Linsenmair, K. E., & Mckey, D. (2002). Extraction and quantification of "condensed tannins" as a measure of plant anti-herbivore de‐

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[12] Harborne, J. B., Palo, R. T., & Robbins, C. T. (1991). Plant defenses against mammali‐

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[17] Chung, K., Wei, C., & Johnson, M. G. (1998). Are tannins a double-edged sword in

[18] Ferreira, P. M., Oliveira, C. N., Oliveira, A. B., Lopes, M. J., Alzamora, F., & Vieira, M. A. R. (2004). A lyophilized aqueous extract of Maytenus ilicifolia leaves inhibits his‐ tamine-mediated acid secretion in isolated frog gastric mucosa. *Planta*, 219(2),

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\*Address all correspondence to: elba@ufpe.br

1 Department of Pharmacy, Federal University Pernambuco, Recife/PE, Brazil

2 Department of Biology, Federal Rural University of Pernambuco, Brazil

3 School of Medicine, Federal University of Tocantins, Palmas/TO, Brazil

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**Author details**

**References**

Joabe Gomes de Melo2

62 Latest Research into Quality Control

Tadeu José da Silva Peixoto Sobrinho3

\*Address all correspondence to: elba@ufpe.br

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court Brace and Company , 318p.

Elba Lúcia Cavalcanti de Amorim1\*, Valérium Thijan Nobre de Almeida de Castro1

1 Department of Pharmacy, Federal University Pernambuco, Recife/PE, Brazil

2 Department of Biology, Federal Rural University of Pernambuco, Brazil

3 School of Medicine, Federal University of Tocantins, Palmas/TO, Brazil

, Allan Jonathan Chernichiarro Corrêa1

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[39] Dias da Silva, J. L. S. (1926). Pharmacopéia dos Estados Unidos do Brasil. 1.ed., São

[40] Osório, A. C., & Martins, J. L. S. (2004). Determinação de cumarina em extrato fluido e tintura de guaco por espectrofotometria derivada de primeira ordem. *Brazilian Jour‐*

[41] Bolina, R. C., Garcia, E. F., & Duarte, M. G. R. (2009). Estudo comparativo da compo‐ sição química das espécies vegetais Mikania glomerata Sprengel e Mikania laevigata

[42] Rocha, L., Lucio, E. M. A., França, H. S., & Sharapin, N. (2008). Mikania glomerata Spreng: Desenvolvimento de um produto fitoterápico. *Revista Brasileira de Farmacog‐*

[43] Silva, C. R., Gomes, V. S., Kulkamp, I. C., & Kanis, L. A. (2008). Metodo espectroscó‐ pico para determinação de cumarina em xarope de Mikania glomerata Sprengel. *Re‐*

[44] Brasil. Ministério da saúde. (2004). Agência Nacional de Vigilância Sanitária. Resolu‐ ção da Diretoria Colegiada (RDC) N° 48, de 16 de março de, Dispõe sobre o registro

[45] Engel, I. C., Ferreira, R. A., Cechinel-Filho, V., & Meyre-Silva, C. (2008). Controle de qualidade de drogas vegetais a base de Bauhinia forficata Link (Fabaceae). *Brazilian*

[46] Marques, G. S., Monteiro, R. P. M., Leão, W. F., Lyra, M. A. M., Peixoto, M. S., Rolim-Neto, P. J., Xavier, H. S., & Soares, L. A. L. (2012). Avaliação de procedimento para quantificação espectrofotomêtrica de flavonóides totais em folhas de Bauhinia forfi‐

[47] Amorim, E. L. C., Nascimento, J. E., Monteiro, J. M., Peixoto, Sobrinho. T. J. S., Araú‐ jo, T. A. S., & Albuquerque, U. P. (2008). A simple and accurate procedure for the de‐ termination of tannin and flavonoid levels and some applications in ethnobotany

and ethnopharmacology. *Functional Ecosystems and Communities*, 2(1), 88-94.

de medicamentos fitoterápicos. Diário Oficial da União de 18.03.2004.

Schultz Bip. ex Baker. *Revista Brasileira de Farmacognosia*, 19(1B), 294-298.

sintético. Dissertação Universidade do Vale do Paraíba, São José dos Campos. [36] Lake, B.G. (1999). Coumarin Metabolism, Toxicity and Carcinogenicity: relevance for

human risk assessment. *Food Chemistry Toxicology*, 37(4), 423.

Paulo:, Editora Nacional.

*nosia*, (18), 744-747.

*nal of Pharmaceutical Sciences*, 40(4), 481-486.

*vista Brasileira de Farmacognosia*, 18(4), 594-599.

*Journal of Pharmacognosy*, 18(2), 258-264.

cata Link. *Química Nova*, 35(3), 517-522.

("guaco") leaves. *Journal Brazilian of Chemical Society*, 12(6), 706-709.

toras de Cumarinas em Angiospermae. *Química Nova*, 25(4), 533-538.


[35] Mello, M.M. (2009). Desenvolvimento de uma metodologia por espectroscopia de fluorescência para quantificação de cumarina e 7hidroxicumarina em Drágeas e soro‐ sintético. Dissertação Universidade do Vale do Paraíba, São José dos Campos.

[23] Nijveldt, R. J., Nood, E., Hoorn, D. E. C., Boelens, P. G., Norren, K., & Leeuwen, P. A. M. (2001). Flavonoids: a review of probable mechanisms of action and potential ap‐

[24] Zuanazzi, J. A. S., & Montanha, J. A. (2004). Flavonóides In: Simões CMO, Schenkel EP, Gosmanm, G, Mello JCP, Mentz LA, Petrovick PR. (ed.) Farmacognosia: da plan‐ ta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul;,

[25] Treutter, D. (2006). Significance of flavonoids in plant resistance: a review. *Environ‐*

[26] Harborne, J. B., & Williams, C. A. (2000). Advances in flavonoids in research since

[27] Argolo, A. C. C., Sant´, Ana. A. E. G., Pletsch, M., & Coelho, C. B. B. (2004). Antioxi‐ dant activity of leaf extracts from Bauhinia monandra. *Bioresource Technology*, 95(2),

[28] Silva, F. R. M. B., Szpoganicz, B., Pizzolatti, M. G., Willrich, M. A. V., & Sousa, E. (2002). Acute effect of Bauhinia forficata on serum glucose levels in normal and allox‐

[29] Sousa, E., Zanatta, L., Seifriz, I., Creczynski-Pasa, T. B., Pizzolatti, M. G., Szpoganicz, B., & Silva, F. R. M. B. (2004). Hypoglycemic effect and antioxidant potential of kaempferol-3,7-O-(α)-dirhammnoside from Bauhinia forficata leaves. *Journal of the*

[30] Vaz, A.M.S.F. (2012). Bauhinia in Lista de Espécies da Flora do Brasil. *Jardim Botânico do Rio de Janeiro.*, http://floradobrasil.jbrj.gov.br/2012/FB082666, accessed 27 June

[31] Damasceno, D. C., Volpato, G. T., Calderon, I. M. P., Aguilar, R., & Rudge, M. V. C. (2004). Effect of Bauhinia forficata extract in diabetic pregnant rats: maternal reper‐

[32] Di Stasi, L. C., Oliveira, G. P., Carvalhaes, M. A., Queiroz-Junior, M., Tien, O. S., Ka‐ kinami, S. H., & Reis, M. S. (2002). Medicinal plants popularly used in the Brazilian

[33] Melo, J. G., Nascimento, V. T., Amorim, E. L. C., Andrade-Lima, C. S., & Albuquer‐ que, U. P. (2004). Avaliação da qualidade de amostras comerciais de boldo (Peumus boldus Molina), pata-de-vaca (Bauhinia spp.) e ginco (Ginkgo biloba L.). *Revista brasi‐*

[34] Kuster, R. M., & Rocha, L. M. (2004). Cumarinas, cromonas e xantonas. In: Simões C M O, Schenkel E P, Gosmanm G, Mello JCP, Mentz LA, Petrovick PR. Farmacogno‐ sia: da planta ao medicamento. 5ª ed. Porto Alegre: Universidade Federal do Rio

an-induced diabetic rats. Journal of Ethnopharmacology., 83(1-2), 33-37.

plications. *The American Journal of Clinical Nutrition*, 74(4), 418-25.

577-614.

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229-233.

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1992. *Phytochemistry*, 55(6), 481-204.

*Natural Products*, 67(5), 829-832.

cussions. *Phytomedicine*, 11(2-3), 196-201.

*leira de farmacognosia*, 14(2), 111-120.

Grande do Sul,, 537-556.

Tropical Atlantic Forest. *Fitoterapia*, 73(1), 69-91.


[48] Peixoto, Sobrinho. T. J. S., Silva, C. H. T. P., Nascimento, J. E., Monteiro, J. M., Albu‐ querque, U. P., & Amorim, E. L. C. (2008). Validação de metodologia espectrofoto‐ métrica para quantificação dos flavonóides de Bauhinia cheilantha (Bongard) Steudel. *Brazilian Journal of Pharmaceutical Sciences*, 44(4), 683-689.

**Chapter 4**

**Microbial Quality of Medicinal Plant Materials**

The use of medicinal plants is continually expanding worldwide. The increasing search for therapeutic agents derived from plant species is justified by the emergence of diseases, yet without proper treatment, and the growth of scientific knowledge about the herbal medi‐ cines as important treatment alternatives. Therefore, the quality and safety of herbal prepa‐ rations are also of great concern [1]. The reference [2] explained that quality is the basis of reproducible efficacy and safety of herbal drugs, and to ensure the standard of research on herbal medicines, the quality of the plant materials or preparations is of utmost importance. With the ever increasing use of herbal medicines and the global expansion of the herbal medicines market, safety has become a concern for both health authorities and the public in many countries. This is because many contaminants and residues that may cause harm to

The microbial load of plants is the result of a series of influences (Figure 1). By their origin, herbal drugs are subject to contamination by microorganisms from soil, air and water may be present potentially pathogenic microorganisms to man. Microbial contamination of me‐ dicinal herbal can be influenced by environmental factors such as temperature, humidity and extent of rainfall during pre-harvesting and post-harvesting periods, handling practices and the storage conditions of crude and processed medicinal-plant materials. In order to im‐ prove the purity and safety of the products, observation of basic hygiene during prepara‐ tion, standardization of some physical characteristic such as moisture content, pH and

The presence of microbial contaminant in non sterile pharmaceutical products can reduce or even inactivate the therapeutic activity of the products and has the potential to adversely af‐ fect patients taking the medicines. As herbal medicinal products are complex mixtures

> © 2012 de Freitas Araújo and Bauab; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 de Freitas Araújo and Bauab; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

Marcelo Gonzaga de Freitas Araújo and

Additional information is available at the end of the chapter

Taís Maria Bauab

**1. Introduction**

http://dx.doi.org/10.5772/51072

the consumers have been reported [3].

microbiological contamination levels are desirable [1,4,5].

properly cited.


## **Microbial Quality of Medicinal Plant Materials**

Marcelo Gonzaga de Freitas Araújo and Taís Maria Bauab

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51072

#### **1. Introduction**

[48] Peixoto, Sobrinho. T. J. S., Silva, C. H. T. P., Nascimento, J. E., Monteiro, J. M., Albu‐ querque, U. P., & Amorim, E. L. C. (2008). Validação de metodologia espectrofoto‐ métrica para quantificação dos flavonóides de Bauhinia cheilantha (Bongard)

[49] Bara, M. T. F., Cirilo, H. N., & Oliveira, V. (2004). Determinação de ginkgoflavo‐ nóides por cromatografia líquida de alta eficiência em matérias-primas e produtos

[50] Williamson, E.M. (2001). Synergy and other interactions in phytomedicines. *Phytome‐*

[51] Capasso, R., Izzo, A. A., Pinto, L., Bifulco, T., Vitobello, C., & Mascolo, N. (2000).

[52] Calixto, J. B. S. (2000). Efficacy, Safety, quality control, marketing and regulatory guidelines for herbal medicines (Phytotherapeutic agents). *Brazilian Journal of Medical*

[53] (2005). Farmacopéia Brasileira. 4. ed. São Paulo: Editora Atheneu, parte II, sexto fascí‐

Steudel. *Brazilian Journal of Pharmaceutical Sciences*, 44(4), 683-689.

Phytotherapy and quality of herbal medicines. *Fitoterapia*, 71-58.

acabados. *Revista Eletrônica de Farmácia*, 1(1), 1-7.

*and Biological Research*, 33(2), 179-189.

*dicine*, 8(5), 401-409.

66 Latest Research into Quality Control

culo.

The use of medicinal plants is continually expanding worldwide. The increasing search for therapeutic agents derived from plant species is justified by the emergence of diseases, yet without proper treatment, and the growth of scientific knowledge about the herbal medi‐ cines as important treatment alternatives. Therefore, the quality and safety of herbal prepa‐ rations are also of great concern [1]. The reference [2] explained that quality is the basis of reproducible efficacy and safety of herbal drugs, and to ensure the standard of research on herbal medicines, the quality of the plant materials or preparations is of utmost importance. With the ever increasing use of herbal medicines and the global expansion of the herbal medicines market, safety has become a concern for both health authorities and the public in many countries. This is because many contaminants and residues that may cause harm to the consumers have been reported [3].

The microbial load of plants is the result of a series of influences (Figure 1). By their origin, herbal drugs are subject to contamination by microorganisms from soil, air and water may be present potentially pathogenic microorganisms to man. Microbial contamination of me‐ dicinal herbal can be influenced by environmental factors such as temperature, humidity and extent of rainfall during pre-harvesting and post-harvesting periods, handling practices and the storage conditions of crude and processed medicinal-plant materials. In order to im‐ prove the purity and safety of the products, observation of basic hygiene during prepara‐ tion, standardization of some physical characteristic such as moisture content, pH and microbiological contamination levels are desirable [1,4,5].

The presence of microbial contaminant in non sterile pharmaceutical products can reduce or even inactivate the therapeutic activity of the products and has the potential to adversely af‐ fect patients taking the medicines. As herbal medicinal products are complex mixtures

properly cited.

© 2012 de Freitas Araújo and Bauab; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2012 de Freitas Araújo and Bauab; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

which originate from biological sources, great efforts are necessary to guarantee a constant and adequate quality. Manipulation and processing factors largely determine the microbio‐ logical quality of the final products [6]. Previous studies have confirmed the presence of po‐ tential contaminants in herbal preparations [7-10]. Thus, manufacturers should ensure the lowest possible level of microorganisms in the raw material, finished dosage forms and the packaging components to maintain appropriate quality, safety and efficacy of the natural products [9].

contaminants [11]. The main microbial contamination of plant materials, in general, are at‐

Microbial Quality of Medicinal Plant Materials

http://dx.doi.org/10.5772/51072

69

The presence of higher numbers of spores bacteria could be explained by the fact that some of these organisms (e.g. *Bacillus* and *Clostridium* spp.) produce spores which are resistant to harsh processing, elevated heat and dry conditions. Therefore, they can survive for a long time on the product in a dormant state. *Bacillus cereus* and *Clostridium perfringens* are recog‐ nized as having potential pathogenicity and have been incriminated in food poisoning [12]. Although bacterial endospores and fungal spores can be regarded as the two dominating groups of contaminants associated with medicinal plants, a broad diversity of bacterial, fun‐ gal cells and viruses can be found either in or on the plant material [4]. *B. cereus* and *C. perfri‐*

Although enterobacteria can be found in nature, this family possesses some indicative value towards faecal contamination. The presence of enterobacteria and *E. coli* reflect the situation regarding faecal contamination [7]. Together with the group of coliforms, it can be taken as an indicator for undesirable hygiene conditions, although this conclusion has to be related to the magnitude of viable count measured [4]. *Staphylococcus aureus* is not common contami‐ nant of this type of plant material and relatively rarely found. However, contamination could provide amount of enterotoxin produced by *S. aureus*, depending on the specific na‐

Herbal medications are likely to be contaminated with a wide variety of others potentially pathogenic bacteria. In a study whose was evaluated the bacterial contamination of pow‐ dered herbal medicinal preparations sourced from identified herbal retail outlets in different parts of Kaduna, Nigeria, the results showed that a number of herbal remedies were conta‐ minated with *Salmonella typhi* and *Shigella* spp., besides *E. coli* and *S. aureus* [1]. In addiction, the presence of pathogenic bacteria like *B. cereus*, *Aeromonas hydrophila, Shigella* spp., *Entero‐ bacter agglomerans*, *E. cloacae*, *Vibrio fluvialis*, *Pasteurella multocida*, *S. epidermidis*, *Acinetobacter iwoffii*, *Klebsiella* spp., *B. subtilis* and *Pseudomonas aeruginosa*, and fungi *Rhizopus stolonifer* also

Because they are widespread in the atmosphere, moulds are common natural contaminants of medicinal herbs. It is known that, under favourable conditions, some fungi can synthesize toxical metabolites – mycotoxins. Among the known mycotoxins, the most toxic one is afla‐ toxin synthesised by species of *A. flavus* and *A. parasiticus*, and a minor number of other fun‐ gi [10]. Contamination by *A. flavus*, the most famous aflatoxin producer, is common in

but causes economic losses by contaminating with aflatoxin [17]. In a study of 91 medicinal herb samples in Brazil [18], were found that 50 % of aerial part samples were contaminated with fungi. Samples of medicinal plants were evaluated by [5] for the fungal contamination, and results indicated that predominant mycoflora (89.9% of the isolates) corresponded to genera *Aspergillus* and *Penicillium*, which are extremely important from the mycotoxicologi‐ cal standpoint. The fungal contamination of powdered herbal medicinal preparations sourced from some herbal retail outlets in some parts of Nigeria was evaluated by [19] and

*avus* colonization does not necessarily reduce yield,

were observed to be present in plant samples analyzed recently [14,15].

fl

tributed to total aerobic mesophilic, enterobacterial, yeast and mould [4].

*gens* were isolated from chamomile and other herbs by [13].

ture of the individual [11].

medicinal plant and herbal tea [16]. *A.* 

This review intends to contribute to knowledge regarding the microbial contamination of medicinal plants by considering the influence of different commonly used pharmaceutical preparation techniques on the microbiological status of the products. Finally, quality stand‐ ards will be discussed, considering the main guidelines of microbial quality control and through quality assurance measures such as good manufacturing practices (GMP) for herbal medicines.

**Figure 1.** Influence of environmental factors and possible pathways of microbial contamination of medicinal herbs (adapted from [4]).

#### **2. Common microbial contaminants associated with medicinal plants**

The growing, harvesting and manipulation methods usually applied cannot avoid microbial contamination of the plant material which therefore reflects the environmental conditions as well as the specific hygiene during the diverse treatments [4]. Biological contamination re‐ fers to impurities in medicinal herbs and their preparations and products, and may involve living microbes such as bacteria and their spores, yeasts and moulds, viruses, protozoa, in‐ sects (their eggs and larvae), and other organisms. However, products of microbial metabo‐ lism such as toxic, low-molecular-weight metabolites from moulds are important chemical contaminants [11]. The main microbial contamination of plant materials, in general, are at‐ tributed to total aerobic mesophilic, enterobacterial, yeast and mould [4].

which originate from biological sources, great efforts are necessary to guarantee a constant and adequate quality. Manipulation and processing factors largely determine the microbio‐ logical quality of the final products [6]. Previous studies have confirmed the presence of po‐ tential contaminants in herbal preparations [7-10]. Thus, manufacturers should ensure the lowest possible level of microorganisms in the raw material, finished dosage forms and the packaging components to maintain appropriate quality, safety and efficacy of the natural

This review intends to contribute to knowledge regarding the microbial contamination of medicinal plants by considering the influence of different commonly used pharmaceutical preparation techniques on the microbiological status of the products. Finally, quality stand‐ ards will be discussed, considering the main guidelines of microbial quality control and through quality assurance measures such as good manufacturing practices (GMP) for herbal

**Figure 1.** Influence of environmental factors and possible pathways of microbial contamination of medicinal herbs

The growing, harvesting and manipulation methods usually applied cannot avoid microbial contamination of the plant material which therefore reflects the environmental conditions as well as the specific hygiene during the diverse treatments [4]. Biological contamination re‐ fers to impurities in medicinal herbs and their preparations and products, and may involve living microbes such as bacteria and their spores, yeasts and moulds, viruses, protozoa, in‐ sects (their eggs and larvae), and other organisms. However, products of microbial metabo‐ lism such as toxic, low-molecular-weight metabolites from moulds are important chemical

**2. Common microbial contaminants associated with medicinal plants**

products [9].

68 Latest Research into Quality Control

medicines.

(adapted from [4]).

The presence of higher numbers of spores bacteria could be explained by the fact that some of these organisms (e.g. *Bacillus* and *Clostridium* spp.) produce spores which are resistant to harsh processing, elevated heat and dry conditions. Therefore, they can survive for a long time on the product in a dormant state. *Bacillus cereus* and *Clostridium perfringens* are recog‐ nized as having potential pathogenicity and have been incriminated in food poisoning [12]. Although bacterial endospores and fungal spores can be regarded as the two dominating groups of contaminants associated with medicinal plants, a broad diversity of bacterial, fun‐ gal cells and viruses can be found either in or on the plant material [4]. *B. cereus* and *C. perfri‐ gens* were isolated from chamomile and other herbs by [13].

Although enterobacteria can be found in nature, this family possesses some indicative value towards faecal contamination. The presence of enterobacteria and *E. coli* reflect the situation regarding faecal contamination [7]. Together with the group of coliforms, it can be taken as an indicator for undesirable hygiene conditions, although this conclusion has to be related to the magnitude of viable count measured [4]. *Staphylococcus aureus* is not common contami‐ nant of this type of plant material and relatively rarely found. However, contamination could provide amount of enterotoxin produced by *S. aureus*, depending on the specific na‐ ture of the individual [11].

Herbal medications are likely to be contaminated with a wide variety of others potentially pathogenic bacteria. In a study whose was evaluated the bacterial contamination of pow‐ dered herbal medicinal preparations sourced from identified herbal retail outlets in different parts of Kaduna, Nigeria, the results showed that a number of herbal remedies were conta‐ minated with *Salmonella typhi* and *Shigella* spp., besides *E. coli* and *S. aureus* [1]. In addiction, the presence of pathogenic bacteria like *B. cereus*, *Aeromonas hydrophila, Shigella* spp., *Entero‐ bacter agglomerans*, *E. cloacae*, *Vibrio fluvialis*, *Pasteurella multocida*, *S. epidermidis*, *Acinetobacter iwoffii*, *Klebsiella* spp., *B. subtilis* and *Pseudomonas aeruginosa*, and fungi *Rhizopus stolonifer* also were observed to be present in plant samples analyzed recently [14,15].

Because they are widespread in the atmosphere, moulds are common natural contaminants of medicinal herbs. It is known that, under favourable conditions, some fungi can synthesize toxical metabolites – mycotoxins. Among the known mycotoxins, the most toxic one is afla‐ toxin synthesised by species of *A. flavus* and *A. parasiticus*, and a minor number of other fun‐ gi [10]. Contamination by *A. flavus*, the most famous aflatoxin producer, is common in medicinal plant and herbal tea [16]. *A.* fl*avus* colonization does not necessarily reduce yield, but causes economic losses by contaminating with aflatoxin [17]. In a study of 91 medicinal herb samples in Brazil [18], were found that 50 % of aerial part samples were contaminated with fungi. Samples of medicinal plants were evaluated by [5] for the fungal contamination, and results indicated that predominant mycoflora (89.9% of the isolates) corresponded to genera *Aspergillus* and *Penicillium*, which are extremely important from the mycotoxicologi‐ cal standpoint. The fungal contamination of powdered herbal medicinal preparations sourced from some herbal retail outlets in some parts of Nigeria was evaluated by [19] and the results showed that all of the herbal preparations had the presence of fungal contami‐ nants with predominance of *Aspergillus* spp. and *Penicillium* spp., but *Mucor* spp., *Candida* spp., *Trichosporium* spp., also were found. The fungal deterioration adversely affects the chemical composition of the raw materials and thereby decreases the medicinal potency of herbal drugs [20].

lyophilization; microwave; or infrared devices. When possible, temperature and humidity should be controlled to avoid damage to the active chemical constituents. In the case of nat‐ ural drying in the open air, efforts should be made to achieve uniform drying of medicinal

Microbial Quality of Medicinal Plant Materials

http://dx.doi.org/10.5772/51072

71

Spray drying technique has been widely used to obtain dried extracts presenting better tech‐ nological characteristics and greater concentration of biological active constituents. This method is widely used in the pharmaceutical industry, despite the high temperature drying (100°C to 200°C), the contact time between the material to be dry and hot air is extremely fast, less than 1 minute, theoretically is not enough to remove the microorganisms [24]. Comparative microbiological analysis of drug pulverized, extraction in liquid phase and the the spray drying extraction, using *Phyllanthus niruri* L., revealed that there is a significant reduction in microbial load, caused by the extraction in liquid process, while the spray dry‐

Drying at high temperature decreases the total aerobic microbial count in herbs. Water is a significant component of biological materials. Drying methods can lower the water activ‐ ity to the level required for preventing growth of *Aspergillus* species and also for ensur‐ ing quality of medicinal herbs which may get destroyed upon over drying [10]. Exposure of herbs to microwaves and warm-air ovens can be efficient to reduce the microbial load, but they are not recommend to medicinal herbs containing volatile oils. The reference [26] evaluated both method of drying of plant, and reduction the microbial load present on the plants was observed but the effect on the volatile oil profile was profound by microwave drying, and warm drying air revealed that at temperatures >60°C, most of the volatile con‐

Other methods such as freeze-drying, oven drying and tray drying have been previously used to preserve medicinal herbs but to date there is little information in the literature on

Once drying is complete, plants are packaged in preparation for shipping or other further

Water is almost universally the solvent used to extract activity. At home, dried plants can be ingested as teas (plants steeped in hot water) or, rarely, tinctures (plants in alcoholic solu‐ tions) or inhaled via steam from boiling suspensions of the parts. Dried plant parts can be added to oils or petroleum jelly and applied externally. Poultices can also be made from concentrated teas or tinctures [28]. These kinds of preparations are usually called medicinal teas and are prepared using natural plants collected, dried and packaged without an effec‐ tive hygienic and sanitary control. In addition, there can be microbiological contamination

and controlling microbial contamination can be difficult in aqueous extracts [13].

the effect of these drying conditions on the decrease of microbial loads [27].

plant materials and so avoid mould formation [23].

stituents were lost.

**3.2. Extraction methods**

processing.

er, despite the high temperature, did not affect the microbial load [25].

The risk of the presence of microorganisms in a plant product depends on this finality of the use, its nature and its potential damage that may be caused to the consumers. Considering natural flora, current production conditions and the need to warrant the quality and the safety of these products, monographs establish a maximum fungal contamination limit for products that contain raw material of natural origin [5]. Although high fungal loads may be accepted due to the natural origin of those products, they indicate the potential for spoilage and mycotoxigenesis.

### **3. Influence of different preparation techniques on the microbiological quality**

The production of an herbal medicine generally involves the steps in which a vegetable is subjected to unfavorable conditions to survival of microorganisms. Next, we introduce some of these processes and their influence on the microbial load.

#### **3.1. Drying process**

Drying is basically defined as the decreasing of plant moisture content, aimed at preventing enzymatic and microbial activity, and consequently preserving the product for extend shelf life [21]. Drying is the most common and fundamental method for post-harvest preservation of medicinal plants because it allows for the quick conservation of the medicinal qualities of the plant material in an uncomplicated manner. This process may also contribute to facili‐ tate the marketing of plants, because drying results in reduction of the weight and volume of the plant with positive consequences for transport and storage [21,22].

The optimization of the drying process contributes to physical, chemical and microbiological stability of the medicinal herbs. The choice of drying conditions depend on the moisture con‐ tent of tissue at harvest, the plant parts used, and the temperature best suited for preservation of the requested ingredients. For this reason, adequate dryers are needed, using temperature, velocity and humidity values for drying air that provides a rapid reduction in the moisture content without affecting the quality of the active ingredients of medicinal plants [21].

Medicinal plants can be dried in a number of ways: in the open air (shaded from direct sun‐ light); placed in thin layers on drying frames, wire-screened rooms or buildings; by direct sunlight, if appropriate; in drying ovens/rooms and solar dryers; by indirect fire; baking; lyophilization; microwave; or infrared devices. When possible, temperature and humidity should be controlled to avoid damage to the active chemical constituents. In the case of nat‐ ural drying in the open air, efforts should be made to achieve uniform drying of medicinal plant materials and so avoid mould formation [23].

Spray drying technique has been widely used to obtain dried extracts presenting better tech‐ nological characteristics and greater concentration of biological active constituents. This method is widely used in the pharmaceutical industry, despite the high temperature drying (100°C to 200°C), the contact time between the material to be dry and hot air is extremely fast, less than 1 minute, theoretically is not enough to remove the microorganisms [24]. Comparative microbiological analysis of drug pulverized, extraction in liquid phase and the the spray drying extraction, using *Phyllanthus niruri* L., revealed that there is a significant reduction in microbial load, caused by the extraction in liquid process, while the spray dry‐ er, despite the high temperature, did not affect the microbial load [25].

Drying at high temperature decreases the total aerobic microbial count in herbs. Water is a significant component of biological materials. Drying methods can lower the water activ‐ ity to the level required for preventing growth of *Aspergillus* species and also for ensur‐ ing quality of medicinal herbs which may get destroyed upon over drying [10]. Exposure of herbs to microwaves and warm-air ovens can be efficient to reduce the microbial load, but they are not recommend to medicinal herbs containing volatile oils. The reference [26] evaluated both method of drying of plant, and reduction the microbial load present on the plants was observed but the effect on the volatile oil profile was profound by microwave drying, and warm drying air revealed that at temperatures >60°C, most of the volatile con‐ stituents were lost.

Other methods such as freeze-drying, oven drying and tray drying have been previously used to preserve medicinal herbs but to date there is little information in the literature on the effect of these drying conditions on the decrease of microbial loads [27].

Once drying is complete, plants are packaged in preparation for shipping or other further processing.

#### **3.2. Extraction methods**

the results showed that all of the herbal preparations had the presence of fungal contami‐ nants with predominance of *Aspergillus* spp. and *Penicillium* spp., but *Mucor* spp., *Candida* spp., *Trichosporium* spp., also were found. The fungal deterioration adversely affects the chemical composition of the raw materials and thereby decreases the medicinal potency of

The risk of the presence of microorganisms in a plant product depends on this finality of the use, its nature and its potential damage that may be caused to the consumers. Considering natural flora, current production conditions and the need to warrant the quality and the safety of these products, monographs establish a maximum fungal contamination limit for products that contain raw material of natural origin [5]. Although high fungal loads may be accepted due to the natural origin of those products, they indicate the potential for spoilage

**3. Influence of different preparation techniques on the microbiological**

The production of an herbal medicine generally involves the steps in which a vegetable is subjected to unfavorable conditions to survival of microorganisms. Next, we introduce some

Drying is basically defined as the decreasing of plant moisture content, aimed at preventing enzymatic and microbial activity, and consequently preserving the product for extend shelf life [21]. Drying is the most common and fundamental method for post-harvest preservation of medicinal plants because it allows for the quick conservation of the medicinal qualities of the plant material in an uncomplicated manner. This process may also contribute to facili‐ tate the marketing of plants, because drying results in reduction of the weight and volume

The optimization of the drying process contributes to physical, chemical and microbiological stability of the medicinal herbs. The choice of drying conditions depend on the moisture con‐ tent of tissue at harvest, the plant parts used, and the temperature best suited for preservation of the requested ingredients. For this reason, adequate dryers are needed, using temperature, velocity and humidity values for drying air that provides a rapid reduction in the moisture

Medicinal plants can be dried in a number of ways: in the open air (shaded from direct sun‐ light); placed in thin layers on drying frames, wire-screened rooms or buildings; by direct sunlight, if appropriate; in drying ovens/rooms and solar dryers; by indirect fire; baking;

content without affecting the quality of the active ingredients of medicinal plants [21].

of these processes and their influence on the microbial load.

of the plant with positive consequences for transport and storage [21,22].

herbal drugs [20].

70 Latest Research into Quality Control

and mycotoxigenesis.

**3.1. Drying process**

**quality**

Water is almost universally the solvent used to extract activity. At home, dried plants can be ingested as teas (plants steeped in hot water) or, rarely, tinctures (plants in alcoholic solu‐ tions) or inhaled via steam from boiling suspensions of the parts. Dried plant parts can be added to oils or petroleum jelly and applied externally. Poultices can also be made from concentrated teas or tinctures [28]. These kinds of preparations are usually called medicinal teas and are prepared using natural plants collected, dried and packaged without an effec‐ tive hygienic and sanitary control. In addition, there can be microbiological contamination and controlling microbial contamination can be difficult in aqueous extracts [13].

Environmental dust settled on different parts of the plant and other contaminations can car‐ ry very significant amounts of bacterial and moulds spores [13]. However, those drugs which are subjected to cold water extraction (herbal maceration) may host a considerable amount of microbes, and the extraction procedure carried out at ambient temperature usual‐ ly enables microbial multiplication [4]. The application of hot water extraction usually com‐ pensates for microbiological contaminations, since it can be expected that boiling water markedly reduces the viable counts by several log units and also inactivates possible patho‐ gens [4]. However, bacterial spores of the Bacillaceae family are resistant to thermal treat‐ ment usually applied in infusion preparation, and this thermal shock may stimulate spore germination. Some of these bacteria like *B. cereus* and *C. perfringens* are recognized as having potential pathogenicity and have been incriminated in food poisoning [12]. Thus, in extrac‐ tions using only water, hot or cold, as extractor liquid, the stability of the extract becomes compromised and the risk of microbiological contamination increases significantly. This contamination can compromise the quality and integrity of the plant material itself, as well as products arising from its use [29-30].

ventilated storehouse usually increases sample moisture content in the bulk due to heat ex‐ change capacity, rendering herbs more susceptible to molds growth and toxin production. Fungi are the predominant contaminants of herbs, but most such microbial populations are probably regarded as commensal residents on the plant that survived drying and storage. Most fungi are present on plants, which develop after harvest if relative humidity is not con‐

Moulds are responsible for biodeterioration of a number of substrates including raw materi‐ als of some medicinal plants. These moulds reduce raw herbal drugs shelf life and market value. The fungal deterioration adversely affects the chemical composition of the raw mate‐ rials and thereby decreases the medicinal potency of herbal drugs [20]. Samples of herbal parts stored for sale in markets located in Ibadan, Nigeria were analysed for mycoflora asso‐ ciated with their storage and twenty eight fungal species were isolated, showing that herbal drug plant pieces are hazardous for human health [36]. Some samples of herbal raw materi‐ als have been reported to contain aflatoxin. The reference [37] determined the incidence of toxigenic fungi and their mycotoxins on 152 dried medicinal and aromatic herbs from Ar‐

dominant species isolated, and high aflatoxin concentrations were detected. There is a potential risk for mycotoxins contamination, especially during prolonged storage in poorly conditions without temperature and moisture control that usually render medicinal plants

The reduction of plant enzyme activity and inactivation of microorganisms is achieved by drying. Dried plant materials tend to be hygroscopic (readily absorbing moisture) and must be stored under controlled humidity. Rehydration can lead to the decomposition of the bio‐ active metabolites by enzymes from microorganisms or the plant itself. Significant contami‐ nation by bacteria and fungi suggest inadequate storage facilities and poor hygienic practice during preparation of these medicinal plants. The storage processes of such products are

Studies on long‐term stability of dried herbal teas and preparations are rare. In a study of [20] was examined the deterioration of herbal drug samples which were stored for 6-9 months by traders after collection. Some of the contaminated materials were found to be de‐ teriorated by toxigenic strains of *A. flavus* and contain aflatoxin B1 which was above the per‐ missible limit. In a study of [38], dried *P. lanceolata* leaves were exposed to atmospheres of different relative humidity (75, 45 and 0%) for 24 weeks and was evaluated the chemical changes of the compounds of interest. It was shown that exposure to water results in loss of bioactive molecules of *P. lanceolata* dried leaves, and that colonising fungi are the key con‐ tributors to this loss. The fungal deterioration adversely affects the chemical composition of the raw materials and thereby decreases the medicinal potency of herbal drugs. Biodeterio‐ ration of herbal products samples by associated fungi during storage has drawn attention

It is common practice for herbalists to prepare herbal medicines and store them in a refrig‐ erator. However, in previous study the effect of microbial contaminants on active com‐

more susceptible to moulds growth and mycotoxins production [5,10,20].

stages during which it is important to avoid even further contamination [38].

regarding quality maintenance of these products [35,38-40].

fl

*avus* and *A. parasiticus* were the pre‐

Microbial Quality of Medicinal Plant Materials

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73

trolled during storage [34-35].

gentina, which are used as raw material for drugs. *A.* 

In addition to extraction temperature, the choice of extraction solvent is another important factor to prevent microbial contamination. The aim of an extraction process should be, of course, to provide for the maximum yield of substances and of the highest quality (concen‐ tration of target compounds and pharmacological power of the extracts). For extraction of active phytochemicals, the most commonly used solvents are methanol, ethanol, hexane, chloroform and diethyl ether [31]. Herbal extraction which made by ethanol or methanol ex‐ traction should, in general, provide good hygiene conditions, but the result depends on the alcoholic concentration applied [4].

#### **3.3. pH influence**

The pH value is one of the main factors influencing the quality of medicine. It always con‐ trols many chemical and microbiological reactions [32]. When the pH value is low (presence of acidic substances), the bacterial count could be low, but at neutral or higher pH the level of contamination of the herbal preparations could observed to be higher. This suggests that a neutral or alkaline pH favoured high contamination levels of the herbal preparations. This agrees with the observation that bacterial growth is optimal at more or less neutral pH, around pH 5-8.5 [1].

#### **3.4. Storage**

Most pre-storage processing of plant material, such as that involving drying, heat, cooling and packaging, can prevent the degradation of plant material during storage [33]. Storage of medicinal herbs is an important part in the process production. During storage, due the fac‐ tors in the outside world and their own physical and chemical properties of the interaction, gradually occurring physical, chemical and biological changes. Prolonged storage in poorly ventilated storehouse usually increases sample moisture content in the bulk due to heat ex‐ change capacity, rendering herbs more susceptible to molds growth and toxin production. Fungi are the predominant contaminants of herbs, but most such microbial populations are probably regarded as commensal residents on the plant that survived drying and storage. Most fungi are present on plants, which develop after harvest if relative humidity is not con‐ trolled during storage [34-35].

Environmental dust settled on different parts of the plant and other contaminations can car‐ ry very significant amounts of bacterial and moulds spores [13]. However, those drugs which are subjected to cold water extraction (herbal maceration) may host a considerable amount of microbes, and the extraction procedure carried out at ambient temperature usual‐ ly enables microbial multiplication [4]. The application of hot water extraction usually com‐ pensates for microbiological contaminations, since it can be expected that boiling water markedly reduces the viable counts by several log units and also inactivates possible patho‐ gens [4]. However, bacterial spores of the Bacillaceae family are resistant to thermal treat‐ ment usually applied in infusion preparation, and this thermal shock may stimulate spore germination. Some of these bacteria like *B. cereus* and *C. perfringens* are recognized as having potential pathogenicity and have been incriminated in food poisoning [12]. Thus, in extrac‐ tions using only water, hot or cold, as extractor liquid, the stability of the extract becomes compromised and the risk of microbiological contamination increases significantly. This contamination can compromise the quality and integrity of the plant material itself, as well

In addition to extraction temperature, the choice of extraction solvent is another important factor to prevent microbial contamination. The aim of an extraction process should be, of course, to provide for the maximum yield of substances and of the highest quality (concen‐ tration of target compounds and pharmacological power of the extracts). For extraction of active phytochemicals, the most commonly used solvents are methanol, ethanol, hexane, chloroform and diethyl ether [31]. Herbal extraction which made by ethanol or methanol ex‐ traction should, in general, provide good hygiene conditions, but the result depends on the

The pH value is one of the main factors influencing the quality of medicine. It always con‐ trols many chemical and microbiological reactions [32]. When the pH value is low (presence of acidic substances), the bacterial count could be low, but at neutral or higher pH the level of contamination of the herbal preparations could observed to be higher. This suggests that a neutral or alkaline pH favoured high contamination levels of the herbal preparations. This agrees with the observation that bacterial growth is optimal at more or less neutral pH,

Most pre-storage processing of plant material, such as that involving drying, heat, cooling and packaging, can prevent the degradation of plant material during storage [33]. Storage of medicinal herbs is an important part in the process production. During storage, due the fac‐ tors in the outside world and their own physical and chemical properties of the interaction, gradually occurring physical, chemical and biological changes. Prolonged storage in poorly

as products arising from its use [29-30].

72 Latest Research into Quality Control

alcoholic concentration applied [4].

**3.3. pH influence**

around pH 5-8.5 [1].

**3.4. Storage**

Moulds are responsible for biodeterioration of a number of substrates including raw materi‐ als of some medicinal plants. These moulds reduce raw herbal drugs shelf life and market value. The fungal deterioration adversely affects the chemical composition of the raw mate‐ rials and thereby decreases the medicinal potency of herbal drugs [20]. Samples of herbal parts stored for sale in markets located in Ibadan, Nigeria were analysed for mycoflora asso‐ ciated with their storage and twenty eight fungal species were isolated, showing that herbal drug plant pieces are hazardous for human health [36]. Some samples of herbal raw materi‐ als have been reported to contain aflatoxin. The reference [37] determined the incidence of toxigenic fungi and their mycotoxins on 152 dried medicinal and aromatic herbs from Ar‐ gentina, which are used as raw material for drugs. *A.* fl*avus* and *A. parasiticus* were the pre‐ dominant species isolated, and high aflatoxin concentrations were detected. There is a potential risk for mycotoxins contamination, especially during prolonged storage in poorly conditions without temperature and moisture control that usually render medicinal plants more susceptible to moulds growth and mycotoxins production [5,10,20].

The reduction of plant enzyme activity and inactivation of microorganisms is achieved by drying. Dried plant materials tend to be hygroscopic (readily absorbing moisture) and must be stored under controlled humidity. Rehydration can lead to the decomposition of the bio‐ active metabolites by enzymes from microorganisms or the plant itself. Significant contami‐ nation by bacteria and fungi suggest inadequate storage facilities and poor hygienic practice during preparation of these medicinal plants. The storage processes of such products are stages during which it is important to avoid even further contamination [38].

Studies on long‐term stability of dried herbal teas and preparations are rare. In a study of [20] was examined the deterioration of herbal drug samples which were stored for 6-9 months by traders after collection. Some of the contaminated materials were found to be de‐ teriorated by toxigenic strains of *A. flavus* and contain aflatoxin B1 which was above the per‐ missible limit. In a study of [38], dried *P. lanceolata* leaves were exposed to atmospheres of different relative humidity (75, 45 and 0%) for 24 weeks and was evaluated the chemical changes of the compounds of interest. It was shown that exposure to water results in loss of bioactive molecules of *P. lanceolata* dried leaves, and that colonising fungi are the key con‐ tributors to this loss. The fungal deterioration adversely affects the chemical composition of the raw materials and thereby decreases the medicinal potency of herbal drugs. Biodeterio‐ ration of herbal products samples by associated fungi during storage has drawn attention regarding quality maintenance of these products [35,38-40].

It is common practice for herbalists to prepare herbal medicines and store them in a refrig‐ erator. However, in previous study the effect of microbial contaminants on active com‐ pounds of African plant extracts was assessed and indicated that after 25 days of storage in low temperature there may be little or no active compounds due to spontaneous biode‐ gradation by naturally-occurring microbes [41]. The World Health Organization (WHO) rec‐ ommends that whenever required and when possible, fresh medicinal plant materials should be stored at appropriate low temperatures, ideally at 2-8°C; frozen products should be stor‐ ed at less than -20°C.

tive compounds to control or inhibit the growth of pathogenic and spoilage microorgan‐

The most widely accepted and used technique is that recommended by WHO for total count of microorganisms in plant materials. According to the methodology of the WHO, 10 g of sample should be suspended in 90 ml of buffer sodium chloride-peptone, adjusting the pH to 7.0. To count total aerobic bacteria, sample should be plated in duplicate, using the official technique of sowing depth on casein-soybean digest agar, and then incubated at 30-35°C for 48h. To count yeast and mold, the technique employed is the sowing depth in Sabourauddextrose plus a solution of 10% tartaric acid to obtain pH 3.0 to 3.5. The dilution is plated in duplicate and incubated at 20-25°C for 5 days [52]. Analysis of specific pathogens, Entero‐ bacteriaceae and other Gram negative bacteria (*E. coli*, *Salmonella* sp., *P. aeruginosa* and *S. aureus*) consists of specific methods of cultivation and through biochemical and serological tests. The specification of WHO for total aerobic microorganisms is not more than 107

for the plant material for use as teas and infusions and at most 105 CFU/g for internal use.

a risk because of the possibility to produce mycotoxin, such as aflatoxin, which is a carcino‐ gen toxin. The WHO also recommends a test to detect the possible presence of aflatoxins,

In Brazil, despite the large consumption of products derived from plants, products sold and consumed were not subject to any kind of quality control. In 1995, the Ministry of Health instituted the ordinance MS/SNVS No. 6, January 31, 1995 [53] that regulated the registra‐ tion of herbal products for commercial purposes. Then came the Resolution RDC No. 17 [54] and, more recently, the RDC No. 48 [55] which confirms definitely that are herbal medicines and thereby rescues the need for the existence of safety studies, efficacy and quality, prior to the registration of these products. The Resolution RDC No. 48 of March 16 of 2004 [55] rec‐ ommends that the contamination analysis on herbal medicines must be in accordance with

Both the Brazilian Pharmacopeia [56], as the United States Pharmacopeia [57] draw the fol‐

absence of *Salmonella* spp, *E. coli* and *S. aureus*. However, the Brazilian Pharmacopeia also indicates the detection of other indicators of increased risk for oral administration, such as *P. aeruginosa*, *B. cereus*, *Enterobacter* spp, *C. albicans*, *A. flavus* and *A. parasiticus*. High microbial

In Europe the evaluation of microbial contamination of medicinal plants has increasingly be‐ come an integral part of Good Agricultural Practice (GAP) and Hazard Analysis and Critical Control Point (HACCP) concepts [33]. The limits of microbial contamination given in Euro‐ pean Pharmacopoeia [58] for herbal medicinal products to which boiling water is added be‐

products to which boiling water is not added before use are: total aerobic bacteria (105

CFU/g), fungi (105

lowing specifications for products for oral use: 104 aerobic bacteria/g or mL, 102

loads are indicative of the possibility of potentially pathogenic microorganisms.

The specification of WHO for yeasts and molds are at most 104

which are highly dangerous contaminants in any material of plant origin.

for use as teas and infusions and at most 103

pharmacopoeial specifications.

fore use are: total aerobic bacteria (107

CFU/g

fungi/g and

CFU/g); for herbal medicinal

CFU/g for the plant material

Microbial Quality of Medicinal Plant Materials

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75

UFC/g for internal use. High counts of fungi are

isms [49-51].

**5. Microbial quality parameters**

Processed medicinal plant materials should be packaged as quickly as possible to prevent deterioration of the product and to protect against unnecessary exposure to potential pest attacks and other sources of contamination.

#### **4. Decontamination of plant materials**

Attempts have always been made to decontaminate and preserve these medicinal plants so as to get more safe, natural and potent medicines. The number of methods has been tried for decontamination such as heat treatment, UV irradiation and fumigation. However, volatility and heat sensitivity of the delicate flavor and aroma components of the medicinal plants do not permit the use of heat treatment [42].

Low penetration power of UV radiations makes this irradiation method unsuitable [42]. Fu‐ migation with gaseous ethylene oxide brings down the microbial burden but this method is now prohibited or restricted in many countries due to the carcinogenic nature of one of its residue in treated medicinal plants [43,44]. Various disinfectant technologies have been sug‐ gested which include electromagnetic radiations, photodynamic pulsing, ultrahigh pressure and CO2 treatment [42].

Gamma irradiation is now getting recognition throughout the world as a phytosanitary treatment of herbal materials. It improves the hygienic quality of various herbal materials and reduces the losses due to microbial contamination and insect damage [45]. Besides, it is a fast, safe, convenient, eco-friendly method which reduces the reliance on chemical fumi‐ gants and preservatives currently used by industries. The chances of recontamination are al‐ so reduced, as it can be done after packaging [46]. Some studies showed that the exposition of plant samples to different doses of gamma radiation can result in reduction in total bacte‐ rial counts and also indicated that the microbial load could be decreased by increasing the radiation-absorbed dose. These studies indicate that gamma irradiation is an effective treat‐ ment for microbial decontamination of medicinal plants [42,47,48].

Certain plants contain natural barriers and antimicrobial substances which exert typical in‐ hibitory effects on microbial growth and stability. It has been estimated that around 1400 herbs and spices may possess antimicrobial agents of different chemical nature as oils, pep‐ tides, liquid and organic extracts [4]. Some medicinal herbs contain essential oils which act as natural antimicrobials and may inhibit mould development and mycotoxin production [11]. Different studies have demonstrated the effectiveness of antimicrobials and their effec‐ tive compounds to control or inhibit the growth of pathogenic and spoilage microorgan‐ isms [49-51].

#### **5. Microbial quality parameters**

pounds of African plant extracts was assessed and indicated that after 25 days of storage in low temperature there may be little or no active compounds due to spontaneous biode‐ gradation by naturally-occurring microbes [41]. The World Health Organization (WHO) rec‐ ommends that whenever required and when possible, fresh medicinal plant materials should be stored at appropriate low temperatures, ideally at 2-8°C; frozen products should be stor‐

Processed medicinal plant materials should be packaged as quickly as possible to prevent deterioration of the product and to protect against unnecessary exposure to potential pest

Attempts have always been made to decontaminate and preserve these medicinal plants so as to get more safe, natural and potent medicines. The number of methods has been tried for decontamination such as heat treatment, UV irradiation and fumigation. However, volatility and heat sensitivity of the delicate flavor and aroma components of the medicinal plants do

Low penetration power of UV radiations makes this irradiation method unsuitable [42]. Fu‐ migation with gaseous ethylene oxide brings down the microbial burden but this method is now prohibited or restricted in many countries due to the carcinogenic nature of one of its residue in treated medicinal plants [43,44]. Various disinfectant technologies have been sug‐ gested which include electromagnetic radiations, photodynamic pulsing, ultrahigh pressure

Gamma irradiation is now getting recognition throughout the world as a phytosanitary treatment of herbal materials. It improves the hygienic quality of various herbal materials and reduces the losses due to microbial contamination and insect damage [45]. Besides, it is a fast, safe, convenient, eco-friendly method which reduces the reliance on chemical fumi‐ gants and preservatives currently used by industries. The chances of recontamination are al‐ so reduced, as it can be done after packaging [46]. Some studies showed that the exposition of plant samples to different doses of gamma radiation can result in reduction in total bacte‐ rial counts and also indicated that the microbial load could be decreased by increasing the radiation-absorbed dose. These studies indicate that gamma irradiation is an effective treat‐

Certain plants contain natural barriers and antimicrobial substances which exert typical in‐ hibitory effects on microbial growth and stability. It has been estimated that around 1400 herbs and spices may possess antimicrobial agents of different chemical nature as oils, pep‐ tides, liquid and organic extracts [4]. Some medicinal herbs contain essential oils which act as natural antimicrobials and may inhibit mould development and mycotoxin production [11]. Different studies have demonstrated the effectiveness of antimicrobials and their effec‐

ment for microbial decontamination of medicinal plants [42,47,48].

ed at less than -20°C.

74 Latest Research into Quality Control

attacks and other sources of contamination.

**4. Decontamination of plant materials**

not permit the use of heat treatment [42].

and CO2 treatment [42].

The most widely accepted and used technique is that recommended by WHO for total count of microorganisms in plant materials. According to the methodology of the WHO, 10 g of sample should be suspended in 90 ml of buffer sodium chloride-peptone, adjusting the pH to 7.0. To count total aerobic bacteria, sample should be plated in duplicate, using the official technique of sowing depth on casein-soybean digest agar, and then incubated at 30-35°C for 48h. To count yeast and mold, the technique employed is the sowing depth in Sabourauddextrose plus a solution of 10% tartaric acid to obtain pH 3.0 to 3.5. The dilution is plated in duplicate and incubated at 20-25°C for 5 days [52]. Analysis of specific pathogens, Entero‐ bacteriaceae and other Gram negative bacteria (*E. coli*, *Salmonella* sp., *P. aeruginosa* and *S. aureus*) consists of specific methods of cultivation and through biochemical and serological tests. The specification of WHO for total aerobic microorganisms is not more than 107 CFU/g for the plant material for use as teas and infusions and at most 105 CFU/g for internal use. The specification of WHO for yeasts and molds are at most 104 CFU/g for the plant material for use as teas and infusions and at most 103 UFC/g for internal use. High counts of fungi are a risk because of the possibility to produce mycotoxin, such as aflatoxin, which is a carcino‐ gen toxin. The WHO also recommends a test to detect the possible presence of aflatoxins, which are highly dangerous contaminants in any material of plant origin.

In Brazil, despite the large consumption of products derived from plants, products sold and consumed were not subject to any kind of quality control. In 1995, the Ministry of Health instituted the ordinance MS/SNVS No. 6, January 31, 1995 [53] that regulated the registra‐ tion of herbal products for commercial purposes. Then came the Resolution RDC No. 17 [54] and, more recently, the RDC No. 48 [55] which confirms definitely that are herbal medicines and thereby rescues the need for the existence of safety studies, efficacy and quality, prior to the registration of these products. The Resolution RDC No. 48 of March 16 of 2004 [55] rec‐ ommends that the contamination analysis on herbal medicines must be in accordance with pharmacopoeial specifications.

Both the Brazilian Pharmacopeia [56], as the United States Pharmacopeia [57] draw the fol‐ lowing specifications for products for oral use: 104 aerobic bacteria/g or mL, 102 fungi/g and absence of *Salmonella* spp, *E. coli* and *S. aureus*. However, the Brazilian Pharmacopeia also indicates the detection of other indicators of increased risk for oral administration, such as *P. aeruginosa*, *B. cereus*, *Enterobacter* spp, *C. albicans*, *A. flavus* and *A. parasiticus*. High microbial loads are indicative of the possibility of potentially pathogenic microorganisms.

In Europe the evaluation of microbial contamination of medicinal plants has increasingly be‐ come an integral part of Good Agricultural Practice (GAP) and Hazard Analysis and Critical Control Point (HACCP) concepts [33]. The limits of microbial contamination given in Euro‐ pean Pharmacopoeia [58] for herbal medicinal products to which boiling water is added be‐ fore use are: total aerobic bacteria (107 CFU/g), fungi (105 CFU/g); for herbal medicinal products to which boiling water is not added before use are: total aerobic bacteria (105 CFU/g), fungi (104 CFU/g); Enterobacteria and other Gram-negative organisms (103 CFU/g); *E. coli* and *Salmonella* sp. should be absent. In general, the tests used to verify the presence of microorganisms in plant drugs and microbial limits show no significant variation and fol‐ low the recommendations used for non-sterile pharmaceutical products (Table 1).

legal permissions are required in many countries. It is evident that more detailed studies of plant species popularly used are needed in order to ensure the quality, an important concept

Microbial Quality of Medicinal Plant Materials

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77

and Taís Maria Bauab

Biological Sciences Department, Faculty of Pharmaceutical Sciences, São Paulo State Univer‐

[1] Abba, D., Inabo, H. I., Yakubu, S. E., & Olonitola, O. S. (2009). Contamination of herbal medicinal products marketed in Kaduna Metropolis with selected pathogenic bacteria. *African Journal of Traditional, Complementary and Alternative Medicines*, 6,

[2] Zhang, X. (1998). Regulatory situation of herbal medicines: a worldwide review. 2003: World Health Organization. Available at:, http://apps.who.int/

[3] Kunle, O. F., Egharevba, H. O., & Ahmadu, P. O. (2012). Standardization of herbal medicines- A review. *International Journal of Biodiversity and Conservation*, 4, 101-112.

[4] Kneifel, W., Czech, E., & Kopp, B. (2002). Microbial contamination of medicinal

[5] Bugno, A., Almodovar, A. A. B., Pereira, T. C., Pinto, T. J. A., & Sabino, M. (2006). Occurrence of toxigenic fungi in herbal drugs. *Brazilian Journal of Microbiology*, 37,

[6] Busse, W. (2000). The significance of quality for efficacy and safety of herbal medici‐

[7] Czech, E., Kneifel, W., & Kopp, B. (2001). Microbiological status of commercially available medicinal herbal drugs- A screening study. *Planta Medica*, 67, 263-269.

[8] Tassaneeyakul, W., Razzazi-Fazeli, E., Porasuphatana, S., & Bohm, J. (2004). Contam‐ ination of aflatoxins in herbal medicinal products in Thailand. *Mycopathologia*, 158,

medicinedocs/en/d/Jwhozip57e/, (accessed 8 June 2012).

plants- A review. *Planta Medica*, 5-15, 68.

nal products. *Drug Information Journal*, 34, 15-23.

for providing the wished security and reliability for its use.

\*Address all correspondence to: mgfaraujo@yahoo.com.br

**Author details**

**References**

70-77.

47-51.

239-244.

Marcelo Gonzaga de Freitas Araújo\*

sity – UNESP, Araraquara, Brazil


a United States Pharmacopoeia: The first value represents dried or powdered botanicals and botanicals to be treated with boiling water before use; The second value represents tinctures, powdered botanicals extracts, fluid extracts and nutritional supplements with botanicals; The third value represents infusions/decoctions. b European Pharmacopoeia: Herbal medicinal products consisting solely of one or more herbal drugs (whole, reduced or powdered): the first value represents herbal medicinal products to which boiling water is added before use; The second value represents herbal medicinal products to which boiling water is not added before use. c WHO: The first value represents contamination of "crude" plant material intended for further processing; The second value represents for plant materials that have been pretreated (e.g. with boiling water as used for herbal teas and infusions) or that are used as topical dosage forms; The third value represents For other plant materials for internal use: d Brazilian Pharmacopoeia: The first value represents herbal drugs to which boiling water is added before use; The second value represents herbal drugs to which the extractive process made in cold temperature; The third value represents final products for oral use. \* Limits are not specified.

**Table 1.** Recommended microbial limits for herbal drugs (values in CFU/g).

#### **6. Conclusion**

Microbial contamination can lead to impaired performance of the product due to disruption of the stability of the formulation, modification of physical characteristics and appearance and lead to inactivation of the active ingredients and excipients in the formulation and also cause loss of confidence in the company. Herbalists should be trained to apply Good Manu‐ facturing Practices, good harvesting practices and the safe handling and storage of herbal medicinal products. Further studies are recommended for herbal products to establish other contaminants and ways in which the contaminants can be reduced to recommended levels. The microbial loads should be established and the contaminants isolated and identified. In addition, alternative methods such as treatment with ethylene oxide or radiation with ionic rays lead to decontamination effects. These methods can be seen as a compromise between ensuring the microbiological safety of the product and avoiding consumer's risk and special legal permissions are required in many countries. It is evident that more detailed studies of plant species popularly used are needed in order to ensure the quality, an important concept for providing the wished security and reliability for its use.

#### **Author details**

CFU/g), fungi (104

76 Latest Research into Quality Control

Enterobacteria and other Gram negative bacteria

**6. Conclusion**

which boiling water is not added before use. c

a

CFU/g); Enterobacteria and other Gram-negative organisms (103 CFU/g);

103 / \* / \* \* / 103 \* / 104 / 103 104 / 103 / 102

WHO: The first value represents contamination of "crude" plant material

**Pharmacopoeiab WHOc Brazilian**

**Pharmacopoeiad**

*E. coli* and *Salmonella* sp. should be absent. In general, the tests used to verify the presence of microorganisms in plant drugs and microbial limits show no significant variation and fol‐

**European**

Aerobic bacteria 105 / 104 / 102 107 / 105 \* / 107 / 105 107 / 105 / 104 Mold and yeast 103 / 102 / 10 105 / 104 105 / 104 / 103 104 / 103 / 102

*E. coli* absent 103 / absent 104 / 102 / 10 absent *Salmonella* absent \* / absent \* / absent /absent absent

 United States Pharmacopoeia: The first value represents dried or powdered botanicals and botanicals to be treated with boiling water before use; The second value represents tinctures, powdered botanicals extracts, fluid extracts and nutritional supplements with botanicals; The third value represents infusions/decoctions. b European Pharmacopoeia: Herbal medicinal products consisting solely of one or more herbal drugs (whole, reduced or powdered): the first value represents herbal medicinal products to which boiling water is added before use; The second value represents herbal medicinal products to

intended for further processing; The second value represents for plant materials that have been pretreated (e.g. with boiling water as used for herbal teas and infusions) or that are used as topical dosage forms; The third value represents For other plant materials for internal use: d Brazilian Pharmacopoeia: The first value represents herbal drugs to which boiling water is added before use; The second value represents herbal drugs to which the extractive process made in cold temperature; The

Microbial contamination can lead to impaired performance of the product due to disruption of the stability of the formulation, modification of physical characteristics and appearance and lead to inactivation of the active ingredients and excipients in the formulation and also cause loss of confidence in the company. Herbalists should be trained to apply Good Manu‐ facturing Practices, good harvesting practices and the safe handling and storage of herbal medicinal products. Further studies are recommended for herbal products to establish other contaminants and ways in which the contaminants can be reduced to recommended levels. The microbial loads should be established and the contaminants isolated and identified. In addition, alternative methods such as treatment with ethylene oxide or radiation with ionic rays lead to decontamination effects. These methods can be seen as a compromise between ensuring the microbiological safety of the product and avoiding consumer's risk and special

low the recommendations used for non-sterile pharmaceutical products (Table 1).

**United States Pharmacopoeiaa**

third value represents final products for oral use. \* Limits are not specified.

**Table 1.** Recommended microbial limits for herbal drugs (values in CFU/g).

Marcelo Gonzaga de Freitas Araújo\* and Taís Maria Bauab

\*Address all correspondence to: mgfaraujo@yahoo.com.br

Biological Sciences Department, Faculty of Pharmaceutical Sciences, São Paulo State Univer‐ sity – UNESP, Araraquara, Brazil

#### **References**


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[36] Efunyoye, M. O. (1996). Fungi associated with herbal drug plants during storage. *Mycopathologia*, 136, 115-118.

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[49] Tiwari, B. K., Valdramidi, V. P., O'Donnell, C. P., Muthukumarappan, K., Bourke, P., & Cullen, P. J. (2009). Application of natural antimicrobials for food preservation.

[50] Wang, Y., Lu, Z., Wu, H., & Lv, F. (2009). Study on the antibiotic activity of microcap‐ sule curcumin against foodborne pathogens. *International Journal of Food Microbiology*,

[51] Negi, P. S. (2012). Plant extracts for the control of bacterial growth: Efficacy, stability and safety issues for food application. *International Journal of Food Microbiology*, 156,

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*Journal of Agricultural and Food Chemistry*, 57, 5987-6000.

plant materials. Geneva, World Health Organization.

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30, 71-74.

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cia Sanitária.

5.1.7.-6.

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[36] Efunyoye, M. O. (1996). Fungi associated with herbal drug plants during storage.

[37] Rizzo, I., Vedoya, G., Maurutto, S., Haidukowski, M., & Varsavsky, E. (2004). Assess‐ ment of toxigenic fungi on Argentinean medicinal herbs. *Microbiological Research*, 159,

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[39] Singh, P., Srivastava, B., Kumar, A., & Dubey, N. K. (2008). Fungal contamination of raw materials of some herbal drugs and recommendation of Cinnamomum cam‐

[40] Shukla, R., Kumar, A., Prasad, C. S., Srivastava, B., & Dubey, N. K. (2008). Antimy‐ cotic and antiaflatoxigenic potency of Adenocalymma alliaceum Miers. on fungi causing biodeterioration of food commodities and raw herbal drugs. *International Bi‐*

[41] Du Plessis-Stoman, D., Downing, T. G., van de Venter, M., & Govender, S. (2009). Traditional herbal medicines: potential degradation of sterols and sterolins by micro‐

[42] Gupta, P. C., Garg, N., & Joshi, P. (2011). Effect of gamma irradiation on the extrac‐ tion yield and microbial contamination of medicinal plants. *Internet Journal of Food*

[43] Kim, M. J., Yook, H. S., & Byun, M. W. (2000). Effects of gamma irradiation on micro‐ bial contamination and extraction yields of Korean medicinal herbs. [2]. *Radiation*

[44] Satomi, L. C., Soriani, R. R., & Pinto, T. J. A. (2005). Descontaminação de drogas vege‐ tais empregando irradiação gama e óxido de etileno: aspectos microbianos e quími‐

[45] Farkas, J. (1998). Irradiation as a method for decontaminating food: A review. Inter‐

[46] Khattak, K. F., Simpson, T. J., & Ihasnullah, . (2009). Effect of gamma irradiation on the microbial load, nutrient composition and free radical scavenging activity of *Ne‐*

[47] Khattak, K. F. (2012). Evaluation of microbial loads, physical characteristics, chemical constituents and biological properties of radiation processed Fagonia arabica. *Radia‐*

phora oil as herbal fungitoxicant. *Microbial Ecology*, 56, 555-560.

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cos. Revista Brasileira de Ciências Farmacêuticas, 41(4), 445-450.

*lumbo nucifera* rhizome. *Radiation Physics and Chemistry*, 78, 206-212.

national Journal of Food Microbiology, 44, 189-204.

*tion Physics and Chemistry*, 81, 679-685.

*odeterioration & Biodegradation*, 62, 348-351.

*Mycopathologia*, 136, 115-118.

*cal Analysis*, 23, 88-93.

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113-120.

80 Latest Research into Quality Control


**Chapter 5**

**Application of ISO 9001 Industrial Standard to Herbal**

We noted earlier [1] that 1978 was the turning point in current public perception of tradi‐ tional medicine (TM) following the famous WHO declaration at Alma-Ata. That declaration ushered in a positive attitude that paved the way for the present global popularity of TM, especially herbal medicine. We noted earlier also [2,3] that whereas herbal remedies are called dietary supplements in the US, thereby shifting emphasis away from their medicinal attributes, the Dietary Supplement Health Education Act of 1994 [4], which occasioned the shift, actually helped to promote herbal medicine in the US, albeit indirectly, through the in‐ novative provision it made for user information [5,6]. A similar situation obtained in Eu‐ rope, where the net effect of the laws and rules passed in 2004 on herbal remedies had been to promote their production and use [7, 8]. In terms of trade and economics of herbal drugs, the following fact is notable: Although, Asia contributed only US\$ 7.3 billion to herbal world trade in 1999 [9], by 2005, a mere 6 years, China's contribution alone rose to US\$ 14 billion [10]. This stupendous growth was due to policies and programmes that favoured herbal medicine – the cornerstone of Traditional Chinese Medicine (TCM). Similar situations as in China held sway in Japan, South Korea and the Indian sub-continent, where government policies also favoured herbal medicine. However, in many developing countries like Niger‐ ia, a totally different picture obtained, not because policies were expressly against herbal medicine, but in these countries there had been a lingering absence of proper policies and laws supportive of traditional remedies. Another key fact on the political economy of herbal drugs is that: Although, about 80% of people in developing countries depended on herbs, these countries contributed only 7.2% to herbal drug trade in 1999. By contrast, the devel‐ oped nations, where people relied less on herbs, contributed 55.2%. Asia, less Japan and

> © 2012 Ameh et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Ameh et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Drug Regulation**

Karniyus Gamaniel

**1. Introduction**

http://dx.doi.org/10.5772/50814

Sunday Ameh, Florence Tarfa, Magaji Garba and

Additional information is available at the end of the chapter

## **Application of ISO 9001 Industrial Standard to Herbal Drug Regulation**

Sunday Ameh, Florence Tarfa, Magaji Garba and Karniyus Gamaniel

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50814

#### **1. Introduction**

We noted earlier [1] that 1978 was the turning point in current public perception of tradi‐ tional medicine (TM) following the famous WHO declaration at Alma-Ata. That declaration ushered in a positive attitude that paved the way for the present global popularity of TM, especially herbal medicine. We noted earlier also [2,3] that whereas herbal remedies are called dietary supplements in the US, thereby shifting emphasis away from their medicinal attributes, the Dietary Supplement Health Education Act of 1994 [4], which occasioned the shift, actually helped to promote herbal medicine in the US, albeit indirectly, through the in‐ novative provision it made for user information [5,6]. A similar situation obtained in Eu‐ rope, where the net effect of the laws and rules passed in 2004 on herbal remedies had been to promote their production and use [7, 8]. In terms of trade and economics of herbal drugs, the following fact is notable: Although, Asia contributed only US\$ 7.3 billion to herbal world trade in 1999 [9], by 2005, a mere 6 years, China's contribution alone rose to US\$ 14 billion [10]. This stupendous growth was due to policies and programmes that favoured herbal medicine – the cornerstone of Traditional Chinese Medicine (TCM). Similar situations as in China held sway in Japan, South Korea and the Indian sub-continent, where government policies also favoured herbal medicine. However, in many developing countries like Niger‐ ia, a totally different picture obtained, not because policies were expressly against herbal medicine, but in these countries there had been a lingering absence of proper policies and laws supportive of traditional remedies. Another key fact on the political economy of herbal drugs is that: Although, about 80% of people in developing countries depended on herbs, these countries contributed only 7.2% to herbal drug trade in 1999. By contrast, the devel‐ oped nations, where people relied less on herbs, contributed 55.2%. Asia, less Japan and

© 2012 Ameh et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ameh et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

South Korea, contributed 37.6%. Equally interesting is the comparison of Brazil with Niger‐ ia. Both are rich in medicinal plants and have high populations that depend substantially on herbs. But, while herbs contributed an unknown amount to the Nigerian economy in 2007, in Brazil it contributed US\$ 160 million. By contrast, Nigeria's entire federal budget for health in 2007 was a mere US\$ 800 million [2]. These findings earlier led us [11,12] to con‐ clude that developing countries need strategies that will enhance the regulation of herbal drugs and promote their trade. The present article is an attempt to enunciate one of such strategies. It is particularly of note that the superior performance of Brazil in comparison with Nigeria indicates that with proper policies and strategies, herbs can indeed contribute substantially to any economy.

application throughout the organization; determine the sequence of the processes and their interactions; determine the criteria and methods for operating and controlling the processes; determine and ensure the availability needed resources and supporting information; check, measure and analyze the processes, where applicable; and implement actions to achieve

Application of ISO 9001 Industrial Standard to Herbal Drug Regulation

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85

planned results and continual improvement of the processes.

**Figure 1.** NIPRD's core business in the context of Plan-Do-Check-Act process-based QMS.1

and Measurement/ analysis/ improvement correspond to clauses 6, 7 and 8 respectively.

specifically notes as follows:

The organization shall manage the processes above in accordance with ISO 9001:2008 re‐ quirements. It shall also define the type and extent of control to be applied to any out‐ sourced process that can affect product conformity to requirements. ISO 9001:2008

1 Management responsibility corresponds to clause 5 of ISO 9001; while Resource management, Product realization

### **2. Methodology: Determinative Review of ISO 9001 and the Mandates of Nigeria's and Europe's DRAs**

#### **2.1. ISO 9001:2008 industrial standard – A synopsis**

ISO 9001:2008 industrial standard or quality management system (QMS) is a document of about 30 pages with 8 clauses, published by and obtainable from the International Organ‐ ization for Standardization (ISO), Basle, Switzerland, or from any of its national affiliates. The standard is designed to be met by any organization that i) needs to demonstrate its ability to consistently provide product or service that meets both customer and applicable statutory and regulatory requirements (collectively legal requirements); ii) aims to en‐ hance customer satisfaction by effectively and continually improving its QMS; and iii) plans to provide continual assurance of conformity to customer and applicable legal re‐ quirements. These aims/ approaches (often called "QMS requirements" or "quality proce‐ dures") are generic and intended to be applicable to all organizations regardless of type, size and product provided. Wherever any requirement cannot be applied due to the na‐ ture of an organization and its product, such can be considered for exclusion. But wher‐ ever exclusions are made, claims of conformity to the standard are not acceptable unless such exclusions are limited to requirements within clause 7 of the standard, and such ex‐ clusions do not affect the organization's ability, or responsibility, to provide product that meets customer and applicable legal requirements. ISO 9001:2008 defines the minimum requirements for a well managed organization. In other words, noncompliance to an ISO 9001:2008 requirement puts at risk an organization's ability to consistently and efficiently satisfy the expectations of its customers/ stakeholders.

#### **2.2. The six QMS requirements or "The Six Quality Procedures"**

These procedures or requirements, as one may choose to call them, actually refer to subclause 4.1 (General requirements) under clause 4 (Quality Management System) of ISO 9001:2008. The sub-clause prescribes that organizations shall establish, document, imple‐ ment, and maintain a QMS, and continually improve its effectiveness. To do so means that the organization shall operate its QMS with a view to carrying out (or meeting) the follow‐ ing six procedures (or requirements): determine the processes needed for the QMS, and their application throughout the organization; determine the sequence of the processes and their interactions; determine the criteria and methods for operating and controlling the processes; determine and ensure the availability needed resources and supporting information; check, measure and analyze the processes, where applicable; and implement actions to achieve planned results and continual improvement of the processes.

South Korea, contributed 37.6%. Equally interesting is the comparison of Brazil with Niger‐ ia. Both are rich in medicinal plants and have high populations that depend substantially on herbs. But, while herbs contributed an unknown amount to the Nigerian economy in 2007, in Brazil it contributed US\$ 160 million. By contrast, Nigeria's entire federal budget for health in 2007 was a mere US\$ 800 million [2]. These findings earlier led us [11,12] to con‐ clude that developing countries need strategies that will enhance the regulation of herbal drugs and promote their trade. The present article is an attempt to enunciate one of such strategies. It is particularly of note that the superior performance of Brazil in comparison with Nigeria indicates that with proper policies and strategies, herbs can indeed contribute

**2. Methodology: Determinative Review of ISO 9001 and the Mandates of**

ISO 9001:2008 industrial standard or quality management system (QMS) is a document of about 30 pages with 8 clauses, published by and obtainable from the International Organ‐ ization for Standardization (ISO), Basle, Switzerland, or from any of its national affiliates. The standard is designed to be met by any organization that i) needs to demonstrate its ability to consistently provide product or service that meets both customer and applicable statutory and regulatory requirements (collectively legal requirements); ii) aims to en‐ hance customer satisfaction by effectively and continually improving its QMS; and iii) plans to provide continual assurance of conformity to customer and applicable legal re‐ quirements. These aims/ approaches (often called "QMS requirements" or "quality proce‐ dures") are generic and intended to be applicable to all organizations regardless of type, size and product provided. Wherever any requirement cannot be applied due to the na‐ ture of an organization and its product, such can be considered for exclusion. But wher‐ ever exclusions are made, claims of conformity to the standard are not acceptable unless such exclusions are limited to requirements within clause 7 of the standard, and such ex‐ clusions do not affect the organization's ability, or responsibility, to provide product that meets customer and applicable legal requirements. ISO 9001:2008 defines the minimum requirements for a well managed organization. In other words, noncompliance to an ISO 9001:2008 requirement puts at risk an organization's ability to consistently and efficiently

These procedures or requirements, as one may choose to call them, actually refer to subclause 4.1 (General requirements) under clause 4 (Quality Management System) of ISO 9001:2008. The sub-clause prescribes that organizations shall establish, document, imple‐ ment, and maintain a QMS, and continually improve its effectiveness. To do so means that the organization shall operate its QMS with a view to carrying out (or meeting) the follow‐ ing six procedures (or requirements): determine the processes needed for the QMS, and their

substantially to any economy.

84 Latest Research into Quality Control

**Nigeria's and Europe's DRAs**

**2.1. ISO 9001:2008 industrial standard – A synopsis**

satisfy the expectations of its customers/ stakeholders.

**2.2. The six QMS requirements or "The Six Quality Procedures"**

**Figure 1.** NIPRD's core business in the context of Plan-Do-Check-Act process-based QMS.1

The organization shall manage the processes above in accordance with ISO 9001:2008 re‐ quirements. It shall also define the type and extent of control to be applied to any out‐ sourced process that can affect product conformity to requirements. ISO 9001:2008 specifically notes as follows:

<sup>1</sup> Management responsibility corresponds to clause 5 of ISO 9001; while Resource management, Product realization and Measurement/ analysis/ improvement correspond to clauses 6, 7 and 8 respectively.

**1.** Processes needed for the QMS include the processes for management activities (clause 5), provision of resources (clause 6), product realization (clause 7), and measurement, analysis, and improvement (clause 8).

**2.4. Key terminologies of ISO 9001:2008**

ment of all ISO 9000 standards, including ISO 9001:2008.

A process is an activity or operation that receives inputs and converts them to outputs. Prac‐ tically all activities or operations involved in generating a product or providing a service are processes. For an organization to function, it must define and manage several inter-linked processes. Most often, the output of one process becomes the input into the next process. The systematic identification and control of the various processes employed within an or‐ ganization, and the interactions between such processes, is termed "process approach" to management. Thus process approach to management is a way of obtaining a desired result, by controlling activities and related resources as a process. Process approach is a key ele‐

Application of ISO 9001 Industrial Standard to Herbal Drug Regulation

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87

System approach to management is based on the premise that the efficiency and effective‐ ness with which an organization achieves its quality objectives are contributed and en‐ hanced by identifying, understanding and managing all the interrelated processes within

Quality policy is a formal statement from the management of an organization that is linked to the nature of its business and its plans to meet the needs of its customers/ stakeholders.

Quality objective is the factual or tangible basis upon which quality policy and plans for im‐ plementing the quality programmes of an organization are built. Quality objective should be SMART (ie: specific, measurable, achievable, realistic and time-bound). Each staff of the or‐

Decision simply means the selection of one or more options from a multitude of options in tackling a given organizational task. As far as the QMS is concerned, an organization should make SMART decisions based on recorded data. An example of a SMART decision is: The QMS must be audited and evaluated regularly for conformance and effectiveness, so as to

The policy is designed to be understood and followed at all levels and by all staff.

ganization is expected to work towards measurable objectives.

assure quality and continual improvement.

*2.4.1. Process approach to management*

*2.4.2. System approach to management*

the organization as a system

*2.4.3. Quality policy*

*2.4.4. Quality objective*

*2.4.5. Decision*


The type and extent of control applied to an outsourced process can be influenced by factors such as: the potential impact of the outsourced process on the organization's capability to provide product that conforms to requirements; the degree to which the control over the process is shared; and the capability of the organization in achieving the necessary control via the application of sub-clause 7.4 (Purchasing). Philosophically, ISO 9001:2008 is formu‐ lated on the basis of management by objectives (MBO) and draws upon eight quality man‐ agement principles. Ideally therefore, quality assurance covers activities in research, development, production and documentation. It embraces the rule: "do it right the first time". It involves regulating the quality of raw materials, the state of production line and works-in-progress, the product and related management processes. One of the most widely used paradigms for quality assurance management (QAM) is the "Shewhart cycle", also called "PDCA approach", meaning, "Plan-Do-Check-Act" [13,14]. The foregoing is illustrat‐ ed in Figure 1 using NIPRD QMS processes as an example.

#### **2.3. The eight quality management principles that underlie ISO 9001:2008**

Like other ISO standards (Example: ISO 9004 - *Managing for Sustained Success*), ISO 9001:2008 is based on 8 quality management principles that are aligned with the philosophy and objec‐ tives of most quality award programmes in the world's most industrialized nations. The 8 principles are associated with the following themes:


#### **2.4. Key terminologies of ISO 9001:2008**

#### *2.4.1. Process approach to management*

**1.** Processes needed for the QMS include the processes for management activities (clause 5), provision of resources (clause 6), product realization (clause 7), and measurement,

**2.** An outsourced process is a process the organization needs for its QMS, and which the

**3.** Ensuring control over outsourced processes does not absolve the organization of the re‐

The type and extent of control applied to an outsourced process can be influenced by factors such as: the potential impact of the outsourced process on the organization's capability to provide product that conforms to requirements; the degree to which the control over the process is shared; and the capability of the organization in achieving the necessary control via the application of sub-clause 7.4 (Purchasing). Philosophically, ISO 9001:2008 is formu‐ lated on the basis of management by objectives (MBO) and draws upon eight quality man‐ agement principles. Ideally therefore, quality assurance covers activities in research, development, production and documentation. It embraces the rule: "do it right the first time". It involves regulating the quality of raw materials, the state of production line and works-in-progress, the product and related management processes. One of the most widely used paradigms for quality assurance management (QAM) is the "Shewhart cycle", also called "PDCA approach", meaning, "Plan-Do-Check-Act" [13,14]. The foregoing is illustrat‐

organization chooses to have performed by an external party.

sponsibility to conform to customer and legal requirements.

ed in Figure 1 using NIPRD QMS processes as an example.

principles are associated with the following themes:

**1.** Customer focus.

**3.** Involvement of people.

**6.** Continual improvement.

**4.** Process approach to management.

**5.** System approach to management.

**7.** Factual approach to decision making.

**8.** Mutually beneficial supplier relationships.

**2.** Leadership.

**2.3. The eight quality management principles that underlie ISO 9001:2008**

Like other ISO standards (Example: ISO 9004 - *Managing for Sustained Success*), ISO 9001:2008 is based on 8 quality management principles that are aligned with the philosophy and objec‐ tives of most quality award programmes in the world's most industrialized nations. The 8

analysis, and improvement (clause 8).

86 Latest Research into Quality Control

A process is an activity or operation that receives inputs and converts them to outputs. Prac‐ tically all activities or operations involved in generating a product or providing a service are processes. For an organization to function, it must define and manage several inter-linked processes. Most often, the output of one process becomes the input into the next process. The systematic identification and control of the various processes employed within an or‐ ganization, and the interactions between such processes, is termed "process approach" to management. Thus process approach to management is a way of obtaining a desired result, by controlling activities and related resources as a process. Process approach is a key ele‐ ment of all ISO 9000 standards, including ISO 9001:2008.

#### *2.4.2. System approach to management*

System approach to management is based on the premise that the efficiency and effective‐ ness with which an organization achieves its quality objectives are contributed and en‐ hanced by identifying, understanding and managing all the interrelated processes within the organization as a system

#### *2.4.3. Quality policy*

Quality policy is a formal statement from the management of an organization that is linked to the nature of its business and its plans to meet the needs of its customers/ stakeholders. The policy is designed to be understood and followed at all levels and by all staff.

#### *2.4.4. Quality objective*

Quality objective is the factual or tangible basis upon which quality policy and plans for im‐ plementing the quality programmes of an organization are built. Quality objective should be SMART (ie: specific, measurable, achievable, realistic and time-bound). Each staff of the or‐ ganization is expected to work towards measurable objectives.

#### *2.4.5. Decision*

Decision simply means the selection of one or more options from a multitude of options in tackling a given organizational task. As far as the QMS is concerned, an organization should make SMART decisions based on recorded data. An example of a SMART decision is: The QMS must be audited and evaluated regularly for conformance and effectiveness, so as to assure quality and continual improvement.

#### *2.4.6. Traceability*

Traceability is concerned with and refers to the fact that typically, recorded data are meant to show how and where raw materials and products were processed, in order to allow prod‐ ucts and problems to be traced to their sources.

cating with customers or stakeholders about product information, inquiries, contracts, orders, feedback, and complaints. All DRAs are "service providers" but some produce and even distribute certain specific items, as mentioned above. Nigeria's National Agency for Food and Drug Administration and Control (NAFDAC) is a national DRA, while the Euro‐

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Mandate is a piece of legislation or instruction from a constituted authority to another con‐ stituted authority or body to carry out a named task. DRAs are mandated by the State to

It is well established that the high state of development in the chemical/ pharmaceutical in‐ dustrial sector in the US, Japan, South Korea, Britain, Germany and other European coun‐ tries owes much to the powerful synergy between regulatory legislations, industrial standards and a focused political will. It is also manifest that the rapid, all-round industrial revolution in China in the past decade or so owes much to China's embrace of ISO stand‐

\*Source ISO Survey 2009 [15]. Most countries have their own national standards in addition to ISO standards. For example the UK is well known for its industrial standards pre-sufixed by BSI (British

It must be stated that countries like Japan, Germany, Britain and US use their own national standards in addition to those of ISO. Based on the foregoing, we state that the specific aim of this article is: To examine the QMS requirements of ISO 9001:2008 and the requirements

pean Medicines Evaluation Agency is a regional DRA.

**2.5. The new industrial revolution and the aim of this chapter**

ards, especially ISO 9001, as shown in Table 1 after a recent [15].

**Country Ranking No. certificates Pertinent remark**

**China** 1 257,076 Relies mostly on ISO standard. **Italy** 2 130,066 Relies mostly on ISO standard **Japan** 3 68,484 Relies only partly on ISO standard **Spain** 4 59,576 Relies substantially on ISO standard **Russia** 5 53,152 Relies substantially on ISO standard **Germany** 6 47,156 Relies only partly on ISO standard **UK** 7 41,193 Relies only partly on ISO standard **India** 8 37,493 Relies substantially on ISO standard **South Korea** 9 28,935 Relies substantially on ISO standard **US** 10 23,400 Relies only partly on ISO standard

regulate drugs and health related products.

Standards Institution).

**Table 1.** The top 10 countries in ISO certification in 2009.

*2.4.13. Mandate*

#### *2.4.7. Product realization*

Product realization refers to the scenario in which, when developing a new product, an or‐ ganization plans the stages of development, with appropriate testing at each stage. The or‐ ganization tests and documents whether the product meets design requirements, legal requirements, and user or customer needs.

#### *2.4.8. Quality plan*

Quality plan refers to a document specifying the QMS processes (including the product real‐ ization processes), and the resources to be applied to a specific product or project.

#### *2.4.9. Monitoring and measurement*

Monitoring and measurement refer to the scenario in which an organization must regularly re‐ view its performance through meetings and internal audits, and determine whether the QMS is working and what improvements can be made. The organization must have a documented procedure for internal audits and a procedure for dealing with past problems and potential problems. It must keep records of these activities and the resulting decisions, and monitor their effectiveness. It must have documented procedures for dealing with actual and potential nonconformances (problems involving suppliers, customers, or internal problems).

#### *2.4.10. Continual improvement*

Continual Improvement refers to the scenario in which an organization 1) makes sure no customer uses a bad product, 2) determines what to do with a bad product, 3) deals with the root cause of problems, and 4) keeps records to use as a tool to improve the QMS.

#### *2.4.11. Customer requirements*

Customer requirements refer to the attributes that the buyer of a product (or user of a serv‐ ice) wants. The core business of an organization is to determine customer requirements and to meet them, in accordance with sub-clause 5.2 (Customer focus).

#### *2.4.12. Drug Regulatory Agencies (DRAs)*

Drug regulatory agencies (DRAs) are organizations set up by the State on behalf of the gen‐ eral public with a Mandate to regulate drugs and related products and services. The Man‐ date of some DRAs may include production and distribution of certain goods like vaccines and orphan drugs. Either the State or the general public can be regarded as customer, stake‐ holder or shareholder. DRAs like all other organizations must have a system for communi‐ cating with customers or stakeholders about product information, inquiries, contracts, orders, feedback, and complaints. All DRAs are "service providers" but some produce and even distribute certain specific items, as mentioned above. Nigeria's National Agency for Food and Drug Administration and Control (NAFDAC) is a national DRA, while the Euro‐ pean Medicines Evaluation Agency is a regional DRA.

#### *2.4.13. Mandate*

*2.4.6. Traceability*

88 Latest Research into Quality Control

*2.4.7. Product realization*

*2.4.8. Quality plan*

ucts and problems to be traced to their sources.

requirements, and user or customer needs.

*2.4.9. Monitoring and measurement*

*2.4.10. Continual improvement*

*2.4.11. Customer requirements*

*2.4.12. Drug Regulatory Agencies (DRAs)*

Traceability is concerned with and refers to the fact that typically, recorded data are meant to show how and where raw materials and products were processed, in order to allow prod‐

Product realization refers to the scenario in which, when developing a new product, an or‐ ganization plans the stages of development, with appropriate testing at each stage. The or‐ ganization tests and documents whether the product meets design requirements, legal

Quality plan refers to a document specifying the QMS processes (including the product real‐

Monitoring and measurement refer to the scenario in which an organization must regularly re‐ view its performance through meetings and internal audits, and determine whether the QMS is working and what improvements can be made. The organization must have a documented procedure for internal audits and a procedure for dealing with past problems and potential problems. It must keep records of these activities and the resulting decisions, and monitor their effectiveness. It must have documented procedures for dealing with actual and potential non-

Continual Improvement refers to the scenario in which an organization 1) makes sure no customer uses a bad product, 2) determines what to do with a bad product, 3) deals with the

Customer requirements refer to the attributes that the buyer of a product (or user of a serv‐ ice) wants. The core business of an organization is to determine customer requirements and

Drug regulatory agencies (DRAs) are organizations set up by the State on behalf of the gen‐ eral public with a Mandate to regulate drugs and related products and services. The Man‐ date of some DRAs may include production and distribution of certain goods like vaccines and orphan drugs. Either the State or the general public can be regarded as customer, stake‐ holder or shareholder. DRAs like all other organizations must have a system for communi‐

ization processes), and the resources to be applied to a specific product or project.

conformances (problems involving suppliers, customers, or internal problems).

root cause of problems, and 4) keeps records to use as a tool to improve the QMS.

to meet them, in accordance with sub-clause 5.2 (Customer focus).

Mandate is a piece of legislation or instruction from a constituted authority to another con‐ stituted authority or body to carry out a named task. DRAs are mandated by the State to regulate drugs and health related products.

#### **2.5. The new industrial revolution and the aim of this chapter**

It is well established that the high state of development in the chemical/ pharmaceutical in‐ dustrial sector in the US, Japan, South Korea, Britain, Germany and other European coun‐ tries owes much to the powerful synergy between regulatory legislations, industrial standards and a focused political will. It is also manifest that the rapid, all-round industrial revolution in China in the past decade or so owes much to China's embrace of ISO stand‐ ards, especially ISO 9001, as shown in Table 1 after a recent [15].


\*Source ISO Survey 2009 [15]. Most countries have their own national standards in addition to ISO standards. For example the UK is well known for its industrial standards pre-sufixed by BSI (British Standards Institution).

**Table 1.** The top 10 countries in ISO certification in 2009.

It must be stated that countries like Japan, Germany, Britain and US use their own national standards in addition to those of ISO. Based on the foregoing, we state that the specific aim of this article is: To examine the QMS requirements of ISO 9001:2008 and the requirements for regulating herbal drugs in Nigeria (a developing economy) and Europe (a developed economy), with a view to devising a framework that will better regulate herbal drugs and facilitate their trade worldwide. Such a framework will greatly benefit developing countries like Nigeria that are yet to benefit optimally from their comparative advantage in the abun‐ dance of spices, herbs and medicinal plants. In addition, marketers and users of herbs in consumer nations like the US, Canada, Germany, UK and France, where consumption now runs in to billions of US dollars, will also profit greatly from an improved and regularized world trade in herbs.

**European Union (EU) – regulated by EMEA Nigeria – regulated by NAFDAC**

**aspect**

Analytical status of the product for registration.

Pre-registration inspection of premises.

Post marketing surveillance plan/ report

and waivers.

\*The Table was drawn based on data gathered from references including Goldman [5]; De Smet [7, 8]; Ann Godsell Regulatory [16]; and various NAFDAC leaflets, including Akunyili [17]. Note that the requirements for registration in Nigeria are not necessarily less tasking, but their lack of explicitness can be a booby trap and a leeway for non-transparency. The necessity for explicitness and transparency is very important because some years ago the Director General of China's drug regulatory agency was sentenced to death for alleged corrupt

But, while the EMEA approach is technically more explicit, though not necessarily more ex‐ erting than NAFDAC's, the latter is administratively much more cumbersome, and therefore more liable to inefficiency and abuse. Table 3 shows the extra bureaucratic demands of NAFDAC. We stated earlier that, although, 80% of people in developing countries like Ni‐ geria depended on herbs, these countries contributed only 7.2% to herbal drug trade in 1999. By contrast, the developed nations, where people relied less on herbs, contributed 55.2%. This scenario is explained by the fact herbal drugs are better regulated in developed regions

practice [18]. In 2000 the entire Management of NAFDAC was sacked in similar grey circumstances.

like the China, India, Japan and South East Asia, the EU and North America [2].

**Requirement**

Application of ISO 9001 Industrial Standard to Herbal Drug Regulation

The product must have: certificate of analysis, dossier containing data on ingredients, method of analysis, stability, dosage and safety precautions.

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91

Manufacturing, storage and distribution premises must be GXP compliant. Marketers must provide convincing evidence of GXP

Applicant may be required to provide a plan for reporting on the use of the product and

Fees are required at several stages of the registration but waivers are not expressly stated, thereby negating the concept and need for transparency (see Table 3 for extra

of any adverse reactions.

requirements).

**Type of data Details of data required Regulatory**

These include: production must be in a GMP compliant, product must be produced with validated formula and method, there must be a product specification, stability studies must be carried out in the container proposed for marketing for purposes storage/ shelflife, and dossiers must be provided for starting materials and

or human studies, review of potential drug-drug interactions, side

15 must be in the EU. Notably, there is no requirement to prove


**Table 2.** Requirements for herbal registration compared between EU and Nigeria.

**Quality control data: Refer to GMP requirements for production.**

**Safety data requirements**

**Traditional use evidence**

finished product.

The data may be assembled from: animal

effects and contraindications. Others include: recognized monographs, data special groups - children, the elderly and mothers.

Evidence that the product has been in use as medicine for 30 years or more (the last

efficacy (De Smet, 2005).

### **3. Results & discussion: A Framework for Efficient Herbal Drug Regulation (HDR)**

#### **3.1. Justification for establishing national or regional DRAs**

Most or all countries have a national or regional agency that regulates the production, distri‐ bution and use of drug products. The process of regulation commences with the registration of the producer, the product, the distributor and in some cases the user. In some countries drugs, foods and dietary supplements are regulated by the same body (eg: Nigeria's NAF‐ DAC and US-FDA). The EU's EMEA however regulates only drug products. States or re‐ gions need to have DRAs in order to ensure order in the production, distribution and use of drugs. Without DRAs utter chaos and pandemonium will result in production (eg: manufac‐ turers will do as they please without a uniform control), distribution (distributors and sup‐ pliers will do as they choose without a uniform order) and use (prescribers and users will do as they think without a uniform regime), which would allow incidences of counterfeit and expired drugs in drug distribution chain, drug abuse and emergence of drug resistant disor‐ ders, especially infective conditions like malaria and TB.

#### **3.2. Comparative analysis of Nigeria's and EU's requirements for herbal drug regulation**

A careful scrutiny of the requirements for registering and regulating herbal drugs in Europe and in Nigeria reveals their basic similarity, as shown in Table 2.



\*The Table was drawn based on data gathered from references including Goldman [5]; De Smet [7, 8]; Ann Godsell Regulatory [16]; and various NAFDAC leaflets, including Akunyili [17]. Note that the requirements for registration in Nigeria are not necessarily less tasking, but their lack of explicitness can be a booby trap and a leeway for non-transparency. The necessity for explicitness and transparency is very important because some years ago the Director General of China's drug regulatory agency was sentenced to death for alleged corrupt practice [18]. In 2000 the entire Management of NAFDAC was sacked in similar grey circumstances.

**Table 2.** Requirements for herbal registration compared between EU and Nigeria.

for regulating herbal drugs in Nigeria (a developing economy) and Europe (a developed economy), with a view to devising a framework that will better regulate herbal drugs and facilitate their trade worldwide. Such a framework will greatly benefit developing countries like Nigeria that are yet to benefit optimally from their comparative advantage in the abun‐ dance of spices, herbs and medicinal plants. In addition, marketers and users of herbs in consumer nations like the US, Canada, Germany, UK and France, where consumption now runs in to billions of US dollars, will also profit greatly from an improved and regularized

**3. Results & discussion: A Framework for Efficient Herbal Drug**

Most or all countries have a national or regional agency that regulates the production, distri‐ bution and use of drug products. The process of regulation commences with the registration of the producer, the product, the distributor and in some cases the user. In some countries drugs, foods and dietary supplements are regulated by the same body (eg: Nigeria's NAF‐ DAC and US-FDA). The EU's EMEA however regulates only drug products. States or re‐ gions need to have DRAs in order to ensure order in the production, distribution and use of drugs. Without DRAs utter chaos and pandemonium will result in production (eg: manufac‐ turers will do as they please without a uniform control), distribution (distributors and sup‐ pliers will do as they choose without a uniform order) and use (prescribers and users will do as they think without a uniform regime), which would allow incidences of counterfeit and expired drugs in drug distribution chain, drug abuse and emergence of drug resistant disor‐

**3.2. Comparative analysis of Nigeria's and EU's requirements for herbal drug regulation**

A careful scrutiny of the requirements for registering and regulating herbal drugs in Europe

**aspect**

Legal status of applicant manufacturer or marketer

**Requirement**

Applicant must be certified by the Corporate Affairs Commission as a business. A marketer must show evidence of Power of Attorney.

**European Union (EU) – regulated by EMEA Nigeria – regulated by NAFDAC**

**3.1. Justification for establishing national or regional DRAs**

ders, especially infective conditions like malaria and TB.

and in Nigeria reveals their basic similarity, as shown in Table 2.

**Type of data Details of data required Regulatory**

form, list of excipients, shelf life, posology, indications, contraindications, and special precautions. These are used as basis for inserts or advertisement, which must undergo a process called

These include: name, strength, dosage

"readability".

world trade in herbs.

90 Latest Research into Quality Control

**Regulation (HDR)**

**Product information: Summary of product characteristics** But, while the EMEA approach is technically more explicit, though not necessarily more ex‐ erting than NAFDAC's, the latter is administratively much more cumbersome, and therefore more liable to inefficiency and abuse. Table 3 shows the extra bureaucratic demands of NAFDAC. We stated earlier that, although, 80% of people in developing countries like Ni‐ geria depended on herbs, these countries contributed only 7.2% to herbal drug trade in 1999. By contrast, the developed nations, where people relied less on herbs, contributed 55.2%. This scenario is explained by the fact herbal drugs are better regulated in developed regions like the China, India, Japan and South East Asia, the EU and North America [2].

#### **3.3. Justification for selecting ISO 9001:2008 for this study**

Although most of the OECD countries and other highly industrialized economies, where herbal drugs are well regulated, have their own national standards, all do embrace ISO standards, especially ISO 9001:2008. For example, the British Standards Institution (BSI) is well known and widely adopted in many other countries worldwide, and although interna‐ tional in application, such national standards do not bear the tag "international". By con‐ trast, the ISO family of standards bear the tag "international". ISO 9001:2008 is an international standard designed to address systemic change (ie: a change that affects an or‐ ganization as a whole). The global popularity of ISO 9001:2000 - the predecessor of ISO 9001:2008, is attributable to the following factors: a) major purchasers require their suppliers to hold ISO 9001 certification [15, 19]; b) studies indicate significant financial benefits for or‐ ganizations certified to ISO 9001 [19,20]; and c) similar superior operational performance of ISO certified firms has been severally confirmed [21-24]. As just noted, ISO 9001:2008 is an update of ISO 9001:2000, and we have selected it for this study by reason of its popularity and versatility, and because it is a process-based QMS that addresses systemic change affect‐ ing whole organizations like a national or regional drug DRA, like Nigeria's National Agen‐ cy for Food and drug Administration and Control (NAFDAC), the US Food and Drug Administration (US-FDA) and the European Medicines Evaluation Agency (EMEA).

**S/No Extra requirement Remark**

10 Certificate of Registration of brand name with

11 The applicable fee payable only if documents are confirmed to be satisfactory

12 Nutraceuticals, medical devices and other regulated drug products have similar requirements, with minor variations. Specific details can be obtained from

reorganization of its Management in 2000.

points/ directing principles of the 8 clauses.

**Table 3.** NAFDAC's extra requirements for registering herbal medicines.

of plant.

NAFDAC.

trademark registry in the Ministry of Commerce here in Nigeria; Letter of invitation from manufacturer to inspect factory abroad, stating full name and location

9 Premises Registration License from PCN Only probably reasonable

\*The information on NAFDAC were drawn from leaflets and NAFDAC's website (2010): www.nafdacnigeria.org/ The remarks are informed by current affairs and public perception of NAFDAC's role and activities including the wholesale

ISO 9001:2008 is the most widely used QMS standard, with over a million certificates issued worldwide. Alas, it was revealed at the SON-NIPRD course in 2011, that only two public in‐ stitutions in Nigeria have ISO 9001 certification! Yet, as stated earlier, ISO 9001:2008 defines the minimum requirements for a well managed organization. The standard is published by the International Organization for Standardization (ISO), Basle, Switzerland. National ac‐ creditation bodies like the Standards Organization of Nigeria (SON) provide accreditation to registrars who issue the ISO 9001 certificates to those they audit. ISO 9001:2008 is set out in eight clauses designated clauses 1 to 8. The structure and salient points/ directing principles of the clauses are tabulated below. A copy of ISO 9001:2008 is a prerequisite for this study. Similarly required, is a grasp of the requirements for registering and regulation herbal drugs in a developed economy like Europe; and in a developing country like Nigeria, as depicted in Table 2. It is well known that herbal drugs are better regulated in the developed than in developing countries. Table 3 suggests that undue bureaucracy or needlessly cumbersome requirements can hinder efficient regulation. Tables 4-13 show the structure and salient

**3.4. A systematic review of the eight clauses of ISO 9001:2008 in relation to DRAs**

*3.4.1. A synopsis of the Mandate of DRAs and the eight clauses of ISO 9001:2008*

Probably unreasonable for all categories of applicants

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Likely to be abused if the amount is high. The fee should be a token amount paid by all

A sketch of the minor variations should be provided in print no matter how brief. Any information provided by NAFDAC should be printable for sake of transparency

applicants

Application of ISO 9001 Industrial Standard to Herbal Drug Regulation



\*The information on NAFDAC were drawn from leaflets and NAFDAC's website (2010): www.nafdacnigeria.org/ The remarks are informed by current affairs and public perception of NAFDAC's role and activities including the wholesale reorganization of its Management in 2000.

**Table 3.** NAFDAC's extra requirements for registering herbal medicines.

**3.3. Justification for selecting ISO 9001:2008 for this study**

92 Latest Research into Quality Control

Although most of the OECD countries and other highly industrialized economies, where herbal drugs are well regulated, have their own national standards, all do embrace ISO standards, especially ISO 9001:2008. For example, the British Standards Institution (BSI) is well known and widely adopted in many other countries worldwide, and although interna‐ tional in application, such national standards do not bear the tag "international". By con‐ trast, the ISO family of standards bear the tag "international". ISO 9001:2008 is an international standard designed to address systemic change (ie: a change that affects an or‐ ganization as a whole). The global popularity of ISO 9001:2000 - the predecessor of ISO 9001:2008, is attributable to the following factors: a) major purchasers require their suppliers to hold ISO 9001 certification [15, 19]; b) studies indicate significant financial benefits for or‐ ganizations certified to ISO 9001 [19,20]; and c) similar superior operational performance of ISO certified firms has been severally confirmed [21-24]. As just noted, ISO 9001:2008 is an update of ISO 9001:2000, and we have selected it for this study by reason of its popularity and versatility, and because it is a process-based QMS that addresses systemic change affect‐ ing whole organizations like a national or regional drug DRA, like Nigeria's National Agen‐ cy for Food and drug Administration and Control (NAFDAC), the US Food and Drug

Administration (US-FDA) and the European Medicines Evaluation Agency (EMEA).

Clearly unreasonable for all categories of applicants

Probably unreasonable for all categories of applicants

Clearly unreasonable for all categories of applicants

Clearly unreasonable for all categories of applicants

Clearly unreasonable for all categories of applicants

Only probably reasonable

**S/No Extra requirement Remark**

3 Notarized original copy of the duly executed Power of Attorney from the product manufacturer.

4 Certificate of Manufacture issued by the competent

5 If contract-manufactured, Contract Manufacturing Agreement, properly executed and notarized by a Notary Public in the country of manufacture.

7 Certificate of Pharmaceutical Products (COOP) duly

8 Current Superintendent Pharmacists license to

practice issued by the Pharmacists Council of Nigeria (PCN).

6 Current World Health Organization Good Manufacturing Practice Certificate for the manufacturer, authenticated by the Nigerian Mission.

issued and authenticated.

authenticate.

health or regulatory authority in country of origin and authenticated by the Nigerian Mission in that country. Where there is no Nigerian mission, The British High Commission or an ECOWAS country Mission will

1 Five (5) copies of the product dossier. Probably unreasonable 2 Three (3) packs of the products samples. Probably reasonable

#### **3.4. A systematic review of the eight clauses of ISO 9001:2008 in relation to DRAs**

#### *3.4.1. A synopsis of the Mandate of DRAs and the eight clauses of ISO 9001:2008*

ISO 9001:2008 is the most widely used QMS standard, with over a million certificates issued worldwide. Alas, it was revealed at the SON-NIPRD course in 2011, that only two public in‐ stitutions in Nigeria have ISO 9001 certification! Yet, as stated earlier, ISO 9001:2008 defines the minimum requirements for a well managed organization. The standard is published by the International Organization for Standardization (ISO), Basle, Switzerland. National ac‐ creditation bodies like the Standards Organization of Nigeria (SON) provide accreditation to registrars who issue the ISO 9001 certificates to those they audit. ISO 9001:2008 is set out in eight clauses designated clauses 1 to 8. The structure and salient points/ directing principles of the clauses are tabulated below. A copy of ISO 9001:2008 is a prerequisite for this study. Similarly required, is a grasp of the requirements for registering and regulation herbal drugs in a developed economy like Europe; and in a developing country like Nigeria, as depicted in Table 2. It is well known that herbal drugs are better regulated in the developed than in developing countries. Table 3 suggests that undue bureaucracy or needlessly cumbersome requirements can hinder efficient regulation. Tables 4-13 show the structure and salient points/ directing principles of the 8 clauses.


**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRA**

\*The Table is to be studied side by side with the contents of ISO 9001:2008 and Table 2, which is on regulatory re‐

well as, maintains the integrity of the QMS.

recommendations for improvement.

Agency's Mandate. Most DRAs have a public relation office.

The DRA must be committed to developing and implementing a QMS, as well as, a commitment to continually improve the effectiveness of the QMS. The DRA can do this by 1) communicating the importance of meeting "legal and customer requirements"; 2) establishing a quality policy and quality objectives; 3) conducting management reviews; and 4) by ensuring the availability of necessary resources. The "legal and customer requirements" of a DRA are implicit in its Mandate – which may be an act, law or decree. In planning, the DRA must 1) ensure that quality objectives are established at the relevant functions and levels within the Agency; 2) ensure that quality objectives are measurable and consistent with the quality policy; and 3) ensure that planning for the QMS meets the general requirements (clause 4.1) and quality objectives (clause 5.4.1), as

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95

In as much as operations must be carried out the DRA must ensure that the responsibilities and authorities for such are defined and communicated appropriately. It is essential that a member of top management, irrespective of other duties, be appointed (as Quality Manager) and given the responsibility to: 1) ensure that the needed processes are established, implemented, and maintained; 2) report to top management on the performance of the QMS; 3) report to top management on any need for improvement; and 4) ensure the promotion of awareness of

For a DRA to be effective it must review its QMS at planned intervals to: 1) ensure an effective QMS; 2) assess possible opportunities for improvement; 3) evaluate the need for any changes; and 4) consider the need for changes to the policy and objectives. The DRA must of course maintain records of reviews as per clause 4.2.4. For a DRA, the inputs for review must include information on: 1) results of audits; 2) feedback from government and the public, eg - incidences of counterfeit drugs; 3) status of preventive and corrective actions, eg – incidences of drug abuse; 4) follow-up actions from earlier reviews; 5) changes that can affect the QMS; and 6)

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRA**

in a permanent form. 2) A record is a permanent

kept for evidence or information. It specifically bears the history of events or arrangements, and is preserved in a

**Table 5.** Clause 4 of ISO 9001:2008 in relation to DRAs.

**5.2 Customer focus** – the organization must ensure that its purpose/ focus (inclusive of customer/ stakeholder requirements) is understood and determined. **5.3 Quality policy** – this should be:

1) appropriate to the purpose of the organization; 2) focused on meeting requirements and continual improvement; 3) used as a framework for quality objectives; 4) publicized and understood at appropriate

5) reviewed for continuing suitability .

**5.5 Responsibility, Authority, and**

**5.5.1 Responsibility and Authority 5.5.2 Management Representative**

processes regarding the effectiveness of the QMS are established and implemented.

**5.6 Management Review 5.6.1 General**

review,

**5.5.3 Internal Communication** – it is crucial that the organization ensures that appropriate communication

**5.6.2 Review Input** – includes audit results, public feedback, process performance, status of preventive/ corrective action, follow-up from previous management

document of something that is

lasting form.

5 **5 Management Responsibility 5.1 Management commitment**

levels; and

**5.4 Planning 5.4.1 Quality Objectives 5.4.2 QMS Planning**

**Communication**

quirements of DRAs.

\*The Table is to be studied side by side with the contents of ISO 9001:2008 and Table 2, which is on regulatory re‐ quirements of DRAs.

**Table 4.** Clauses 1-3 of ISO 9001:2008 in relation to DRAs.



in a permanent form. 2) A record is a permanent document of something that is kept for evidence or information. It specifically bears the history of events or arrangements, and is preserved in a lasting form.

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

associated therewith.

specialized health products.

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRA**

\*The Table is to be studied side by side with the contents of ISO 9001:2008 and Table 2, which is on regulatory re‐

ISO 9001:2008 can be used to establish, and to update a DRA's QMS. A DRA, like other parastatals or private organizations must consider its unique operational environment and the dynamics and risks

A normative reference implies, unless otherwise stated, that the most recent versions of the separate documents should be referenced. DRAs would benefit immensely from such key references and compendia such as the *International Pharmacopoeia* and others like the *BP* and *USP*, and the WHO

The term "product" may also mean "service". "Legal requirements" means "statutory and regulatory requirements". Most DRAs are service providers only, while others may produce and distribute certain

Clause 4.1 implies that the DRA must identify, manage and document the processes that make up its QMS – ie: the DRA must address the so called "Six Quality Procedures" and generate relevant documents, including: 1) quality manual, 2) quality policy, 3) quality objective, 4) process flowchart, and 5) work instructions. The DRA can achieve this by using a management strategy called "process approach", which means that it must manage: 1) the processes that make up its organization, 2) the interaction between these processes, and 3) the inputs and outputs that glue these processes together. The quality manual should: 1) describe how the QMS processes interact; 2) define the scope of the QMS (it should explain any reductions in the scope of the QMS and justify all exclusions/ reductions); and 3) document all procedures in the QMS or refer to them. It is most crucial that the DRA

The DRA must establish SOPs to define the controls needed: 1) to approve, review, update and re-approve documents prior to use; 2) to ensure that changes, current status, relevant versions of documents are identified; and 3) to prevent the unintended use of obsolete documents. The DRA must establish records to provide: 1) evidence that operations conform to QMS requirements; and 2) evidence that operations of the QMS are effective. Records

prepares, establishes and maintains a quality manual.

must be ensured to be legible, readily identifiable and retrievable.

manual on *Quality Control Methods for Medicinal Plant Materials*.

1 **1. Scope**

**1.1 General 1.2 Application**

94 Latest Research into Quality Control

2 **2. Normative references**

*Sustained Success*)

3 **3. Terms and definitions**

quirements of DRAs.

(eg: ISO 9000:2005 is devoted to *QMS Fundamentals and Vocabulary*; and ISO 9004:2009 is devoted to *Managing for*

(see section 2.4 of this article on "Key terminologies of ISO 9001:2008)

4 **4. Quality Management System (QMS) 4.1 General requirements 4.2 Documentation requirements: 4.2.1 General** – QMS documents must include: quality policy, quality objective, quality manual, documented procedures/ records specified by ISO 9001:2008, and documents/ records determined by the organization to be relevant for effective planning, operation and control of the QMS.

**4.2.2 Quality manual** – this should

**4.2.4 Control of records** – records are a

the QMS processes.

controls needed.

controlled. Here too, SOPs

It may or may exist

include the scope of the QMS, SOPs and a description of

**4.2.3 Control of documents** - the documents required by the QMS must be established and controlled. This means that SOPs are to be established to define the

special type of documents and must be established and

are to be established to define the controls needed. **Note:**1) A document is a piece of written, printed, or electronic matter that provides information or evidence.

**Table 4.** Clauses 1-3 of ISO 9001:2008 in relation to DRAs.

\*The Table is to be studied side by side with the contents of ISO 9001:2008 and Table 2, which is on regulatory re‐ quirements of DRAs.

**Table 5.** Clause 4 of ISO 9001:2008 in relation to DRAs.

feedback, process performance, status of preventive/ corrective action, follow-up from previous management

review,


ments of DRAs.


**Clause Title and subtitles, with remark Salient points/ directing principles/ application to DRAs**

Mandate.

The DRA must determine and provide the resources needed: 1) to implement, maintain and continually improve the effectiveness of its QMS; and 2) to enhance the fulfilment of its

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By virtue of its role as a highly specialized agency, the DRA must ensure that all staff irrespective of department whose work can impact DRA's Mandate are competent based on

The DRA must, of course, provide appropriate work environment for all staff whose work

appropriate education, skills, experience and abide by their professional ethics. This implies that the DRA must: 1) determine the competency of staff; 2) provide training as needed; 3) evaluate the effectiveness of the actions taken on training and skills acquisition ; 4) inform staff of their relevance within the QMS; 5) ensure staff know their contributions to achieving quality objectives; and 6) maintain staff records of education, training, skill, and

experience in accordance with clause 4.2.4.

impacts the DRA's Mandate.

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on regulatory require‐

In most countries where DRAs are not a department of the Ministry of Health, they exist as a parastatal or as a special department within the Ministry (as in Japan), with conditions of service being slightly more favourable than in the rest of the Ministry. The idea is to give special incentives to the staff on account of hazards perceived to be peculiar to the job. In Nigeria, NAFDAC is well housed both at the federal and state levels and the staff earn about the same remuneration as the universities and research institutes. In most countries the DRAs have well equipped offices and laboratories, and those DRAs that produce and dis‐

In Nigeria, NAFDAC previously handled certain aspects of manufacture/ distribution of vaccines until certain developments (or rather controversies over quality/ effectiveness of polio vaccines during the late 1990s/ early 2000s) led, first to the creation of a National Pro‐ gramme on Immunization (NPI); and latter to the transfer of the same functions from NPI back to the Federal Ministry of Health. It is obvious from the foregoing that institutions like NAFDAC and NPI would have performed better had they been certified. It seems also that one of the keys to ending the cycle of poverty and underdevelopment in some countries is to ensure that elite institutions like the DRAs are certified to appropriate ISO standards. Certif‐ ications of agencies like the Health Insurance Scheme and the Pension Commission will def‐

*3.4.3. The making of stronger DRAs and the need for clearer demarcation of responsibilities*

tribute goods are equipped with the necessary plant and storage facilities.

*3.4.4. The inevitability of confusion in the absence of regulatory standardization*

initely reduce perceive current levels of corruption in such institution.

6 **6 Resource management 6.1 Provision of resources 6.2 Human resources 6.2.1 General**

> **Awareness 6.3 Infrastructure**

services.

ments of DRAs.

**6.4 Work Environment**

and noise pollution.

**6.2.2 Competence, Training, and**

Like any other public outfit the DRA must determine, provide, and maintain infrastructure like buildings, workspace and associated utilities, and essential support

"Work environment" implies conditions under which work is performed, and includes physical aspects like weather

**Table 7.** Clause 6 of ISO 9001:2008 in relation to DRAs.

**Table 6.** Clause 5 of ISO 9001:2008 in relation to DRAs.

#### *3.4.2. The immediate historical antecedent of NAFDAC and the continuing relevance of ISO 9001*

Nigeria's NAFDAC was created by decree in 1992/93 following the ethylene glycol disas‐ ter of 1991/92 in Langtang General Hospital, Plateau State, where ethylene glycol was used in the place of propylene glycol in preparing paracetamol elixir. The glycol had been purchased from a hitherto popular pharmacy shop located at Masalachin-Jumai Street, Jos. Prior to 1992/93, a department in the Federal Ministry of Health handled food and drug administration in Nigeria. Alas, in 2009 another ethylene glycol disaster occurred in Lagos. In this latter disaster a hitherto popular brand of paediatric mixture ("My Pikin") was found to contain ethylene glycol that had been purchased from an unregulated source. It is important to note that whereas the glycol implicated in the Lantang disaster was purchased from pharmaceutically regulated source, the glycol in the case of the La‐ gos disaster was purchased from a company that dealt in industrial chemicals associated with automobiles and cooling systems. The occurrence of this kind of disaster within less than two decades is matter of concern that calls for a more efficient programme for regu‐ lating drugs and industrial chemicals in developing countries. The US-FDA after whose image and likeness NAFDAC was created is known for efficiency mostly because it is supported by proper laws and strong institutions. Better laws and stronger institutions, including DRAs, are required to avoid or minimize this kind of disaster as seen in Niger‐ ia. It seems instructive to mention the "Tylenol case" in the US, and how that case led to a new legislation. Between late September and early October 1982, seven persons in Chi‐ cago died after taking capsules of Tylenol (a brand of paracetamol), to which cyanide crystals had been added. The crystals had apparently been introduced into the capsules by someone who had removed bottles of Tylenol from several drugstores and then re‐ placed them on the shelves. It took an intensive investigation by a team of over 100 agents, including FDA staff, to discover the mischief, which led to a 1982-legislation that required all over-the-counter drugs and medicines sold in the county (and later elsewhere in the US and beyond) carry manufacturers' seals which broken would be obvious. The rapid conclusion of the investigation led by the Illinois Attorney General himself (Tyrone C. Fahner) and the dispatch with which the new law was issued collectively testify to the inner workings of strong institutions – which developing nations lack.


\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on regulatory require‐ ments of DRAs.

**Table 7.** Clause 6 of ISO 9001:2008 in relation to DRAs.

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRA**

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on regulatory require‐

*3.4.2. The immediate historical antecedent of NAFDAC and the continuing relevance of ISO 9001*

inner workings of strong institutions – which developing nations lack.

Nigeria's NAFDAC was created by decree in 1992/93 following the ethylene glycol disas‐ ter of 1991/92 in Langtang General Hospital, Plateau State, where ethylene glycol was used in the place of propylene glycol in preparing paracetamol elixir. The glycol had been purchased from a hitherto popular pharmacy shop located at Masalachin-Jumai Street, Jos. Prior to 1992/93, a department in the Federal Ministry of Health handled food and drug administration in Nigeria. Alas, in 2009 another ethylene glycol disaster occurred in Lagos. In this latter disaster a hitherto popular brand of paediatric mixture ("My Pikin") was found to contain ethylene glycol that had been purchased from an unregulated source. It is important to note that whereas the glycol implicated in the Lantang disaster was purchased from pharmaceutically regulated source, the glycol in the case of the La‐ gos disaster was purchased from a company that dealt in industrial chemicals associated with automobiles and cooling systems. The occurrence of this kind of disaster within less than two decades is matter of concern that calls for a more efficient programme for regu‐ lating drugs and industrial chemicals in developing countries. The US-FDA after whose image and likeness NAFDAC was created is known for efficiency mostly because it is supported by proper laws and strong institutions. Better laws and stronger institutions, including DRAs, are required to avoid or minimize this kind of disaster as seen in Niger‐ ia. It seems instructive to mention the "Tylenol case" in the US, and how that case led to a new legislation. Between late September and early October 1982, seven persons in Chi‐ cago died after taking capsules of Tylenol (a brand of paracetamol), to which cyanide crystals had been added. The crystals had apparently been introduced into the capsules by someone who had removed bottles of Tylenol from several drugstores and then re‐ placed them on the shelves. It took an intensive investigation by a team of over 100 agents, including FDA staff, to discover the mischief, which led to a 1982-legislation that required all over-the-counter drugs and medicines sold in the county (and later elsewhere in the US and beyond) carry manufacturers' seals which broken would be obvious. The rapid conclusion of the investigation led by the Illinois Attorney General himself (Tyrone C. Fahner) and the dispatch with which the new law was issued collectively testify to the

changes that can affect the QMS, and recommendations

**5.6.3 Review Output** - includes decisions/ actions related to: 1) improvement of the QMS; 2) improvement in

**Table 6.** Clause 5 of ISO 9001:2008 in relation to DRAs.

for improvement.

96 Latest Research into Quality Control

meeting Mandate; and 3) resource needs

ments of DRAs.

#### *3.4.3. The making of stronger DRAs and the need for clearer demarcation of responsibilities*

In most countries where DRAs are not a department of the Ministry of Health, they exist as a parastatal or as a special department within the Ministry (as in Japan), with conditions of service being slightly more favourable than in the rest of the Ministry. The idea is to give special incentives to the staff on account of hazards perceived to be peculiar to the job. In Nigeria, NAFDAC is well housed both at the federal and state levels and the staff earn about the same remuneration as the universities and research institutes. In most countries the DRAs have well equipped offices and laboratories, and those DRAs that produce and dis‐ tribute goods are equipped with the necessary plant and storage facilities.

#### *3.4.4. The inevitability of confusion in the absence of regulatory standardization*

In Nigeria, NAFDAC previously handled certain aspects of manufacture/ distribution of vaccines until certain developments (or rather controversies over quality/ effectiveness of polio vaccines during the late 1990s/ early 2000s) led, first to the creation of a National Pro‐ gramme on Immunization (NPI); and latter to the transfer of the same functions from NPI back to the Federal Ministry of Health. It is obvious from the foregoing that institutions like NAFDAC and NPI would have performed better had they been certified. It seems also that one of the keys to ending the cycle of poverty and underdevelopment in some countries is to ensure that elite institutions like the DRAs are certified to appropriate ISO standards. Certif‐ ications of agencies like the Health Insurance Scheme and the Pension Commission will def‐ initely reduce perceive current levels of corruption in such institution.

#### **Clause Title and subtitles, with remark Salient points/ directing principles / application to DRAs**

Some DRAs produce/ store/ distribute specialized and non-profit products like vaccines and

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

for the product or the type of service.

include details for product preservation.

The same principle followed in planning a physical product is followed in planning a service. The interfaces between the different groups involved must be managed to ensure effective communication/ clear assignment of responsibility. Design and development review, verification and validation have distinct purposes. They can be conducted and recorded separately or in any combination, as the DRA deems suitable

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A DRA would review the selected inputs for adequacy and resolve any incomplete, ambiguous, or conflicting requirements. Examples of application inputs include: (1 applicable information derived from similar designs; and (2 requirements essential for design and development. If a DRA is designing a policy to curb drug abuse in a particular locality, useful inputs for the design would include statistics like 1) the age, gender and occupation of abusers; 2) the type of drugs abused; and 3) the success rate of similar policies elsewhere. NOTE: Information for production and service can

A DRA must perform systematic reviews of design and development at suitable stages in accordance with planned arrangements (7.3.1) so as to: 1) evaluate the ability of the results to meet requirements; and 2) identify problems and propose necessary actions. Reviews must include representatives of the functions concerned. Results of reviews and subsequent follow-up actions must be maintained as per 4.2.4. A DRA would perform design and development verification in accordance with 7.3.1 to ensure that output meets the design and development input requirements; and maintain the results of such verification and subsequent follow-up actions. When practical and desirable, validation must be completed before delivery or

implementation of the product. Results of the validation and of subsequent follow-up actions must be maintained as per 4.2.4. Just as some DRAs produce or distribute physical products, some DRA have their own testing facilities while others contract out such tests. Thus the purchase needs of DRAs differ with their Mandate. However, whenever purchasing is indicated the DRA must 1) ensure that purchased items conform to specified purchase requirements (Note: The type and extent of control applied to the supplier and purchased product depends upon the effect of the product on the subsequent realization processes or the final product); 2) evaluate and

select suppliers based on their ability to supply goods in accordance with requirements; 3) establish the criteria for selection, evaluation, and re-evaluation; and 4) maintain the results of such evaluations and subsequent follow-up actions in

accordance with sub-clauses 4.2.3 and 4.2.4.

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐

7 **7.3 Design and Development**

development.

**7.3.1 Design/ Development Planning**

**7.3.2 Design /Development Inputs**

essential for design and development. **7.3.3 Design/ Development Outputs** Where applicable, DRAs must

requirements; 2) provide information

**7.3.4 Design/ Development Review 7.3.5 Design/ Dev. Verification 7.3.6 Design/ Dev. Validation**

**7.3.7 Control of Des./ Dev Changes** For either physical goods or policy,

quirements of DRAs.

DRAs that produce or distribute must plan and control product design/ development. They must determine 1) the stages of design/development; 2) appropriate testing, review and validation for each stage; and 3) responsibility/authority for design/

In designing/ developing a physical good or a service, the DRA must determine the needed inputs and keep records as per 4.2.4. The inputs must include: 1) functional and performance requirements; 2) applicable legal requirements; 3) applicable information derived from similar designs; and 4) requirements

document the outputs of the design/ development process in a form suitable for verification against the inputs to the process. The outputs must 1) meet or match design and development input

for purchasing, production and service; 3) contain or reference product acceptance criteria; 4) define essential characteristics for safe and proper use; 5) be approved before their release

Validation activities are performed in accordance with 7.3.1 to confirm that the resulting product is capable of meeting the requirements for its specified application or intended use.

DRAs must 1) identify design and development changes and maintain records as per 4.2.4; 2) review, verify, validate and approve changes before implementation; 3) evaluate the changes in terms of their effect on constituent parts (raw material) and products (or policies) already delivered (or implemented).

**Table 9.** Clause 7 of ISO 9001:2008 in relation to DRAs (Design and Development).

7 **7 Product realization**

**7.1 Planning of product** realization Product realization typically implies that manufacturers 1) plan and develop the QMS processes needed for product realization; 2) keep the planning consistent with other requirements of the QMS; 3) document the plan in a suitable form; and 4) determine through the planning, the following: a) quality objectives and product requirements; b) need for processes, documents, and resources; c) verification (establishment of truth/ confirmatory evidence), validation (formal registration/ obtainment of official sanction), monitoring, measurement, inspection, and test activities; d) criteria for product acceptance; and e) records providing evidence that the processes and resulting product meet requirements. Since DRAs regulate manufacturers they too must be acquainted with clause 7. NOTE 1: Recall that "quality plan" (2.4.8 of this article) is a document specifying the processes, and the resources to be applied to a specific product, project, or contract. NOTE 2: An organization can apply the requirements of subclause 7.3 (vide infra) to the development of product realization processes7.2 Customer-Related Processes **7.2.1 Determination of Requirements Related to the Product 7.2.2 Review of Requirements Related to the Product 7.2.3 Customer Communication** The intensity and scope of communication depends on the product and the associated orphan drugs. For such, all aspects of clause 7 apply. The DRAs of developed economies concentrate on regulating manufacturers, distributors and use of products. Different processes are involved in drug regulation but these often have some aspects in common. For example, the process of registering a manufacturer and that of registering a product are essentially the same, but they differ in their aims, point of action, who by, and so on. Some of the processes involved in "planning of service realization", which is the core business of a typical DRA, require a wide range of differing concepts, technicalities, approaches, specializations, and so on. For example, although the technical aspects of producing tablets of aspirin, diazepam, B-complex, erythromycin, and orphan drugs may be similar, the modes of their regulation and distribution are different. Given the involved Mandate of DRAs, different strategies must be developed to grapple with the differing nuances and intricacies associated with the regulation of the five products. Typically, questions that have answer buried in culture/ society rather than the lab do arise in drug regulation. Why, for example, despite the similarities between NAFDAC and EMEA, it is impossible to buy erythromycin or diazepam over-the-counter in Europe but not in Nigeria? To what extent do political, social and economic factors affect "planning of service realization" in different social environments? It well known that regulatory strategies that work in Europe often fail to work outside despite obvious legislative similarities between nations. The customers/ stakeholders of a typical DRA are the general public, manufacturers, suppliers and the government. DRAs that engage in production and distribution must determine customer requirements, which invariably include specified and unspecified but desirable attributes. Such DRAs must also determine the legal requirements applicable to the product. Other desirable requirements, including post-delivery activities like maintenance services, may be considered. DRAs that produce or distribute would normally review the product requirements before committing to supply in order to: 1) ensure that product requirements are defined; 2) resolve any requirements differing from those previously expressed; and 3) ensure its ability to meet the requirements. In the same vein when a DRA plans a regulatory strategy or legislation the plan should be graduated and made reasonable to its purpose and scope and with reference to the operating socioeconomic environment. The DRA must maintain the results of reviews, and any

mandate. Thus the DRA must determine and implement the necessary arrangements for communicating with on aspects like 1) product information; 2) inquiries and subsequent follow-up actions in accordance with 4.2.4. When the requirements are not documented, they must be confirmed before acceptance. But if product requirements are changed, the DRA must ensure relevant documents are amended and relevant personnel are made aware of the changed requirements. NOTE: In some situations a formal review is impractical for each order. In such cases reviews

contracts; 3) customer/ stakeholder feedbacks -positive or can cover relevant product information such as catalogues or adverts.

negative

stakeholders

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs.

**Table 8.** Clause 7 of ISO 9001 in relation to DRAs (Product planning and Customer-Related processes).


**Clause Title and subtitles, with remark Salient points/ directing principles / application to DRAs**

Some DRAs produce/ store/ distribute specialized and non-profit products like vaccines and orphan drugs. For such, all aspects of clause 7 apply. The DRAs of developed economies concentrate on regulating manufacturers, distributors and use of products. Different processes are involved in drug regulation but these often have some aspects in common. For example, the process of registering a manufacturer and that of registering a product are essentially the same, but they differ in their aims, point of action, who by, and so on. Some of the processes involved in "planning of service realization", which is the core business of a typical DRA, require a wide range of differing concepts, technicalities, approaches, specializations, and so on. For example, although the technical aspects of producing tablets of aspirin, diazepam, B-complex, erythromycin, and orphan drugs may be similar, the modes of their regulation and distribution are different. Given the involved Mandate of DRAs, different strategies must be developed to grapple with the differing nuances and intricacies associated with the regulation of the five products. Typically, questions that have answer buried in culture/ society rather than the lab do arise in drug regulation. Why, for example, despite the similarities between NAFDAC and EMEA, it is impossible to buy erythromycin or diazepam over-the-counter in Europe but not in Nigeria? To what extent do political, social and economic factors affect "planning of service realization" in different social environments? It well known that regulatory strategies that work in Europe often fail to

work outside despite obvious legislative similarities between nations.

are made aware of the changed requirements.

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐

**Table 8.** Clause 7 of ISO 9001 in relation to DRAs (Product planning and Customer-Related processes).

The customers/ stakeholders of a typical DRA are the general public, manufacturers, suppliers and the government. DRAs that engage in production and distribution must determine customer requirements, which invariably include specified and unspecified but desirable attributes. Such DRAs must also determine the legal requirements applicable to the product. Other desirable requirements, including post-delivery activities like maintenance services, may be considered. DRAs that produce or distribute would normally review the product requirements before committing to supply in order to: 1) ensure that product requirements are defined; 2) resolve any requirements differing from those previously expressed; and 3) ensure its ability to meet the requirements. In the same vein when a DRA plans a regulatory strategy or legislation the plan should be graduated and made reasonable to its purpose and scope and with reference to the operating socioeconomic environment. The DRA must maintain the results of reviews, and any subsequent follow-up actions in accordance with 4.2.4. When the requirements are not documented, they must be confirmed before acceptance. But if product requirements are changed, the DRA must ensure relevant documents are amended and relevant personnel

NOTE: In some situations a formal review is impractical for each order. In such cases reviews

can cover relevant product information such as catalogues or adverts.

7 **7 Product realization**

98 Latest Research into Quality Control

**7.1 Planning of product**

document the plan

acceptance; and e) records

product, project, or contract.

**7.2.1 Determination of Requirements Related to the**

**7.2.2 Review of Requirements Related to the Product 7.2.3 Customer Communication**

product and the associated

stakeholders

negative

quirements of DRAs.

**Product**

realization Product realization typically implies that manufacturers 1) plan and develop the QMS processes needed for product realization; 2) keep the planning consistent with other requirements of the QMS; 3)

in a suitable form; and 4) determine through the planning, the following: a) quality objectives and product requirements; b) need for processes, documents, and resources; c) verification (establishment of truth/ confirmatory evidence), validation (formal registration/ obtainment of official sanction), monitoring, measurement, inspection, and test activities; d) criteria for product

providing evidence that the processes and resulting product meet requirements. Since DRAs regulate manufacturers they too must be acquainted with clause 7. NOTE 1: Recall that "quality plan" (2.4.8 of this article) is a document specifying the processes, and the resources to be applied to a specific

NOTE 2: An organization can apply the requirements of subclause 7.3 (vide infra) to the development of product realization processes7.2 Customer-Related Processes

The intensity and scope of communication depends on the

mandate. Thus the DRA must determine and implement the necessary arrangements for communicating with

on aspects like 1) product information; 2) inquiries and contracts; 3) customer/ stakeholder feedbacks -positive or

> \*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs.

**Table 9.** Clause 7 of ISO 9001:2008 in relation to DRAs (Design and Development).

#### *3.4.5. Some causes and signs of a malfunctioning DRA*

Once the staff recruitment system can be skewed to favour persons, a serious non-compli‐ ance exists. Once the purchase processes can be demonstrated to have vested interest, a serious flaw exists in the QMS. Once there is a convincing evidence of maladministration, arbitrary treatment of personnel or executive high handed, a serious condition against performance exists.

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

3. Adjust or re-adjust as necessary.

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐

**Table 10.** Clause 7 of ISO 9001:2008 in relation to DRAs (Purchasing/ Production/ Control of Equipment).

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

ensure that manufacturers/ distributors comply.

1. Maintain records of the audits and their results. 2. Ensure control of the audited areas.

4. Verify through follow-up actions.

To institute thoroughness and effectiveness, producing/ distributing DRAs must:

3. Take actions without undue delay to eliminate detected nonconformities and their causes.

5. Safeguard equipment from improper adjustments. 6. Protect equipment from damage and deterioration

basis).

2. Calibrate the equipment to national or international standards (or record other appropriate

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4. Identify the measuring equipment in order to determine its calibration status

Given the overwhelming importance of measurement, analysis and improvement to the Mandate of DRAs, a DRA would typically want to be sure, thorough and effective in the application of clause 8. To ensure effectiveness therefore, the DRA would routinely, or as may be necessary, determine through planning the need for, use of, and extent of use of applicable methods, including statistical techniques. DRAs should view customer/ stakeholder perception as a key performance measurement of its QMS. For producing and/ or distributing DRAs especially, monitoring customer/ stakeholder perception can be obtained from: 1) customer/ stakeholder satisfaction surveys; 2) customer data on delivered product quality; 3) user opinion surveys; 4) lost business analysis; 5) compliments; 6) warranty claims; and 7) dealer reports. For thoroughness and effectiveness a producing/ distributing DRA must: 1) plan the audit program; 2) consider the status and importance of the audited areas; 3) consider the results of prior audits; 4) define the audit criteria, scope, frequency, and methods; and 5) select and use impartial and objective auditors. Non-producing/ distributing DRAs must have the ability to

**7.5.5 Preservation of Product**

3) packaging, 4) storage, and 5) protection **7.6 Control of Measuring and Monitoring Equipment** As may be applicable, a DRA would: 1) Determine the type of monitoring and measurements to be made, and the equipment/ method to be used in providing evidence of conformity

2) handling,

requirements.

8 **8 Measurement, analysis and Improvement 8.1 General**

to planned arrangements;

distributors comply.

meeting

Non-producing/ distributing DRAs

**8.2 Monitoring and measurement 8.2.1 Customer Satisfaction**

its Mandate; and 2) define the

**8.2.2 Internal Audit**

quirements of DRAs.

Preservation of product broadly includes: 1) identification,

2) Use and control the monitoring and measuring devices in order to ensure that measurement capability is consistent with monitoring and measurement

A producing/ distributing DRA would plan and implement the monitoring, measurement, analysis, and improvement processes it needs to: 1) demonstrate conformity to product requirements; 2) ensure conformity of the QMS

and 3) continually improve the effectiveness of the QMS.

must have the ability to ensure that manufacturers/

DRAs must routinely: 1) monitor information on customer/ stakeholder perception as to whether it is

methods for obtaining and using that information.



*3.4.5. Some causes and signs of a malfunctioning DRA*

All organizations irrespective of type of business will have cause to purchase a multitude of goods for the business. If such goods are to meet their purposes criteria and processes must be developed their purchase. Thus purchasing information should contain: 1) explicit description of goods: 2) approval criteria for the goods, procedures, processes, and associated equipment or accessories; and 3) profession/ qualification of staff

performance exists.

100 Latest Research into Quality Control

7 **7.4 Purchasing**

**7.4.1 Purchasing Process 7.4.2 Purchasing Information**

associated with the goods.

**Service Provision**

and measurement

QMS requirements.

**7.5.2 Validation of Processes for Production and Service Provision 7.5.3 Identification and Traceability 7.5.4 Customer Property**

**7.4.3 Verification of Purchased Product 7.5 Production and Service Provision 7.5.1 Control of Production and**

conditions, which include: 1) availa-

A producing/ distributing DRA must plan and carry out production and service provision under controlled

bility of data on needed inputs; 2) availability of necessary work instructions; 3) availability/ usability of essential equipment; 4) availability/ usability of monitoring and measuring equipment; 4) ability to implement monitoring

activities; and 5) ability to implement product release, delivery, and post-delivery activities. DRAs that do not produce/ distribute must nevertheless have possess the ability to ensure that manufacturer/ distributors have all it takes to adequately meet

Once the staff recruitment system can be skewed to favour persons, a serious non-compli‐ ance exists. Once the purchase processes can be demonstrated to have vested interest, a serious flaw exists in the QMS. Once there is a convincing evidence of maladministration, arbitrary treatment of personnel or executive high handed, a serious condition against

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

records.

otherwise.

of results, a DRA would normally:

Whether a DRA produces/ distributes it will have cause to purchase various items of commerce hence QMS requirements for purchases are required. Like other organizations a DRA would require and ensure the adequacy of the specifications of items to be purchased before communicating the purchasing information to the supplier. Typically a DRA would establish and implement inspection or other necessary activities for ensuring that purchased goods meet the specified purchase requirements. If a DRA or its customers/ stakeholders propose to verify a good or service at the supplier's location, the intended verification arrangements/

It is typically pertinent that a DRA 1) validates any production or service provision that subsequent monitoring cannot verify. Such validations include processes where deficiencies may become apparent only after product use or service delivery; 2) demonstrates through the validation the ability of processes to achieve the planned results; and 3) establishes validation arrangements including, as applicable: a) criteria for process review and approval, b) approval of equipment, c) qualification of staff, d) use of defined methods and procedures, e) requirements for records, and f) re-validation. DRAs that produce/ distribute or have their own test facilities must 1) identify, where appropriate, the product by suitable means during product realization; and 2) identify the product status with respect to monitoring and measurement requirements throughout product realization. DRAs may require that manufacturers of herbal products have the following where necessary and feasible: a) chemically defined reference active crude extract (RACE), b) chemically defined marker substance (DMS) and TLC, HPLC or GC-MS fingerprints of RACE and DMS. Since traceability is a key requirement, DRAs need to enforce manufacturers to have the means of controlling the unique identification of the product at various stages of development, and of course maintain

Obviously, DRAs that produce/ distribute products or run test laboratories/ facilities must exercise care with any customer property under their control. They must record and promptly report any loss or damage to the customer. NOTE: Customer property may be physical or

As a standard practice, a DRA would: 1) assess and record the validity of prior results if the equipment/ method are found not to conform to requirements; 2) maintain records of the results of calibration and verification; and 3) confirm or re-confirm the ability of any software or programme used for monitoring or measurement before its initial use. To ensure the validity

1. Calibrate and/or verify the measuring equipment at specified intervals or prior to use.

method must be stated in the purchasing information.

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs.

**Table 10.** Clause 7 of ISO 9001:2008 in relation to DRAs (Purchasing/ Production/ Control of Equipment).


**8.2.2 Internal Audit**


\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs.

**Table 11.** Clause 8 of ISO 9001:2008 in relation to DRAs (Monitoring and measurement).

#### *3.4.6. Remediation of a malfunctioning DRA*

A national or regional DRA is a critical factor in socioeconomic development and wellbeing in at least two ways: i) by "guaranteeing the health of the nation" (as trumpeted in NAF‐ DAC's adverts); and by supporting the emergence of responsible manufacturers of regulat‐ ed products. It is well known that the US-FDA more than any US organization has made the US the world leader in manufacture of health products. The prominence of India and China in world drug trade owes much to the vibrancy and relative efficiency of their DRAs. There is therefore a critical need for DRAs to be vibrant and responsible. The gravity with which China views the role of her DRA can be gauged by the death sentence passed on the Direc‐ tor General in 2007 for accepting a bribe [15].


**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

following ways:

product.

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs. Although a DRA may not possess certain facilities for measurements and monitoring, it should possess the ability or the means necessary to ensure that manufacturers/ distributors possess and use them in accord‐

In Nigeria, the entire NAFDAC Management was sacked on alleged acts of corruption in 2000.

**1.** The laws creating/ amending a DRA should be well articulated as is the case with the US-

**2.** DRAs should be so well funded as not to rely on a plethora of frivolous fees as with NAF‐

**3.** Staffing of DRAs must be transparent - competence and integrity must be the decisive

**Table 12.** Clause 8 of ISO 9001 in relation to DRAs (Product characteristics/ Control of nonconformities).

It seems to us that the following are essential for a DRA to perform optimally:

**4.** DRAs should be audited frequently, at least yearly or twice yearly.

**5.** DRAs should have a Board of Governors to whom the Management reports.

manufacturers/ distributors comply.

relevant authority, and where applicable, the customer.

1. Take action to eliminate the detected nonconformity. 2. Authorize its use, release, or acceptance by concession. 3. Take action to preclude its original intended use or application.

nonconforming product is detected after delivery or use has started

It is again stressed that non-producing/ distributing DRAs must have the ability to ensure that

Application of ISO 9001 Industrial Standard to Herbal Drug Regulation

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103

DRAs must ensure that product release and service delivery cannot proceed until all planned arrangements (see 7.1) have been satisfactorily completed, unless otherwise approved by a

Where applicable, DRAs must deal with the nonconforming product by one or more of the

4. Take action appropriate to the effects, or potential effects, of the nonconformity when

To better fulfil their Mandate, DRAs must: 1) Ensure that any nonconforming product is identified and controlled to prevent its unintended use or delivery. 2) Establish a documented procedure to define the controls and related responsibilities and authorities for dealing with nonconforming

1) Monitor and measure product characteristics to verify if product requirements are being met. 2) Carry out the monitoring and measuring at the

of product realization in accordance with planned

3) Maintain evidence of conformity with the

4) Record the person responsible for authorizing release of product for delivery to the customer.

DRAs must: 1) Ensure any nonconforming product is identified and controlled to prevent its unintended use

2) Establish a documented procedure to define the controls and the related responsibilities/authorities for

dealing with nonconforming product. DRAs must maintain records of the nature of the nonconformity, and any subsequent actions, (including

any concessions). When the nonconformity is corrected, DRAs must re-verify it to prove or show evidence of

ance with approved QMS guidelines.

appropriate stages

arrangements (see 7.1).

acceptance criteria.

**product**

or delivery.

conformity.

FDA.

DAC.

criteria.

**8.3 Control of nonconforming**


**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

auditing.

distributors comply.

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐

A national or regional DRA is a critical factor in socioeconomic development and wellbeing in at least two ways: i) by "guaranteeing the health of the nation" (as trumpeted in NAF‐ DAC's adverts); and by supporting the emergence of responsible manufacturers of regulat‐ ed products. It is well known that the US-FDA more than any US organization has made the US the world leader in manufacture of health products. The prominence of India and China in world drug trade owes much to the vibrancy and relative efficiency of their DRAs. There is therefore a critical need for DRAs to be vibrant and responsible. The gravity with which China views the role of her DRA can be gauged by the death sentence passed on the Direc‐

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

satisfy its intended purpose.

on the effectiveness of the QMS.

distributors comply.

**Table 11.** Clause 8 of ISO 9001:2008 in relation to DRAs (Monitoring and measurement).

Again, non-producing/ distributing DRAs must have the ability to ensure that manufacturers/

NOTE: ISO 19011 (Audit guidance) should be consulted for further enlightenment quality

Producing/ distributing DRAs need to 1) apply suitable methods for monitoring and measuring QMS processes; and 2) confirm through these methods the continuing ability of each process to

Non-producing/ distributing DRAs must have the ability to ensure that manufacturers/

NOTE: When determining "suitable" methods, consideration is given to the type and extent of monitoring or measurement for each process in relation to its impact on product conformity and

To better fulfil their Mandate producing/ distributing DRAs must 1) monitor and measure product characteristics so as to verify if product requirements are being met; 2) carry out the monitoring and measurements at the appropriate stages of product realization in accordance with planned arrangements; and 3) maintain evidence of conformity with the acceptance criteria.

Internal audits must be conducted at planned intervals so that DRAs can determine if their QMS: 1) conforms to requirements of ISO 9001:2008; 2) conforms to planned arrangements as per sub-clause 7.1; and 3) is effectively implemented and maintained. In order to thoroughly address staff responsibilities and the requirements to be met by the audit exercise, DRAs must establish: 1) a documented procedure for planning audit; 2) a documented procedure for conducting audits; and 3) a documented procedure for recording and reporting audit

*3.4.6. Remediation of a malfunctioning DRA*

tor General in 2007 for accepting a bribe [15].

1) Apply suitable methods to monitor and, where applicable, measure the QMS processes. 2) Confirm through these methods

the continuing ability of each process to satisfy its

3) When the planned results are not achieved, take correction and corrective action, as appropriate.

results.

102 Latest Research into Quality Control

quirements of DRAs.

8 **8.2.3 Monitoring and**

intended purpose.

**8.2.4 Monitoring and Measurement of Product**

**Measurement of Processes**

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs. Although a DRA may not possess certain facilities for measurements and monitoring, it should possess the ability or the means necessary to ensure that manufacturers/ distributors possess and use them in accord‐ ance with approved QMS guidelines.

**Table 12.** Clause 8 of ISO 9001 in relation to DRAs (Product characteristics/ Control of nonconformities).

In Nigeria, the entire NAFDAC Management was sacked on alleged acts of corruption in 2000. It seems to us that the following are essential for a DRA to perform optimally:


**6.** Parliamentary health committees should view DRAs as critical to socioeconomic well‐ being of the nation.

*3.4.7. Further remarks on clauses 7 and 8 of ISO 9001:2008*

of ISO 9001or some other ISO standard provide an answer?

**4. Conclusions**

Although the principles of clauses 7 and 8 apply to all organizations, they are strictly speak‐ ing, the deeds and stuff intended for high profile institutions with elaborate concern and fa‐ cilities for design and R&D, and with tall entrepreneurial ambition. Such organizations include the most successful pharmaceutical and biotechnology companies of the US, Eu‐ rope, Japan and India; NASA, aircraft manufacturers, international airlines and 5-star hospi‐ tality concerns. However, in as much as DRAs must regulate the work and product of advanced pharmaceutical manufacturers, the onus is upon the DRAs themselves to be con‐ versant with the entire provisions of these clauses and be as intellectually equipped as the manufacturer. This explains why it is often desirable that regulators have a stint in both aca‐ demia and industry. In many countries, especially the US and India, top rate biomedical fa‐ cilities/ institutions and personnel are to be found in the following four circles: i) the DRAs (eg: US-FDA); ii) health research institutions (eg: NIH); iii) the universities/ R&D institutions patronized by the DRAs; and iv) big transnational drug manufacturers (eg: Pfizer). We once again refer to the Nigeria polio vaccine controversy of the late 1990s/ early 2000s mentioned earlier, and ask the following question: When a DRA produces or distributes product as is the case in many developing economies, who regulates the DRA? Can subsequent revisions

Application of ISO 9001 Industrial Standard to Herbal Drug Regulation

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105

It is evident from the foregoing that all the eight clauses of ISO 9001:2008 apply to the Man‐ date of DRAs. However, most of what appears in clauses 7 and 8, the lengthiest of the claus‐ es, relates more pertinently to high stake pharmaceutical manufacturers that have elaborate R&D than they do to the average DRA, which nevertheless should be thoroughly acquaint‐ ed with the clauses. Some DRAs like the US-FDA and EMEA that have advance laboratories or access to such or that heavily fund R&D must be guided by the rigorous provisions of clauses 7 and 8. Needless to say, those DRAs that produce/ distribute products must be simi‐ larly guided to the extent of their relevance to the scope and size of their operations. The US-FDA, Japan's Ministry of Health and Social Services and EMEA are certified to appropriate performance standards and are known for their efficiency. By contrast NAFDAC and other developing national DRAs are not similarly certified and are less well known for efficiency, considering the rampancy of counterfeit drugs and other ills in their drug delivery systems. The DRAs of China, India and Southeast Asian countries compare quite well in many as‐ pects with those of Europe, Canada and the US, and are by far more efficient than those of many African and South American countries. From the foregoing, and in view of the histori‐ cal and international dimensions of phytotherapy, especially its galloping global patronage in recent times [1,2,11,12], it is necessary that there to be a minimum global standard to which DRAs should be certified. We propose ISO 9001 because of its global popularity, ap‐ plicability and suitability. The standard provides the general climate for DRAs to efficiently discharge their Mandate. We project that a carefully planned application of ISO 9001 to herbal drug regulation will improve the production, distribution and usage of herbal drugs.


\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs. It must be stressed once again that if DRAs are to persuade manufacturers/ distributors to com‐ ply with the provisions of this and other clauses of ISO 9001:2008 industrial standard, they too must be conversant with and adept in them.

**Table 13.** Clause 8 of ISO 9001:2008 in relation to DRAs (Analysis of data/ Improvement).

#### *3.4.7. Further remarks on clauses 7 and 8 of ISO 9001:2008*

Although the principles of clauses 7 and 8 apply to all organizations, they are strictly speak‐ ing, the deeds and stuff intended for high profile institutions with elaborate concern and fa‐ cilities for design and R&D, and with tall entrepreneurial ambition. Such organizations include the most successful pharmaceutical and biotechnology companies of the US, Eu‐ rope, Japan and India; NASA, aircraft manufacturers, international airlines and 5-star hospi‐ tality concerns. However, in as much as DRAs must regulate the work and product of advanced pharmaceutical manufacturers, the onus is upon the DRAs themselves to be con‐ versant with the entire provisions of these clauses and be as intellectually equipped as the manufacturer. This explains why it is often desirable that regulators have a stint in both aca‐ demia and industry. In many countries, especially the US and India, top rate biomedical fa‐ cilities/ institutions and personnel are to be found in the following four circles: i) the DRAs (eg: US-FDA); ii) health research institutions (eg: NIH); iii) the universities/ R&D institutions patronized by the DRAs; and iv) big transnational drug manufacturers (eg: Pfizer). We once again refer to the Nigeria polio vaccine controversy of the late 1990s/ early 2000s mentioned earlier, and ask the following question: When a DRA produces or distributes product as is the case in many developing economies, who regulates the DRA? Can subsequent revisions of ISO 9001or some other ISO standard provide an answer?

#### **4. Conclusions**

**6.** Parliamentary health committees should view DRAs as critical to socioeconomic well‐

**Clause Title and subtitles, with remarks Salient points/ directing principles/ application to DRAs**

DRAs should as a matter of practice:

relevant sources in their analyses.

a. Customer satisfaction as per 8.2.1.

action as per 8.2.3, 8.2.4, and 8.5.3.

5. Corrective and preventive action 6. Management review

2. Determine the causes of nonconformities. 3. Evaluate the need for actions to prevent recurrence. 4. Determine and implementing the needed action. 5. Maintain records of the results of the action taken. 6. Review the effectiveness of corrective action taken

\*The Table is to be studied side by side with contents of ISO 9001:2008 and Table 2, which is on the regulatory re‐ quirements of DRAs. It must be stressed once again that if DRAs are to persuade manufacturers/ distributors to com‐ ply with the provisions of this and other clauses of ISO 9001:2008 industrial standard, they too must be conversant

**Table 13.** Clause 8 of ISO 9001:2008 in relation to DRAs (Analysis of data/ Improvement).

d. Suppliers as per 7.4

1. Quality policy 2. Quality objectives 3. Audit results 4. Analysis of data

2. Analyze such primary data to provide secondary data on:

DRAs are to continually improve the effectiveness of their QMS through:

corrective action. Such a procedure must define requirements to: 1. Review nonconformities (including customer complaints).

preventive action. Such a procedure must define requirements to: 1. Determine potential nonconformities and their causes. 2. Evaluate the need for actions to prevent occurrence. 3. Determine and implementing the needed action. 4. Maintain records of the results of the action taken. Review the effectiveness of preventive action taken

b. Conformity to product requirements as per 8.2.4.

1. Include the primary data generated by monitoring and measuring activities, and from other

c. Characteristics and trends of processes and products, including opportunities for preventive

For thoroughness and effectiveness, DRAs must establish a documented procedure (SOP) for

For thoroughness and effectiveness, DRAs must establish a documented procedure for

being of the nation.

104 Latest Research into Quality Control

Whether producing/ distributing or not, as a rule, DRAs must determine, collect, and analyze appropriate data to demonstrate the suitability and effectiveness of their QMS, as well as, evaluate where continual improvement of the QMS can be made. DRAs cannot enforce compliance among manufacturers/ distributors in an aspect of quality management in which they are themselves deficient.

8 **8.4 Analysis of data**

**8.5 Improvement**

appropriate to the

**8.5.3 Preventive Action** Organizations must:

1. Determine in advance the action that needs to be taken to eliminate the causes of potential non-

conformity, in order to prevent its occurrence. 2. Ensure that preventive actions are appropriate to the anticipated effects of the potential problem.

breach.

with and adept in them.

**8.5.1 Continual Improvement 8.5.2 Corrective Action**

The standard practice for organizations is that: 1. Wherever a nonconformity or breach is detected, corrective action must be taken to eliminate the cause of the nonconformity and to prevent its recurrence. 2. Wherever corrective action is taken by an organization, such action must be such as is

effects of the problem caused by the nonconformity or

It is evident from the foregoing that all the eight clauses of ISO 9001:2008 apply to the Man‐ date of DRAs. However, most of what appears in clauses 7 and 8, the lengthiest of the claus‐ es, relates more pertinently to high stake pharmaceutical manufacturers that have elaborate R&D than they do to the average DRA, which nevertheless should be thoroughly acquaint‐ ed with the clauses. Some DRAs like the US-FDA and EMEA that have advance laboratories or access to such or that heavily fund R&D must be guided by the rigorous provisions of clauses 7 and 8. Needless to say, those DRAs that produce/ distribute products must be simi‐ larly guided to the extent of their relevance to the scope and size of their operations. The US-FDA, Japan's Ministry of Health and Social Services and EMEA are certified to appropriate performance standards and are known for their efficiency. By contrast NAFDAC and other developing national DRAs are not similarly certified and are less well known for efficiency, considering the rampancy of counterfeit drugs and other ills in their drug delivery systems. The DRAs of China, India and Southeast Asian countries compare quite well in many as‐ pects with those of Europe, Canada and the US, and are by far more efficient than those of many African and South American countries. From the foregoing, and in view of the histori‐ cal and international dimensions of phytotherapy, especially its galloping global patronage in recent times [1,2,11,12], it is necessary that there to be a minimum global standard to which DRAs should be certified. We propose ISO 9001 because of its global popularity, ap‐ plicability and suitability. The standard provides the general climate for DRAs to efficiently discharge their Mandate. We project that a carefully planned application of ISO 9001 to herbal drug regulation will improve the production, distribution and usage of herbal drugs. It will also boost the economy of developing economies that rely to a large extent on herbal drugs. But since the DRAs of many developing economies produce/ distribute certain prod‐ ucts, there is a need for subsequent revisions of ISO 9001 to take cognisance of the question of who regulates the regulator that produces/ distributes? In the meantime we recommend that the Minister/ Secretary of Health and/ or the Parliamentary Committees of Health take note of this significant lacuna.

*In: Eldin AB, editor. Modern Approaches to Quality Control*, Rijeka, InTech,

Application of ISO 9001 Industrial Standard to Herbal Drug Regulation

http://dx.doi.org/10.5772/50814

107

[4] DSHEA (1994). Dietary Supplements Health Education Act of 1994. Available: http://fda/Food/DietarySupplements/ucm109764.htm Accessed 2012 April 25. [5] Goldman, P. (2001). Herbal medicines today and the roots of modern

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367-382.

#### **Acknowledgements**

We gratefully acknowledge a copy of ISO 9001:2008 kindly furnished by the SON and the enlightenment offered by Engineer Timothy N. Abner, Dr. Justin B. Nickaf and Engineer. Shehu I. Maik during the NIPRD-SON workshop on ISO 9001:2008 held at Bolton White Apartments, Abuja, in November-December 2011.

#### **Author details**

Sunday Ameh1\*, Florence Tarfa1 , Magaji Garba2 and Karniyus Gamaniel3

\*Address all correspondence to: sjitodo@yahoo.com

1 Department of Medicinal Chemistry & Quality Control, National Institute for Pharmaceut‐ ical Research and Development (NIPRD), Idu Indusrial Area, Nigeria

2 Department of Pharmaceutical & Medicinal Chemistry, Ahmadu Bello University, Nigeria

3 Office of the Director General, NIPRD, Idu Indusrial Area, Nigeria

#### **References**


*In: Eldin AB, editor. Modern Approaches to Quality Control*, Rijeka, InTech, 367-382.

[4] DSHEA (1994). Dietary Supplements Health Education Act of 1994. Available: http://fda/Food/DietarySupplements/ucm109764.htm Accessed 2012 April 25.

It will also boost the economy of developing economies that rely to a large extent on herbal drugs. But since the DRAs of many developing economies produce/ distribute certain prod‐ ucts, there is a need for subsequent revisions of ISO 9001 to take cognisance of the question of who regulates the regulator that produces/ distributes? In the meantime we recommend that the Minister/ Secretary of Health and/ or the Parliamentary Committees of Health take

We gratefully acknowledge a copy of ISO 9001:2008 kindly furnished by the SON and the enlightenment offered by Engineer Timothy N. Abner, Dr. Justin B. Nickaf and Engineer. Shehu I. Maik during the NIPRD-SON workshop on ISO 9001:2008 held at Bolton White

1 Department of Medicinal Chemistry & Quality Control, National Institute for Pharmaceut‐

2 Department of Pharmaceutical & Medicinal Chemistry, Ahmadu Bello University, Nigeria

[1] Ameh, S. J., Obodozie, O., Inyang, U., Abubakar, M., & Garba, M. (2010). Current phytotherapy- a perspective on the science and regulation of herbal

[2] Ameh, S. J., Obodozie, O., Inyang, U., Abubakar, M., & Garba, M. (2010). Current phytotherapy- an inter-regional perspective on policy, research and development of herbal medicine. *Journal of Medicinal Plants Research*, 4(15),

[3] Ameh, S. J., Obodozie, O., Gamaniel, K., Abubakar, M., & Garba, M. (2011). Herbal Drug Regulation Illustrated with Niprifan® Antifungal Phytomedicine.

and Karniyus Gamaniel3

, Magaji Garba2

ical Research and Development (NIPRD), Idu Indusrial Area, Nigeria

3 Office of the Director General, NIPRD, Idu Indusrial Area, Nigeria

medicine. *Journal of Medicinal Plants Research*, 4(2), 072-081.

note of this significant lacuna.

Apartments, Abuja, in November-December 2011.

\*Address all correspondence to: sjitodo@yahoo.com

**Acknowledgements**

106 Latest Research into Quality Control

**Author details**

**References**

1508-1516.

Sunday Ameh1\*, Florence Tarfa1


[17] Akunyili, D. (2002). Herbal Preparations. *NAFDAC Consumer Safety Bulletin*, 1(2), 5-6.

**Section 2**

**Quality Control in Food Science**


**Quality Control in Food Science**

[17] Akunyili, D. (2002). Herbal Preparations. *NAFDAC Consumer Safety Bulletin*,

[18] Gross, A., & Minot, J. (2007). Chinese Manufacturing: Scandals and Opportu‐ nities. Published in MX, November/ December, Pacific Bridge Medicals. Available: http://www.pacificbridgemedicals.com/ Accessed 2012 April 25. [19] Corbett, C. J., Montes-sancho, M. J., & Kirsch, D. A. (2005). The financial impact of ISO 9000 certification in the United States: An empirical analysis. Management Science Available: http://personal.anderson.ucla.edu/charles.corbett/

[20] Heras, I., Dick, G. P., & Casadesus, M. (2002). ISO 9000 registration's impact on sales and profitability - A longitudinal analysis of performance before and after accreditation. International Journal of Quality and Reliability Management Available: http://eps.udg.es/oe/webmarti/p774.pdf Accessed 2012

[21] Naveh, E., & Marcus, A. (2007). Financial performance, ISO 9000 standard and safe driving practices effects on accident rate in the U.S. motor carrier industry. Accident Analysis & Prevention PMID 17166474Accessed 2012 April

[22] Sharma, D. S. (2005). The association between ISO 9000 certification and financial performance. The international Journal of Accounting Available: http://masp.bus.ku.ac.th/files/ISO%209000%20and%20performamce.pdf Accessed

[23] Chow-chua, C., Goh, M., & Wan, T. B. (2002). Does ISO 9000 certification improve business performance? The International Journal of Quality & Reliability Management Available: http://www.emeraldinsight.com/journals.htm?

[24] Rajan, M., & Tamimi, N. (2003). Payoff to ISO 9000 registration. Available: http://www.iijournals.com/doi/abs/10.3905/joi.2003.319536Accessed 2012 April 25.

articleid=840633&show=abstract Accessed 2012 April 25., 20(8), 936-953.

paper/does\_iso\_pay.pdf Accessed 2012 April 25., 51(7), 1046-1059.

1(2), 5-6.

108 Latest Research into Quality Control

April 25., 19(6), 774-791.

2012 April 25., 40, 151-172.

25., 39(4), 731-742.

**Chapter 6**

**QA: Fraud Control for Foods and Other Biomaterials by**

Fraud can be generally defined as "the intentional deception made for personal gain or to damage another individual". In particular, *food fraud consists in the deliberate misdescription in order to deceive the consumers about the real nature of the product or of any of its ingredients*. It results in the mismatch between what a food product is and what it is claimed to be. Food fraud is a broad term that also involve criminal acts such as tax-avoidance and smuggling. In the following we will mainly discuss about economically motivated adulteration and mis‐ labelling i.e. food fraud issues falling in one of the following categories: 1) the substitution of an ingredient with a cheaper alternative (e.g., substitution of ethanol with methanol in wine or proteins with melamine in milk powders), 2) misdescription of the real nature of the product or one of its ingredients (e.g. counterfeiting, conventional products that are sold as added value products such as organic, fair trade, biodynamic), 3) incorrect quantitative in‐ gredient declaration and 4) implementation of non-acceptable process practices such as irra‐

Food fraud can be implemented in any step of the food chain but it is mainly a food indus‐ try issue. In criminology, there are 3 elements of fraud opportunity (the crime triangle): vic‐ tim, fraudster and guardian [1]. The typical set-up is that where final consumers play the role of victims, food industry (but in general food producers, processors, traders or retailers) plays the role of the fraudsters and governmental control authorities, non-governmental and certification organizations play the role of the guardian. However, food producers can also be victim or guardian. They are victims when, for example, their products are counterfeited or simulated and guardian when they implements QA systems for the assessment of the au‐ thenticity of the raw materials. Nowadays food fraud represents a major problem that costs the EU food industry and governments hundreds of millions of euros every year. But the

> © 2012 Capuano and van Ruth; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Capuano and van Ruth; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Product Fingerprinting**

http://dx.doi.org/10.5772/51109

**1. Introduction**

Edoardo Capuano and Saskia M. van Ruth

Additional information is available at the end of the chapter

diation, heating or freezing (e.g. thawed fish sold as fresh).

## **QA: Fraud Control for Foods and Other Biomaterials by Product Fingerprinting**

Edoardo Capuano and Saskia M. van Ruth

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51109

#### **1. Introduction**

Fraud can be generally defined as "the intentional deception made for personal gain or to damage another individual". In particular, *food fraud consists in the deliberate misdescription in order to deceive the consumers about the real nature of the product or of any of its ingredients*. It results in the mismatch between what a food product is and what it is claimed to be. Food fraud is a broad term that also involve criminal acts such as tax-avoidance and smuggling. In the following we will mainly discuss about economically motivated adulteration and mis‐ labelling i.e. food fraud issues falling in one of the following categories: 1) the substitution of an ingredient with a cheaper alternative (e.g., substitution of ethanol with methanol in wine or proteins with melamine in milk powders), 2) misdescription of the real nature of the product or one of its ingredients (e.g. counterfeiting, conventional products that are sold as added value products such as organic, fair trade, biodynamic), 3) incorrect quantitative in‐ gredient declaration and 4) implementation of non-acceptable process practices such as irra‐ diation, heating or freezing (e.g. thawed fish sold as fresh).

Food fraud can be implemented in any step of the food chain but it is mainly a food indus‐ try issue. In criminology, there are 3 elements of fraud opportunity (the crime triangle): vic‐ tim, fraudster and guardian [1]. The typical set-up is that where final consumers play the role of victims, food industry (but in general food producers, processors, traders or retailers) plays the role of the fraudsters and governmental control authorities, non-governmental and certification organizations play the role of the guardian. However, food producers can also be victim or guardian. They are victims when, for example, their products are counterfeited or simulated and guardian when they implements QA systems for the assessment of the au‐ thenticity of the raw materials. Nowadays food fraud represents a major problem that costs the EU food industry and governments hundreds of millions of euros every year. But the

© 2012 Capuano and van Ruth; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Capuano and van Ruth; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

problem that food fraud poses is not merely an economic one: It is also a problem of public health because the adulteration can pose toxicological and hygienic risks to purchasers and consumers. In 2008, for example, Chinese milk was adulterated with melamine, an hazard‐ ous chemical, to increase milk nitrogen content causing 900 infants to be hospitalized with six deaths. Several other such examples can be given.

Since no one likes to be swindled, neither producers, traders, importers, retailers, and con‐ sumers, fraud prevention and detection is an important issue. Nowadays, authenticity of in‐ gredients or products is mainly warranted by paper trailing. Analytical tests which can help to confirm the authenticity of ingredients/products compose a very useful complementary approach to paper trailing.

#### **2. Fingerprinting approach: generalities and tools**

Traditional strategies for the food fraud control have relied on the determination of the amount of a marker compound or compounds and the comparison of the obtained values for the test material with those established for the genuine material. The presence of an un‐ desired adulterant can be uncovered by checking for its presence in the food material whereas the compliance of the food composition with the established legislative standards or with the amount of an ingredient as declared on the label can be simply proved by meas‐ uring the target compound or compounds. However, some aspects of food authenticity such as the geographical origin, the farming management systems (organic, free range..), or the application of some specific processes cannot be dealt with those traditional approaches. No single marker exists for the unequivocal authentication of an organic egg or a Dutch special‐ ty cheese. Furthermore, based on conventional target analyses, an adulteration can be de‐ tected only if the adulterant is known beforehand and explicitly searched for by the analyst. Traditional quality control strategies are not designed to look for a near infinite number of potential contaminants so that new adulterants will not be unveiled until their presence in food is first acknowledged. For those reasons a more holistic approach is needed that is based on the measurement and the evaluation of several compounds at once, i.e. a finger‐ printing approach. Moreover, in industrial and laboratory settings, there is always the need of implement screening methods that are able to reliably identify, in large numbers of sam‐ ples, those that are potentially non-compliant before more detailed and accurate analysis with confirmatory methods are performed. A fingerprinting approach may, in many cases, provide rapid and high-throughput analyses well suited for screening purposes.

**Figure 1.** Analytical fingerprints: A mass spectrum (a), a chromatogram (b), a schematic representation of a DNA fin‐

QA: Fraud Control for Foods and Other Biomaterials by Product Fingerprinting

http://dx.doi.org/10.5772/51109

113

In a mass spectrum (Figure 1a), a collection of m/z and relative signal intensities is a chemi‐ cal fingerprint of the material. Similarly, a chromatographic profile (Figure 1b) is a finger‐ print of a more or less broad class of constituents of the material. The specific distribution of the restriction fragments of a selected DNA sequence on the electrophoretic gel (Figure 1c) is a genetic fingerprint of the test material. A NIR spectrum (Figure 1d) is a representation of the interaction of a test material with the infrared radiation whereas a thermogram (Figure 1e) is a representation of its interaction with thermal energy. (a) and (b) can be referred to as *chemical fingerprints*. They may be composed of as many groups of compounds as possible or alternatively of a specific group of compounds which requires higher level of purification and a selective extraction from the sample. (d) and (e) can be referred to as *physical finger‐*

A fingerprinting approach implies that the whole information contained in the fingerprint (or a selected part of it) is used to infer about the properties of the system under study. To do that, a special statistical tool is necessary, i.e. chemometrics. Chemometrics can be defined as the science of extracting chemically relevant information from multivariate data by using statistical techniques to reduce the dimensionality of the dataset. It offers a tool to graphical‐ ly summarise the analytical data to reveal relationships between samples and to detect char‐ acteristic patterns that can be used to identify a certain material. As a first step, an exploratory analysis is carried out in order to investigate the natural relations between the samples. This is carried out by so called unsupervised pattern recognition techniques because they do not require any prior knowledge of the properties of the samples. Examples of such techniques

gerprint on gel electrophoresis (c), an infrared spectrum (d) and a thermogram (e).

*prints* even though chemical information can be obtained as well.

*Fingerprint refers to the characteristic spectrum or image of a test material which can be related to its properties and thus to its authenticity in the same way as a human fingerprint is specific of a certain person and unequivocally identify him/her.* The term thus recalls a comprehensive description of a test material that is carried out in a non-selective (or untargeted) way. Fingerprints can be generated through many analytical techniques. They can be obtained from chromatograms, spectroscopic measurements, spectral measurements or any other specific signal of complete spectra (Figure 1).

problem that food fraud poses is not merely an economic one: It is also a problem of public health because the adulteration can pose toxicological and hygienic risks to purchasers and consumers. In 2008, for example, Chinese milk was adulterated with melamine, an hazard‐ ous chemical, to increase milk nitrogen content causing 900 infants to be hospitalized with

Since no one likes to be swindled, neither producers, traders, importers, retailers, and con‐ sumers, fraud prevention and detection is an important issue. Nowadays, authenticity of in‐ gredients or products is mainly warranted by paper trailing. Analytical tests which can help to confirm the authenticity of ingredients/products compose a very useful complementary

Traditional strategies for the food fraud control have relied on the determination of the amount of a marker compound or compounds and the comparison of the obtained values for the test material with those established for the genuine material. The presence of an un‐ desired adulterant can be uncovered by checking for its presence in the food material whereas the compliance of the food composition with the established legislative standards or with the amount of an ingredient as declared on the label can be simply proved by meas‐ uring the target compound or compounds. However, some aspects of food authenticity such as the geographical origin, the farming management systems (organic, free range..), or the application of some specific processes cannot be dealt with those traditional approaches. No single marker exists for the unequivocal authentication of an organic egg or a Dutch special‐ ty cheese. Furthermore, based on conventional target analyses, an adulteration can be de‐ tected only if the adulterant is known beforehand and explicitly searched for by the analyst. Traditional quality control strategies are not designed to look for a near infinite number of potential contaminants so that new adulterants will not be unveiled until their presence in food is first acknowledged. For those reasons a more holistic approach is needed that is based on the measurement and the evaluation of several compounds at once, i.e. a finger‐ printing approach. Moreover, in industrial and laboratory settings, there is always the need of implement screening methods that are able to reliably identify, in large numbers of sam‐ ples, those that are potentially non-compliant before more detailed and accurate analysis with confirmatory methods are performed. A fingerprinting approach may, in many cases,

provide rapid and high-throughput analyses well suited for screening purposes.

*Fingerprint refers to the characteristic spectrum or image of a test material which can be related to its properties and thus to its authenticity in the same way as a human fingerprint is specific of a certain person and unequivocally identify him/her.* The term thus recalls a comprehensive description of a test material that is carried out in a non-selective (or untargeted) way. Fingerprints can be generated through many analytical techniques. They can be obtained from chromatograms, spectroscopic measurements, spectral measurements or any other specific signal of complete

six deaths. Several other such examples can be given.

**2. Fingerprinting approach: generalities and tools**

approach to paper trailing.

112 Latest Research into Quality Control

spectra (Figure 1).

**Figure 1.** Analytical fingerprints: A mass spectrum (a), a chromatogram (b), a schematic representation of a DNA fin‐ gerprint on gel electrophoresis (c), an infrared spectrum (d) and a thermogram (e).

In a mass spectrum (Figure 1a), a collection of m/z and relative signal intensities is a chemi‐ cal fingerprint of the material. Similarly, a chromatographic profile (Figure 1b) is a finger‐ print of a more or less broad class of constituents of the material. The specific distribution of the restriction fragments of a selected DNA sequence on the electrophoretic gel (Figure 1c) is a genetic fingerprint of the test material. A NIR spectrum (Figure 1d) is a representation of the interaction of a test material with the infrared radiation whereas a thermogram (Figure 1e) is a representation of its interaction with thermal energy. (a) and (b) can be referred to as *chemical fingerprints*. They may be composed of as many groups of compounds as possible or alternatively of a specific group of compounds which requires higher level of purification and a selective extraction from the sample. (d) and (e) can be referred to as *physical finger‐ prints* even though chemical information can be obtained as well.

A fingerprinting approach implies that the whole information contained in the fingerprint (or a selected part of it) is used to infer about the properties of the system under study. To do that, a special statistical tool is necessary, i.e. chemometrics. Chemometrics can be defined as the science of extracting chemically relevant information from multivariate data by using statistical techniques to reduce the dimensionality of the dataset. It offers a tool to graphical‐ ly summarise the analytical data to reveal relationships between samples and to detect char‐ acteristic patterns that can be used to identify a certain material. As a first step, an exploratory analysis is carried out in order to investigate the natural relations between the samples. This is carried out by so called unsupervised pattern recognition techniques because they do not require any prior knowledge of the properties of the samples. Examples of such techniques are: Hierarchical cluster analysis (HCA), cluster analysis (CA) and principal component anal‐ ysis (PCA). PCA is the most widespread of those explorative tools. In a PCA model the original variables are transformed in new uncorrelated variables that arise from the linear combina‐ tion of the original variables: the principal components (PCs). A number of PCs are extract‐ ed in sequence with each principal component accounting for the maximum of the residual variance in the data. The PCs extraction stops when most of the variance in the original data (typically around 90%) is explained. The new set of PCs define therefore a new space where the contribution of each original variables to each PC can be easily represented and the relationships between the original samples highlighted (Figure 2).

ing of regression models, multivariate regression methods such as principal component re‐

The building of a classification (or regression) model comprises four essential steps: 1) Selec‐ tion of a training set, which consist of objects of known class membership (or known quan‐ titative values of a sample property) for which variables are measured. (2) Variable selection. Variables that contain information for the aimed classification are retained, whereas those encoding noise and/or with no discriminating power are eliminated. (3) Building of a mod‐ el using the training set. A mathematical model is derived between the selected variables measured on the training set and their known categories (or quantitative values of the sam‐ ple property). (4) Validation of the model. The model is validated in order to evaluate the reliability of the classification achieved either using an independent test set of samples (ex‐

The fingerprinting techniques are gaining more and more popularity over the past years thanks to advancements in the analytical instruments that are able to generate enormous amount of data at once and the application of chemometrics techniques. Herewith, finger‐ printing techniques are classified in five broad categories according to the kind of finger‐ print that can be obtained: *Mass spectrometry* (*MS) fingerprinting*, *chromatographic fingerprinting, electrophoretic fingerprinting, spectroscopic fingerprinting, and other fingerprinting*. This classifica‐

> **Electrophoretic fingerprinting**

Gel electrophoresis (isoelectric focusing,

electrophoresis…)

MS is a powerful analytical technique that measures the mass-to-charge ratio of ions. The samples are first ionised, the ions are separated and their relative abundance assessed based

PTR-MS LC (HPLC, LC-MS..) CE (CZE, CIF…) NMR DSC

DNA

IR-MS IR (NIR, MIR,

**Spectroscopic fingerprinting**

QA: Fraud Control for Foods and Other Biomaterials by Product Fingerprinting

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115

Fluorescence spectroscopy

FTIR)

**Other fingerprinting**

Electronic nose

Microarray technologies, reverse PCR

gression (PCR) and partial-least square regression (PLSR) are used.

ternal validation) or the training set (cross validation).

**Chromatographic fingerprinting**

**Table 1.** Classification of fingerprinting techniques. For abbreviations, see text.

tion is shown in Table 1.

**MS**

**fingerprinting**

Direct infusion and ambient MS (ESI, MALDI-TOF, DART…)

**3. MS fingerprinting**

ICP-MS GC (GC-FID, GC-MS..)

**Figure 2.** Plot of the first two dimensions in a typical PCA model. In a scores plot (panel a), samples are plotted in the space defined by the PCs. Similarities and differences between samples can be highlighted (in this case, two well separated groups of samples, red and green are apparent). In a loading plot (panel b) each variable (analytical response) is plot‐ ted on the new space defined by PCs. When score plot and loading plots are superimposed, information on the effect of the variable on samples properties can be obtained. When a variable is plotted close to a sample, this indicates that this variable shows relatively high concentration in this particular sample compared to the other samples. In the figure, variables 1,4,12 are higher in the samples of the red group and variables 6,7 and 11 are higher in the green group.

As a further step, multivariate methods are applied to either classify a certain product or quantify a certain property of the product. In the *classification models*, information about the class membership of the samples to a certain group (class or category) is used to classify new unknown samples in one of the known classes on the basis of its pattern of measurements. Classification models are useful, for instance, for the authentication of organic products or of geographical origin. Supervised pattern recognition techniques are used such: k nearest neigh‐ bours (kNN), soft independent modelling of class analogy (SIMCA), partial least square regression discriminant analysis (PLS-DA), linear discriminant analysis (LDA), support vec‐ tor machine (SVM) and artificial neural network (ANN). Classification models may either build a delimiter between the classes so that they always assign a new object to the class to which it most probably belongs (suitable for limited and defined number of possible classes) or build a model for each class studied and then evaluate the fitting of new objects to each model (suitable for unlimited number of classes). In the *regression models*, a functional relation‐ ship is established between some quantitative sample property, the dependent variable, and a multivariate independent variables such as a raw chromatogram or a IR spectrum and the model is used to predict the property of interest in the unknown sample. Regression model are used, for instance, to quantify the level of adulteration in a food sample. For the build‐ ing of regression models, multivariate regression methods such as principal component re‐ gression (PCR) and partial-least square regression (PLSR) are used.

The building of a classification (or regression) model comprises four essential steps: 1) Selec‐ tion of a training set, which consist of objects of known class membership (or known quan‐ titative values of a sample property) for which variables are measured. (2) Variable selection. Variables that contain information for the aimed classification are retained, whereas those encoding noise and/or with no discriminating power are eliminated. (3) Building of a mod‐ el using the training set. A mathematical model is derived between the selected variables measured on the training set and their known categories (or quantitative values of the sam‐ ple property). (4) Validation of the model. The model is validated in order to evaluate the reliability of the classification achieved either using an independent test set of samples (ex‐ ternal validation) or the training set (cross validation).

The fingerprinting techniques are gaining more and more popularity over the past years thanks to advancements in the analytical instruments that are able to generate enormous amount of data at once and the application of chemometrics techniques. Herewith, finger‐ printing techniques are classified in five broad categories according to the kind of finger‐ print that can be obtained: *Mass spectrometry* (*MS) fingerprinting*, *chromatographic fingerprinting, electrophoretic fingerprinting, spectroscopic fingerprinting, and other fingerprinting*. This classifica‐ tion is shown in Table 1.


**Table 1.** Classification of fingerprinting techniques. For abbreviations, see text.

#### **3. MS fingerprinting**

are: Hierarchical cluster analysis (HCA), cluster analysis (CA) and principal component anal‐ ysis (PCA). PCA is the most widespread of those explorative tools. In a PCA model the original variables are transformed in new uncorrelated variables that arise from the linear combina‐ tion of the original variables: the principal components (PCs). A number of PCs are extract‐ ed in sequence with each principal component accounting for the maximum of the residual variance in the data. The PCs extraction stops when most of the variance in the original data (typically around 90%) is explained. The new set of PCs define therefore a new space where the contribution of each original variables to each PC can be easily represented and the

**Figure 2.** Plot of the first two dimensions in a typical PCA model. In a scores plot (panel a), samples are plotted in the space defined by the PCs. Similarities and differences between samples can be highlighted (in this case, two well separated groups of samples, red and green are apparent). In a loading plot (panel b) each variable (analytical response) is plot‐ ted on the new space defined by PCs. When score plot and loading plots are superimposed, information on the effect of the variable on samples properties can be obtained. When a variable is plotted close to a sample, this indicates that this variable shows relatively high concentration in this particular sample compared to the other samples. In the figure, variables 1,4,12 are higher in the samples of the red group and variables 6,7 and 11 are higher in the green group.

As a further step, multivariate methods are applied to either classify a certain product or quantify a certain property of the product. In the *classification models*, information about the class membership of the samples to a certain group (class or category) is used to classify new unknown samples in one of the known classes on the basis of its pattern of measurements. Classification models are useful, for instance, for the authentication of organic products or of geographical origin. Supervised pattern recognition techniques are used such: k nearest neigh‐ bours (kNN), soft independent modelling of class analogy (SIMCA), partial least square regression discriminant analysis (PLS-DA), linear discriminant analysis (LDA), support vec‐ tor machine (SVM) and artificial neural network (ANN). Classification models may either build a delimiter between the classes so that they always assign a new object to the class to which it most probably belongs (suitable for limited and defined number of possible classes) or build a model for each class studied and then evaluate the fitting of new objects to each model (suitable for unlimited number of classes). In the *regression models*, a functional relation‐ ship is established between some quantitative sample property, the dependent variable, and a multivariate independent variables such as a raw chromatogram or a IR spectrum and the model is used to predict the property of interest in the unknown sample. Regression model are used, for instance, to quantify the level of adulteration in a food sample. For the build‐

relationships between the original samples highlighted (Figure 2).

114 Latest Research into Quality Control

MS is a powerful analytical technique that measures the mass-to-charge ratio of ions. The samples are first ionised, the ions are separated and their relative abundance assessed based on the intensity of the ions flux. MS produces therefore a mass spectrum representing the fingerprint of the sample components (Figure 1a). A number of different MS set-ups are pos‐ sible based on the ionisation technique and the mass analyser used. MS can be used alone or they can be coupled with separation techniques. In this section, the stand-alone MS techni‐ ques will be dealt with in details whereas the application of MS as coupled with separation techniques will be dealt with in the next sections.

correctly accepted by the model) and specificity (% of object, extraneous from the modelled

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117

In conclusion, PTR-MS is a rapid and low cost analytical technique that can be also fully au‐ tomated and implemented on-line. Recently, the coupling of the time of flight (TOF, see be‐ low in this section) mass analyser to PTR-MS instruments has generated PTR-TOF-MS which is characterized by a high sensitivity with limits of detection down to few pptv and a high time resolution. The technique has been recently applied to discriminate among PDO

ICP-MS provides quantitative measurements of a wide range of metals and non-metals (inorganic elements) at trace and ultratrace concentration level (ppt). In this technique, the sample (even solid or liquid) is decomposed to neutral elements in a high-temperature argon plasma and the single elements are separated based on their mass/charge ratio and ana‐ lysed. The great advantage of this technique compared to others (e.g. atomic spectroscopy) is that more than one element can be analysed at once so that a multi-elemental fingerprint is obtained in a very fast and sensitive way. The multi-elemental composition of animal and vegetal tissues can provide valuable information on the characteristics of the soil where a crop has been cultivated and on plants composition of the animal diet. The multi-element fingerprint is thus a valuable marker of the geographical origin of food. For instance, the authenticity of Tropea red onion, an onion Italian variety that achieved the PGI certifica‐ tion by the European Union as "Cipolla Rossa di Tropea Calabria" can be proved by means of multi-elemental analysis by ICP-MS and multivariate statistics [6]. All the statistical mod‐ els applied (LDA, stepwise LDA, SIMCA, ANN), allowed a success rate of prediction >90% for the genuine samples. Moreover, the availability of nutrients from the soil strictly de‐ pends on the fertilization strategies and the pest and weed control management systems. In organic farming synthetic fertilizers are not permitted and the pest and weed control is based exclusively on natural products. It has been thus proposed that the multi-elemental finger‐ print might be a marker for organically cultivated crops as compared to conventionally cultivated ones. Laursen *et al.* managed to discriminate between organic and conventional wheat, barley and faba beans (but not potatoes) based on the profile of 25 elements meas‐

IRMS is a technique that can measure the ratio of the stable isotopes of the constituents of a biological material. Light elements like carbon, nitrogen, hydrogen, oxygen and sulphur sta‐ ble isotopes ratios are most frequently assessed with this technique. Those ratios vary ac‐ cording to specific food production factors and geo-climatic conditions. Carbon stable isotope ratio depends, for example, from the plant composition of ruminant diets and can then be used to authenticate feeding regime or the farming management system (organic, free-range). Nitrogen stable isotope ratio is on the other hand depending on the type of fer‐ tilizers used in agriculture and is thus much useful for the authentication of farming practi‐ ces for vegetal products and crops. Oxygen isotope ratio is instead highly dependent on the

class, correctly refused by the model) close to 100% [4].

*Inductively coupled plasma mass spectrometry (ICP-MS)*

labelled hams from Spain and Italy [5].

ured by ICP-MS [7].

*Isotope ratio mass spectrometry (IRMS)*

Stand-alone MS fingerprinting techniques that proved to be very useful for the fraud control and prevention are: proton transfer reaction MS (PTR-MS), inductively coupled MS (ICP-MS), isotope ratio mass spectrometry (IRMS), and direct infusion MS techniques.

#### *Proton Transfer Reaction Mass Spectrometry (PTR-MS)*

PTR-MS is a relatively new technique that is rapidly gaining popularity in the food analysis. PTR-MS allows quantitative on-line monitoring of volatile organic compounds. The volatile compounds are softly ionized by means of hydroxonium ions that are generated in an exter‐ nal ion source operating in pure water vapour. Only the volatile compounds that have a higher affinity for the ions are protonated and then accelerated by an electric field to the re‐ action chamber where they are separated and quantified. Because of this soft chemical ioni‐ sation the fragmentation of the parent compounds is limited and the interpretation of the spectra are much easier. Other major advantages of this technique are the great sensitivity with detection limits as low as few part per trillion, volume (pptv) and the possibility to monitor the food samples in real time, without any work up procedure. As a result, a finger‐ print of all the volatile compounds comprised in a well definite mass range is obtained. The main disadvantage of this technique is that compounds are characterized only via their masses which is insufficient for their unequivocal identification.

PTR-MS has been extensively used in several aspects of food fraud control. It proved, for example, very successful for the geographical authentication of foods. The EU has long rec‐ ognized the importance of differentiating food products on a regional basis. The normative framework introduced by the EU comprises the EU Regulations 509/2006 and 510/2006 and the EU Regulation 1898/2006. The EU Regulation introduced three geographical indications to a food product: protected designation of origin (PDO), protected geographical indication (PGI) and traditional specialities (TSG). In a study of 2008, the geographical origin (country) of butter samples was successfully predicted in 88% of the cases based on PTR-MS finger‐ print and PLS-DA [2]. Recently, volatile fingerprint generated by PTR-MS has been used to discriminate between the Boeren Leidse specialty cumin cheeses with EU PDO from other 29 cumin cheese manufactured in the Netherlands [3]. The volatile fingerprint coupled with a PLS-DA model allowed the correct classification of 96% of the traditional boeren leidse cheese samples and 100% of the other commercial cheese samples. Another typical added value that is protected by the EU regulations is represented by the monovarietal extra virgin olive oil (EVOO), i.e. oil that is produced out of just one variety of olive trees. Frauds can be committed by mixing the more valuable monovarietal virgin olive oil with cheaper oils or by mixing different monovarietal olive oils. Volatile fingerprint of virgin olive oil obtained by PTR-MS and subjected to PLS-DA proved successful in discriminating among 5 different monovarietal EVOO from Spain with 100% sensitivity (% of objects of the modelled class correctly accepted by the model) and specificity (% of object, extraneous from the modelled class, correctly refused by the model) close to 100% [4].

In conclusion, PTR-MS is a rapid and low cost analytical technique that can be also fully au‐ tomated and implemented on-line. Recently, the coupling of the time of flight (TOF, see be‐ low in this section) mass analyser to PTR-MS instruments has generated PTR-TOF-MS which is characterized by a high sensitivity with limits of detection down to few pptv and a high time resolution. The technique has been recently applied to discriminate among PDO labelled hams from Spain and Italy [5].

#### *Inductively coupled plasma mass spectrometry (ICP-MS)*

on the intensity of the ions flux. MS produces therefore a mass spectrum representing the fingerprint of the sample components (Figure 1a). A number of different MS set-ups are pos‐ sible based on the ionisation technique and the mass analyser used. MS can be used alone or they can be coupled with separation techniques. In this section, the stand-alone MS techni‐ ques will be dealt with in details whereas the application of MS as coupled with separation

Stand-alone MS fingerprinting techniques that proved to be very useful for the fraud control and prevention are: proton transfer reaction MS (PTR-MS), inductively coupled MS (ICP-

PTR-MS is a relatively new technique that is rapidly gaining popularity in the food analysis. PTR-MS allows quantitative on-line monitoring of volatile organic compounds. The volatile compounds are softly ionized by means of hydroxonium ions that are generated in an exter‐ nal ion source operating in pure water vapour. Only the volatile compounds that have a higher affinity for the ions are protonated and then accelerated by an electric field to the re‐ action chamber where they are separated and quantified. Because of this soft chemical ioni‐ sation the fragmentation of the parent compounds is limited and the interpretation of the spectra are much easier. Other major advantages of this technique are the great sensitivity with detection limits as low as few part per trillion, volume (pptv) and the possibility to monitor the food samples in real time, without any work up procedure. As a result, a finger‐ print of all the volatile compounds comprised in a well definite mass range is obtained. The main disadvantage of this technique is that compounds are characterized only via their

PTR-MS has been extensively used in several aspects of food fraud control. It proved, for example, very successful for the geographical authentication of foods. The EU has long rec‐ ognized the importance of differentiating food products on a regional basis. The normative framework introduced by the EU comprises the EU Regulations 509/2006 and 510/2006 and the EU Regulation 1898/2006. The EU Regulation introduced three geographical indications to a food product: protected designation of origin (PDO), protected geographical indication (PGI) and traditional specialities (TSG). In a study of 2008, the geographical origin (country) of butter samples was successfully predicted in 88% of the cases based on PTR-MS finger‐ print and PLS-DA [2]. Recently, volatile fingerprint generated by PTR-MS has been used to discriminate between the Boeren Leidse specialty cumin cheeses with EU PDO from other 29 cumin cheese manufactured in the Netherlands [3]. The volatile fingerprint coupled with a PLS-DA model allowed the correct classification of 96% of the traditional boeren leidse cheese samples and 100% of the other commercial cheese samples. Another typical added value that is protected by the EU regulations is represented by the monovarietal extra virgin olive oil (EVOO), i.e. oil that is produced out of just one variety of olive trees. Frauds can be committed by mixing the more valuable monovarietal virgin olive oil with cheaper oils or by mixing different monovarietal olive oils. Volatile fingerprint of virgin olive oil obtained by PTR-MS and subjected to PLS-DA proved successful in discriminating among 5 different monovarietal EVOO from Spain with 100% sensitivity (% of objects of the modelled class

MS), isotope ratio mass spectrometry (IRMS), and direct infusion MS techniques.

techniques will be dealt with in the next sections.

116 Latest Research into Quality Control

*Proton Transfer Reaction Mass Spectrometry (PTR-MS)*

masses which is insufficient for their unequivocal identification.

ICP-MS provides quantitative measurements of a wide range of metals and non-metals (inorganic elements) at trace and ultratrace concentration level (ppt). In this technique, the sample (even solid or liquid) is decomposed to neutral elements in a high-temperature argon plasma and the single elements are separated based on their mass/charge ratio and ana‐ lysed. The great advantage of this technique compared to others (e.g. atomic spectroscopy) is that more than one element can be analysed at once so that a multi-elemental fingerprint is obtained in a very fast and sensitive way. The multi-elemental composition of animal and vegetal tissues can provide valuable information on the characteristics of the soil where a crop has been cultivated and on plants composition of the animal diet. The multi-element fingerprint is thus a valuable marker of the geographical origin of food. For instance, the authenticity of Tropea red onion, an onion Italian variety that achieved the PGI certifica‐ tion by the European Union as "Cipolla Rossa di Tropea Calabria" can be proved by means of multi-elemental analysis by ICP-MS and multivariate statistics [6]. All the statistical mod‐ els applied (LDA, stepwise LDA, SIMCA, ANN), allowed a success rate of prediction >90% for the genuine samples. Moreover, the availability of nutrients from the soil strictly de‐ pends on the fertilization strategies and the pest and weed control management systems. In organic farming synthetic fertilizers are not permitted and the pest and weed control is based exclusively on natural products. It has been thus proposed that the multi-elemental finger‐ print might be a marker for organically cultivated crops as compared to conventionally cultivated ones. Laursen *et al.* managed to discriminate between organic and conventional wheat, barley and faba beans (but not potatoes) based on the profile of 25 elements meas‐ ured by ICP-MS [7].

#### *Isotope ratio mass spectrometry (IRMS)*

IRMS is a technique that can measure the ratio of the stable isotopes of the constituents of a biological material. Light elements like carbon, nitrogen, hydrogen, oxygen and sulphur sta‐ ble isotopes ratios are most frequently assessed with this technique. Those ratios vary ac‐ cording to specific food production factors and geo-climatic conditions. Carbon stable isotope ratio depends, for example, from the plant composition of ruminant diets and can then be used to authenticate feeding regime or the farming management system (organic, free-range). Nitrogen stable isotope ratio is on the other hand depending on the type of fer‐ tilizers used in agriculture and is thus much useful for the authentication of farming practi‐ ces for vegetal products and crops. Oxygen isotope ratio is instead highly dependent on the distance from the ocean and the altitude above sea level and could then be used for the au‐ thentication of the geographical origin of a food product.

The adulteration with hazelnut oil was predicted at a level of 10% v/v [10]. This adultera‐ tion is difficult to detect at levels below 20% by conventional methods due to the composi‐ tional similarity between the two oils. Similarly, PCA and HCA methodologies, applied to the ESI(+)-MS data were able to readily detect adulteration of EVOO with ordinary olive oils, at levels as low as 1% w/w [11]. Mono-, di- and triglycerides together with vitamins and antioxidants were detected and quantified with this method. Direct infusion ESI-MS has been used to authenticate other food commodities. The chemical fingerprint generated by direct infusion ESI-Q-TOF-MS in the negative mode can be used to discriminate between genuine whisky from Scotland and US, from counterfeited whisky produced in Brazil [12] and between alembic (the most valuable) and industrial cachaças (Brazilian sugarcane spi‐ rit) as well as the fraudulent addition of sucrose to the spirit [13]. Finally, direct-infusion ESI-QqQ-TOF-MS and atmospheric pressure photoionization (APPI)-QqQ-TOF-MS have been used for Iberian ham typification. APPI is a soft ionization technique based on a photoioni‐ sation mechanism. Five types of Iberian hams were successfully classified. Applying a PLS-

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MALDI is another soft ionisation technique that proved very useful in the analysis of macro‐ molecules, especially proteins. In MALDI the molecules are desorbed from the support ma‐ trix and ionised by means of a UV laser beam in a complex process mediated by the support matrix itself. MALDI is mainly coupled with a time-of-flight (TOF) mass analyser which separate the ions based on their flying time to the detector, which on turns depends on their m/z ratio. An example of application of MALDI-TOF-MS for authentication issues is repre‐ sented by the fast method developed by Wang *et al.* for the fingerprinting of honey proteins [15]. The mass spectra were used to build up a database library to be used for authentication purpose. The protein fingerprint was thus successfully used to authenticate the geographical origin of commercial honeys produced in the US and other countries. In a similar fashion, peptide fingerprinting obtained by MALDI-TOF has been converted in a biological bar code

An innovative technique for food fingerprinting is represented by the direct analysis in real time (DART)-MS. DART is an ambient ionisation technique i.e. in which ions are formed outside the mass spectrometer without sample preparation or separation. The samples, ei‐ ther gaseous, liquid or solid are ionised in open air under ambient conditions. This means that organic compounds can be directly, and in real time, determined without time-consum‐ ing analytical protocols and thus with high sample throughput. DART coupled with TOF-MS has been used to obtain the fingerprint of the triglycerides from olive oil [17]. This method, coupled with LDA allowed the discrimination between EVOO, olive oil and olive oil po‐ mace and the detection of hazelnut oil in EVOO at 6% v/v. DART-TOF-MS with solid phase micro extraction (SPME) pre-concentration of the analytes has been also reported to allow discrimination between trappist and non-trappist beers based on volatiles and phenolic com‐ pounds [18]. A combination of DART-TOF-MS and chemometrics was used for animal fat (lard and beef tallow) authentication [19]. TGs and polar compounds were extracted and analyzed. Mass spectral records were processed by PCA and stepwise LDA. The LDA mod‐ el developed using TAG data enabled the classification of lard and beef tallow samples but

also detection of admixed lard and tallow at adulteration levels of 5 and 10% w/w.

for the authentication of high quality Campania white wines [16].

DA model [14].

The stable isotope fingerprint has been successfully used for the authentication of geograph‐ ical origin and the farming practice. Normally the data are measured for many different ele‐ ments and analysed with multivariate statistics. As an example, Fontina PDO cheese can be discriminated with good success from other cheeses based on stable isotopes 2 H/1 H, 13C/12C, 15N/14N and 34S/32S and PCA analysis [8]. However, frequently the isotope ratios (or a selec‐ tion of them) are combined with other markers (elements) to improve the accuracy of the classification models. IRMS is often combined with ICP-MS for simultaneous elemental analysis. Stable isotope analysis combined with multi-elemental analysis has proven ideally suited to determine geographical origin of foods. The most accurate measurements of the isotope ratios is obtained by dual inlet (DI) IRMS. However, the purchasing and operating costs of a DI-IRMS instrument coupled with the time-consuming sample preparation are major disadvantages for the diffusion of this technique. The introduction of continuous flow (CF) IRMS instruments offers on-line, rapid and automated sample preparation, greater cost-effectiveness and easier interfacing with other preparation techniques.

#### *Direct infusion mass spectrometry*

Direct infusion MS techniques are based on the direct injection of the sample in the ion source without or with small sample pre-treatment. This allows for rapid analysis suited for high-throughput screenings. Electrospray ionisation (ESI), matrix assisted laser desorption ionization (MALDI) and direct analysis in real time (DART) are typical ionization techni‐ ques used for direct infusion MS. They are coupled with a variety of mass analysers, e.g. time of flight (TOF), Fourier Transform Ion Cyclotron Resonance (FT-ICR), single quadru‐ pole (Q) and ion trap (IT) in many different set-ups.

ESI is a typical soft ionisation technique that is particularly suited for the determination of the molecular mass of large molecules (proteins, peptides, polysaccharides, triglycerides), because the ionisation does not bring about the fragmentation of the molecule. The liquid in which the analyte is contained is dispersed by electrospray to a fine aerosol. The drop‐ lets shrink as the solvent evaporates till solvated ions desorb from their surface. ESI-MS has proven to be very helpful in the authentication of vegetable oils. Lipid composition of veg‐ etable oils depends on their botanical origin and the way they are processed. Fatty acids (FAs) and/or triglycerides (TGs) profile can thus help authenticate the type of oil, its ori‐ gin, its quality grade and potential adulteration. Direct infusion ESI-MS has been for exam‐ ple used to predict the olive oil quality according to European Union marketing standards based on fatty acids and LDA analysis [9]. In the same research, the percentage of either EVOO and VOO in binary mixture with other lower grade oils was predicted with 5–11% average prediction errors by using PLS and multilinear regression (MLR). Samples were 1:50 diluted in an alkaline 85:15 (v/v) propanol/methanol mixture and directly infused into the MS system. Triglycerides analysis has some advantage over the analysis of the fatty acids profile for authentication or fraud control. Indeed, different oils can have specific TG finger‐ print despite showing the same fatty acids composition. The triglyceride profiles, obtained using Q-TOF-ESI-MS was used to predict adulteration of olive oils with other vegetable oils. The adulteration with hazelnut oil was predicted at a level of 10% v/v [10]. This adultera‐ tion is difficult to detect at levels below 20% by conventional methods due to the composi‐ tional similarity between the two oils. Similarly, PCA and HCA methodologies, applied to the ESI(+)-MS data were able to readily detect adulteration of EVOO with ordinary olive oils, at levels as low as 1% w/w [11]. Mono-, di- and triglycerides together with vitamins and antioxidants were detected and quantified with this method. Direct infusion ESI-MS has been used to authenticate other food commodities. The chemical fingerprint generated by direct infusion ESI-Q-TOF-MS in the negative mode can be used to discriminate between genuine whisky from Scotland and US, from counterfeited whisky produced in Brazil [12] and between alembic (the most valuable) and industrial cachaças (Brazilian sugarcane spi‐ rit) as well as the fraudulent addition of sucrose to the spirit [13]. Finally, direct-infusion ESI-QqQ-TOF-MS and atmospheric pressure photoionization (APPI)-QqQ-TOF-MS have been used for Iberian ham typification. APPI is a soft ionization technique based on a photoioni‐ sation mechanism. Five types of Iberian hams were successfully classified. Applying a PLS-DA model [14].

distance from the ocean and the altitude above sea level and could then be used for the au‐

The stable isotope fingerprint has been successfully used for the authentication of geograph‐ ical origin and the farming practice. Normally the data are measured for many different ele‐ ments and analysed with multivariate statistics. As an example, Fontina PDO cheese can be

15N/14N and 34S/32S and PCA analysis [8]. However, frequently the isotope ratios (or a selec‐ tion of them) are combined with other markers (elements) to improve the accuracy of the classification models. IRMS is often combined with ICP-MS for simultaneous elemental analysis. Stable isotope analysis combined with multi-elemental analysis has proven ideally suited to determine geographical origin of foods. The most accurate measurements of the isotope ratios is obtained by dual inlet (DI) IRMS. However, the purchasing and operating costs of a DI-IRMS instrument coupled with the time-consuming sample preparation are major disadvantages for the diffusion of this technique. The introduction of continuous flow (CF) IRMS instruments offers on-line, rapid and automated sample preparation, greater

Direct infusion MS techniques are based on the direct injection of the sample in the ion source without or with small sample pre-treatment. This allows for rapid analysis suited for high-throughput screenings. Electrospray ionisation (ESI), matrix assisted laser desorption ionization (MALDI) and direct analysis in real time (DART) are typical ionization techni‐ ques used for direct infusion MS. They are coupled with a variety of mass analysers, e.g. time of flight (TOF), Fourier Transform Ion Cyclotron Resonance (FT-ICR), single quadru‐

ESI is a typical soft ionisation technique that is particularly suited for the determination of the molecular mass of large molecules (proteins, peptides, polysaccharides, triglycerides), because the ionisation does not bring about the fragmentation of the molecule. The liquid in which the analyte is contained is dispersed by electrospray to a fine aerosol. The drop‐ lets shrink as the solvent evaporates till solvated ions desorb from their surface. ESI-MS has proven to be very helpful in the authentication of vegetable oils. Lipid composition of veg‐ etable oils depends on their botanical origin and the way they are processed. Fatty acids (FAs) and/or triglycerides (TGs) profile can thus help authenticate the type of oil, its ori‐ gin, its quality grade and potential adulteration. Direct infusion ESI-MS has been for exam‐ ple used to predict the olive oil quality according to European Union marketing standards based on fatty acids and LDA analysis [9]. In the same research, the percentage of either EVOO and VOO in binary mixture with other lower grade oils was predicted with 5–11% average prediction errors by using PLS and multilinear regression (MLR). Samples were 1:50 diluted in an alkaline 85:15 (v/v) propanol/methanol mixture and directly infused into the MS system. Triglycerides analysis has some advantage over the analysis of the fatty acids profile for authentication or fraud control. Indeed, different oils can have specific TG finger‐ print despite showing the same fatty acids composition. The triglyceride profiles, obtained using Q-TOF-ESI-MS was used to predict adulteration of olive oils with other vegetable oils.

H/1

H, 13C/12C,

discriminated with good success from other cheeses based on stable isotopes 2

cost-effectiveness and easier interfacing with other preparation techniques.

thentication of the geographical origin of a food product.

*Direct infusion mass spectrometry*

118 Latest Research into Quality Control

pole (Q) and ion trap (IT) in many different set-ups.

MALDI is another soft ionisation technique that proved very useful in the analysis of macro‐ molecules, especially proteins. In MALDI the molecules are desorbed from the support ma‐ trix and ionised by means of a UV laser beam in a complex process mediated by the support matrix itself. MALDI is mainly coupled with a time-of-flight (TOF) mass analyser which separate the ions based on their flying time to the detector, which on turns depends on their m/z ratio. An example of application of MALDI-TOF-MS for authentication issues is repre‐ sented by the fast method developed by Wang *et al.* for the fingerprinting of honey proteins [15]. The mass spectra were used to build up a database library to be used for authentication purpose. The protein fingerprint was thus successfully used to authenticate the geographical origin of commercial honeys produced in the US and other countries. In a similar fashion, peptide fingerprinting obtained by MALDI-TOF has been converted in a biological bar code for the authentication of high quality Campania white wines [16].

An innovative technique for food fingerprinting is represented by the direct analysis in real time (DART)-MS. DART is an ambient ionisation technique i.e. in which ions are formed outside the mass spectrometer without sample preparation or separation. The samples, ei‐ ther gaseous, liquid or solid are ionised in open air under ambient conditions. This means that organic compounds can be directly, and in real time, determined without time-consum‐ ing analytical protocols and thus with high sample throughput. DART coupled with TOF-MS has been used to obtain the fingerprint of the triglycerides from olive oil [17]. This method, coupled with LDA allowed the discrimination between EVOO, olive oil and olive oil po‐ mace and the detection of hazelnut oil in EVOO at 6% v/v. DART-TOF-MS with solid phase micro extraction (SPME) pre-concentration of the analytes has been also reported to allow discrimination between trappist and non-trappist beers based on volatiles and phenolic com‐ pounds [18]. A combination of DART-TOF-MS and chemometrics was used for animal fat (lard and beef tallow) authentication [19]. TGs and polar compounds were extracted and analyzed. Mass spectral records were processed by PCA and stepwise LDA. The LDA mod‐ el developed using TAG data enabled the classification of lard and beef tallow samples but also detection of admixed lard and tallow at adulteration levels of 5 and 10% w/w.

Additional ambient ionisation techniques have been recently proposed for authentication and fraud control by product fingerprinting. For instance, easy sonic spray ionisation (EASI)- MS fingerprinting of fatty acids and phenolic compounds have been used for the authentica‐ tion of olive oil geographical origin [20].

*Liquid chromatography*

*Gas Chromatography*

In liquid chromatography, the mixture components are separated as they pass through a column based on their selective partition behaviour between a stationary phase (column ma‐ terial) and a mobile liquid phase. Depending on the type of stationary phase, compounds can be separated based on their size, charge, molecular mass, hydrophobicity etc. The most popular LC technique is high performance liquid chromatography (HPLC) that is a straight‐ forward, robust and reproducible technique. HPLC has been used for the analysis of a wide range of food compounds such as vitamins, proteins, carbohydrates, TGs, additives, secon‐ dary plant metabolites. A typical example of the application of HPLC based fingerprint for fraud control is the authentication of the organic eggs by means of the carotenoids profile [21]. Carotenoids are a group of fat-soluble pigments that occur in the egg yolk in concentra‐ tions of about 10 mg kg-1. In animals carotenoids are entirely of dietary origin. Since the feeding regime of organic hens is clearly different from that of conventionally reared hens, the carotenoids fingerprint in eggs were used to discriminate between production systems. The carotenoids profile was determined by HPLC with UV detection, the single carotenoids quantified and the resulting concentrations used to build up a classification model by kNN. Almost all the conventional eggs and all the organic ones were correctly classified in exter‐ nal validation. The robustness of the method has been recently improved by testing eggs produced in several EU and non-EU countries. An example of HPLC fingerprint based on raw chromatographic data is in [22]. The authors applied PLS to the full TGs chromatogram profiles of vegetable oils to predict the % of olive oil in the mixtures with errors not exceed‐ ing 10%. Liquid chromatography can also be coupled with MS which allows higher resolu‐ tion and higher sensitivity for metabolites occurring in relatively low amount. As an example, an untargeted method for proteins analysis based on LC-QTOF-MS has been de‐ veloped which allowed to detect the fraudulent addition of cheaper vegetal proteins (from

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soy and pea) to skimmed milk powders based on the different peptides profile [23].

In gas chromatography, the mixture is first vaporised in a heated chamber and then the mix‐ ture components are separated as they travel through the column transported by the flow of an inert gas (helium, nitrogen or hydrogen) based on their selective interaction with the col‐ umn material. GC is a very popular separation technique mainly used for the analysis of volatile compounds. However a wide spectrum of compounds can be rendered volatile by proper derivatisation and thus analysed by GC. The analysis of fatty acids and triglycerides is usually carried out by GC with flame ionisation detector (FID) previous derivatisation in fatty acids methyl esters (FAME) and TG trimethylethers, respectively. FID is a general de‐ tector capable of high sensitivity and robustness. Fatty acids composition of animal tissues and animal products strongly depends on the feeding regime. FAs composition of fish mus‐ cle fat is affected by animal diet/feeding, the geographical area of catch and the marine con‐ ditions and is thus different between farmed and wild fishes. The discrimination between wild and farmed Atlantic salmon (*Salmo salar* L.) and Wild Turbot (*Psetta maxima*) has been reported based on FAs analysis and chemometrics [24-25]. The fatty acid fingerprint ob‐ tained by GC-FID followed by PLS-DA analysis has been also reported for the authentica‐

#### **4. Chromatographic fingerprinting**

Chromatographic techniques aim at resolving complex mixtures in well separated com‐ pound. Based on the detection system, each single compounds generates a signal that can be used for the qualitative and quantitative analysis of the mixture. The graphical representa‐ tion of such signal as a function of time is referred to as a chromatogram and can be thought of as the fingerprint of one or more classes of compounds occurring in the sample. Differ‐ ent strategies are available to obtained multivariate data matrices from chromatographic analyses (Figure 3). The fingerprint can be composed by the set of concentrations of the separated compounds based on an identification/calibration/quantification procedure as de‐ picted in the path (a) of Figure 3. Alternatively, the fingerprint can be represented by the set of peak areas/heights (b). In this case the identification of each single peak is not necessary. Finally, it can be represented by the whole chromatogram that is handled as a continuous signal (c). In this case, the multivariate dataset is composed by as many variables as the sampling points the chromatogram is made up of (each data point of the chromatogram represents an individual variable). However, the application of chemometrics on raw chro‐ matographic data requires specific data pre-processing techniques. In fact, problems related to the peak alignment or baseline shifts are particularly critical when a raw chromatogram is used as a data set.

**Figure 3.** Schematic representation of the strategies to obtain multivariate dataset from a chromatogram for chemo‐ metric analysis.

#### *Liquid chromatography*

Additional ambient ionisation techniques have been recently proposed for authentication and fraud control by product fingerprinting. For instance, easy sonic spray ionisation (EASI)- MS fingerprinting of fatty acids and phenolic compounds have been used for the authentica‐

Chromatographic techniques aim at resolving complex mixtures in well separated com‐ pound. Based on the detection system, each single compounds generates a signal that can be used for the qualitative and quantitative analysis of the mixture. The graphical representa‐ tion of such signal as a function of time is referred to as a chromatogram and can be thought of as the fingerprint of one or more classes of compounds occurring in the sample. Differ‐ ent strategies are available to obtained multivariate data matrices from chromatographic analyses (Figure 3). The fingerprint can be composed by the set of concentrations of the separated compounds based on an identification/calibration/quantification procedure as de‐ picted in the path (a) of Figure 3. Alternatively, the fingerprint can be represented by the set of peak areas/heights (b). In this case the identification of each single peak is not necessary. Finally, it can be represented by the whole chromatogram that is handled as a continuous signal (c). In this case, the multivariate dataset is composed by as many variables as the sampling points the chromatogram is made up of (each data point of the chromatogram represents an individual variable). However, the application of chemometrics on raw chro‐ matographic data requires specific data pre-processing techniques. In fact, problems related to the peak alignment or baseline shifts are particularly critical when a raw chromatogram is

**Figure 3.** Schematic representation of the strategies to obtain multivariate dataset from a chromatogram for chemo‐

tion of olive oil geographical origin [20].

120 Latest Research into Quality Control

**4. Chromatographic fingerprinting**

used as a data set.

metric analysis.

In liquid chromatography, the mixture components are separated as they pass through a column based on their selective partition behaviour between a stationary phase (column ma‐ terial) and a mobile liquid phase. Depending on the type of stationary phase, compounds can be separated based on their size, charge, molecular mass, hydrophobicity etc. The most popular LC technique is high performance liquid chromatography (HPLC) that is a straight‐ forward, robust and reproducible technique. HPLC has been used for the analysis of a wide range of food compounds such as vitamins, proteins, carbohydrates, TGs, additives, secon‐ dary plant metabolites. A typical example of the application of HPLC based fingerprint for fraud control is the authentication of the organic eggs by means of the carotenoids profile [21]. Carotenoids are a group of fat-soluble pigments that occur in the egg yolk in concentra‐ tions of about 10 mg kg-1. In animals carotenoids are entirely of dietary origin. Since the feeding regime of organic hens is clearly different from that of conventionally reared hens, the carotenoids fingerprint in eggs were used to discriminate between production systems. The carotenoids profile was determined by HPLC with UV detection, the single carotenoids quantified and the resulting concentrations used to build up a classification model by kNN. Almost all the conventional eggs and all the organic ones were correctly classified in exter‐ nal validation. The robustness of the method has been recently improved by testing eggs produced in several EU and non-EU countries. An example of HPLC fingerprint based on raw chromatographic data is in [22]. The authors applied PLS to the full TGs chromatogram profiles of vegetable oils to predict the % of olive oil in the mixtures with errors not exceed‐ ing 10%. Liquid chromatography can also be coupled with MS which allows higher resolu‐ tion and higher sensitivity for metabolites occurring in relatively low amount. As an example, an untargeted method for proteins analysis based on LC-QTOF-MS has been de‐ veloped which allowed to detect the fraudulent addition of cheaper vegetal proteins (from soy and pea) to skimmed milk powders based on the different peptides profile [23].

#### *Gas Chromatography*

In gas chromatography, the mixture is first vaporised in a heated chamber and then the mix‐ ture components are separated as they travel through the column transported by the flow of an inert gas (helium, nitrogen or hydrogen) based on their selective interaction with the col‐ umn material. GC is a very popular separation technique mainly used for the analysis of volatile compounds. However a wide spectrum of compounds can be rendered volatile by proper derivatisation and thus analysed by GC. The analysis of fatty acids and triglycerides is usually carried out by GC with flame ionisation detector (FID) previous derivatisation in fatty acids methyl esters (FAME) and TG trimethylethers, respectively. FID is a general de‐ tector capable of high sensitivity and robustness. Fatty acids composition of animal tissues and animal products strongly depends on the feeding regime. FAs composition of fish mus‐ cle fat is affected by animal diet/feeding, the geographical area of catch and the marine con‐ ditions and is thus different between farmed and wild fishes. The discrimination between wild and farmed Atlantic salmon (*Salmo salar* L.) and Wild Turbot (*Psetta maxima*) has been reported based on FAs analysis and chemometrics [24-25]. The fatty acid fingerprint ob‐ tained by GC-FID followed by PLS-DA analysis has been also reported for the authentica‐ tion of organic eggs and of organic feeds [26-27]. In the last case 90% of the analysed samples were correctly classified in their proper group in external validation. GC-FID can also be used for the TG profiling. TG fingerprinting by GC-FID has been for example report‐ ed for the authentication of three fat classes (animal fats, fish oils, recycled cooking oils) [28]. The TGs fingerprint was subjected to multivariate analysis (PLS-DA) and allowed the cor‐ rect classification of 96% of the fat samples.

Genomic fingerprinting mainly relies on polymerase chain reaction (PCR) based techniques. PCR is based on the amplification of a target DNA sequence by means of a thermostable DNA polymerase. The process consists of several cycles where the DNA molecule is denatu‐ rated, specific primers (small DNA sequences) anneal to the target DNA sequence and the DNA polymerase synthetizes a new DNA fragment delimited by the two primers. In each cycle the number of DNA molecules increases exponentially. A PCR-derived fingerprint can be obtained in different ways. In PCR-RFLP (restriction fragment length polymorphism) the amplified region is digested with an endonuclease and the resulting DNA fragments are separated by electrophoresis and properly visualised. The pattern of fragment represents a fingerprint of the DNA sequence that has been amplified. In multiplex PCR, two or more DNA fragments are simultaneously amplified by means of different target primer pairs, sep‐ arated by electrophoresis and visualised. In RAPD (random amplification of polymorphic DNA) random DNA fragments are amplified by means of arbitrarily created primers. After separation, the DNA fragments will give rise specific patterns on the gel. Finally, in singlestrand conformation polymorphism (SSCP), DNA sequences are amplified, denatured and the resulting single strand DNA molecules separated by electrophoresis based on their spe‐

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PCR-based fingerprinting techniques have been widely used for species identification. For instance, the identification of ten species of salmon genus in a wide range of commercial products can be accomplished by PCR-RFLP based on the amplification of a specific region of the mitochondrial *cytochrome b* gene followed by polyacrylamide gel electrophoresis (PAGE) [32]. Similarly, PCR-RFLP has been used to identify 15 species of gadoid fishes based on the amplification of a small region of the *cytochrome b* gene and three restriction enzymes [33]. Gadidae family is one of the most commercially important fish family com‐ prising species as Atlantic cod (*Gadus morhua*), the pollack (*Pollachius pollachius*) and the had‐ dock (*Melanogrammus aeglenus*). Duplex PCR targeting the *cytochrome b* gene can be used to detect cow milk in buffalo mozzarella at a level of 1%. Buffalo mozzarella is labelled with PDO and can be produced only with pure water buffalo milk (*Bubalus bubalis*) [34]. In an original approach, the multiplex PCR fingerprint of the 16S and 23S rDNA genes of the lac‐ tic bacteria naturally occurring in milk has been used to discriminate the geographical origin of PDO mozzarella cheese [35]. The PCR fingerprint was subjected to cluster analysis (neigh‐

bour-joining algorithm) which allowed a fair discrimination of the samples.

Genomic fingerprinting shows a unique potential for the species or variety authentication in food products. The introduction of PCR has notably increased the potential of this approach. However, compared to other fingerprinting techniques, genomic fingerprinting is relatively time-consuming and labour-intensive. Its applicability to fraud issues other than genetic identification is limited. Furthermore, food processing may degrade the DNA molecule and lower its recovery thus negatively affecting the results of a analysis when applied to heavily

Capillary electrophoresis (CE) is the electrophoretic technique that shows a notable potential for food fraud detection based on product fingerprint. CE is a family of separation techni‐

cific secondary structures.

processed foods.

*Capillary electrophoresis*

GC coupled with MS represents the method of choice for the analysis of volatile compounds because of its high reproducibility. On the other hand, GC-MS analysis requires careful sam‐ ple cleaning and is quite expensive and time-consuming. The volatile fingerprint of coffee obtained by GC-TOF-MS after SPME has been reported for the geographical authentication of coffee [29]. SPME preconcentration of volatiles followed by GC-MS analysis coupled with PCA analysis allowed the detection of adulteration of ground roasted coffee with roasted barley [30]. The adulteration is detectable at level of 1% w/w in mixtures of dark roasted bar‐ ley and coffees. Metabolomics studies can be also fruitfully performed by GC-MS. The fin‐ gerprint of a large range of metabolites obtained by GC-MS has been used to discriminate between mechanical separated meat (MSM) from hand-deboned meat [31]. MSM could be detected in raw meat mixtures down to a level of 10%.

#### **5. Electrophoretic fingerprinting**

Electrophoretic techniques are able to separate a complex mixture under a spatially uniform electric field, based on electrophoretic mobility of its components that depends, in turn, from their hydrodynamic properties and charge. Positively charged molecules move to‐ wards the anode and negatively charged molecules towards the cathode at a different rate based mainly on their mass to charge ratio. Smaller molecules move faster than larger ones.

#### *Gel electrophoresis*

In a gel electrophoresis, a gel is used as a medium for the movement of the charged particles under the applied electric field. Agar and polyacrylamide are typical medium used in gel electrophoresis. Proteins and nucleic acid fragments are usually separated by gel electropho‐ resis. Gel electrophoresis is of major importance for the genomic fingerprint of a sample ma‐ terial. Genomic fingerprints are obtained when properly amplified targeted or untargeted DNA or RNA fragments are separated by electrophoresis thus providing patterns that can be associated to sample properties (specie, variety and the like). Unlike the fingerprints dis‐ cussed in the previous (and the next) sections, DNA fingerprint shows somehow different characteristics. The single features of the fingerprint are not quantitative variables (physical or chemical variables allowed to take on quantitative values, e.g. area of a peak in a chroma‐ togram, signal intensity for a m/z or absorbance at a fixed wavelength in a IR spectra) but rather categorical variables, i.e. electrophoretic bands that can be either present or absent (see Figure 1 (c)). The sample identification is thus mainly carried out by checking for the presence (or absence) of one or more target bands. Multivariate analysis of the DNA frag‐ ments patterns is rarely performed.

Genomic fingerprinting mainly relies on polymerase chain reaction (PCR) based techniques. PCR is based on the amplification of a target DNA sequence by means of a thermostable DNA polymerase. The process consists of several cycles where the DNA molecule is denatu‐ rated, specific primers (small DNA sequences) anneal to the target DNA sequence and the DNA polymerase synthetizes a new DNA fragment delimited by the two primers. In each cycle the number of DNA molecules increases exponentially. A PCR-derived fingerprint can be obtained in different ways. In PCR-RFLP (restriction fragment length polymorphism) the amplified region is digested with an endonuclease and the resulting DNA fragments are separated by electrophoresis and properly visualised. The pattern of fragment represents a fingerprint of the DNA sequence that has been amplified. In multiplex PCR, two or more DNA fragments are simultaneously amplified by means of different target primer pairs, sep‐ arated by electrophoresis and visualised. In RAPD (random amplification of polymorphic DNA) random DNA fragments are amplified by means of arbitrarily created primers. After separation, the DNA fragments will give rise specific patterns on the gel. Finally, in singlestrand conformation polymorphism (SSCP), DNA sequences are amplified, denatured and the resulting single strand DNA molecules separated by electrophoresis based on their spe‐ cific secondary structures.

PCR-based fingerprinting techniques have been widely used for species identification. For instance, the identification of ten species of salmon genus in a wide range of commercial products can be accomplished by PCR-RFLP based on the amplification of a specific region of the mitochondrial *cytochrome b* gene followed by polyacrylamide gel electrophoresis (PAGE) [32]. Similarly, PCR-RFLP has been used to identify 15 species of gadoid fishes based on the amplification of a small region of the *cytochrome b* gene and three restriction enzymes [33]. Gadidae family is one of the most commercially important fish family com‐ prising species as Atlantic cod (*Gadus morhua*), the pollack (*Pollachius pollachius*) and the had‐ dock (*Melanogrammus aeglenus*). Duplex PCR targeting the *cytochrome b* gene can be used to detect cow milk in buffalo mozzarella at a level of 1%. Buffalo mozzarella is labelled with PDO and can be produced only with pure water buffalo milk (*Bubalus bubalis*) [34]. In an original approach, the multiplex PCR fingerprint of the 16S and 23S rDNA genes of the lac‐ tic bacteria naturally occurring in milk has been used to discriminate the geographical origin of PDO mozzarella cheese [35]. The PCR fingerprint was subjected to cluster analysis (neigh‐ bour-joining algorithm) which allowed a fair discrimination of the samples.

Genomic fingerprinting shows a unique potential for the species or variety authentication in food products. The introduction of PCR has notably increased the potential of this approach. However, compared to other fingerprinting techniques, genomic fingerprinting is relatively time-consuming and labour-intensive. Its applicability to fraud issues other than genetic identification is limited. Furthermore, food processing may degrade the DNA molecule and lower its recovery thus negatively affecting the results of a analysis when applied to heavily processed foods.

#### *Capillary electrophoresis*

tion of organic eggs and of organic feeds [26-27]. In the last case 90% of the analysed samples were correctly classified in their proper group in external validation. GC-FID can also be used for the TG profiling. TG fingerprinting by GC-FID has been for example report‐ ed for the authentication of three fat classes (animal fats, fish oils, recycled cooking oils) [28]. The TGs fingerprint was subjected to multivariate analysis (PLS-DA) and allowed the cor‐

GC coupled with MS represents the method of choice for the analysis of volatile compounds because of its high reproducibility. On the other hand, GC-MS analysis requires careful sam‐ ple cleaning and is quite expensive and time-consuming. The volatile fingerprint of coffee obtained by GC-TOF-MS after SPME has been reported for the geographical authentication of coffee [29]. SPME preconcentration of volatiles followed by GC-MS analysis coupled with PCA analysis allowed the detection of adulteration of ground roasted coffee with roasted barley [30]. The adulteration is detectable at level of 1% w/w in mixtures of dark roasted bar‐ ley and coffees. Metabolomics studies can be also fruitfully performed by GC-MS. The fin‐ gerprint of a large range of metabolites obtained by GC-MS has been used to discriminate between mechanical separated meat (MSM) from hand-deboned meat [31]. MSM could be

Electrophoretic techniques are able to separate a complex mixture under a spatially uniform electric field, based on electrophoretic mobility of its components that depends, in turn, from their hydrodynamic properties and charge. Positively charged molecules move to‐ wards the anode and negatively charged molecules towards the cathode at a different rate based mainly on their mass to charge ratio. Smaller molecules move faster than larger ones.

In a gel electrophoresis, a gel is used as a medium for the movement of the charged particles under the applied electric field. Agar and polyacrylamide are typical medium used in gel electrophoresis. Proteins and nucleic acid fragments are usually separated by gel electropho‐ resis. Gel electrophoresis is of major importance for the genomic fingerprint of a sample ma‐ terial. Genomic fingerprints are obtained when properly amplified targeted or untargeted DNA or RNA fragments are separated by electrophoresis thus providing patterns that can be associated to sample properties (specie, variety and the like). Unlike the fingerprints dis‐ cussed in the previous (and the next) sections, DNA fingerprint shows somehow different characteristics. The single features of the fingerprint are not quantitative variables (physical or chemical variables allowed to take on quantitative values, e.g. area of a peak in a chroma‐ togram, signal intensity for a m/z or absorbance at a fixed wavelength in a IR spectra) but rather categorical variables, i.e. electrophoretic bands that can be either present or absent (see Figure 1 (c)). The sample identification is thus mainly carried out by checking for the presence (or absence) of one or more target bands. Multivariate analysis of the DNA frag‐

rect classification of 96% of the fat samples.

122 Latest Research into Quality Control

detected in raw meat mixtures down to a level of 10%.

**5. Electrophoretic fingerprinting**

ments patterns is rarely performed.

*Gel electrophoresis*

Capillary electrophoresis (CE) is the electrophoretic technique that shows a notable potential for food fraud detection based on product fingerprint. CE is a family of separation techni‐ ques that separate charged analytes based on their electrophoretic mobility: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIF), capillary gel electrophoresis, ca‐ pillary electrochromatography. An electric field is applied to the ends of a capillary column. The ions migrate through the column in the same direction pulled by the electrosmotic flow and are separated based on their electrophoretic mobility. The signal that is generated when the mixture components are detected as they are eluted from the column is referred to as a capillary electropherogram. Multivariate dataset can be obtained from electropherogram in the same way as depicted in Figure 3. However, only strategy (a) has been used so far for authentication and fraud control purposes. CE are capable of rapid, low cost and high reso‐ lution analysis with little consumption of mobile phase. Main disadvantages of the techni‐ que are the low reproducibility (compared to other separation techniques) and low sensitivity that makes CE not suitable for the analysis of compounds occurring in trace amounts. CE represents a good alternative for the multiple detection of inorganic and organ‐ ic acids. Many fruits and vegetables is rich in organic acids occurring in varying quantities in different fruits types, giving each fruit a unique organic acid profile. These profiles can be thus used to authenticate a vegetable product or identify the addition of another fruit type. For example the organic acids content measured by CE and LDA has been used to classify Spanish white wines [36].

Generally, NMR is superior to other spectroscopic technique because of the much richer and more detailed information that can be gathered from the NMR spectra, at least with high resolution instruments that use frequencies above 100 MHz. Those information can be used for the simultaneous quantitative determination of a number of compounds without any prior separation. Furthermore the NMR spectrum can be considered a molecular fingerprint of the test material and subjected to multivariate analysis. The main disadvantage of this technique is the elevated costs of the instruments and the running costs. Nowadays, low res‐ olution NMR instruments are available that use frequency ranging from 10 to 40 MHz. Those instruments are much cheaper and easy to use but do not provide the same detailed information as the high resolution instruments. NMR instruments are also capable of good

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A recent study on the quality control of cola beverages using NMR is exemplar of the poten‐

spectroscopy was used to discriminate with high success between premium and discount cola brands. This is important in the light of possible counterfeiting. The whole NMR spec‐ tra were used in combination with PCA. In addition, the information contained in specific regions of the NMR spectra combined with multivariate calibration (PLS) allowed the quan‐ tification of several cola ingredients (caffeine, aspartame, acesulfame-K, and benzoate) which concentration must comply with regulatory limits. NMR has been also used for the

its corresponding unsaponifiable fraction, and a subfractions of the unsaponifiable fraction (alcohol, sterol, hydrocarbon, and tocopherol fractions) were used to classify olive oils ac‐ cording to their origin [38]. The unsaponifiable fraction had to be extracted to avoid the sig‐ nal to be masked by that from the TGs in the bulk oil. The adulteration of virgin olive oil with a wide range of seed oils can be detected at level as low as 5% by means of combined

H and 31P NMR spectra and discriminant analysis provided that the virgin olive oil are fresh (as reflected by their high 1,2-diglycerides to total diglycerides ratio) [39]. In this case the multivariate analysis was performed on 13 compositional parameters derived from the spectra rather than on the whole NMR spectral fingerprint. 13C NMR spectra have been used for the authentication of fish and fish products. Discrimination between farmed and wild salmon is possible based on the NMR spectra of the muscle lipids and neural networks (PNN) and support vector machines (SVM) multivariate analysis [40]. Using the peak inten‐ sities of 12 selected chemical shifts an excellent discrimination is obtained by using PNN and SVM (98.5 and 100.0%, respectively). The authentication of different gadoid species was also achieved based on the NMR spectra of muscle lipids and Bayesian belief networks

provide useful information for the authentication of wild fish. In Figure 4, the PLS-DA

ned salmons both wild and farmed is presented. The score plot shows a clear separation of the two groups in distinct regions of the three dimensional plot. The results of the classifica‐ tion model (leave 5 out internal validation) were extremely positive with 100% of the wild samples (29 samples) and almost 100% of the farmed samples (60 out of 62 samples) correct‐

H NMR data measured in the authors' group on frozen, smoked and can‐

H NMR

H NMR spectra of the bulk olive oil,

H NMR spectroscopy can also

tial application of this technique for food authentication and fraud control [37]. 1

accuracy but the sensitivity is lower compared to MS.

authentication of the geographical origin of olive oils. 1

(BBN) with successful classification of 100% [41]. However, 1

1

scores plot for the 1

ly classified.

#### **6. Spectroscopic fingerprinting**

Spectroscopy is the study of the interaction between a material and radiated energy. The graphical representation of such interaction is what is referred to as a spectrum i.e. a plot of the response of interest as a function of the wavelength or the frequency of the radiation used (see Figure 1d). Such a spectrum is by its very nature a fingerprint of the target materi‐ al and contains information that are multivariate in nature. The extraction of the chemically relevant information from such a fingerprint requires the application of multivariate statisti‐ cal techniques. The whole spectrum is used (or part of it) to obtain a multivariate dataset for further chemometric analysis in the same way as described for raw chromatograms (see Fig‐ ure 3c). Spectral fingerprinting can be used either to classify and discriminate between sam‐ ples or to quantify a certain compounds. According to the nature of the radiating energy (infrared, visible, ultraviolet, x-rays) and the nature of the interaction between energy and matter (absorbance, emission, scattering, resonance) different kind of spectra can be ob‐ tained. In the following we will mainly focus on nuclear magnetic resonance (NMR), fluo‐ rescence spectra and infrared (IR) spectra.

#### *NMR*

NMR spectra are generated by the absorption of radiofrequency radiation by atomic nuclei with non-zero spin in a strong magnetic field. Such absorption is affected by the surround‐ ings of the atomic nucleus so that precise information about the molecular structure of a sample can be obtained. The atomic nuclei with non-zero spin that are most frequently used in NMR are 1 H, 13C even though 15N, 17O, 19F and 31P can be also employed.

Generally, NMR is superior to other spectroscopic technique because of the much richer and more detailed information that can be gathered from the NMR spectra, at least with high resolution instruments that use frequencies above 100 MHz. Those information can be used for the simultaneous quantitative determination of a number of compounds without any prior separation. Furthermore the NMR spectrum can be considered a molecular fingerprint of the test material and subjected to multivariate analysis. The main disadvantage of this technique is the elevated costs of the instruments and the running costs. Nowadays, low res‐ olution NMR instruments are available that use frequency ranging from 10 to 40 MHz. Those instruments are much cheaper and easy to use but do not provide the same detailed information as the high resolution instruments. NMR instruments are also capable of good accuracy but the sensitivity is lower compared to MS.

ques that separate charged analytes based on their electrophoretic mobility: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIF), capillary gel electrophoresis, ca‐ pillary electrochromatography. An electric field is applied to the ends of a capillary column. The ions migrate through the column in the same direction pulled by the electrosmotic flow and are separated based on their electrophoretic mobility. The signal that is generated when the mixture components are detected as they are eluted from the column is referred to as a capillary electropherogram. Multivariate dataset can be obtained from electropherogram in the same way as depicted in Figure 3. However, only strategy (a) has been used so far for authentication and fraud control purposes. CE are capable of rapid, low cost and high reso‐ lution analysis with little consumption of mobile phase. Main disadvantages of the techni‐ que are the low reproducibility (compared to other separation techniques) and low sensitivity that makes CE not suitable for the analysis of compounds occurring in trace amounts. CE represents a good alternative for the multiple detection of inorganic and organ‐ ic acids. Many fruits and vegetables is rich in organic acids occurring in varying quantities in different fruits types, giving each fruit a unique organic acid profile. These profiles can be thus used to authenticate a vegetable product or identify the addition of another fruit type. For example the organic acids content measured by CE and LDA has been used to classify

Spectroscopy is the study of the interaction between a material and radiated energy. The graphical representation of such interaction is what is referred to as a spectrum i.e. a plot of the response of interest as a function of the wavelength or the frequency of the radiation used (see Figure 1d). Such a spectrum is by its very nature a fingerprint of the target materi‐ al and contains information that are multivariate in nature. The extraction of the chemically relevant information from such a fingerprint requires the application of multivariate statisti‐ cal techniques. The whole spectrum is used (or part of it) to obtain a multivariate dataset for further chemometric analysis in the same way as described for raw chromatograms (see Fig‐ ure 3c). Spectral fingerprinting can be used either to classify and discriminate between sam‐ ples or to quantify a certain compounds. According to the nature of the radiating energy (infrared, visible, ultraviolet, x-rays) and the nature of the interaction between energy and matter (absorbance, emission, scattering, resonance) different kind of spectra can be ob‐ tained. In the following we will mainly focus on nuclear magnetic resonance (NMR), fluo‐

NMR spectra are generated by the absorption of radiofrequency radiation by atomic nuclei with non-zero spin in a strong magnetic field. Such absorption is affected by the surround‐ ings of the atomic nucleus so that precise information about the molecular structure of a sample can be obtained. The atomic nuclei with non-zero spin that are most frequently used

H, 13C even though 15N, 17O, 19F and 31P can be also employed.

Spanish white wines [36].

124 Latest Research into Quality Control

**6. Spectroscopic fingerprinting**

rescence spectra and infrared (IR) spectra.

*NMR*

in NMR are 1

A recent study on the quality control of cola beverages using NMR is exemplar of the poten‐ tial application of this technique for food authentication and fraud control [37]. 1 H NMR spectroscopy was used to discriminate with high success between premium and discount cola brands. This is important in the light of possible counterfeiting. The whole NMR spec‐ tra were used in combination with PCA. In addition, the information contained in specific regions of the NMR spectra combined with multivariate calibration (PLS) allowed the quan‐ tification of several cola ingredients (caffeine, aspartame, acesulfame-K, and benzoate) which concentration must comply with regulatory limits. NMR has been also used for the authentication of the geographical origin of olive oils. 1 H NMR spectra of the bulk olive oil, its corresponding unsaponifiable fraction, and a subfractions of the unsaponifiable fraction (alcohol, sterol, hydrocarbon, and tocopherol fractions) were used to classify olive oils ac‐ cording to their origin [38]. The unsaponifiable fraction had to be extracted to avoid the sig‐ nal to be masked by that from the TGs in the bulk oil. The adulteration of virgin olive oil with a wide range of seed oils can be detected at level as low as 5% by means of combined 1 H and 31P NMR spectra and discriminant analysis provided that the virgin olive oil are fresh (as reflected by their high 1,2-diglycerides to total diglycerides ratio) [39]. In this case the multivariate analysis was performed on 13 compositional parameters derived from the spectra rather than on the whole NMR spectral fingerprint. 13C NMR spectra have been used for the authentication of fish and fish products. Discrimination between farmed and wild salmon is possible based on the NMR spectra of the muscle lipids and neural networks (PNN) and support vector machines (SVM) multivariate analysis [40]. Using the peak inten‐ sities of 12 selected chemical shifts an excellent discrimination is obtained by using PNN and SVM (98.5 and 100.0%, respectively). The authentication of different gadoid species was also achieved based on the NMR spectra of muscle lipids and Bayesian belief networks (BBN) with successful classification of 100% [41]. However, 1 H NMR spectroscopy can also provide useful information for the authentication of wild fish. In Figure 4, the PLS-DA scores plot for the 1 H NMR data measured in the authors' group on frozen, smoked and can‐ ned salmons both wild and farmed is presented. The score plot shows a clear separation of the two groups in distinct regions of the three dimensional plot. The results of the classifica‐ tion model (leave 5 out internal validation) were extremely positive with 100% of the wild samples (29 samples) and almost 100% of the farmed samples (60 out of 62 samples) correct‐ ly classified.

matrix as in standard multivariate data sets). In the right-angle fluorescence spectroscopy the incidence angle between the excitation and the emission radiation is 90°. Only liquids and diluted solutions can be analysed and an attenuation of the signal intensity at high ab‐ sorbance (> 0.1) is observed. To overcome this problem the front-face fluorescence (FFF) has been developed where only the surface of the material is analysed and the incidence angle is around 56° to minimise the artefacts from the excitation radiation reflected or scattered by the sample. Solid and powdered samples as well as bulk liquids can be analysed by FFF.

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Fluorescence spectra are a promising tool to verify the egg freshness. Albumen samples stor‐ ed for 1,2,3 and 4 weeks can be discriminated by means of the Maillard reaction products fluorescence and factorial discriminant analysis (FDA) with high success rate [42]. Vitamin A fluorescence together with FDA allows discrimination among egg yolk samples stored for different times [43]. Similarly, the freshness of fish can be predicted based on NADH and tryptophan fluorescence spectra. NADH fluorescence spectra can be also considered as a promising tool for the discrimination between frozen-thawed fish and fresh fish. The NADH emission spectra show a typical maximum at 455 nm in fresh fish and at 379 nm in frozenthawed fish. The multivariate analysis (FDA) of the NADH spectrum allowed the correct classification of 100% of the analysed samples [44]. The authenticity of edible oils has also been extensively investigated by fluorescence spectroscopy. Chlorophyll and vitamin E are important fluorophores in olive oils and contribute greatly to oil colour and stability during storage. Refining processes decreases the content of chlorophyll and vitamin E with a corre‐ sponding change in the fluorescence spectrum. However, the discrimination between virgin and refined olive oil is mainly based on the fluorescence of lipid oxidation products (more abundant in the less resistant refined oils). A fast screening method has been also developed to detect adulteration of EVOO with olive-pomace oil [45]. It is based on the EEMs and it is able to detect adulteration at a level of 5%. Similarly, the discrimination between olive oils according to their overall acidity are also possible with fluorescence spectroscopy [46]. In this case, the maximum differentiation between the oils was obtained in the region 429-545 nm of the spectrum and allowed 100% correct classification of lampante olive oil (acidity >3.3%, not edible) from virgin olive oil with lower acidity (<3.3%). Finally, SFS with multiple regression analysis has been reported to for the detection of adulteration of EVOO with

olive oil to a level as low as 8.4% when a 80 nm wavelength interval is used [47].

techniques because the fluorescence signal has in principle no background.

*Infrared spectroscopy*

The great advantage of fluorescence spectroscopy is the rapidity, the limited costs and the non-destructive nature of the analysis. The sensitivity is also much greater than that of other

Infrared spectra are produced by measuring the intensity of the absorbance of infrared light by a sample as function of the wavelength. The absorption of infrared light is ascribed to transitions in the vibrational energies of the molecules contained in a sample. Each function‐ al group of a molecule shows characteristic IR absorption at specific frequency ranges re‐ gardless of the interaction of the functional group with the rest of the molecule. However, interaction between atoms within a molecule may sometimes affect the position of charac‐ teristic bands in a IR spectra depending on the surroundings of the functional group. IR

**Figure 4.** PLS-DA scores plot for the 1H NMR data on wild and farmed salmon (frozen, canned and smoked). Red=farmed salmons; green=wild salmons

#### *Fluorescence spectroscopy*

Fluorescence spectra are normally obtained by exciting the test material. With radiations at a fixed wavelength and recording the intensity of the emitted radiation over a range of wave‐ lengths. However, a 3D spectrum can be obtained by recording the emission spectra at dif‐ ferent excitation wavelengths: the so called fluorescence excitation-emission matrix (EEM) which provides more information about the fluorescent compounds occurring in the sam‐ ple. In synchronous fluorescence scan (SFS) the whole fluorescence landscape can also be achieved by scanning the excitation and the emission wavelengths simultaneously keeping a fixed wavelength interval (the so-called offset). Products that contains natural (or added) fluorophores are suitable for Fluorescence spectroscopy. Tryptophan, tyrosine and phenyla‐ lanine residues are fluorophores typically present in a variety of foods along with vitamin A, riboflavin (vit B2), NAD, NADH and compounds originating from Maillard reaction/ lipid oxidation. Fluorescence spectra provide information about the amount of those com‐ pounds and on the way the fluorophore environment interacts with them. Traditionally, the fluorescence spectra have been treated by means of univariate approaches i.e. taking ad‐ vantage of one specific wavelength or the derived fluorescence peak features. Nowadays the multivariate information contained in the fluorescence spectra is processed by chemometric techniques. When EEMs or SFS spectra are produced, decomposition methods such as twoway PCA, TUCKER and parallel factorial analysis (PARAFAC) are necessary to extract in‐ formation from such a multi-way dataset (the data can be arranged in a cube instead of a matrix as in standard multivariate data sets). In the right-angle fluorescence spectroscopy the incidence angle between the excitation and the emission radiation is 90°. Only liquids and diluted solutions can be analysed and an attenuation of the signal intensity at high ab‐ sorbance (> 0.1) is observed. To overcome this problem the front-face fluorescence (FFF) has been developed where only the surface of the material is analysed and the incidence angle is around 56° to minimise the artefacts from the excitation radiation reflected or scattered by the sample. Solid and powdered samples as well as bulk liquids can be analysed by FFF.

Fluorescence spectra are a promising tool to verify the egg freshness. Albumen samples stor‐ ed for 1,2,3 and 4 weeks can be discriminated by means of the Maillard reaction products fluorescence and factorial discriminant analysis (FDA) with high success rate [42]. Vitamin A fluorescence together with FDA allows discrimination among egg yolk samples stored for different times [43]. Similarly, the freshness of fish can be predicted based on NADH and tryptophan fluorescence spectra. NADH fluorescence spectra can be also considered as a promising tool for the discrimination between frozen-thawed fish and fresh fish. The NADH emission spectra show a typical maximum at 455 nm in fresh fish and at 379 nm in frozenthawed fish. The multivariate analysis (FDA) of the NADH spectrum allowed the correct classification of 100% of the analysed samples [44]. The authenticity of edible oils has also been extensively investigated by fluorescence spectroscopy. Chlorophyll and vitamin E are important fluorophores in olive oils and contribute greatly to oil colour and stability during storage. Refining processes decreases the content of chlorophyll and vitamin E with a corre‐ sponding change in the fluorescence spectrum. However, the discrimination between virgin and refined olive oil is mainly based on the fluorescence of lipid oxidation products (more abundant in the less resistant refined oils). A fast screening method has been also developed to detect adulteration of EVOO with olive-pomace oil [45]. It is based on the EEMs and it is able to detect adulteration at a level of 5%. Similarly, the discrimination between olive oils according to their overall acidity are also possible with fluorescence spectroscopy [46]. In this case, the maximum differentiation between the oils was obtained in the region 429-545 nm of the spectrum and allowed 100% correct classification of lampante olive oil (acidity >3.3%, not edible) from virgin olive oil with lower acidity (<3.3%). Finally, SFS with multiple regression analysis has been reported to for the detection of adulteration of EVOO with olive oil to a level as low as 8.4% when a 80 nm wavelength interval is used [47].

The great advantage of fluorescence spectroscopy is the rapidity, the limited costs and the non-destructive nature of the analysis. The sensitivity is also much greater than that of other techniques because the fluorescence signal has in principle no background.

#### *Infrared spectroscopy*

Factor 1 (19.8%)

Factor 2 (37.2%)

Factor 3 (14.8%)

Red=farmed salmons; green=wild salmons

*Fluorescence spectroscopy*

126 Latest Research into Quality Control

**Figure 4.** PLS-DA scores plot for the 1H NMR data on wild and farmed salmon (frozen, canned and smoked).

Fluorescence spectra are normally obtained by exciting the test material. With radiations at a fixed wavelength and recording the intensity of the emitted radiation over a range of wave‐ lengths. However, a 3D spectrum can be obtained by recording the emission spectra at dif‐ ferent excitation wavelengths: the so called fluorescence excitation-emission matrix (EEM) which provides more information about the fluorescent compounds occurring in the sam‐ ple. In synchronous fluorescence scan (SFS) the whole fluorescence landscape can also be achieved by scanning the excitation and the emission wavelengths simultaneously keeping a fixed wavelength interval (the so-called offset). Products that contains natural (or added) fluorophores are suitable for Fluorescence spectroscopy. Tryptophan, tyrosine and phenyla‐ lanine residues are fluorophores typically present in a variety of foods along with vitamin A, riboflavin (vit B2), NAD, NADH and compounds originating from Maillard reaction/ lipid oxidation. Fluorescence spectra provide information about the amount of those com‐ pounds and on the way the fluorophore environment interacts with them. Traditionally, the fluorescence spectra have been treated by means of univariate approaches i.e. taking ad‐ vantage of one specific wavelength or the derived fluorescence peak features. Nowadays the multivariate information contained in the fluorescence spectra is processed by chemometric techniques. When EEMs or SFS spectra are produced, decomposition methods such as twoway PCA, TUCKER and parallel factorial analysis (PARAFAC) are necessary to extract in‐ formation from such a multi-way dataset (the data can be arranged in a cube instead of a

Infrared spectra are produced by measuring the intensity of the absorbance of infrared light by a sample as function of the wavelength. The absorption of infrared light is ascribed to transitions in the vibrational energies of the molecules contained in a sample. Each function‐ al group of a molecule shows characteristic IR absorption at specific frequency ranges re‐ gardless of the interaction of the functional group with the rest of the molecule. However, interaction between atoms within a molecule may sometimes affect the position of charac‐ teristic bands in a IR spectra depending on the surroundings of the functional group. IR spectra can thus provide qualitative information about the nature of the functional group present in a food sample and quantitative information on their amount. When the effect of all the functional groups is taken together, the whole spectrum represents a molecular fin‐ gerprint that can be used to verify the nature of the sample. The IR region of the electromag‐ netic spectrum can be divided in 3 portion: The far IR (FIR, 400-10 cm-1) has the lower energy and induces rotational transitions in the molecules. The mid IR (MIR, 4000-400 cm-1) induces fundamental vibrational transitions in the molecules. The near IR region (NIR, 14000-4000 cm-1) also induces transitions in the vibrational energies of the molecules. How‐ ever, the transitions of the vibrational energy induced by the NIR portion of the spectrum are more complex than those induced by the MIR region. Overtones (transitions from the fundamental vibrational level over two or higher energy levels) and combination modes (arising from the interaction of two or more vibrations taking place simultaneously in differ‐ ent functional groups) give rise to very complex bands in the NIR spectrum that can give more complex structural information than MIR. On the other hand, NIR spectra are less se‐ lective than MIR spectra because of the superposition of different overtones and combina‐ tion bands. A raw spectrum contains background information and noise beside valuable information. To remove those interferences as well as those coming from scattering, to nor‐ malise the effect of particle size and light distance, pre-processing methods such as smooth‐ ing, derivative, standard normal variate transformation (SNC), multiplicative scatter correction (MSC) or wavelet transforms (WT) are required. Recently, the introduction of the Fourier transform technique in IR (FTIR) has further increased the application range of the IR spectroscopy in the food field. In such a case the spectrum is obtained by mean of an in‐ terferogram in which multiple frequencies are measured simultaneously. The resulting in‐ terferogram is then deconvoluted using proper algorithms in order to have the original spectrum. The advantages of that technology is a faster analysis and a higher throughput and a better alignment of spectrum obtained by repetitive scans.

olive oils has also attracted much attention due to the economic value of the product. Adul‐ teration of EVOO with palm oil can be detected by FTIR and PLS in concentration varying from 1.0 to 50.0% w/w [56]. The region 1500-1000 cm-1 of the MIR spectra was used for the regression model. The adulteration of EVOO with sunflower, corn, soyabean and hazelnut oil can be detected at level as low as 5% by using FTIR and LDA [57]. In this latter case, the normalized absorbance of peaks and shoulders areas were used in the model as predictors. The standard of identity for butter require that no vegetal oil nor margarine is added to the product. The presence of margarine can be detected by NIR coupled with PLS in the range 0-100% with a standard error of calibration after validation (SECV) <1.2% [58]. IR spectro‐ scopy has been also widely used to predict and control meat quality. The discrimination be‐ tween fresh and frozen-thawed beef can be accomplished by IR spectroscopy due to modification of the myofibrillar proteins and the corresponding change in their water hold‐

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Infrared spectroscopy is a well-established technique for fast, high-throughput and non-de‐ structive analysis of food and other biological samples. The analysis can be easily imple‐ mented on-line, can be automated and does not requires trained personnel to be carried out. It is little expensive and environmental friendly since does not require solvents, chemicals and does not produce waste. However, even though the analysis per se (collection of the spectrum) is fast, post-processing (pre-processing of the spectra and model building) of the input data can be laborious and time-consuming. The calibration models are usually built against reference analytical methods so that the measurement errors accumulated and the total predictive error increases. Finally, the classification or regression models are theoreti‐ cally valid only on the instruments with which the training and the calibration has been car‐ ried out. The transfer of a multivariate model to other instruments affects its precision and

As an example of the potential of the NIR Spectroscopy for the detection of adulteration, the results of an investigation that has been carried out in the authors' research group will be shown. NIR spectra were used to detect the presence of nitrogen replacers in milk powders. The compositional standards for milk powders require that the amount of milk proteins in milk solids-not-fat should be at least equal to 34% m/m, unless declared. The low prices of some nitrogen containing compounds make them attractive as potential adulterants to in‐ crease the level of apparent proteins in milk powders. The Kjeldahl method (official refer‐ ence method for proteins content) measures the total amount of N irrespective of whether it comes from proteins or not. Expensive and time-consuming analytical methods such as en‐ zyme-linked immunosorbent assay (ELISA), LC-MS/MS and GC-MS/MS are necessary for confirmatory analysis of melamine and its analogues in milk powders. To prove the poten‐ tial of NIR spectroscopy for the detection of such adulteration, 33 skim milk powders were randomly adulterated with adulterants ammonium chloride, caprolactam, diammonium phosphate and polyvinylpyrrolidone (PVP) in order to produce an increment of 0.10, 0.50, 1.00 and 2.00% in the (apparent) proteins content of the milk powder. The samples were measured by NIR spectroscopy and the spectra subjected to PLS-DA analysis. A few milk

ing capacity [59].

accuracy compared to the original ones.

*A case study on NIRS and adulteration*.

IR spectroscopy have been successfully applied to detect adulteration of juices, purees and syrups with cheaper juice concentrates. Adulteration of orange juice with orange pulpwash, grapefruit juice or synthetic sugars/acids mixture can be detected at a level as low as 50 g/kg by NIR [48]. Similarly, the adulteration of strawberry or raspberry juice with apple juice can be detected at level> 10% by transmittance NIR coupled with PLS [49]. MIR spectra have been used to detect adulteration of pure pomegranate juice with grape juice (2%-14% v/v) [50] and to predict the percent fruit content in strawberry jam [51]. Adulteration of honey and maple syrup can also be detected by NIR and MIR spectroscopy. NIR and FTIR have been successfully applied for the detection and quantification of cane and beet sugars in ma‐ ple syrup [52]. Attenuated total reflectance (ATR)-FTIR coupled with LDA and PLS was used to discriminate the type of adulterant in three different honey varieties. A success rate of prediction of 100% was achieved for honey samples adulterated with 7-25% w/w of sim‐ ple (glucose, fructose, sucrose) and complex (beet and cane invert) sugars [53]. NIR and MIR have also been employed for the authentication of lard and fats. Lard adulteration can be detected in cake [54] and in chocolate [55]. The adulteration of shortening with lard can be detected at levels ranging from 0 to 100% and a standard error of calibration (SEC) of 1.75 by using the regions 1.117-1.097 cm-1 and 990-950 cm-1 of the NIR spectrum. Adulteration of olive oils has also attracted much attention due to the economic value of the product. Adul‐ teration of EVOO with palm oil can be detected by FTIR and PLS in concentration varying from 1.0 to 50.0% w/w [56]. The region 1500-1000 cm-1 of the MIR spectra was used for the regression model. The adulteration of EVOO with sunflower, corn, soyabean and hazelnut oil can be detected at level as low as 5% by using FTIR and LDA [57]. In this latter case, the normalized absorbance of peaks and shoulders areas were used in the model as predictors. The standard of identity for butter require that no vegetal oil nor margarine is added to the product. The presence of margarine can be detected by NIR coupled with PLS in the range 0-100% with a standard error of calibration after validation (SECV) <1.2% [58]. IR spectro‐ scopy has been also widely used to predict and control meat quality. The discrimination be‐ tween fresh and frozen-thawed beef can be accomplished by IR spectroscopy due to modification of the myofibrillar proteins and the corresponding change in their water hold‐ ing capacity [59].

Infrared spectroscopy is a well-established technique for fast, high-throughput and non-de‐ structive analysis of food and other biological samples. The analysis can be easily imple‐ mented on-line, can be automated and does not requires trained personnel to be carried out. It is little expensive and environmental friendly since does not require solvents, chemicals and does not produce waste. However, even though the analysis per se (collection of the spectrum) is fast, post-processing (pre-processing of the spectra and model building) of the input data can be laborious and time-consuming. The calibration models are usually built against reference analytical methods so that the measurement errors accumulated and the total predictive error increases. Finally, the classification or regression models are theoreti‐ cally valid only on the instruments with which the training and the calibration has been car‐ ried out. The transfer of a multivariate model to other instruments affects its precision and accuracy compared to the original ones.

#### *A case study on NIRS and adulteration*.

spectra can thus provide qualitative information about the nature of the functional group present in a food sample and quantitative information on their amount. When the effect of all the functional groups is taken together, the whole spectrum represents a molecular fin‐ gerprint that can be used to verify the nature of the sample. The IR region of the electromag‐ netic spectrum can be divided in 3 portion: The far IR (FIR, 400-10 cm-1) has the lower energy and induces rotational transitions in the molecules. The mid IR (MIR, 4000-400 cm-1) induces fundamental vibrational transitions in the molecules. The near IR region (NIR, 14000-4000 cm-1) also induces transitions in the vibrational energies of the molecules. How‐ ever, the transitions of the vibrational energy induced by the NIR portion of the spectrum are more complex than those induced by the MIR region. Overtones (transitions from the fundamental vibrational level over two or higher energy levels) and combination modes (arising from the interaction of two or more vibrations taking place simultaneously in differ‐ ent functional groups) give rise to very complex bands in the NIR spectrum that can give more complex structural information than MIR. On the other hand, NIR spectra are less se‐ lective than MIR spectra because of the superposition of different overtones and combina‐ tion bands. A raw spectrum contains background information and noise beside valuable information. To remove those interferences as well as those coming from scattering, to nor‐ malise the effect of particle size and light distance, pre-processing methods such as smooth‐ ing, derivative, standard normal variate transformation (SNC), multiplicative scatter correction (MSC) or wavelet transforms (WT) are required. Recently, the introduction of the Fourier transform technique in IR (FTIR) has further increased the application range of the IR spectroscopy in the food field. In such a case the spectrum is obtained by mean of an in‐ terferogram in which multiple frequencies are measured simultaneously. The resulting in‐ terferogram is then deconvoluted using proper algorithms in order to have the original spectrum. The advantages of that technology is a faster analysis and a higher throughput

128 Latest Research into Quality Control

and a better alignment of spectrum obtained by repetitive scans.

IR spectroscopy have been successfully applied to detect adulteration of juices, purees and syrups with cheaper juice concentrates. Adulteration of orange juice with orange pulpwash, grapefruit juice or synthetic sugars/acids mixture can be detected at a level as low as 50 g/kg by NIR [48]. Similarly, the adulteration of strawberry or raspberry juice with apple juice can be detected at level> 10% by transmittance NIR coupled with PLS [49]. MIR spectra have been used to detect adulteration of pure pomegranate juice with grape juice (2%-14% v/v) [50] and to predict the percent fruit content in strawberry jam [51]. Adulteration of honey and maple syrup can also be detected by NIR and MIR spectroscopy. NIR and FTIR have been successfully applied for the detection and quantification of cane and beet sugars in ma‐ ple syrup [52]. Attenuated total reflectance (ATR)-FTIR coupled with LDA and PLS was used to discriminate the type of adulterant in three different honey varieties. A success rate of prediction of 100% was achieved for honey samples adulterated with 7-25% w/w of sim‐ ple (glucose, fructose, sucrose) and complex (beet and cane invert) sugars [53]. NIR and MIR have also been employed for the authentication of lard and fats. Lard adulteration can be detected in cake [54] and in chocolate [55]. The adulteration of shortening with lard can be detected at levels ranging from 0 to 100% and a standard error of calibration (SEC) of 1.75 by using the regions 1.117-1.097 cm-1 and 990-950 cm-1 of the NIR spectrum. Adulteration of

As an example of the potential of the NIR Spectroscopy for the detection of adulteration, the results of an investigation that has been carried out in the authors' research group will be shown. NIR spectra were used to detect the presence of nitrogen replacers in milk powders. The compositional standards for milk powders require that the amount of milk proteins in milk solids-not-fat should be at least equal to 34% m/m, unless declared. The low prices of some nitrogen containing compounds make them attractive as potential adulterants to in‐ crease the level of apparent proteins in milk powders. The Kjeldahl method (official refer‐ ence method for proteins content) measures the total amount of N irrespective of whether it comes from proteins or not. Expensive and time-consuming analytical methods such as en‐ zyme-linked immunosorbent assay (ELISA), LC-MS/MS and GC-MS/MS are necessary for confirmatory analysis of melamine and its analogues in milk powders. To prove the poten‐ tial of NIR spectroscopy for the detection of such adulteration, 33 skim milk powders were randomly adulterated with adulterants ammonium chloride, caprolactam, diammonium phosphate and polyvinylpyrrolidone (PVP) in order to produce an increment of 0.10, 0.50, 1.00 and 2.00% in the (apparent) proteins content of the milk powder. The samples were measured by NIR spectroscopy and the spectra subjected to PLS-DA analysis. A few milk powders were randomly selected and adulterated by melamine, ammelide and urea to test the robustness of the predictive models.

tration) and 3 adulterated samples could not be classified. Samples adulterated with mela‐

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131


> **Adulterated powder**

Internal validation

0 68 8

1 22 3

External validation

**No match**

**Figure 5.** PLS-DA scores plot of IR spectra for genuine and adulterated milk powders. Red = adulterated powders;

**Genuine powder**

Genuine powder 24 2 -

Genuine powder 6 1 0

NIRS combined with chemometrics proved to be a promising tool for the cost-effective de‐ tection of adulteration of milk powders with a range of nitrogen replacers. The reliability of the classification models can be improved by the careful selection of those regions of the NIR-visible spectrum which showed the best discrimination power between genuine and

mine, ammelide and urea were all correctly predicted as adulterated.


**Class Item**

> Adulterated powder

> Adulterated powder

**Table 3.** Prediction results of PLS-DA model for genuine versus adulterated milk powders.

0

Factor 2 (14.8%)

green=genuine powders.

adulterated samples.

20

A PLS-DA model was first developed to predict the type of adulterant. The training set con‐ sisted of 80% genuine milk powders (26 samples) and 80% adulterated samples (19 samples from each adulterant) which were randomly selected. The remaining 20% of the samples, and those adulterated with the non-modelled adulterants melamine, ammelide and urea were used for external validation. The success rate of prediction was 100% in cross-valida‐ tion and 78% for the external validation set (Table 2). Three out of 6 samples adulterated with melamine, ammelide and urea were correctly predicted as adulterated.


**Table 2.** Prediction results of PLS-DA model for the type of adulterant in milk powders

A PLS-DA model was then developed to discriminate generally between genuine and adul‐ terated samples. The training set and the validation set were built in the same way as previ‐ ously described. The PLS-DA scores plot is presented in Figure 4. The prediction results are reported in Table 3.

In external validation only one genuine sample was wrongly predicted as adulterated. One adulterated sample was incorrectly predicted as genuine (PVP added at its lowest concen‐ tration) and 3 adulterated samples could not be classified. Samples adulterated with mela‐ mine, ammelide and urea were all correctly predicted as adulterated.

powders were randomly selected and adulterated by melamine, ammelide and urea to test

A PLS-DA model was first developed to predict the type of adulterant. The training set con‐ sisted of 80% genuine milk powders (26 samples) and 80% adulterated samples (19 samples from each adulterant) which were randomly selected. The remaining 20% of the samples, and those adulterated with the non-modelled adulterants melamine, ammelide and urea were used for external validation. The success rate of prediction was 100% in cross-valida‐ tion and 78% for the external validation set (Table 2). Three out of 6 samples adulterated

> Genuine powder 26 - - - - - + NH4Cl - 19 - - - - +caprolactam - - 19 - - - + (NH4)2HPO4 - - - 19 - - +PVP - - - - 19 - External validation

> Genuine powder 7 - - - - - + NH4Cl - 4 - - 1 - +caprolactam - - 3 - 1 1 + (NH4)2HPO4 - - - 4 - 1 +PVP - - - - 3 2

+melamine - - - - - 2 +ammelide 2 - - - - - + urea 1 - - - - 1

A PLS-DA model was then developed to discriminate generally between genuine and adul‐ terated samples. The training set and the validation set were built in the same way as previ‐ ously described. The PLS-DA scores plot is presented in Figure 4. The prediction results are

In external validation only one genuine sample was wrongly predicted as adulterated. One adulterated sample was incorrectly predicted as genuine (PVP added at its lowest concen‐

**Table 2.** Prediction results of PLS-DA model for the type of adulterant in milk powders

**+ NH4Cl + caprolactam + (NH4)2HPO4 + PVP No**

PLS-DA model based on training set

External validation based on melamine, ammelide and urea

**match**

with melamine, ammelide and urea were correctly predicted as adulterated.

**Genuine powder**

the robustness of the predictive models.

130 Latest Research into Quality Control

**Class item**

reported in Table 3.

**Figure 5.** PLS-DA scores plot of IR spectra for genuine and adulterated milk powders. Red = adulterated powders; green=genuine powders.


**Table 3.** Prediction results of PLS-DA model for genuine versus adulterated milk powders.

NIRS combined with chemometrics proved to be a promising tool for the cost-effective de‐ tection of adulteration of milk powders with a range of nitrogen replacers. The reliability of the classification models can be improved by the careful selection of those regions of the NIR-visible spectrum which showed the best discrimination power between genuine and adulterated samples.

#### **7. Other fingerprintings**

#### *Differential scanning calorimetry (DSC)*

Differential scanning calorimetry is a themoanalytical technique that measures the amount of heat required to increase the temperature of a sample relative to a reference material. When the amount of heat absorbed/released is plotted as a function of temperature a ther‐ mal spectrum of the sample is obtained from which kinetic and thermodynamic information such as the heat capacity and the enthalpy of any phase transition (fusion, evaporation, glass transition etc) the sample undergoes in the temperature span of the experiments can be de‐ termined. Those physical properties depends in turn on compositional and structural prop‐ erties of the samples.

*Transcriptomics*

respect is still underexplored.

**8. Fingerprinting options for other biomaterials**

oil can be predicted with good accuracy when a PLS model is used [68].

Transcriptomics is a post-genomic technique that consists in the simultaneous measure‐ ments of all the transcripts (mRNA molecules) in a given organism, or of a specific subset of transcripts present in a particular cell type. Unlike genome, transcriptome varies according to the environmental conditions and represents the genes that are actively expressed in a certain cell at a certain time. Transcriptome is usually obtained by DNA microarray technol‐ ogies and reverse PCR. The set of all the mRNA produced (and hence of the genes actively expressed) represents a fingerprint of that target cell/organism and can thus use for fraud detection and authentication purposes. At the present the potential of transcriptomics in this

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Biofuels are an important environmental-friendly alternative to fossil fuels. The term biodie‐ sel refers primarily to FAME obtained after transesterification of triglycerides with metha‐ nol. The methyl esters can be produced from many different triglyceride sources, primarily rapeseed oil but also sunflower oil, soybean oil, palm oil, linseed oil, tallow, and used frying oil. Blends of biodiesel with conventional petrodiesel fuel represent a common utilization of biodiesel. The ability to predict retail biodiesel blend percent composition is important to detect adulterations. It has been reported that biodiesel from different sources can be discri‐ minated by direct infusion ESI-MS and multivariate statistics [66]. In addition, the % of rape‐ seed or salmon biodiesel in petrodiesel in concentrations ranging from 0.5% to 10% can be predicted with good accuracy by applying a PLS model. Similarly, the feedstock source of blends of biodiesel and conventional diesel, as well as the % composition of the blend can be predicted applying a kNN and a PLS model respectively to the total ion current chromato‐ grams from gas chromatography–quadrupole mass spectrometry (GC–qMS) using a polar column [67]. The precision of the prediction was between 4-5%. Furthermore, the addition of residual oil (non-transesterified residual vegetable oil) is one of the easiest ways of adulter‐ ating biofuels. Synchronous fluorescence combined to LDA can be used to discriminate be‐ tween diesel oils, biodiesels and biodiesel adulterated with residual oil and the % of residual

Perfume counterfeiting is an illegal practice that causes huge economical loss to the perfume industry and pose potential health risk to consumers who might be exposed to harmful chemical from counterfeited products. Traditionally, the quality control analyses for per‐ fume focuses on volatile and semi-volatile compounds and are performed by GC based tech‐ niques. Recently, more straightforward methods have been proposed for the fast authentication of premium perfumes and detection of adulteration/counterfeiting. A finger‐ print of the polar compounds can be achieved by direct infusion EASI-MS (see section 3) [69]. The samples are sprayed onto a glass rod and directly exposed to the ionisation source of the MS system. An almost instantaneous, simple and reproducible fingerprint of the polar compounds in the product is obtained that allows a complete discrimination between au‐

DSC has been mainly applied for the authentication of fats/oils. The level of adulteration of canola oil with lard, beef tallow and chicken fat as well as that of virgin coconut oil with palm kernel oil (PKO) and soybean oil can be predicted by the analysis of the DSC thermo‐ gram and stepwise multilinear regression (SMLR) [60]. The melting profiles of cow, goat, sheep, camel, horse and water buffalo milk fat samples were also determined by DSC. Dif‐ ferences in the DSC profiles for the fat fraction of milk of different species can be attributed to their fatty acid/TAG composition. In general, the melting point of the fats decreases with decreasing chain length and increasing degree of unsaturation of the fatty acids in the milk. The thermograms were subjected to PCA analysis which showed a clear distinction of the four milk samples analysed. The loadings plots of the heat flow data showed that the data are most influenced between the temperatures of 13° and 24° C [61].

#### *Sensor technology*

Sensor technology is often referred to as electronic nose technology. In this technology the volatile compounds present in the headspace of a sample are detected by an array of semiselective sensors. Normally each sensor is sensitive to all the volatile but each in a peculiar way. There are many sensors for EN instruments such as metal oxide sensors, conducting polymer sensors, quartz crystal membrane sensors, or mass spectrometers (MS-EN) availa‐ ble on the market. The sensor's response is then transformed in a spectrum that represents a fingerprint of the volatile compounds of the tested sample. Electronic nose analysis are often cheaper and faster than GC analysis and the sample preparation is usually quite simple. The technique, however, does not have the same sensitivity of other techniques. Furthermore, single volatile compounds cannot be identified and the signal is sensitive to water vapour.

Electronic nose has been used mainly for the detection of adulteration in fats/oils. Rapid de‐ tection of pork and lard in food samples for halal authentication (compliance with Islamic dietary rules) are reported with electronic nose [62]. Under Jewish and Islamic dietary laws, foods containing porcine-based ingredients such as lard are strictly prohibited from con‐ sumption. Electronic nose and chemometric analysis was applied for the detection of adul‐ teration of olive oil samples with sunflower and olive-pomace oil at levels as low as 5% [63]. Application are also reported for the authentication of the geographical origin of Italian wines [64], and emmenthal cheese [65].

#### *Transcriptomics*

**7. Other fingerprintings**

132 Latest Research into Quality Control

erties of the samples.

*Sensor technology*

wines [64], and emmenthal cheese [65].

*Differential scanning calorimetry (DSC)*

Differential scanning calorimetry is a themoanalytical technique that measures the amount of heat required to increase the temperature of a sample relative to a reference material. When the amount of heat absorbed/released is plotted as a function of temperature a ther‐ mal spectrum of the sample is obtained from which kinetic and thermodynamic information such as the heat capacity and the enthalpy of any phase transition (fusion, evaporation, glass transition etc) the sample undergoes in the temperature span of the experiments can be de‐ termined. Those physical properties depends in turn on compositional and structural prop‐

DSC has been mainly applied for the authentication of fats/oils. The level of adulteration of canola oil with lard, beef tallow and chicken fat as well as that of virgin coconut oil with palm kernel oil (PKO) and soybean oil can be predicted by the analysis of the DSC thermo‐ gram and stepwise multilinear regression (SMLR) [60]. The melting profiles of cow, goat, sheep, camel, horse and water buffalo milk fat samples were also determined by DSC. Dif‐ ferences in the DSC profiles for the fat fraction of milk of different species can be attributed to their fatty acid/TAG composition. In general, the melting point of the fats decreases with decreasing chain length and increasing degree of unsaturation of the fatty acids in the milk. The thermograms were subjected to PCA analysis which showed a clear distinction of the four milk samples analysed. The loadings plots of the heat flow data showed that the data

Sensor technology is often referred to as electronic nose technology. In this technology the volatile compounds present in the headspace of a sample are detected by an array of semiselective sensors. Normally each sensor is sensitive to all the volatile but each in a peculiar way. There are many sensors for EN instruments such as metal oxide sensors, conducting polymer sensors, quartz crystal membrane sensors, or mass spectrometers (MS-EN) availa‐ ble on the market. The sensor's response is then transformed in a spectrum that represents a fingerprint of the volatile compounds of the tested sample. Electronic nose analysis are often cheaper and faster than GC analysis and the sample preparation is usually quite simple. The technique, however, does not have the same sensitivity of other techniques. Furthermore, single volatile compounds cannot be identified and the signal is sensitive to water vapour. Electronic nose has been used mainly for the detection of adulteration in fats/oils. Rapid de‐ tection of pork and lard in food samples for halal authentication (compliance with Islamic dietary rules) are reported with electronic nose [62]. Under Jewish and Islamic dietary laws, foods containing porcine-based ingredients such as lard are strictly prohibited from con‐ sumption. Electronic nose and chemometric analysis was applied for the detection of adul‐ teration of olive oil samples with sunflower and olive-pomace oil at levels as low as 5% [63]. Application are also reported for the authentication of the geographical origin of Italian

are most influenced between the temperatures of 13° and 24° C [61].

Transcriptomics is a post-genomic technique that consists in the simultaneous measure‐ ments of all the transcripts (mRNA molecules) in a given organism, or of a specific subset of transcripts present in a particular cell type. Unlike genome, transcriptome varies according to the environmental conditions and represents the genes that are actively expressed in a certain cell at a certain time. Transcriptome is usually obtained by DNA microarray technol‐ ogies and reverse PCR. The set of all the mRNA produced (and hence of the genes actively expressed) represents a fingerprint of that target cell/organism and can thus use for fraud detection and authentication purposes. At the present the potential of transcriptomics in this respect is still underexplored.

#### **8. Fingerprinting options for other biomaterials**

Biofuels are an important environmental-friendly alternative to fossil fuels. The term biodie‐ sel refers primarily to FAME obtained after transesterification of triglycerides with metha‐ nol. The methyl esters can be produced from many different triglyceride sources, primarily rapeseed oil but also sunflower oil, soybean oil, palm oil, linseed oil, tallow, and used frying oil. Blends of biodiesel with conventional petrodiesel fuel represent a common utilization of biodiesel. The ability to predict retail biodiesel blend percent composition is important to detect adulterations. It has been reported that biodiesel from different sources can be discri‐ minated by direct infusion ESI-MS and multivariate statistics [66]. In addition, the % of rape‐ seed or salmon biodiesel in petrodiesel in concentrations ranging from 0.5% to 10% can be predicted with good accuracy by applying a PLS model. Similarly, the feedstock source of blends of biodiesel and conventional diesel, as well as the % composition of the blend can be predicted applying a kNN and a PLS model respectively to the total ion current chromato‐ grams from gas chromatography–quadrupole mass spectrometry (GC–qMS) using a polar column [67]. The precision of the prediction was between 4-5%. Furthermore, the addition of residual oil (non-transesterified residual vegetable oil) is one of the easiest ways of adulter‐ ating biofuels. Synchronous fluorescence combined to LDA can be used to discriminate be‐ tween diesel oils, biodiesels and biodiesel adulterated with residual oil and the % of residual oil can be predicted with good accuracy when a PLS model is used [68].

Perfume counterfeiting is an illegal practice that causes huge economical loss to the perfume industry and pose potential health risk to consumers who might be exposed to harmful chemical from counterfeited products. Traditionally, the quality control analyses for per‐ fume focuses on volatile and semi-volatile compounds and are performed by GC based tech‐ niques. Recently, more straightforward methods have been proposed for the fast authentication of premium perfumes and detection of adulteration/counterfeiting. A finger‐ print of the polar compounds can be achieved by direct infusion EASI-MS (see section 3) [69]. The samples are sprayed onto a glass rod and directly exposed to the ionisation source of the MS system. An almost instantaneous, simple and reproducible fingerprint of the polar compounds in the product is obtained that allows a complete discrimination between au‐ thentic and counterfeited products. A fast discrimination between authentic and counterfeit‐ ed products can be also obtained by fingerprinting of the polar compounds by ESI-MS in the positive ion mode and chemometric analysis previous extraction of few μL of the sample in a 1:1 methanol/water solution [70]. Even more recently, a fast, simple and low-cost method for the authentication of perfume based on a commercial electronic olfactory system (EOS) equipped with thin film metal oxide semiconductors has been proposed [71]. The PCA anal‐ ysis of the R/R0 values (resistance of the sensor in the presence of the volatile compounds relative to that of the sensor balanced in air) generated by 6 sensors based on different metal oxide semiconductors can unequivocally discriminate between authentic and counterfeited perfumes. The prior removal of ethanol from the samples is necessary for the correct dis‐ crimination of the samples.

cate herbal products. For instance, different species of *Echinacea*, *e.g*., *E. purpurea*, *E. angustifolia*, and *E. pallida* are used for commercial preparations to prevent or cure the com‐ mon cold, flu, and several other diseases due to their nonspecific stimulating effect of the immune system. It is also well known that *Echinacea* preparation are commonly adulterated with roots of *Parthenium integrifolium* L. This adulteration can be detected by NIRS at a mini‐ mum of 10% of adulteration [75]. The method requires just the milling of the sample and can

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The list of potential applications of products fingerprinting is not limited to the cases dis‐ cussed above and many other examples may be provided. In the authors' research group for instance, PTR-MS and ICP-MS are used for the authentication of the geographical origin of flower bulbs. Counterfeiting of pharmaceuticals is another area where the application of fin‐ gerprinting techniques has proved of great help. The topic is so huge that we would address

Product fingerprinting combined with chemometrics represents a valuable tool for fraud de‐ tection and control for food products and other biomaterials. A fingerprinting approach is

**•** For the authentication of products for which target analyses based on specific markers are

**•** For a fast and high-throughput screening of the samples before more elaborated confir‐

At the same time a fingerprinting approach may substantiate nutritional, sensory or other

LC, GC, and IR spectroscopy are already common instrumental platforms available in most QA laboratories and they will continue to provide valuable support for food fraud preven‐ tion. IR and other spectroscopic techniques have the great advantage of providing fast, highthroughput and non-destructive analyses with limited costs. They can be easily automated and adapted for *in-line* or *in-situ* analysis which makes these techniques well suited for im‐ plementation in the industrial setting. MS and NMR are not as common in QA laboratories, principally because of their high costs but they may become more important in routine QA testing because of their superior performances. However, even though the costs of MS and NMR instruments is still very high, the cost per sample can be very low if a high samples

From an analytical point of view, a further improvement is expected in the future from the broader application of multi-dimensional separation techniques such as GCxGC or LCxLC which provide enhanced resolution and an higher number of peaks. Ultra-performance liq‐

**•** For the detection of adulteration based of yet unknown adulterants

be carried out within 1 minute.

**9. Conclusions**

particularly useful:

not available

product qualities.

turnover can be achieved.

matory analysis are applied.

the interested readers to specialized publications.

Essential oils are also widely employed for their fragrance in perfumery but also in cosmet‐ ics and household products. One of the most common fraud is the blend of valuable essen‐ tial oils with other less valuable alternatives. As an example, the valuable Rosewood essential oil, obtained from the trees of *Aniba rosaeodora* Ducke and employed in fine per‐ fumery, can be blended with the far cheaper synthetic linaool, obtained by re-distillation of Rosewood leaf oil. The ESI-MS fingerprint in the positive ions mode of the polar compounds extracted by an acidified 1:1 water methanol solution can easily detect adulteration of Rose‐ wood oil with synthetic linaool at concentration as low as 10% v/v [72]. A PCA model is nec‐ essary to extract the relevant information from the fingerprint.

Direct infusion MS can be used to authenticate wood as well. Venturi easy ambient sonic spray ionization (V-EASI)-MS fingerprint of a very simple methanolic extract of wood chips or directly acquired from the freshly scratched wood surface may help to control the illegal logging and trade of the noble Mahogany tropical wood and its falsification [73]. V-EASI-MS is a novel ambient ionization MS technique characterized by sonic spray ionization and a self-pumping system based on the Venturi effect. It allows the direct analysis of solid or liquid samples. Ionization is assisted by compressed nitrogen and the apparatus is thus free of electrical discharge, thermal interferences since no heating, voltage or radiation is used. The introduction of fast and high-throughput analytical techniques for wood authentication is especially valuable since the classical controls are based on time-consuming morphologi‐ cal evaluations. Recently an original and fast approach for the authentication of wood spe‐ cies has been proposed. It is based on the analysis of the volatile compounds measured by a low-cost conductive polymer-based portable electronic nose formed by an array of only three gas sensors and/or the elemental fingerprint measured by laser-induced breakdown spectrom‐ etry (LIBS) which performs a multielemental and direct analysis even in solid samples [74].

The assurance of quality of herbal supplements and medicines is a major concern for the phytopharmaceutical and the food industry. The identification of the herbal drug and the presence of adulterant is a mandatory test to ensure the quality, the efficacy and the safety of a medical preparation or an herbal supplement. Among a variety of quality control meth‐ ods, chromatographic fingerprinting has gained more and more attention recently and have been used to authenticate a large number of herbal products. They are accepted by many international organization for the quality control of herbal medicine but are relatively timeconsuming. More recently, spectroscopic techniques have been explored to rapidly authenti‐ cate herbal products. For instance, different species of *Echinacea*, *e.g*., *E. purpurea*, *E. angustifolia*, and *E. pallida* are used for commercial preparations to prevent or cure the com‐ mon cold, flu, and several other diseases due to their nonspecific stimulating effect of the immune system. It is also well known that *Echinacea* preparation are commonly adulterated with roots of *Parthenium integrifolium* L. This adulteration can be detected by NIRS at a mini‐ mum of 10% of adulteration [75]. The method requires just the milling of the sample and can be carried out within 1 minute.

The list of potential applications of products fingerprinting is not limited to the cases dis‐ cussed above and many other examples may be provided. In the authors' research group for instance, PTR-MS and ICP-MS are used for the authentication of the geographical origin of flower bulbs. Counterfeiting of pharmaceuticals is another area where the application of fin‐ gerprinting techniques has proved of great help. The topic is so huge that we would address the interested readers to specialized publications.

#### **9. Conclusions**

thentic and counterfeited products. A fast discrimination between authentic and counterfeit‐ ed products can be also obtained by fingerprinting of the polar compounds by ESI-MS in the positive ion mode and chemometric analysis previous extraction of few μL of the sample in a 1:1 methanol/water solution [70]. Even more recently, a fast, simple and low-cost method for the authentication of perfume based on a commercial electronic olfactory system (EOS) equipped with thin film metal oxide semiconductors has been proposed [71]. The PCA anal‐ ysis of the R/R0 values (resistance of the sensor in the presence of the volatile compounds relative to that of the sensor balanced in air) generated by 6 sensors based on different metal oxide semiconductors can unequivocally discriminate between authentic and counterfeited perfumes. The prior removal of ethanol from the samples is necessary for the correct dis‐

Essential oils are also widely employed for their fragrance in perfumery but also in cosmet‐ ics and household products. One of the most common fraud is the blend of valuable essen‐ tial oils with other less valuable alternatives. As an example, the valuable Rosewood essential oil, obtained from the trees of *Aniba rosaeodora* Ducke and employed in fine per‐ fumery, can be blended with the far cheaper synthetic linaool, obtained by re-distillation of Rosewood leaf oil. The ESI-MS fingerprint in the positive ions mode of the polar compounds extracted by an acidified 1:1 water methanol solution can easily detect adulteration of Rose‐ wood oil with synthetic linaool at concentration as low as 10% v/v [72]. A PCA model is nec‐

Direct infusion MS can be used to authenticate wood as well. Venturi easy ambient sonic spray ionization (V-EASI)-MS fingerprint of a very simple methanolic extract of wood chips or directly acquired from the freshly scratched wood surface may help to control the illegal logging and trade of the noble Mahogany tropical wood and its falsification [73]. V-EASI-MS is a novel ambient ionization MS technique characterized by sonic spray ionization and a self-pumping system based on the Venturi effect. It allows the direct analysis of solid or liquid samples. Ionization is assisted by compressed nitrogen and the apparatus is thus free of electrical discharge, thermal interferences since no heating, voltage or radiation is used. The introduction of fast and high-throughput analytical techniques for wood authentication is especially valuable since the classical controls are based on time-consuming morphologi‐ cal evaluations. Recently an original and fast approach for the authentication of wood spe‐ cies has been proposed. It is based on the analysis of the volatile compounds measured by a low-cost conductive polymer-based portable electronic nose formed by an array of only three gas sensors and/or the elemental fingerprint measured by laser-induced breakdown spectrom‐ etry (LIBS) which performs a multielemental and direct analysis even in solid samples [74]. The assurance of quality of herbal supplements and medicines is a major concern for the phytopharmaceutical and the food industry. The identification of the herbal drug and the presence of adulterant is a mandatory test to ensure the quality, the efficacy and the safety of a medical preparation or an herbal supplement. Among a variety of quality control meth‐ ods, chromatographic fingerprinting has gained more and more attention recently and have been used to authenticate a large number of herbal products. They are accepted by many international organization for the quality control of herbal medicine but are relatively timeconsuming. More recently, spectroscopic techniques have been explored to rapidly authenti‐

essary to extract the relevant information from the fingerprint.

crimination of the samples.

134 Latest Research into Quality Control

Product fingerprinting combined with chemometrics represents a valuable tool for fraud de‐ tection and control for food products and other biomaterials. A fingerprinting approach is particularly useful:


At the same time a fingerprinting approach may substantiate nutritional, sensory or other product qualities.

LC, GC, and IR spectroscopy are already common instrumental platforms available in most QA laboratories and they will continue to provide valuable support for food fraud preven‐ tion. IR and other spectroscopic techniques have the great advantage of providing fast, highthroughput and non-destructive analyses with limited costs. They can be easily automated and adapted for *in-line* or *in-situ* analysis which makes these techniques well suited for im‐ plementation in the industrial setting. MS and NMR are not as common in QA laboratories, principally because of their high costs but they may become more important in routine QA testing because of their superior performances. However, even though the costs of MS and NMR instruments is still very high, the cost per sample can be very low if a high samples turnover can be achieved.

From an analytical point of view, a further improvement is expected in the future from the broader application of multi-dimensional separation techniques such as GCxGC or LCxLC which provide enhanced resolution and an higher number of peaks. Ultra-performance liq‐ uid chromatography (UPLC) and CE may also provide rapid separation with limited costs. The application of novel (or still underexploited) ambient ionisation MS techniques that al‐ low the rapid analysis of liquid and solid samples with little, if any, preparation will be par‐ ticularly valuable in the future.

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The applicability and the reliability of a fingerprint approach also depends from the correct and tailored usage of the relevant and appropriate chemometric tools. For the development of regression and/or classification models, special care should be devoted to ensure:


Another key aspect is represented by the validation of methods based on fingerprinting and chemometrics that is essential for their application in a commercial context. Whereas stand‐ ards exist for the validation of regular analytical methods (see for instance, Commission De‐ cision 2002/657/EC and ISO 17025), internationally accepted protocols for the validation of methods based on fingerprinting techniques and chemometric classification models are lack‐ ing at the present. Such protocols should indicate the performance characteristics that have to be checked and the criteria to be met in order to verify the compliance of the method with the performance characteristics.

#### **Acknowledgements**

We acknowledge Martin Alewijn, Grishja van der Veer and Pan Weijing for the work on NIR spectroscopy and milk powder and Angela Dura de Miguel, Arjen Lommen and Maikel Rozijn for the work on 1 H NMR and fish. We also acknowledge the Dutch ministry of agri‐ culture, economic affair and innovation for the funding of this book chapter.

#### **Author details**

Edoardo Capuano1\* and Saskia M. van Ruth1

\*Address all correspondence to: edoardo.capuano@wur.nl

1 RIKILT – Institute of Food Safety, Wageningen University and Research Centre,, The Netherlands

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uid chromatography (UPLC) and CE may also provide rapid separation with limited costs. The application of novel (or still underexploited) ambient ionisation MS techniques that al‐ low the rapid analysis of liquid and solid samples with little, if any, preparation will be par‐

The applicability and the reliability of a fingerprint approach also depends from the correct and tailored usage of the relevant and appropriate chemometric tools. For the development

**•** The representativeness of the classes considered, in order to cover all the possible source

**•** A robust validation of the model (external validation to be preferred over internal valida‐

**•** The use of the appropriate chemometric tools depending on the problem at stake. Where‐ as a few pattern recognition techniques are frequently used (PLS-DA, SIMCA, LDA..), some other such as classification and regression trees (CART), quadratic discriminant analysis (QDA) are still underexploited despite the good results that they can provide. Another key aspect is represented by the validation of methods based on fingerprinting and chemometrics that is essential for their application in a commercial context. Whereas stand‐ ards exist for the validation of regular analytical methods (see for instance, Commission De‐ cision 2002/657/EC and ISO 17025), internationally accepted protocols for the validation of methods based on fingerprinting techniques and chemometric classification models are lack‐ ing at the present. Such protocols should indicate the performance characteristics that have to be checked and the criteria to be met in order to verify the compliance of the method with

We acknowledge Martin Alewijn, Grishja van der Veer and Pan Weijing for the work on NIR spectroscopy and milk powder and Angela Dura de Miguel, Arjen Lommen and Maikel

1 RIKILT – Institute of Food Safety, Wageningen University and Research Centre,, The

culture, economic affair and innovation for the funding of this book chapter.

H NMR and fish. We also acknowledge the Dutch ministry of agri‐

of regression and/or classification models, special care should be devoted to ensure:

ticularly valuable in the future.

136 Latest Research into Quality Control

tion).

of variability for the class at stake.

the performance characteristics.

**Acknowledgements**

Rozijn for the work on 1

Edoardo Capuano1\* and Saskia M. van Ruth1

\*Address all correspondence to: edoardo.capuano@wur.nl

**Author details**

Netherlands


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**Chapter 7**

**Principle of Meat Aroma Flavors and Future Prospect**

The population growth fact of the world has been much quickly increasing through the years. As reported by the United Nations Population Fund (UNFPA) the estimated world population of 6.1 billion in the year 2000 and reached to 7 billion in the year 2011, increased 0.9 billion people only after 10 years. The population increases always proportionally ac‐ company to the consumption demands in which including foods. Calculating the global meat consumption only and based on the data collected from IFPRI/ FAO/ILRI by Delgado et al (1999) [1] suggested that global production and consumption of meat will continue to raise from 233 million metric tons in the year 2000 to 300 million metric tons in 2020. On the other hand, income growth of people in most of the countries especially in the developed countries has been significantly increasing in the recent years. Combination of the large pop‐ ulations together with a high-income that will give a big pressure for the food producers in general and meat producers in particular. As a consequence, higher income growth in coun‐ tries has led to an increase in living standards and changes in consumer diets to include a higher proportion of meat and meat products. While, productivity and provision of meats on the markets has been limited and rising costs of production resulting in not keeping pace

Although, a strong demand for meat amounts but consumers are getting quite fastidious to choose meat and meat products since consumer's preference for meat buying is strongly based on quality, freshness and hygiene. Quality factors are very important in the meat pur‐ chasing behavior of consumers including marbling (intramuscular fat tissues), texture, color,

Aroma flavor characteristics of cooked meat in particular play the most important level in eating quality of meat, acceptance and preference by consumers. The aroma flavor charac‐

> © 2012 Ba et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Ba et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

with the strong growth in demand, that has caused a rise in meat prices.

tenderness and especially flavor characteristics.

Hoa Van Ba, Inho Hwang, Dawoon Jeong and

Additional information is available at the end of the chapter

Amna Touseef

**1. Introduction**

http://dx.doi.org/10.5772/51110

## **Principle of Meat Aroma Flavors and Future Prospect**

Hoa Van Ba, Inho Hwang, Dawoon Jeong and Amna Touseef

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51110

#### **1. Introduction**

The population growth fact of the world has been much quickly increasing through the years. As reported by the United Nations Population Fund (UNFPA) the estimated world population of 6.1 billion in the year 2000 and reached to 7 billion in the year 2011, increased 0.9 billion people only after 10 years. The population increases always proportionally ac‐ company to the consumption demands in which including foods. Calculating the global meat consumption only and based on the data collected from IFPRI/ FAO/ILRI by Delgado et al (1999) [1] suggested that global production and consumption of meat will continue to raise from 233 million metric tons in the year 2000 to 300 million metric tons in 2020. On the other hand, income growth of people in most of the countries especially in the developed countries has been significantly increasing in the recent years. Combination of the large pop‐ ulations together with a high-income that will give a big pressure for the food producers in general and meat producers in particular. As a consequence, higher income growth in coun‐ tries has led to an increase in living standards and changes in consumer diets to include a higher proportion of meat and meat products. While, productivity and provision of meats on the markets has been limited and rising costs of production resulting in not keeping pace with the strong growth in demand, that has caused a rise in meat prices.

Although, a strong demand for meat amounts but consumers are getting quite fastidious to choose meat and meat products since consumer's preference for meat buying is strongly based on quality, freshness and hygiene. Quality factors are very important in the meat pur‐ chasing behavior of consumers including marbling (intramuscular fat tissues), texture, color, tenderness and especially flavor characteristics.

Aroma flavor characteristics of cooked meat in particular play the most important level in eating quality of meat, acceptance and preference by consumers. The aroma flavor charac‐

© 2012 Ba et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ba et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

teristics of cooked meats are derived from volatile flavor components which derive from thermally induced reactions occurring during heating via the four pathways including (1) Maillard reaction of amino acid or peptides with reducing sugars, (2) Lipid oxidation, (3) in‐ teraction between Maillard reaction products with lipid-oxidized products and (4) vitamin degradation during cooking [2]. Aroma flavor is perceived through the nostrils (orthonasal aroma) it gives the first impression of a certain food. When the food is placed in the mouth, the volatile flavor compounds will be transferred through the pharynx to the olfactory re‐ ceptors (retronasal aroma). It has been reported that flavors together with other sensory at‐ tributes such as tenderness and juiciness are specially considered the most important criterion of acceptability and the palatability of meat that affects consumer's purchasing de‐ cisions [3,4]. It has been well known that all volatile flavor components are organic and they have low molecular weight [5]. The chemical structures of volatile flavor classes are varied widely including aldehydes, ketones, hydrocarbons, pyrazines, acids, esters, alcohols, nitro‐ gen and sulfur-containing compounds and other heterocyclic compounds as well. Due to the differences in chemical structures therefore their volatility is also quite different.

As mentioned above, regarding the chemical structures of volatile flavor classes, among that the heterocyclic compounds especially those containing sulfur are the important flavor com‐ pounds produced in the Maillard reaction providing savory, meaty, roasty and boiled fla‐ vor characteristics. While, lipid-degraded- compounds which give 'fatty' aromas to cooked meat and compounds which determine some of the aroma flavor differences between meats from different species [20]. The individual volatile compounds have been found to deter‐ mine distinct aroma flavors of cooked meat represent; dimethylsulfide, 2-butanone, ethyl acetate, 2- and 3-methylbutanal, 2-heptanone, dimethyl trisulphide and nonanal were detect‐ ed as key flavor compounds of cooked Irish Angus beef, while methional, 2,4-nonadienal and bezothiazole were characterized as meaty, oily notes in cooked Belgian Blue, Limousin and Aberdeen Angus beefs [21]. Kerscher & Grosch, (1997) [22] reported that 2-furfurylthiol, 4 hydroxy-2,5-dimethyl-3(2*H*)-furanone and 2-methyl-3-furanthiol were the most important odorants of boiled beef. 2-ethyl-3,5-dimethyl pyrazine and 2,3-diethyl-5-methylpyrazine pos‐ sess roasty, caramel-like, burnt and earthy notes of roasted beef [23]. Other carbonyl com‐ pounds such as methional, E-2-undecenal, E-2-dodecenal, decanal, heptanal and 2 methylbutanal also were found to be associated with roasty, sweet, fruity and fatty odor notes of cooked beef [14,17]. Also, a great number of studies considered on the objective volatile flavor components in cooked pork, chicken, lamb, ham and etc… have been documented over the last years [24, 25, 26]. In fact, although thousands of volatile compounds identified but not all of them are important because their high odor detection threshold, only some of them play a significant role in the overall aroma flavor characteristics of cooked meat. An aroma flavor compound with its distinct odor note can be defined as its flavor dilution factor indi‐ cating that at the lowest concentration at which the compound still can be detected by the sense of smell. Some represent volatile flavors active-compounds have been detected in cooked meats by using gas chromatography-olfactometry technique (GC-O) are showed in Table 1.

Principle of Meat Aroma Flavors and Future Prospect

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147

**Compound name Aroma flavor characteristics**

Decanal Sweet, fruity, like aldehydes, roasty

Methional Cooked potato, meaty

Heptanal Fruity, fatty, sweet, oil Nonanal Sweet, fatty, green Undecanal Sweet, pungent, green

E,2-nonenal Fatty E,E,2,4-decadienal Fatty

E,E,2,4-nonedienal Fatty

E,2-heptenal Fatty E,2-heptenal Fatty

Benzenacetaldehyde Sweet, honey

*Aldehydes*

Many factors have been found to be as influences on the aroma flavors of cooked meat. Rabe et al (2003) [6] found that among all food constituents, lipids generally have the greatest influence on production of aroma flavor components, as they not only reduce the vapour pressure of most flavor compounds. Otherwise, Kinsella (1990) [7] showed that aroma com‐ pounds are more lipophilic than hydrophilic therefore fats act as a solvents for aroma com‐ pounds reducing their volatility. In addition to these effects, other factors such as diets, breed, sex, chiller ageing, meat pH, cooking conditions which all also affect the flavor [8, 9, 40, 11]

With the crucial importance of aroma flavor of meat for the acceptance and preference of consumers and as well as the factors influencing the generation of aroma flavor compounds as mentioned above, the present chapter aims to highlight the basic information regarding aroma flavor components in terms of mechanisms of formation pathways; current techni‐ ques being used for detection; factors that affect aroma flavors; and final ideas and as well as suggestions are also given out to improve flavor quality attributes according to criterion of acceptability, satisfaction and the palatability for consumer.

#### **2. Meat aroma flavor**

#### **2.1.The importance of volatile flavor compounds in contributing to the flavor characteristics of cooked meat**

Flavor characteristics of cooked meat are directly detected by the nose (i.e., olfactory recep‐ tors) before and during chewing. Raw meat has little aroma and only blood-like taste, meat develops its aroma flavor characteristics during cooking as the result of complex interaction of precursors derived from both the lean and fat compositions of meat generating volatile flavor compounds that contribute to meat flavor [12]. To date, approximately thousands of volatile flavor compounds have been detected and identified in cooked meat. There is a large number of these compounds contributing to the flavor characteristics of cooked meat have been identified in previous works [13, 14, 15, 16, 17, 18, 19].

As mentioned above, regarding the chemical structures of volatile flavor classes, among that the heterocyclic compounds especially those containing sulfur are the important flavor com‐ pounds produced in the Maillard reaction providing savory, meaty, roasty and boiled fla‐ vor characteristics. While, lipid-degraded- compounds which give 'fatty' aromas to cooked meat and compounds which determine some of the aroma flavor differences between meats from different species [20]. The individual volatile compounds have been found to deter‐ mine distinct aroma flavors of cooked meat represent; dimethylsulfide, 2-butanone, ethyl acetate, 2- and 3-methylbutanal, 2-heptanone, dimethyl trisulphide and nonanal were detect‐ ed as key flavor compounds of cooked Irish Angus beef, while methional, 2,4-nonadienal and bezothiazole were characterized as meaty, oily notes in cooked Belgian Blue, Limousin and Aberdeen Angus beefs [21]. Kerscher & Grosch, (1997) [22] reported that 2-furfurylthiol, 4 hydroxy-2,5-dimethyl-3(2*H*)-furanone and 2-methyl-3-furanthiol were the most important odorants of boiled beef. 2-ethyl-3,5-dimethyl pyrazine and 2,3-diethyl-5-methylpyrazine pos‐ sess roasty, caramel-like, burnt and earthy notes of roasted beef [23]. Other carbonyl com‐ pounds such as methional, E-2-undecenal, E-2-dodecenal, decanal, heptanal and 2 methylbutanal also were found to be associated with roasty, sweet, fruity and fatty odor notes of cooked beef [14,17]. Also, a great number of studies considered on the objective volatile flavor components in cooked pork, chicken, lamb, ham and etc… have been documented over the last years [24, 25, 26]. In fact, although thousands of volatile compounds identified but not all of them are important because their high odor detection threshold, only some of them play a significant role in the overall aroma flavor characteristics of cooked meat. An aroma flavor compound with its distinct odor note can be defined as its flavor dilution factor indi‐ cating that at the lowest concentration at which the compound still can be detected by the sense of smell. Some represent volatile flavors active-compounds have been detected in cooked meats by using gas chromatography-olfactometry technique (GC-O) are showed in Table 1.

teristics of cooked meats are derived from volatile flavor components which derive from thermally induced reactions occurring during heating via the four pathways including (1) Maillard reaction of amino acid or peptides with reducing sugars, (2) Lipid oxidation, (3) in‐ teraction between Maillard reaction products with lipid-oxidized products and (4) vitamin degradation during cooking [2]. Aroma flavor is perceived through the nostrils (orthonasal aroma) it gives the first impression of a certain food. When the food is placed in the mouth, the volatile flavor compounds will be transferred through the pharynx to the olfactory re‐ ceptors (retronasal aroma). It has been reported that flavors together with other sensory at‐ tributes such as tenderness and juiciness are specially considered the most important criterion of acceptability and the palatability of meat that affects consumer's purchasing de‐ cisions [3,4]. It has been well known that all volatile flavor components are organic and they have low molecular weight [5]. The chemical structures of volatile flavor classes are varied widely including aldehydes, ketones, hydrocarbons, pyrazines, acids, esters, alcohols, nitro‐ gen and sulfur-containing compounds and other heterocyclic compounds as well. Due to

the differences in chemical structures therefore their volatility is also quite different.

acceptability, satisfaction and the palatability for consumer.

been identified in previous works [13, 14, 15, 16, 17, 18, 19].

**2.1.The importance of volatile flavor compounds in contributing to the flavor**

Flavor characteristics of cooked meat are directly detected by the nose (i.e., olfactory recep‐ tors) before and during chewing. Raw meat has little aroma and only blood-like taste, meat develops its aroma flavor characteristics during cooking as the result of complex interaction of precursors derived from both the lean and fat compositions of meat generating volatile flavor compounds that contribute to meat flavor [12]. To date, approximately thousands of volatile flavor compounds have been detected and identified in cooked meat. There is a large number of these compounds contributing to the flavor characteristics of cooked meat have

**2. Meat aroma flavor**

146 Latest Research into Quality Control

**characteristics of cooked meat**

Many factors have been found to be as influences on the aroma flavors of cooked meat. Rabe et al (2003) [6] found that among all food constituents, lipids generally have the greatest influence on production of aroma flavor components, as they not only reduce the vapour pressure of most flavor compounds. Otherwise, Kinsella (1990) [7] showed that aroma com‐ pounds are more lipophilic than hydrophilic therefore fats act as a solvents for aroma com‐ pounds reducing their volatility. In addition to these effects, other factors such as diets, breed, sex, chiller ageing, meat pH, cooking conditions which all also affect the flavor [8, 9, 40, 11] With the crucial importance of aroma flavor of meat for the acceptance and preference of consumers and as well as the factors influencing the generation of aroma flavor compounds as mentioned above, the present chapter aims to highlight the basic information regarding aroma flavor components in terms of mechanisms of formation pathways; current techni‐ ques being used for detection; factors that affect aroma flavors; and final ideas and as well as suggestions are also given out to improve flavor quality attributes according to criterion of



1-heptanol Fragrant, woody, oily, green, fatty, winey, sap 1-hexanol Woody, cut grass, chemical-winey, fatty, fruity

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149

1-octanol Penetrating aromatic odor, fatty, waxy, citrus, oily,

2-Ethyl 1-hexanol Resin, flower, green

1-pentanol Mild odor, fuel oil, fruit, balsamic

Ethenylbenzene Pungent, aromatic, fragrant, roasty

Pentane Very slight warmed-over flavor, oxidized

1-undecen Fatty, burnt, nutty, rubbery

Styrene Penetrating odor, sweet smell

Ethenylbenzene Aromatic, fragrant, roasty

2-ethyl-3,5-dimethylpyrazin Burnt, fragrant, meaty, green 2-ethenyl-3,6(5)-dimethylpyrazine Sweet, cooked rice, fatty

2,3-diethyl-5-methylpyrazine meaty, roasty, fragrant, sweet 2,5-dimethylpyrazine Fried rice, popcorn, pungent, green 2-ethenyl-5(6)-methylpyrazine Roasty break-like, cooked rice, coffee-like 2,5-dimethylpyrazine Fried rice, popcorn, pungent, green

2-ethyl-5-methylpyrazine Fruity, sweet, pungent

2-ethyl-3,6-dimethylpyrazine Burnt, pungent, roasty

Sulfur & nitrogen containing compounds 2-fufurylthiol Roasty

2-ethenyl-3,6(5)-dimethylpyrazine Pungent, sweet, cooked rice, fatty 2,3-diethyl-5-methylpyrazine Meaty, roasty, fragrant, sweet

2-isopentyl-3,6-dimethylpyrazine Sweet, fragrant, fatty, fruity, pungent

2-ethenyl-5(6)-methylpyrazine Smoky, roasty, break-like, cooked rice, popcorn

Hexane Faint peculiar odor (Z)-3-Octene Fruity, old apples

2-Octen-1-ol Green citrus

Propanol Alcoholic

Tridecane Alkane Tetradecane Alkane

2-ethyl-3,6-dimethylpyrazine Burnt, roasty

*Hydrocarbons*

*Pyrazines*


Hexanal Green, fatty E,2-hexenal Green E,Z,2,6-nonadienal Cucumber

Undecanal Sweet, pungent, green 2-methylbutanal Pungent, sweet, roasty E,2-undecenal Sweet, fruity, fatty

2,E-dodecenal Sweet, fruity, roasty, pungent Ethanol Grilled (weak), acetaldehyde-like

Octanal Green, lemon, citrus, aldehyde E,E,2,4-heptadienal Aldehyde, green, broth, spicy

1-octen-3-one fresh, mushrooms, pungent, rubbery 3-octanone Fruity, nutty, moldy, fatty, earthy

4,5-dihydro-5-propyl-2(3H)-furanone Fruity, fatty, sweet, pungent, roasty

2,3-pentanedione buttery, lemon-like, sweet, fruity 2-nonanone Hot milk, soap, green, fruity, floral

2-heptanone Citrus grapefruit, limonene, floral, cheese

3-octen-2-one Nut, crushed bug, earthy, spicy, sweet, mushroom,

furanone Roasted almonds, sweet

2,3-butanedione Sweet, buttery

6-Methyl 2-heptanone Cloves, menthol

2,2,6-Trimethylcyclohexanone Mint, acetone

1-octen-3-ol Mushroom Cyclobutanol Roasted

2-undecanone Fruity

2-octanone Fruity, musty 2-decanone Fruity, musty 2-dodecanone Fruity, musty

2,5-dimethyl-4-hydroxy-3(2H)-

*Ketones*

148 Latest Research into Quality Control

*Alcohols*

3-methylbutanal Meaty, fish, rotten, aldehyde,valeric acid, fatty

Propanal caramel, sweet, alcoholic, "cooked", broth, spicy Butanal smoky, fish, amylic, aldehyde-enal or dienal


of cooked meat are not similar. Macey et al (1964) [28] found some sugars present in beef such as glucose, fructose, mannose and ribose, in that ribose was the most heat-labile sugar among these whereas fructose was the most stable. Among the amino acids present in meat, systein and systine are two sulfur-containing amino acids, the reaction of these with other sugars lead to formation of many sulfur-containing flavor compounds [34], while the reaction of other nonsulfur containing amino acids with sugars dominated by the nitrogen-containing products such as pyrazines [72]. In the recent years, researchers have found that the flavor precursor compo‐ nents in meats are influenced by several factors. Koutsidis et al (2008) [31] indicated that diets significantly affected the reducing sugars in beef *longissimus lumborum* muscle, higher total reducing sugars was obtained in beef from concentrate feeding group compared to the grass silage feeding group whereas beef from cattle fed with grass silage had higher level of free amino acids. When the beef was chiller aged for several days at chilling condition resulted in several times increase in free sugars such as ribose, free amino acids also increased with conditioning especially phenylalanine, methionine, lysine, leucine and isoleucine were the amino acids showing the greatest increase with conditioning time [32]. Meinert et al (2009) [35] have found that feeding, fasting and post-mortem ageing factors significantly influenced the concentra‐ tion of flavor precursors of beef *longissimus dorsi* muscle. Additionally, the recent works also showed that fat-supplemented diets had large effect on the fatty acid compositions, for in‐ stance, dietary linseed oil and soybean oil significantly increased the contents of C18:3 and C18:2 in the neutral lipids and phospholipids in both *longissimus* and *biceps brachii* muscles [36], and

subsequently influence the volatile flavor compounds of cooked beef [19, 25, 37].

Free amino acids

Reducing sugars

Fats/ lipids

Nucleotides and peptides

**Table 2.** The representative precursors of meat flavor.

arginine.

etc.

**Flavor precursors Names in detail Reference**

Ribose; glucose; xylose; starch; mannose; fructose; maltose; mannose 6-phosphate, glucose 6-phosphate;

fructose 6-phosphate; ribose 6-phosphate.

Vitamin Thiamin [33,44]

inosine 5'-monophosphate; guanosine 5-

Glutathione; carnosine inosine; inosine monophosphate;

monophosphate; creatine; creatinine; Hypoxanthine and

Triglycerides and phospholipids

Linolenic acid (C18:3n-3) and etc.

Oleic acid (C18:1n-9) Linoleic acid (C18:2n-6)

Systine; systeine; glycine; lysine; alanine; valine; isoleucine; leucine; threonine; serine; proline; asparagines; aspartic acid; methionine; glutamic acid; phenylalanine; glutamine; ornithine; histidine; tyrosine; tryptophan;

[38,39,40, 31,3232]

Principle of Meat Aroma Flavors and Future Prospect

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151

[38,39,72, 41,31,32]

[42,19, 43,34]

[45,44, 31,32]

**Table 1.** The representative volatile flavor compounds with their aroma flavor characteristics found in cooked meat. [References: 13, 14, 20, 15, 27]

#### **2.2. Precursors of meat flavor**

Earlier studies on meat flavor, researchers recognized that the low molecular weight, watersoluble compounds and fats in meat constituents are the most important precursor of aroma flavor characteristics of cooked meat [28, 29]. The flavor precursor of meat namely, free sug‐ ars, free amino acids, peptides, vitamin, sugar phosphate, nucleotide-bound sugars and nucleo‐ tides [30, 31, 32, 33], all of them are able to either participate the Maillard reaction or oxidation/ degradation and interaction on heating to generate volatile flavor compounds then create the final aroma flavor characteristics of cooked meat. It is suggested that these precursor compo‐ nents found to contribute to the development of meaty flavor, while the adipose tissues and intramuscular fat not only occupy an important role in development of flavor characteristics of cooked meat but also contribute to the characteristic-specific species flavors. This means that the distinct flavor characteristics between the meats from different species are due to the intramuscular fat content and not from water-soluble precursor compounds. The details on flavor precursors of meat found in the past years are showed in Table 2. However, research‐ ers found that the roles of these flavor precursors in the development of flavor characteristics of cooked meat are not similar. Macey et al (1964) [28] found some sugars present in beef such as glucose, fructose, mannose and ribose, in that ribose was the most heat-labile sugar among these whereas fructose was the most stable. Among the amino acids present in meat, systein and systine are two sulfur-containing amino acids, the reaction of these with other sugars lead to formation of many sulfur-containing flavor compounds [34], while the reaction of other nonsulfur containing amino acids with sugars dominated by the nitrogen-containing products such as pyrazines [72]. In the recent years, researchers have found that the flavor precursor compo‐ nents in meats are influenced by several factors. Koutsidis et al (2008) [31] indicated that diets significantly affected the reducing sugars in beef *longissimus lumborum* muscle, higher total reducing sugars was obtained in beef from concentrate feeding group compared to the grass silage feeding group whereas beef from cattle fed with grass silage had higher level of free amino acids. When the beef was chiller aged for several days at chilling condition resulted in several times increase in free sugars such as ribose, free amino acids also increased with conditioning especially phenylalanine, methionine, lysine, leucine and isoleucine were the amino acids showing the greatest increase with conditioning time [32]. Meinert et al (2009) [35] have found that feeding, fasting and post-mortem ageing factors significantly influenced the concentra‐ tion of flavor precursors of beef *longissimus dorsi* muscle. Additionally, the recent works also showed that fat-supplemented diets had large effect on the fatty acid compositions, for in‐ stance, dietary linseed oil and soybean oil significantly increased the contents of C18:3 and C18:2 in the neutral lipids and phospholipids in both *longissimus* and *biceps brachii* muscles [36], and subsequently influence the volatile flavor compounds of cooked beef [19, 25, 37].


2-acetyl-1-pyrroline Roasted, sweet 2-formyl-5-methylthiophene Sulfurous

Benzylthiol Sulphurous

2-acetylthiazole Roasted

Bis(2-methyl-3-furyl)disulfide Meaty-like Benzothiazole Metallic

2-acethylthiophene Sulphurous, sweet

2-methyl-3-furanthiol Meaty, sweet, sulfurous

2,4-dimethylthiazole Rubber y, moldy, fruity, pungent

Dimethyltrisulfide Fragrant, musty, roasty, rubbery

Dimethyldisulfide Moldy, pungent, rubbery, onion-like 2,4-dimethylthiazole Rubbery, moldy, fruity, pungent 4,5-dimethylthiazole Smoky, roasty, fragrant, nutty

3-mercapto-2-butanone Fried onion, sulfury, cooked meat

2-[(methyldithio)methyl]furan Brothy, spices, roast, fatty

3-[(2-furanylmethyl)dithio]-2-

butanone

**2.2. Precursors of meat flavor**

[References: 13, 14, 20, 15, 27]

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2-methylchinoxaline Aromatic, roasted, nutty, sweet, fruity, fatty

2-mercapto-3-pentanone Brothy, mashed potatoes meaty, roast meat

**Table 1.** The representative volatile flavor compounds with their aroma flavor characteristics found in cooked meat.

Earlier studies on meat flavor, researchers recognized that the low molecular weight, watersoluble compounds and fats in meat constituents are the most important precursor of aroma flavor characteristics of cooked meat [28, 29]. The flavor precursor of meat namely, free sug‐ ars, free amino acids, peptides, vitamin, sugar phosphate, nucleotide-bound sugars and nucleo‐ tides [30, 31, 32, 33], all of them are able to either participate the Maillard reaction or oxidation/ degradation and interaction on heating to generate volatile flavor compounds then create the final aroma flavor characteristics of cooked meat. It is suggested that these precursor compo‐ nents found to contribute to the development of meaty flavor, while the adipose tissues and intramuscular fat not only occupy an important role in development of flavor characteristics of cooked meat but also contribute to the characteristic-specific species flavors. This means that the distinct flavor characteristics between the meats from different species are due to the intramuscular fat content and not from water-soluble precursor compounds. The details on flavor precursors of meat found in the past years are showed in Table 2. However, research‐ ers found that the roles of these flavor precursors in the development of flavor characteristics

onion, burnt rubber, burnt wood

#### **2.3. Pathways for the formation of volatile flavor compounds**

#### *2.3.1. Maillard reaction*

Maillard reaction, a non-enzymatic browning which plays an important role in generation of volatile flavor compounds and appearances of the cooked foods, it is due to most of impor‐ tant volatile flavor compounds found in cooked foods are originated from this reaction. Oth‐ erwise, Maillard reaction also can produce antioxidative components and toxicological implications as well. However, in the present chapter we are focusing on the Maillard reac‐ tion in relation to aroma flavor characteristics, particularly the formation of volatile flavor compounds in cooked meat. Maillard reaction was firstly mentioned in the early time, 1912 by Maillard [46] since he wanted to investigate the browning reaction between glucose and glycine. After that many studies focused on determining the fundaments and mechanisms of this reaction [47, 48, 49].

tion, polymeration and cyclization reactions. A general scheme of the Maillard reaction is

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given in Figure 1.

**Figure 2.** Strecker degradation mechanisms, a part of Maillard reaction

**Figure 3.** The formation of H2S from the Strecker degradation of cysteine

weight end flavor compounds.

Among events occurring in the Maillard reaction, Strecker degradation is one of the quite important events, in which amino acids are undergone degradation processes (oxidative de‐ amination and decarboxylation) in the presence of a dicarbonyls compound formed from Maillard reaction. The Strecker degradation processes lead to formation of aldehydes (e.g., fufural) and aminoketone (Figure 2). Especially the other important intermediate products such as H2S, NH3, etc are also formed from the Strecker degradation by sulphur-containing amino acids such as cystein and systine (Figure 3); all of these intermediate products can further react with other compounds or with each other to produce low and high molecular

**Figure 1.** General stages of Maillard reaction showing the formations of flavor compounds (based on van Boekel, 2006) [51].

The Maillard reaction is taken placed with the participation of reducing sugars (e.g., ribose, glucose) and free amino compounds (e.g., amino acids, amines, peptides, proteins, ammo‐ nia) at certain heating condition to produce the Maillard products, and usually this reaction is divided into three main stages. In which the firstly initial stage starts with a condensation between a reducing sugar and an amino group, the loss of water from this molecule produ‐ ces an amine that is able to cyclise resulting in formation of an N-glycosylamine (a sugar attached to NR2 group) or called Amadori product. The next intermediate stage involves the rearrangement and decomposition of the Amadori product to release amino group and sug‐ ar fragmentation. The final stage of Maillard reaction is leading to dehydration, fragmenta‐ tion, polymeration and cyclization reactions. A general scheme of the Maillard reaction is given in Figure 1.

**Figure 2.** Strecker degradation mechanisms, a part of Maillard reaction

**2.3. Pathways for the formation of volatile flavor compounds**

Maillard reaction, a non-enzymatic browning which plays an important role in generation of volatile flavor compounds and appearances of the cooked foods, it is due to most of impor‐ tant volatile flavor compounds found in cooked foods are originated from this reaction. Oth‐ erwise, Maillard reaction also can produce antioxidative components and toxicological implications as well. However, in the present chapter we are focusing on the Maillard reac‐ tion in relation to aroma flavor characteristics, particularly the formation of volatile flavor compounds in cooked meat. Maillard reaction was firstly mentioned in the early time, 1912 by Maillard [46] since he wanted to investigate the browning reaction between glucose and glycine. After that many studies focused on determining the fundaments and mechanisms

**Figure 1.** General stages of Maillard reaction showing the formations of flavor compounds (based on van Boekel,

The Maillard reaction is taken placed with the participation of reducing sugars (e.g., ribose, glucose) and free amino compounds (e.g., amino acids, amines, peptides, proteins, ammo‐ nia) at certain heating condition to produce the Maillard products, and usually this reaction is divided into three main stages. In which the firstly initial stage starts with a condensation between a reducing sugar and an amino group, the loss of water from this molecule produ‐ ces an amine that is able to cyclise resulting in formation of an N-glycosylamine (a sugar attached to NR2 group) or called Amadori product. The next intermediate stage involves the rearrangement and decomposition of the Amadori product to release amino group and sug‐ ar fragmentation. The final stage of Maillard reaction is leading to dehydration, fragmenta‐

*2.3.1. Maillard reaction*

152 Latest Research into Quality Control

of this reaction [47, 48, 49].

2006) [51].

**Figure 3.** The formation of H2S from the Strecker degradation of cysteine

Among events occurring in the Maillard reaction, Strecker degradation is one of the quite important events, in which amino acids are undergone degradation processes (oxidative de‐ amination and decarboxylation) in the presence of a dicarbonyls compound formed from Maillard reaction. The Strecker degradation processes lead to formation of aldehydes (e.g., fufural) and aminoketone (Figure 2). Especially the other important intermediate products such as H2S, NH3, etc are also formed from the Strecker degradation by sulphur-containing amino acids such as cystein and systine (Figure 3); all of these intermediate products can further react with other compounds or with each other to produce low and high molecular weight end flavor compounds.

alytic condition (e.g., heating temperature, moisture, pH). For the type of reducing sugars and amino acids which determine the kinds of flavor compounds generated for instance, many sulfur-containing flavor compounds are formed from the Maillard reaction between systeine and ribose [34] whereas, the nitrogen-containing compounds (e.g., pyrazines) domi‐ nated in the Maillard reaction containing glucose and lysine [72]. Therefore, it should be noted that nature of reactants will require the kinds of Maillard products. For the catalytic condition of Maillard reaction, it usually influences the kinetics of flavor compound genera‐ tion by Maillard reaction in that depending on each catalytic condition (temperature, pH and etc) will determine the yields and also kinds of Maillard products. The kinetic of flavor compound formation resembles the scheme in Figure 4 with 11 determining steps [50]. Based on the kinetic scheme it shows that there are many chemical classes of flavors are formed via the Maillard reaction, some of the representative classes associated with odor

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Lipids and fatty acids play an important role in direct and indirect generating the volatile flavor compounds and some of them contributing to the aroma flavor characteristics of cooked meat. Therefore, the levels of fat contents and as well as fatty acids of meats should be concerned, and it has been reported that the fatty acids of meat are influenced by several factors but almost are the pre-harvest factors such as diets, feed regimes and breeds [52, 53, 54]. Based on our surveillance it seems that the fatty acid profiles significantly vary across the breeds even these breeds are fed with the same diets [55, 56, 57]. Both adipose tissue and intramuscular fat contents are constituted by fatty acids including saturated and unsaturat‐ ed fatty acids which all are capable to get oxidized and degraded under a certain condition to create a prolific number of volatile flavor compounds [2]. Hundreds of volatile flavor compounds derived from lipid degradation have been found in cooked meat including ali‐ phatic hydrocarbons, aldehydes, ketones, alcohols, carboxylic acids and esters. In general, the odor detection threshold values for the lipid-derived compounds are much higher than those for the sulfur and nitrogen-containing heterocyclic compounds which are formed from the water-soluble precursors via the Maillard reaction. Therefore, the aroma significance of many of these lipid-derived compounds is not as great as that for relatively low concentra‐ tions of the heterocyclic compounds. However, certain classes of compounds such as partic‐ ular aldehydes included saturated and unsaturated aldehydes which containing from 6 to 10 carbons in the structures are major volatile components of all cooked meats and, therefore, they probably play an important part in meat aroma [20]. The oxidation of subcutaneous fat, adipose tissues and intramuscular fat occur in raw meat and continues under the catalysis of

Among the oxidation-induced factors for instance, lights (e.g., ultraviolet) is thought to be thermodynamically capable of production of free radicals directly in lipids, the principles of light-absorbing groups of lipids are double bonds, peroxide bonds and carbonyls which subsequently under the other steps to generate volatiles. And other factors such as oxygen, lypoxygenase, metals and etc which all also affect the lipid oxidation however that is anoth‐

notes are showed in Table 3.

**2.3.2 Lipid oxidation and degradation**

many factors such as metals, oxygen, light, heating and etc.

**Figure 4.** Kinetic scheme of flavor formation by Maillard reaction (Jousse et al., 2002) [50]


**Table 3.** Some representative classes of flavor compounds formed from the Maillard reaction (based on van Boekel, 2006) [51].

The formation of volatile flavor compounds in the Maillard reaction largely depend on the reactants (e.g., the nature of reducing sugars and amino acids participated) and also the cat‐ alytic condition (e.g., heating temperature, moisture, pH). For the type of reducing sugars and amino acids which determine the kinds of flavor compounds generated for instance, many sulfur-containing flavor compounds are formed from the Maillard reaction between systeine and ribose [34] whereas, the nitrogen-containing compounds (e.g., pyrazines) domi‐ nated in the Maillard reaction containing glucose and lysine [72]. Therefore, it should be noted that nature of reactants will require the kinds of Maillard products. For the catalytic condition of Maillard reaction, it usually influences the kinetics of flavor compound genera‐ tion by Maillard reaction in that depending on each catalytic condition (temperature, pH and etc) will determine the yields and also kinds of Maillard products. The kinetic of flavor compound formation resembles the scheme in Figure 4 with 11 determining steps [50]. Based on the kinetic scheme it shows that there are many chemical classes of flavors are formed via the Maillard reaction, some of the representative classes associated with odor notes are showed in Table 3.

#### **2.3.2 Lipid oxidation and degradation**

**Figure 4.** Kinetic scheme of flavor formation by Maillard reaction (Jousse et al., 2002) [50]

Pyrazines Cooked, roasted, toasted, baked cereals

Alkylpyrazines Nutty, roasted

Acetylpyridines Caracker-like Pyrroles Cereal –like

Oxazoles Green, nutty, sweet

Furan, furanones, pyranone

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2006) [51].

**Flavor class Characterized Flavor/aroma notes Remark**

Alkylpyridines Green, bitter, astringent, burnt Unpleasant flavor

Sweet, burnt, pungent, caramel-like

Thiophenes Meaty Formed from heated

**Table 3.** Some representative classes of flavor compounds formed from the Maillard reaction (based on van Boekel,

The formation of volatile flavor compounds in the Maillard reaction largely depend on the reactants (e.g., the nature of reducing sugars and amino acids participated) and also the cat‐

meat by the reaction of systein and ribose

Lipids and fatty acids play an important role in direct and indirect generating the volatile flavor compounds and some of them contributing to the aroma flavor characteristics of cooked meat. Therefore, the levels of fat contents and as well as fatty acids of meats should be concerned, and it has been reported that the fatty acids of meat are influenced by several factors but almost are the pre-harvest factors such as diets, feed regimes and breeds [52, 53, 54]. Based on our surveillance it seems that the fatty acid profiles significantly vary across the breeds even these breeds are fed with the same diets [55, 56, 57]. Both adipose tissue and intramuscular fat contents are constituted by fatty acids including saturated and unsaturat‐ ed fatty acids which all are capable to get oxidized and degraded under a certain condition to create a prolific number of volatile flavor compounds [2]. Hundreds of volatile flavor compounds derived from lipid degradation have been found in cooked meat including ali‐ phatic hydrocarbons, aldehydes, ketones, alcohols, carboxylic acids and esters. In general, the odor detection threshold values for the lipid-derived compounds are much higher than those for the sulfur and nitrogen-containing heterocyclic compounds which are formed from the water-soluble precursors via the Maillard reaction. Therefore, the aroma significance of many of these lipid-derived compounds is not as great as that for relatively low concentra‐ tions of the heterocyclic compounds. However, certain classes of compounds such as partic‐ ular aldehydes included saturated and unsaturated aldehydes which containing from 6 to 10 carbons in the structures are major volatile components of all cooked meats and, therefore, they probably play an important part in meat aroma [20]. The oxidation of subcutaneous fat, adipose tissues and intramuscular fat occur in raw meat and continues under the catalysis of many factors such as metals, oxygen, light, heating and etc.

Among the oxidation-induced factors for instance, lights (e.g., ultraviolet) is thought to be thermodynamically capable of production of free radicals directly in lipids, the principles of light-absorbing groups of lipids are double bonds, peroxide bonds and carbonyls which subsequently under the other steps to generate volatiles. And other factors such as oxygen, lypoxygenase, metals and etc which all also affect the lipid oxidation however that is anoth‐ er concern, in the present work we only consider on the heat effect that similar to cooking condition to induce the oxidation and degradation of fatty acids in producing volatile flavor compounds of cooked meat. The degrees of heating temperatures have been reported to af‐ fect variously lipid oxidation, in that high heating temperatures (e.g., frying, roasting) can have highly sufficient energies to break the single bonds (e.g., C-C or C-H) in the acyl back bonds to generate a lot of lipid alkyl radicals that participate the radical chain formation of oxidation [58]. Lower heating temperatures have lower energies which can break O-O bonds in traces of ROOH. Mottram (1985) [59] also stated that meat is cooked under boiled and lightly roasted conditions, lipid oxidation products dominated the detected compounds, and many of among them such as aldehydes, alcohols, ketones and lactones which have suf‐ ficiently low odor threshold to be contributors of meat aroma flavors.

and thiophenes were the dominant aroma volatile compounds. But the levels of these meaty compounds decrease when increasing pH to 9.0 [64]. Similarly, a recent study by Dreher et al (2003) [65] also showed that the most significant thiamin thermal degradation products in the model reaction of orange juice containing 0.024 mM thiamin are 2-methyl-3-furanthiol and bis(2-methyl-3-furyl) disulfide produce intense meaty aromas. Otherwise, some other aroma-active compounds also were found such as 4, 5-dimethylthiazole (skunky, earthy), 3 thiophenethiol (meaty, cooked), 2-methyl-4, 5-dihydro-3(2H)-thiophenone (sour-fruity, mus‐ ty, green), 2-acethylthiophene (burnt), 2-formyl-5-methylthiophene (meaty), and 2-methyl-3-

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(methyldithio) furan (meaty).

**Figure 5.** The thermal degradation of thiamin under basic condition

*2.3.4. Interaction between lipid-oxidized products with Maillard products*

ucts that are able to react with the different reactive protein residues.

The interaction between oxidized lipids and amino acids or proteins is very complex, in term of a consequence of the contribution of both lipid hydroperoxide and its secondaryoxidized products. This interaction may imply both the formation of physical complexes be‐ tween the oxidized lipids and the amino acids or protein and the formation of various types of covalent bonds. Protein polymerizarion produced by reaction with peroxy free radicals generated during lipid peroxidation is known to occur during nonenzymatic browning [66, 67, 68]. However, in term of flavor study, the interaction between lipid-oxidized products (secondary products) with amino acids or proteins is the most concerned. Lipid-oxidized products are generic terms used to describe a mixture of aldehydes, alcohols, ketones and other products obtained by the decomposition of lipid hydroperoxides. Although it is not widely recognized, this decomposition does not necessarily imply the breakage of the lipid chain, and the formation of covalent bonds in the reaction between long chain oxidized lip‐ ids and amino acids and proteins has been described [69, 70]. This is a consequence of the existence of fatty acids that produce a complex and diverse mixture of lipid oxidation prod‐

In the Maillard reaction, amino acids can undergo the Strecker degradation process that sub‐ sequently generates some reactive radicals such as ammonia, hydrosulfide and etc which al‐

Early work of Mottram et al (1982) [60] found that lipid has a considerable role in meat fla‐ vor, when the adipose tissue is added to lean meat does not affect the lipid-derived flavor compounds. A later study by Mottram and Edwards (1983) [42] found that the removal of intramuscular fats and phospholipids from beef caused marked differences in flavor com‐ pounds and sensory characteristics as well. So that the intramuscular fat contents (marbling fats) and membrane lipids are the main source of volatile flavor components and make spe‐ cies-specific flavors. However, it has been demonstrated that high levels of lipids especially polyunsaturated fatty acid contents (PUFA) cause undesirable aroma flavors due to their PUFA-derived products lower or inhibit the formation of some heterocyclic Maillard prod‐ ucts [42]. This phenomenon has recently been elucidated by researchers when they used model systems. In the model systems containing systeine, ribose and lipid (e.g., lecithin or individual fatty acids) the concentrations of heterocyclic compounds and especially sulfurcontaining compounds were lower several times compared with the model system without lipid content [43, 38, 61, 34]. However, the interaction between the lipid-derived products with Maillard products to form volatile flavor components has been much considered in the previous studies and thought as the important pathway for formation of flavor compounds.

#### *2.3.3. Thiamin degradation*

Thiamin is considered as a source of meat flavor generated on heating. Researchers found that the thermal degradation of thiamin produces some ended and intermediate flavor com‐ pounds [62,63]. It was assumed that thermal degradation of thiamin is a quite complex reac‐ tion including various degradation pathways to produce interesting flavor compounds in which most of them contain one or more sulfur and/or nitrogen atoms, and many of them are heterocyclic structures. The thermal degradation of thiamin under the basic condition to produce several flavor compounds is illustrated in Figure 5.

It was reported that the primary products of thermally-degraded thiamin including 4-meth‐ yl-5-(2-hydroxyethyl)thiazole which subsequently responds for formation of thiazoles and other sulfur compounds such as 5-hydroxy-3-mercaptopentan-2-one which then gives some sulfur-containing compounds such as thiophenes and furans as well [62]. Heating tempera‐ ture and pH conditions have been showed to affect the degradation products of thiamin. At pH 5.0 and 7.0 the 2-methyl-3furanthiol and bis (2-methyl3-furyl) disulfide (meaty aroma) and thiophenes were the dominant aroma volatile compounds. But the levels of these meaty compounds decrease when increasing pH to 9.0 [64]. Similarly, a recent study by Dreher et al (2003) [65] also showed that the most significant thiamin thermal degradation products in the model reaction of orange juice containing 0.024 mM thiamin are 2-methyl-3-furanthiol and bis(2-methyl-3-furyl) disulfide produce intense meaty aromas. Otherwise, some other aroma-active compounds also were found such as 4, 5-dimethylthiazole (skunky, earthy), 3 thiophenethiol (meaty, cooked), 2-methyl-4, 5-dihydro-3(2H)-thiophenone (sour-fruity, mus‐ ty, green), 2-acethylthiophene (burnt), 2-formyl-5-methylthiophene (meaty), and 2-methyl-3- (methyldithio) furan (meaty).

**Figure 5.** The thermal degradation of thiamin under basic condition

er concern, in the present work we only consider on the heat effect that similar to cooking condition to induce the oxidation and degradation of fatty acids in producing volatile flavor compounds of cooked meat. The degrees of heating temperatures have been reported to af‐ fect variously lipid oxidation, in that high heating temperatures (e.g., frying, roasting) can have highly sufficient energies to break the single bonds (e.g., C-C or C-H) in the acyl back bonds to generate a lot of lipid alkyl radicals that participate the radical chain formation of oxidation [58]. Lower heating temperatures have lower energies which can break O-O bonds in traces of ROOH. Mottram (1985) [59] also stated that meat is cooked under boiled and lightly roasted conditions, lipid oxidation products dominated the detected compounds, and many of among them such as aldehydes, alcohols, ketones and lactones which have suf‐

Early work of Mottram et al (1982) [60] found that lipid has a considerable role in meat fla‐ vor, when the adipose tissue is added to lean meat does not affect the lipid-derived flavor compounds. A later study by Mottram and Edwards (1983) [42] found that the removal of intramuscular fats and phospholipids from beef caused marked differences in flavor com‐ pounds and sensory characteristics as well. So that the intramuscular fat contents (marbling fats) and membrane lipids are the main source of volatile flavor components and make spe‐ cies-specific flavors. However, it has been demonstrated that high levels of lipids especially polyunsaturated fatty acid contents (PUFA) cause undesirable aroma flavors due to their PUFA-derived products lower or inhibit the formation of some heterocyclic Maillard prod‐ ucts [42]. This phenomenon has recently been elucidated by researchers when they used model systems. In the model systems containing systeine, ribose and lipid (e.g., lecithin or individual fatty acids) the concentrations of heterocyclic compounds and especially sulfurcontaining compounds were lower several times compared with the model system without lipid content [43, 38, 61, 34]. However, the interaction between the lipid-derived products with Maillard products to form volatile flavor components has been much considered in the previous studies and thought as the important pathway for formation of flavor compounds.

Thiamin is considered as a source of meat flavor generated on heating. Researchers found that the thermal degradation of thiamin produces some ended and intermediate flavor com‐ pounds [62,63]. It was assumed that thermal degradation of thiamin is a quite complex reac‐ tion including various degradation pathways to produce interesting flavor compounds in which most of them contain one or more sulfur and/or nitrogen atoms, and many of them are heterocyclic structures. The thermal degradation of thiamin under the basic condition to

It was reported that the primary products of thermally-degraded thiamin including 4-meth‐ yl-5-(2-hydroxyethyl)thiazole which subsequently responds for formation of thiazoles and other sulfur compounds such as 5-hydroxy-3-mercaptopentan-2-one which then gives some sulfur-containing compounds such as thiophenes and furans as well [62]. Heating tempera‐ ture and pH conditions have been showed to affect the degradation products of thiamin. At pH 5.0 and 7.0 the 2-methyl-3furanthiol and bis (2-methyl3-furyl) disulfide (meaty aroma)

produce several flavor compounds is illustrated in Figure 5.

ficiently low odor threshold to be contributors of meat aroma flavors.

*2.3.3. Thiamin degradation*

156 Latest Research into Quality Control

#### *2.3.4. Interaction between lipid-oxidized products with Maillard products*

The interaction between oxidized lipids and amino acids or proteins is very complex, in term of a consequence of the contribution of both lipid hydroperoxide and its secondaryoxidized products. This interaction may imply both the formation of physical complexes be‐ tween the oxidized lipids and the amino acids or protein and the formation of various types of covalent bonds. Protein polymerizarion produced by reaction with peroxy free radicals generated during lipid peroxidation is known to occur during nonenzymatic browning [66, 67, 68]. However, in term of flavor study, the interaction between lipid-oxidized products (secondary products) with amino acids or proteins is the most concerned. Lipid-oxidized products are generic terms used to describe a mixture of aldehydes, alcohols, ketones and other products obtained by the decomposition of lipid hydroperoxides. Although it is not widely recognized, this decomposition does not necessarily imply the breakage of the lipid chain, and the formation of covalent bonds in the reaction between long chain oxidized lip‐ ids and amino acids and proteins has been described [69, 70]. This is a consequence of the existence of fatty acids that produce a complex and diverse mixture of lipid oxidation prod‐ ucts that are able to react with the different reactive protein residues.

In the Maillard reaction, amino acids can undergo the Strecker degradation process that sub‐ sequently generates some reactive radicals such as ammonia, hydrosulfide and etc which al‐ so are able to further react with the secondary oxidized products of lipid to produce volatile flavor compounds such as thiols, thiophenes, thiazoles and etc as showed in Figure 6.

colleagues (2002) [34] concluded that breakdown products of polyunsaturated fatty acids es‐ pecially are *n*-3 acids have a shorter chain length have lower odor thresholds will also be present at relatively high concentrations and are more reactive. These breakdown products will affect meat flavor by interacting with the Maillard reaction reducing levels of meaty ar‐ oma compounds, such as sulfur-substituted thiophenes and furans. As *n*-3 PUFAs are readi‐ ly oxidized, they could initiate the free radical oxidation of more saturated acids, increasing levels of breakdown products of *n*-6 and *n*-9 fatty acids, which may also alter the aroma

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Diet is as an important indicator to show the growth rate, performance, reproducibility ef‐ fects and as well as meat quality of cattle. There has been an existed hypothesis of meat fla‐ vor changes due to feeding diets in which some works stated a large difference in meat flavor characteristics of the same cattle breed but fed on different diets. Early work by Mel‐ ton (1983) [71] stated that steers fed with corn-based diets had more intense beef flavor (de‐ sirable flavor) than the same age steers fed based pasture or Bermuda pellets. A later study of Melton (1990) [10] found that the less desirable flavor of meat from cattle is mainly caused by several grass species. Conversely, no significant difference in flavors existed between the grass and grain diets-fed animals [73]. The less desirable flavors were also seen on meats from the hay diets-fed animals compared to corn silage diets [74], while Oltjen et al (1971) [75] showed the opposite results. It has been hypothesized that majority of flavor effects due to feeding of forages is mainly due to changes in fatty acid compositions. Fishy off-flavor was significantly higher in meat from grass-finished cattle with increasing unsaturated fatty acids [76]. Recently, researchers have attempted to higher level of PUFA in meat aiming to increase the health benefits by using the fat supplemented-diets (e.g., linseed, sunflower oil and fish oil) to cattle [77, 78], although these works have achieved an increase in several ben‐ efit fatty acids however, the detrimental effects on meat flavor characteristics appeared due to higher levels of PUFA [79]. A large number of studies regarding the effect of diets on vol‐ atile flavor compounds of cooked meat have been performed. Melton (1983) [71] also noted that the greatest difference in the flavors of meat from cattle fed on grass and grain-based diets is due to fatty acid concentration and type as fatty acids are the primary source of car‐ bonyl. Suzuki and Bailey (1985) [80] indicated that higher concentrations of pentanoic,hepta‐ noic, octanoic, nonanoic, decanoic, and dodecanoic acids were formed in the meat fat from grass-fed animals while heptanal, 2,3-octanedione, 3-hydroxyoctan-2-one, 2-decenal, 2-tride‐ canone, hexadecane, heptadecane, octodecane, d-dodecalactone, phyt-1-ene, neophytadiene, phyt-2-ene, an isomer of neophytadiene, 2-heptadecanone, dihydrophytol, and phytol with the terpenoids in much higher concentration due to rumen-fermented chlorophyll. Individu‐ al volatile flavor compounds like 4-heptanal, 2, 4-heptadienal and 2, 6-nonadienal (derived

compounds of the cooked meat.

**3.1 Effect of diets**

**3. The factors affect aroma flavors**

The interaction between lipid and Maillard reaction have extensively been studied in a num‐ ber of studies using model systems containing amino acids and sugars in the presence of lip‐ id [43, 61, 39, 34]. In these studies, systeine and ribose were used for Maillard reaction and in the presence of phospholipids from various sources including egg-yolk and beef. The reac‐ tion mixtures produced a lot of aroma volatiles which dominated by sulfur-containing com‐ ponents especially heterocyclics such as thiols and thiophenes. These studies also observed that the presence of phospholipids made a great reduction in amounts of these compounds. Famer and Mottram (1990) [61] also noted that beef-originated triglyceride has much less in‐ fluence on amounts of heterocyclics than the phospholipids from beef do. The study also found that the addition of beef triglyceride to the Maillard reaction did not influence the sul‐ furous and rubbery aroma but when beef phospholipids were added resulting in higher meaty aroma note whereas the sulfurous notes were less. However, the Maillard reaction systems containing phospholipids usually had lower level of some meaty compounds espe‐ cially 2-methyl-3-furanthiol this is due to the lipid limits generation of these compounds and only maintain theme at an optimum level in the reaction mixture.

**Figure 6.** The interaction between lipid-oxidized products with Maillard products.

In general, in Maillard reaction mixtures containing lipids produce a lot of lipids-derived volatile compounds such as aldehydes, furans, hydrocarbons, alcohols and ketones. Further‐ more, the reaction mixtures also containing the Maillard products such as H2S, NH3, etc. Which all are able to interact with each other to form new heterocyclic aroma volatile com‐ pounds as the consequences of the interactions between lipids with Maillard products. The most abundant compounds have been detected as results of the interactions are thiophene class such as 2-pentylthiophene, 2-hexylthiophene and thiol class such as 2-thiophenethiol, 2-furylmethanethiol, 2-methyl-3-furanthiol and etc [38, 61]. A recent study by Elmore and colleagues (2002) [34] concluded that breakdown products of polyunsaturated fatty acids es‐ pecially are *n*-3 acids have a shorter chain length have lower odor thresholds will also be present at relatively high concentrations and are more reactive. These breakdown products will affect meat flavor by interacting with the Maillard reaction reducing levels of meaty ar‐ oma compounds, such as sulfur-substituted thiophenes and furans. As *n*-3 PUFAs are readi‐ ly oxidized, they could initiate the free radical oxidation of more saturated acids, increasing levels of breakdown products of *n*-6 and *n*-9 fatty acids, which may also alter the aroma compounds of the cooked meat.

#### **3. The factors affect aroma flavors**

#### **3.1 Effect of diets**

so are able to further react with the secondary oxidized products of lipid to produce volatile

The interaction between lipid and Maillard reaction have extensively been studied in a num‐ ber of studies using model systems containing amino acids and sugars in the presence of lip‐ id [43, 61, 39, 34]. In these studies, systeine and ribose were used for Maillard reaction and in the presence of phospholipids from various sources including egg-yolk and beef. The reac‐ tion mixtures produced a lot of aroma volatiles which dominated by sulfur-containing com‐ ponents especially heterocyclics such as thiols and thiophenes. These studies also observed that the presence of phospholipids made a great reduction in amounts of these compounds. Famer and Mottram (1990) [61] also noted that beef-originated triglyceride has much less in‐ fluence on amounts of heterocyclics than the phospholipids from beef do. The study also found that the addition of beef triglyceride to the Maillard reaction did not influence the sul‐ furous and rubbery aroma but when beef phospholipids were added resulting in higher meaty aroma note whereas the sulfurous notes were less. However, the Maillard reaction systems containing phospholipids usually had lower level of some meaty compounds espe‐ cially 2-methyl-3-furanthiol this is due to the lipid limits generation of these compounds and

flavor compounds such as thiols, thiophenes, thiazoles and etc as showed in Figure 6.

only maintain theme at an optimum level in the reaction mixture.

158 Latest Research into Quality Control

**Figure 6.** The interaction between lipid-oxidized products with Maillard products.

In general, in Maillard reaction mixtures containing lipids produce a lot of lipids-derived volatile compounds such as aldehydes, furans, hydrocarbons, alcohols and ketones. Further‐ more, the reaction mixtures also containing the Maillard products such as H2S, NH3, etc. Which all are able to interact with each other to form new heterocyclic aroma volatile com‐ pounds as the consequences of the interactions between lipids with Maillard products. The most abundant compounds have been detected as results of the interactions are thiophene class such as 2-pentylthiophene, 2-hexylthiophene and thiol class such as 2-thiophenethiol, 2-furylmethanethiol, 2-methyl-3-furanthiol and etc [38, 61]. A recent study by Elmore and

Diet is as an important indicator to show the growth rate, performance, reproducibility ef‐ fects and as well as meat quality of cattle. There has been an existed hypothesis of meat fla‐ vor changes due to feeding diets in which some works stated a large difference in meat flavor characteristics of the same cattle breed but fed on different diets. Early work by Mel‐ ton (1983) [71] stated that steers fed with corn-based diets had more intense beef flavor (de‐ sirable flavor) than the same age steers fed based pasture or Bermuda pellets. A later study of Melton (1990) [10] found that the less desirable flavor of meat from cattle is mainly caused by several grass species. Conversely, no significant difference in flavors existed between the grass and grain diets-fed animals [73]. The less desirable flavors were also seen on meats from the hay diets-fed animals compared to corn silage diets [74], while Oltjen et al (1971) [75] showed the opposite results. It has been hypothesized that majority of flavor effects due to feeding of forages is mainly due to changes in fatty acid compositions. Fishy off-flavor was significantly higher in meat from grass-finished cattle with increasing unsaturated fatty acids [76]. Recently, researchers have attempted to higher level of PUFA in meat aiming to increase the health benefits by using the fat supplemented-diets (e.g., linseed, sunflower oil and fish oil) to cattle [77, 78], although these works have achieved an increase in several ben‐ efit fatty acids however, the detrimental effects on meat flavor characteristics appeared due to higher levels of PUFA [79]. A large number of studies regarding the effect of diets on vol‐ atile flavor compounds of cooked meat have been performed. Melton (1983) [71] also noted that the greatest difference in the flavors of meat from cattle fed on grass and grain-based diets is due to fatty acid concentration and type as fatty acids are the primary source of car‐ bonyl. Suzuki and Bailey (1985) [80] indicated that higher concentrations of pentanoic,hepta‐ noic, octanoic, nonanoic, decanoic, and dodecanoic acids were formed in the meat fat from grass-fed animals while heptanal, 2,3-octanedione, 3-hydroxyoctan-2-one, 2-decenal, 2-tride‐ canone, hexadecane, heptadecane, octodecane, d-dodecalactone, phyt-1-ene, neophytadiene, phyt-2-ene, an isomer of neophytadiene, 2-heptadecanone, dihydrophytol, and phytol with the terpenoids in much higher concentration due to rumen-fermented chlorophyll. Individu‐ al volatile flavor compounds like 4-heptanal, 2, 4-heptadienal and 2, 6-nonadienal (derived from C18:3n-3) and hexanal, 2-heptanal and 2, 4-decadienal (derived from C18:2n-6) found to be higher concentration in meats from grass and grain-fed animals, respectively [81]. El‐ more et al (1997) [82] also reported that cooked meat from the animals that had been fed fish oil had considerably higher concentrations of saturated and unsaturated aldehydes than meat from the control. While, Descalzo et al (2005) [83] found that some classes of volatile flavor components affected by diets in which aldehydes increased in meat from concentrate diets-fed animals. In general, we can see that diets have a large influence on meat flavors due to directly affect the meat contents especially the intramuscular fat contents which play an important role in interaction and generation of volatile flavor compounds. On the other hand, it is worth noting that the uses of fat –supplemented diets to feed cattle may result in increases of important polyunsaturated fatty acids (e.g., n-3 fatty acids, DHA, EPA) which known to positively affect on consumers health however, a negative effect on meat flavors may appear due to these fatty acids not only produce some unexpected volatile compounds but also inhibit production of other Maillard products.

the differences in meat flavors existing between breeds or sexes are probably due to the dif‐ ferences in the levels of flavor precursors especially the fat contents which large affect the formation of aroma flavor compounds and also interact with other contents in determining

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161

Ageing has been become a universal method widely used to improve eating quality of meat (e.g. tenderness, juiciness, flavor). Un-aged beef has a weak, bland odor while aged beef has a strong, savory, roasted odor. Ageing of meat makes an increase in fatty flavor characteris‐ tics however; long term ageing (e.g., > 3 weeks) could cause a decrease in positive flavor notes and increase liver-like aroma, bloody, bitter and off-flavor [87, 85, 88]. Ismail et al (2008) [89] stated that ethanol was responsible for the increase in alcohols caused by the mi‐ crobial growth in beef during storage furthermore, the levels of aldehydes significantly in‐ creased after 7 days of storage. Beef from various muscles including gluteus medius, rectus femoris, vastus lateralis, vatsus medialis, teres major, complexus, serratus ventralis, psoas major and longissimus dorsi of heifer carcasses were chiller aged for 7 or 14 days the results showed that flavor-active volatiles included nonanal, 2,3-octanedione, pentanal, 3-hy‐ droxy-2-butanone, 2-pentyl furan, 1-octen-3-ol, butanoic acid, pentanal and hexanoic acid which all often associated with lipid oxidation were affected by enhancement and ageing in the various muscles [90]. Additionally, ageing of beef achieved an increase in characteristic flavor and also aftertaste intensity, making an appreciable improvement of its flavor. After slaughter, loss of circulatory competency results in the accumulation of metabolic by-prod‐ ucts, including lactic acid, in the muscle, that induces pH decline. The endogenous enzymes (e.g., cathespins B and L) are activated at near pH 5.4. Spanier and Miller (1993) and Spanier et al (1990) [91, 92] suggested that these thiol proteinases can hydrolyze more peptide bonds than any other group of enzymes, are redistributed during ageing period. Proteolytic en‐ zyme activity is temperature-dependent; some enzymes retain high activity levels even at cooking temperatures. The combined effect of postmortem ageing and cooking, via enzyme redistribution and activity can influence the production of aroma flavor compounds. Toldrá and Flores (2000) [93] stated that enzymes known primarily for textural changes (e.g., μ- and m-calpain) during the postmortem period affect flavors by producing peptides, but it was observed that these enzymes correlate with increases in rancid, sour and salty flavors. The ageing conditions (e.g., oxygen availability, temperature, humidity and aging time) under which beef is aged influences the ultimate flavors of the meat particularly ageing in a higher oxygen environment cause a burnt, toasted off-odor. In addition, dry-ageing increases beef flavor attributes more than ageing in vacuum or in carbon dioxide [94, 95]. Based on the re‐ sults reported in the previous studies it could be concluded that chiller ageing of meat re‐ sulted in increases of most of flavor compounds however a long ageing period (e.g., > 3 weeks) may negatively influences the flavor quality of cooked meat due to increase in amounts of some unexpected compounds which associated with undesirable flavors and de‐

crease in the some important compounds which associated with desirable flavors.

flavor characteristics of cooked meat.

**3.3. Effect of chiller ageing**

#### **3.2. Effect of breeds and sex**

Researchers have reported that breed also affects volatile flavor components and then influ‐ ence overall flavor notes of cooked meat. Elmore et al (2000) [25] stated that fifty-four com‐ pounds were affected by breed, 75% of which was Maillard reaction products. Over 40 compounds were present at higher levels in the Soay breed than in the Suffolk breed. Other sulfur-containing compounds present at higher levels in the Suffolks than the Soays were bis-(2-furylmethyl) disulfide and 2-methyl-4,5-dihydro-thiophene and the differences in sul‐ fur and nitrogen-containing compounds could contribute to flavor differences between the two breeds. A study on pork flavors as affected by breeds also have found that twenty-three among the detected flavor compounds were significantly affected by breed. Based on senso‐ ry analysis indicated that cooked *longissimus* muscle from hybrid breed (Duroc x Landrace x Large White) had the lowest pork flavor intensity and flavor-liking compared with the Chi‐ nese indigenous breeds. Laiwu and Dahuabai breeds showed the highest pork flavor inten‐ sity and flavor-liking in cooked longissimus muscle [36]. In beef, Insausti and colleagues (2005) [84] also found the differences in volatile flavor compounds among the breeds were considerable and may contributed to the perception of flavor differences in the cooked beef. Particularly, level of dimethyl sulfide probably related to cauliflower notes, was highest for meat from the Pirenaica breed. While, levels of the sulfur-compounds in cooked beef from the Asturiana breed were low-intermediate and potentially related to blood and liver notes and unpleasant flavors.

For the sex effect, it has been reported that meat from bulls has a strong livery and blood flavors while meat from heifers has a strong characteristic flavor. The differences could be associated with the differences in amounts of certain volatile compounds such as hydrocar‐ bons, aldehydes, alcohols and ketones [85]. On the other hand, the differences in meat fla‐ vors between bulls and heifers could be explained by the genetic control of animal development and production of sex hormones and their influence the lipid composition which affects the kinds of volatile flavor compounds [86]. Overall, it may be assumed that the differences in meat flavors existing between breeds or sexes are probably due to the dif‐ ferences in the levels of flavor precursors especially the fat contents which large affect the formation of aroma flavor compounds and also interact with other contents in determining flavor characteristics of cooked meat.

#### **3.3. Effect of chiller ageing**

from C18:3n-3) and hexanal, 2-heptanal and 2, 4-decadienal (derived from C18:2n-6) found to be higher concentration in meats from grass and grain-fed animals, respectively [81]. El‐ more et al (1997) [82] also reported that cooked meat from the animals that had been fed fish oil had considerably higher concentrations of saturated and unsaturated aldehydes than meat from the control. While, Descalzo et al (2005) [83] found that some classes of volatile flavor components affected by diets in which aldehydes increased in meat from concentrate diets-fed animals. In general, we can see that diets have a large influence on meat flavors due to directly affect the meat contents especially the intramuscular fat contents which play an important role in interaction and generation of volatile flavor compounds. On the other hand, it is worth noting that the uses of fat –supplemented diets to feed cattle may result in increases of important polyunsaturated fatty acids (e.g., n-3 fatty acids, DHA, EPA) which known to positively affect on consumers health however, a negative effect on meat flavors may appear due to these fatty acids not only produce some unexpected volatile compounds

Researchers have reported that breed also affects volatile flavor components and then influ‐ ence overall flavor notes of cooked meat. Elmore et al (2000) [25] stated that fifty-four com‐ pounds were affected by breed, 75% of which was Maillard reaction products. Over 40 compounds were present at higher levels in the Soay breed than in the Suffolk breed. Other sulfur-containing compounds present at higher levels in the Suffolks than the Soays were bis-(2-furylmethyl) disulfide and 2-methyl-4,5-dihydro-thiophene and the differences in sul‐ fur and nitrogen-containing compounds could contribute to flavor differences between the two breeds. A study on pork flavors as affected by breeds also have found that twenty-three among the detected flavor compounds were significantly affected by breed. Based on senso‐ ry analysis indicated that cooked *longissimus* muscle from hybrid breed (Duroc x Landrace x Large White) had the lowest pork flavor intensity and flavor-liking compared with the Chi‐ nese indigenous breeds. Laiwu and Dahuabai breeds showed the highest pork flavor inten‐ sity and flavor-liking in cooked longissimus muscle [36]. In beef, Insausti and colleagues (2005) [84] also found the differences in volatile flavor compounds among the breeds were considerable and may contributed to the perception of flavor differences in the cooked beef. Particularly, level of dimethyl sulfide probably related to cauliflower notes, was highest for meat from the Pirenaica breed. While, levels of the sulfur-compounds in cooked beef from the Asturiana breed were low-intermediate and potentially related to blood and liver notes

For the sex effect, it has been reported that meat from bulls has a strong livery and blood flavors while meat from heifers has a strong characteristic flavor. The differences could be associated with the differences in amounts of certain volatile compounds such as hydrocar‐ bons, aldehydes, alcohols and ketones [85]. On the other hand, the differences in meat fla‐ vors between bulls and heifers could be explained by the genetic control of animal development and production of sex hormones and their influence the lipid composition which affects the kinds of volatile flavor compounds [86]. Overall, it may be assumed that

but also inhibit production of other Maillard products.

**3.2. Effect of breeds and sex**

160 Latest Research into Quality Control

and unpleasant flavors.

Ageing has been become a universal method widely used to improve eating quality of meat (e.g. tenderness, juiciness, flavor). Un-aged beef has a weak, bland odor while aged beef has a strong, savory, roasted odor. Ageing of meat makes an increase in fatty flavor characteris‐ tics however; long term ageing (e.g., > 3 weeks) could cause a decrease in positive flavor notes and increase liver-like aroma, bloody, bitter and off-flavor [87, 85, 88]. Ismail et al (2008) [89] stated that ethanol was responsible for the increase in alcohols caused by the mi‐ crobial growth in beef during storage furthermore, the levels of aldehydes significantly in‐ creased after 7 days of storage. Beef from various muscles including gluteus medius, rectus femoris, vastus lateralis, vatsus medialis, teres major, complexus, serratus ventralis, psoas major and longissimus dorsi of heifer carcasses were chiller aged for 7 or 14 days the results showed that flavor-active volatiles included nonanal, 2,3-octanedione, pentanal, 3-hy‐ droxy-2-butanone, 2-pentyl furan, 1-octen-3-ol, butanoic acid, pentanal and hexanoic acid which all often associated with lipid oxidation were affected by enhancement and ageing in the various muscles [90]. Additionally, ageing of beef achieved an increase in characteristic flavor and also aftertaste intensity, making an appreciable improvement of its flavor. After slaughter, loss of circulatory competency results in the accumulation of metabolic by-prod‐ ucts, including lactic acid, in the muscle, that induces pH decline. The endogenous enzymes (e.g., cathespins B and L) are activated at near pH 5.4. Spanier and Miller (1993) and Spanier et al (1990) [91, 92] suggested that these thiol proteinases can hydrolyze more peptide bonds than any other group of enzymes, are redistributed during ageing period. Proteolytic en‐ zyme activity is temperature-dependent; some enzymes retain high activity levels even at cooking temperatures. The combined effect of postmortem ageing and cooking, via enzyme redistribution and activity can influence the production of aroma flavor compounds. Toldrá and Flores (2000) [93] stated that enzymes known primarily for textural changes (e.g., μ- and m-calpain) during the postmortem period affect flavors by producing peptides, but it was observed that these enzymes correlate with increases in rancid, sour and salty flavors. The ageing conditions (e.g., oxygen availability, temperature, humidity and aging time) under which beef is aged influences the ultimate flavors of the meat particularly ageing in a higher oxygen environment cause a burnt, toasted off-odor. In addition, dry-ageing increases beef flavor attributes more than ageing in vacuum or in carbon dioxide [94, 95]. Based on the re‐ sults reported in the previous studies it could be concluded that chiller ageing of meat re‐ sulted in increases of most of flavor compounds however a long ageing period (e.g., > 3 weeks) may negatively influences the flavor quality of cooked meat due to increase in amounts of some unexpected compounds which associated with undesirable flavors and de‐ crease in the some important compounds which associated with desirable flavors.

#### **3.4. Effect of cooking temperature and pH conditions**

Cooking temperature is one of the important factors impacting the development of fla‐ vors through the Maillard reactions and lipid oxidation. Amino acids can undergo Streck‐ er degradation to produce Strecker products. Degradation of sulfur-containing amino acids (e.g., cysteine, cystine and methionine) generates sulfur that contributes to subsequent proc‐ esses of Maillard reaction. These compounds can react with amines and amino acids to produce a number of flavor-contributing compounds and potent cooked meat odorants such as pyrazines, oxazoles, thiophenes, thiazoles and other heterocyclic sulfur containing com‐ pounds [20]. It was well seen that cooking temperatures affect these reactions and then determine flavor characteristics, for instance the stewed meat lacks flavors of the roasted products because of stewed meat has a water activity of approximately 1.0 and not ex‐ ceed temperature of 100o C while roasted meat has dried surfaces and temperature may exceed 100o C therefore, the conditions like low water activity and high surface tempera‐ ture will increase production of flavor compounds which give roasted odor notes rather than meat is stewed. Ames et al (2001) [40] concluded that the amounts of most volatile flavor compounds increased with cooking temperature. Cooking at lower temperatures (<165 o C) versus higher temperatures (>180 o C) results in differences in the concentrations of a number of compounds such as 2, 4, 5-trimethyl- 3-oxazoline; 2, 4-dimethyl-5-ethyl-3-oxazo‐ line; 2, 5- dimethyl-4-ethyl-3-oxazoline; 2, 4-dimethyl-3-thiazoline; 2, 4, 5-trimethyl-3-thiazo‐ line [96]. Previous works found that a strong relationship existing between cooking temperature, concentration of free amino acids, carnosine, pyrazines and hexanol, and roasted, burnt and beefy flavor intensity [97, 98]. Cooking beef generates urea content which can also reduce sulfur-containing compounds generating important nitrogen-containing compounds like pyrazines and thiazoles in which pyrazines are formed mostly on the surface of meat and having nutty and roasty odor notes [99]. In general, the higher degree of heating, the high‐ er the concentration of aliphatic aldehydes, benzenoids, polysulfides, heterocyclic com‐ pounds and lipid-derived volatiles. Ketones, alcohols sulfur-containing components make smaller contributions.

showed decrease as the pH increased, and an increase in the disulphide was observed. It was observed that a large number of sulphur-containing compounds such as 2-methyl-3-fur‐ anthiol a strong meaty aroma, whose formation was greatly favored by lower pH condition. Ames et al (2001) [40] used model reactions containing cysteine and reducing sugar at var‐ ied pH conditions 5.5, 6.5 and 7.5, results showed that amounts of most of compounds in‐ creased with pH especially are pyrazines. Cerny and Biffod (2007) [103] recently found that pH determined strongly which volatile flavors were formed and to what extent. In general, based on the results of the previous studies which all found that pH condition strongly in‐

Principle of Meat Aroma Flavors and Future Prospect

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163

Irradiation is a food safety technology designed to eliminate disease-causing germs from foods. Depending on the dose levels of irradiation applying on the raw meat and poultry, or ready-to-eat meats that can eliminate bacteria commonly found such as E. coli, Salmonella and Listeria; virus; or parasites. However, irradiation may result in off-odors and flavors. The odors vary with the type of meat, temperature during irradiation, oxygen exposure dur‐ ing and/or after the irradiation process, packaging and presence of antioxidative substances [104]. Most of studies have reported that the aroma flavors of irradiated meat associated with rotten egg, sweet, bloody, cooked meat, barbecued corn, burnt, sulfur, metallic, alcohol, acetic acid, liver-like serumy and bloody [105, 106, 107]. Irradiation can initiate or promote lipid oxidation resulting in undesirable off-odors and flavors [108, 109]. Jo and Ahn, (2000) [110] showed that reactions of sulfur-containing amino acids with radiolytic products of wa‐ ter appear to be the source of hydrogen sulfide and other volatile sulfur-containing com‐ pounds which contribute to off-flavor. On the other hand, irradiation may result in the formation of free radicals from unsaturated fatty acids at double bond positions [109]. An increase in lipid peroxidation products such as hexanal and (E)-4,5-epoxy-(E)-2-decenal in combination with a loss of desirable meaty odorants (4-hydroxy-2,5-dimethyl-3(2H)-fura‐ none and 3-hydroxy-4,5-dimethyl-2(5H)-furanone) result in development of warmed over flavor of cooked, refrigerated beef [16]. However, the effects of irradiation on aroma flavors are also depended on: (1) Dose levels of irradiation, it has been demonstrated that the dose levels of irradiation influence variedly on volatile flavor components of cooked meat, as re‐ ported by Jo and Ahn (2000) [110] who indicated some of hydrocarbons included 1-heptene and 1-nonene increased with irradiation dose immediately after irradiation of beef. A simi‐ lar observation also was reported by Yong et al (2000) [111] who indicated that among the 150 flavor compounds indentified in beef the cyclodecene, (E)-2-hexenal, nonene and 2 nonenal showed an increase in a dose-dependent fashion. For the effect of irradiation on chicken flavors, Yong e al (2000) [112] showed that among the 129 identified volatile flavor compounds the cyclotetradecene, 2-methylpentanal and 4-methylcyclohexene were formed specifically in response to irradiation, and level of cyclotetradecene increased in a dose-de‐ pendent fashion; (2) Oxygen presence, the presence of oxygen around meats during irradiat‐ ing can diffuse into the meats, and then results in radiolytic changes which precipitate oxidation and unacceptable secondary breakdown products. As well known, lipid oxidation needs oxygen presence to produce oxidized-products such as aldehydes, Nam and Ahn

fluence the formation of flavor components.

**3.5. Effect of irradiation on meat flavors**

pH is one of the important factors that influence the kind of volatile flavor compounds formed in the Maillard reaction, and then determine the final flavor characteristics of cooked food. Madruga and Mottram (1995) [8] showed that as pH increases, color and polymeric compounds increase and nitrogen-containing compounds like pyrazines are favored, there‐ fore it was assumed that higher ultimate pH in meat from grass-fed animals may favor the formation of thiazoles and thiophenones due to the availability of amino acid degradation products while decreasing other sulfur volatiles that favor lower pH. A number of early studies have been performed to investigate the effect of pH changes on volatile flavor com‐ pounds using model systems (El'Gde et al., 1966; Shu et *al.,* 1985; Meynier and Mottram, 1995) [100, 101, 102]. These studies found that high pH values also favor the formation of many volatile compounds but other compounds are only favored at low pH condition. Mey‐ nier and Mottram (1995) [102] used meat-like model systems containing amino acids and ri‐ bose on different pH 4.5 and 6.5, results showed that nitrogen-containing compounds such as pyrazines were detected at higher pHs. While, dimethyldisulphide and methional showed decrease as the pH increased, and an increase in the disulphide was observed. It was observed that a large number of sulphur-containing compounds such as 2-methyl-3-fur‐ anthiol a strong meaty aroma, whose formation was greatly favored by lower pH condition. Ames et al (2001) [40] used model reactions containing cysteine and reducing sugar at var‐ ied pH conditions 5.5, 6.5 and 7.5, results showed that amounts of most of compounds in‐ creased with pH especially are pyrazines. Cerny and Biffod (2007) [103] recently found that pH determined strongly which volatile flavors were formed and to what extent. In general, based on the results of the previous studies which all found that pH condition strongly in‐ fluence the formation of flavor components.

#### **3.5. Effect of irradiation on meat flavors**

**3.4. Effect of cooking temperature and pH conditions**

ceed temperature of 100o

162 Latest Research into Quality Control

smaller contributions.

C) versus higher temperatures (>180 o

exceed 100o

o

Cooking temperature is one of the important factors impacting the development of fla‐ vors through the Maillard reactions and lipid oxidation. Amino acids can undergo Streck‐ er degradation to produce Strecker products. Degradation of sulfur-containing amino acids (e.g., cysteine, cystine and methionine) generates sulfur that contributes to subsequent proc‐ esses of Maillard reaction. These compounds can react with amines and amino acids to produce a number of flavor-contributing compounds and potent cooked meat odorants such as pyrazines, oxazoles, thiophenes, thiazoles and other heterocyclic sulfur containing com‐ pounds [20]. It was well seen that cooking temperatures affect these reactions and then determine flavor characteristics, for instance the stewed meat lacks flavors of the roasted products because of stewed meat has a water activity of approximately 1.0 and not ex‐

C while roasted meat has dried surfaces and temperature may

C) results in differences in the concentrations of a

C therefore, the conditions like low water activity and high surface tempera‐

ture will increase production of flavor compounds which give roasted odor notes rather than meat is stewed. Ames et al (2001) [40] concluded that the amounts of most volatile flavor compounds increased with cooking temperature. Cooking at lower temperatures (<165

number of compounds such as 2, 4, 5-trimethyl- 3-oxazoline; 2, 4-dimethyl-5-ethyl-3-oxazo‐ line; 2, 5- dimethyl-4-ethyl-3-oxazoline; 2, 4-dimethyl-3-thiazoline; 2, 4, 5-trimethyl-3-thiazo‐ line [96]. Previous works found that a strong relationship existing between cooking temperature, concentration of free amino acids, carnosine, pyrazines and hexanol, and roasted, burnt and beefy flavor intensity [97, 98]. Cooking beef generates urea content which can also reduce sulfur-containing compounds generating important nitrogen-containing compounds like pyrazines and thiazoles in which pyrazines are formed mostly on the surface of meat and having nutty and roasty odor notes [99]. In general, the higher degree of heating, the high‐ er the concentration of aliphatic aldehydes, benzenoids, polysulfides, heterocyclic com‐ pounds and lipid-derived volatiles. Ketones, alcohols sulfur-containing components make

pH is one of the important factors that influence the kind of volatile flavor compounds formed in the Maillard reaction, and then determine the final flavor characteristics of cooked food. Madruga and Mottram (1995) [8] showed that as pH increases, color and polymeric compounds increase and nitrogen-containing compounds like pyrazines are favored, there‐ fore it was assumed that higher ultimate pH in meat from grass-fed animals may favor the formation of thiazoles and thiophenones due to the availability of amino acid degradation products while decreasing other sulfur volatiles that favor lower pH. A number of early studies have been performed to investigate the effect of pH changes on volatile flavor com‐ pounds using model systems (El'Gde et al., 1966; Shu et *al.,* 1985; Meynier and Mottram, 1995) [100, 101, 102]. These studies found that high pH values also favor the formation of many volatile compounds but other compounds are only favored at low pH condition. Mey‐ nier and Mottram (1995) [102] used meat-like model systems containing amino acids and ri‐ bose on different pH 4.5 and 6.5, results showed that nitrogen-containing compounds such as pyrazines were detected at higher pHs. While, dimethyldisulphide and methional

Irradiation is a food safety technology designed to eliminate disease-causing germs from foods. Depending on the dose levels of irradiation applying on the raw meat and poultry, or ready-to-eat meats that can eliminate bacteria commonly found such as E. coli, Salmonella and Listeria; virus; or parasites. However, irradiation may result in off-odors and flavors. The odors vary with the type of meat, temperature during irradiation, oxygen exposure dur‐ ing and/or after the irradiation process, packaging and presence of antioxidative substances [104]. Most of studies have reported that the aroma flavors of irradiated meat associated with rotten egg, sweet, bloody, cooked meat, barbecued corn, burnt, sulfur, metallic, alcohol, acetic acid, liver-like serumy and bloody [105, 106, 107]. Irradiation can initiate or promote lipid oxidation resulting in undesirable off-odors and flavors [108, 109]. Jo and Ahn, (2000) [110] showed that reactions of sulfur-containing amino acids with radiolytic products of wa‐ ter appear to be the source of hydrogen sulfide and other volatile sulfur-containing com‐ pounds which contribute to off-flavor. On the other hand, irradiation may result in the formation of free radicals from unsaturated fatty acids at double bond positions [109]. An increase in lipid peroxidation products such as hexanal and (E)-4,5-epoxy-(E)-2-decenal in combination with a loss of desirable meaty odorants (4-hydroxy-2,5-dimethyl-3(2H)-fura‐ none and 3-hydroxy-4,5-dimethyl-2(5H)-furanone) result in development of warmed over flavor of cooked, refrigerated beef [16]. However, the effects of irradiation on aroma flavors are also depended on: (1) Dose levels of irradiation, it has been demonstrated that the dose levels of irradiation influence variedly on volatile flavor components of cooked meat, as re‐ ported by Jo and Ahn (2000) [110] who indicated some of hydrocarbons included 1-heptene and 1-nonene increased with irradiation dose immediately after irradiation of beef. A simi‐ lar observation also was reported by Yong et al (2000) [111] who indicated that among the 150 flavor compounds indentified in beef the cyclodecene, (E)-2-hexenal, nonene and 2 nonenal showed an increase in a dose-dependent fashion. For the effect of irradiation on chicken flavors, Yong e al (2000) [112] showed that among the 129 identified volatile flavor compounds the cyclotetradecene, 2-methylpentanal and 4-methylcyclohexene were formed specifically in response to irradiation, and level of cyclotetradecene increased in a dose-de‐ pendent fashion; (2) Oxygen presence, the presence of oxygen around meats during irradiat‐ ing can diffuse into the meats, and then results in radiolytic changes which precipitate oxidation and unacceptable secondary breakdown products. As well known, lipid oxidation needs oxygen presence to produce oxidized-products such as aldehydes, Nam and Ahn (2003) [113] indicated that irradiation of meat in aerobic packaging promoted production of aldehydes such as propanal and hexanal which is assumed as a good indicator of lipid oxi‐ dation. The similar observation also was reported by Nam et al (2001) [114] who also showed that irradiation increased TBARS values and off-flavor in aerobically-packaged pork (3) Temperature effect, temperature during irradiating meats has a large effect on aro‐ ma flavors of irradiated meat because temperature affects what radiolytic products are formed and what ratios [104]. Using lower temperature during irradiation of meat by freez‐ ing meat before irradiation can reduce detrimental effects via retarding autoxidation and ex‐ tending shelf life; (4) pH effect, it has also been demonstrated that the ultimate pH of meat at the time of irradiation influences lipid oxidation. Nam et al (2001) [114] recently showed that irradiation increased lipid oxidation of normal and pale-soft-exudative (low pH group) muscles, whereas dark-firm-dry (high pH group) muscle was very stable and resistant to ox‐ idative changes. Therefore, to minimize the detrimental effects of irradiation on aroma fla‐ vor characteristics we can modify atmosphere packaging by using vacuum packaging (anaerobic packaging) or replacement with inert gases (i.e. nitrogen, helium, hydrogen, car‐ bon dioxide) to eliminate oxygen. Reducing the temperature (freezing) prior to irradiation and addition of antioxidants. Vacuum packaging retains irradiation-generated sulfur-con‐ taining compounds, however re-packaging meat in oxygen-permeable materials allows for dissipation of these flavor compounds.

er these conditions might promote the liver-like flavor [27]. James and Calkins, (2005) [121] also hypothesized that the slower cooking and longer hold time allow the undesirable

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165

Up to present time, various techniques have been designed, combined with gas chromatog‐ raphy and mass spectrometry (GC/MS) or Flame ionizing detector (GC/FID) and applied to evaluate volatile flavor components in cooked meat. Of which, simultaneous steam distilla‐ tion-extraction (SDE), dynamic headspace entrainment on Tenax TA, and solid-phase micro‐ extraction (SPME) are the techniques widely used for the extraction of volatile compounds in cooked meat [19, 122, 123, 85, 90, 124, 18,125]. SDE is a simple technique which involves small volumes of solvent, efficient stripping of volatiles and quantitative recovery of many compounds. The sample is dispersed in water which is heated to boiling. The steam that is generated carries volatiles with it into a section of the apparatus where the steam condenses in the presence of extracting solvent vapor. The co-condensation of volatile-laden steam and extracting solvent results in an effective extraction of volatiles [123]. The Dynamic head‐ space entrainment on Tenax has been used in the studies regarding cooked meat volatile fla‐ vor compounds since the 1980s. This technique probably has been used more than any other aroma extraction technique for the analysis of meat aroma and continues to be widely used. The action mechanism of this technique involving purging the headspace of a sample with a purified inert gas (e.g., nitrogen or helium), followed by collection of the volatiles onto a trap containing a suitable adsorbent, which will retain the volatile analytes carried there by the purge gas. Finally, the volatiles of meat samples collected on this trap are desorbed onto a GC or GC-MS column using a modified injection port. In the recent years, SPME technique has been widely adopted and considered as an alternative to isolate volatile flavor compo‐ nents in cooked meat. In SPME, the needle is coated with an absorbent material (e.g. CAR/ PDMS), is placed above the cooked meat samples. Volatiles will migrate from the sample matrix to the needle coating and be absorbed. Volatile components will then be desorbed

**5. The current techniques used for extraction and detection of aroma**

from the needle coating by inserting the needle in GC injection port.

The extraction techniques as mentioned above in combination with GC/MS or GSC/FID can help researchers to tentatively detect the volatile flavor compounds in experimented meat samples but it could not identify the aroma flavors or odor characteristics of detected com‐ pounds. It would be advantageous to combine two or more different techniques, such as gas chromatography (GC) and olfactometry, the combination of measuring odor notes is called gas chromatography-olfactometry (GC/O). Gas chromatography-olfactometry (GC-O) is a bioassay that measures human response to odorants separated by gas chromatography. The superior sensitivity and selectivity of human olfaction make GC-O a powerful and meaning‐ ful tool for flavor chemistry. In the recent year, GC-O is one of the main techniques which have been used to determine intensity of aroma (odor) characteristics of volatile compounds

volatile flavor compounds to dissipate.

**flavor components**

in cooked meat [15, 21, 125].

#### **4. Warm-off flavor and liver-like off flavor in cooked meat**

Warm-off flavor and liver-like off flavor are undesirable flavors that result from the fla‐ vor changes and deterioration in meats that have been pre-cooked, chilled-stored and re‐ heated. The warm-off flavor includes odors and tastes commonly described as stale, cardboardlike, painty, rancid, bitter and sour [115], and together with liver-like off flavor they both are the main factors that negatively affect eating sensory quality, purchase, economic im‐ pact of meat industry, and consumer complaint. Researchers have found that warm-off flavor appearing in cooked meat is mainly caused by oxidation of membrane phospholi‐ pids [116,115]. A recent report of Byrne et al (2001) [117], which also demonstrated that warm-off flavor associated with the development of lipid oxidation derived nuance offflavor and odor notes such as rancid-like flavor and linseed oil-like odor, in association with a concurrent decrease in cooked pork meat-like flavor. The development of warmoff flavor usually results in loss of meaty flavor due to mask by lipid-oxidized products. Additionally, processes which involve any action that disrupts the muscle fiber mem‐ brane, such as chopping, restructuring, or heating which all can enhance warm-off flavor of meat product [118]. Previous works also suggested that reactions involving sulphydryl– disulfide interchanges in proteins and the degradation of sulfur-containing heteroatomic compounds, leading to a decrease in the ''meatiness'' of freshly cooked meat may also be an integral part of warm-off flavor [119,120]. For the liver-like off flavor of cooked meat, it was hypothesized that since foodservice preparation traditionally cooked the meat quick‐ ly and then held the product in warming ovens until the food was presented to the consum‐ er these conditions might promote the liver-like flavor [27]. James and Calkins, (2005) [121] also hypothesized that the slower cooking and longer hold time allow the undesirable volatile flavor compounds to dissipate.

### **5. The current techniques used for extraction and detection of aroma flavor components**

(2003) [113] indicated that irradiation of meat in aerobic packaging promoted production of aldehydes such as propanal and hexanal which is assumed as a good indicator of lipid oxi‐ dation. The similar observation also was reported by Nam et al (2001) [114] who also showed that irradiation increased TBARS values and off-flavor in aerobically-packaged pork (3) Temperature effect, temperature during irradiating meats has a large effect on aro‐ ma flavors of irradiated meat because temperature affects what radiolytic products are formed and what ratios [104]. Using lower temperature during irradiation of meat by freez‐ ing meat before irradiation can reduce detrimental effects via retarding autoxidation and ex‐ tending shelf life; (4) pH effect, it has also been demonstrated that the ultimate pH of meat at the time of irradiation influences lipid oxidation. Nam et al (2001) [114] recently showed that irradiation increased lipid oxidation of normal and pale-soft-exudative (low pH group) muscles, whereas dark-firm-dry (high pH group) muscle was very stable and resistant to ox‐ idative changes. Therefore, to minimize the detrimental effects of irradiation on aroma fla‐ vor characteristics we can modify atmosphere packaging by using vacuum packaging (anaerobic packaging) or replacement with inert gases (i.e. nitrogen, helium, hydrogen, car‐ bon dioxide) to eliminate oxygen. Reducing the temperature (freezing) prior to irradiation and addition of antioxidants. Vacuum packaging retains irradiation-generated sulfur-con‐ taining compounds, however re-packaging meat in oxygen-permeable materials allows for

dissipation of these flavor compounds.

164 Latest Research into Quality Control

**4. Warm-off flavor and liver-like off flavor in cooked meat**

Warm-off flavor and liver-like off flavor are undesirable flavors that result from the fla‐ vor changes and deterioration in meats that have been pre-cooked, chilled-stored and re‐ heated. The warm-off flavor includes odors and tastes commonly described as stale, cardboardlike, painty, rancid, bitter and sour [115], and together with liver-like off flavor they both are the main factors that negatively affect eating sensory quality, purchase, economic im‐ pact of meat industry, and consumer complaint. Researchers have found that warm-off flavor appearing in cooked meat is mainly caused by oxidation of membrane phospholi‐ pids [116,115]. A recent report of Byrne et al (2001) [117], which also demonstrated that warm-off flavor associated with the development of lipid oxidation derived nuance offflavor and odor notes such as rancid-like flavor and linseed oil-like odor, in association with a concurrent decrease in cooked pork meat-like flavor. The development of warmoff flavor usually results in loss of meaty flavor due to mask by lipid-oxidized products. Additionally, processes which involve any action that disrupts the muscle fiber mem‐ brane, such as chopping, restructuring, or heating which all can enhance warm-off flavor of meat product [118]. Previous works also suggested that reactions involving sulphydryl– disulfide interchanges in proteins and the degradation of sulfur-containing heteroatomic compounds, leading to a decrease in the ''meatiness'' of freshly cooked meat may also be an integral part of warm-off flavor [119,120]. For the liver-like off flavor of cooked meat, it was hypothesized that since foodservice preparation traditionally cooked the meat quick‐ ly and then held the product in warming ovens until the food was presented to the consum‐ Up to present time, various techniques have been designed, combined with gas chromatog‐ raphy and mass spectrometry (GC/MS) or Flame ionizing detector (GC/FID) and applied to evaluate volatile flavor components in cooked meat. Of which, simultaneous steam distilla‐ tion-extraction (SDE), dynamic headspace entrainment on Tenax TA, and solid-phase micro‐ extraction (SPME) are the techniques widely used for the extraction of volatile compounds in cooked meat [19, 122, 123, 85, 90, 124, 18,125]. SDE is a simple technique which involves small volumes of solvent, efficient stripping of volatiles and quantitative recovery of many compounds. The sample is dispersed in water which is heated to boiling. The steam that is generated carries volatiles with it into a section of the apparatus where the steam condenses in the presence of extracting solvent vapor. The co-condensation of volatile-laden steam and extracting solvent results in an effective extraction of volatiles [123]. The Dynamic head‐ space entrainment on Tenax has been used in the studies regarding cooked meat volatile fla‐ vor compounds since the 1980s. This technique probably has been used more than any other aroma extraction technique for the analysis of meat aroma and continues to be widely used. The action mechanism of this technique involving purging the headspace of a sample with a purified inert gas (e.g., nitrogen or helium), followed by collection of the volatiles onto a trap containing a suitable adsorbent, which will retain the volatile analytes carried there by the purge gas. Finally, the volatiles of meat samples collected on this trap are desorbed onto a GC or GC-MS column using a modified injection port. In the recent years, SPME technique has been widely adopted and considered as an alternative to isolate volatile flavor compo‐ nents in cooked meat. In SPME, the needle is coated with an absorbent material (e.g. CAR/ PDMS), is placed above the cooked meat samples. Volatiles will migrate from the sample matrix to the needle coating and be absorbed. Volatile components will then be desorbed from the needle coating by inserting the needle in GC injection port.

The extraction techniques as mentioned above in combination with GC/MS or GSC/FID can help researchers to tentatively detect the volatile flavor compounds in experimented meat samples but it could not identify the aroma flavors or odor characteristics of detected com‐ pounds. It would be advantageous to combine two or more different techniques, such as gas chromatography (GC) and olfactometry, the combination of measuring odor notes is called gas chromatography-olfactometry (GC/O). Gas chromatography-olfactometry (GC-O) is a bioassay that measures human response to odorants separated by gas chromatography. The superior sensitivity and selectivity of human olfaction make GC-O a powerful and meaning‐ ful tool for flavor chemistry. In the recent year, GC-O is one of the main techniques which have been used to determine intensity of aroma (odor) characteristics of volatile compounds in cooked meat [15, 21, 125].

#### **6. Conclusion and Implication**

In order to have a cooked meat product with its desirable aroma flavors as expectation of consumer, it is important to understand how aroma flavors are derived, the mechanisms by which flavor components are generated, and the factors affect formation of flavor com‐ pounds then determine the final aroma flavor characteristics of cooked meat. Regarding the effects of factors on aroma flavors of cooked meat and to minimize the detrimental effects it is suggested if increasing the polyunsaturated fatty acids (e.g. C18:3n-3, DHA, EPA) to in‐ crease nutritional benefits to the consumer by using fat-supplemented diets however the un‐ desirable flavors may result. Because the breakdown products of these fatty acids have a shorter chain length therefore are more volatile and they affect meat flavors by interacting with the Maillard reaction results in reducing levels of meaty aroma compounds such as sul‐ fur-substituted thiophenes. Therefore, diets, feeding regimes, welfare and management of animals should be taken into account. Cooking conditions such as temperature, holding time and cooking methods play an important role in determining the formation volatile fla‐ vor compounds. In general, it has been demonstrated that cooking meat at high temperature (by roasting, grilling) will produce better aroma flavor characteristics due to the important Maillard products are formed. In addition to the cooking effect, it is suggested that a slow cooking and longer hold time can allow the undesirable volatile flavor compounds to dissi‐ pate, thus reduce warm-off flavor. Irradiation of meat can eliminate pathogens however, offflavor may result therefore, and to minimize the detrimental effect of this method we can lower temperature during irradiation of meat by freezing meat before irradiation. Modify‐ ing atmosphere packaging by using vacuum packaging (anaerobic packaging) or replace‐ ment with inert gases (i.e. nitrogen, helium, hydrogen, carbon dioxide) to eliminate oxygen in meat during irradiation are also the alternatives. Chiller ageing of meat should be applied to improve eating quality however should not age for a long time (3 week period in maxi‐ mum is encouraged) because chiller ageing meat for a too long period may result in flavor deterioration and decreasing desirable flavors.

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#### **Author details**

Hoa Van Ba1\*, Inho Hwang1 , Dawoon Jeong2 and Amna Touseef2

\*Address all correspondence to: ba\_cnu1981@yahoo.com

1 Department of Animal Science and Biotechnology, Lab of Muscle Biology and Meat Sci‐ ence, Chonbuk National University, South Korea

2 Department of Animal Science and Biotechnology, Lab of Muscle Biology and Meat Sci‐ ence, Chonbuk National University, South Korea

#### **References**

**6. Conclusion and Implication**

166 Latest Research into Quality Control

deterioration and decreasing desirable flavors.

, Dawoon Jeong2

\*Address all correspondence to: ba\_cnu1981@yahoo.com

ence, Chonbuk National University, South Korea

ence, Chonbuk National University, South Korea

and Amna Touseef2

1 Department of Animal Science and Biotechnology, Lab of Muscle Biology and Meat Sci‐

2 Department of Animal Science and Biotechnology, Lab of Muscle Biology and Meat Sci‐

**Author details**

Hoa Van Ba1\*, Inho Hwang1

In order to have a cooked meat product with its desirable aroma flavors as expectation of consumer, it is important to understand how aroma flavors are derived, the mechanisms by which flavor components are generated, and the factors affect formation of flavor com‐ pounds then determine the final aroma flavor characteristics of cooked meat. Regarding the effects of factors on aroma flavors of cooked meat and to minimize the detrimental effects it is suggested if increasing the polyunsaturated fatty acids (e.g. C18:3n-3, DHA, EPA) to in‐ crease nutritional benefits to the consumer by using fat-supplemented diets however the un‐ desirable flavors may result. Because the breakdown products of these fatty acids have a shorter chain length therefore are more volatile and they affect meat flavors by interacting with the Maillard reaction results in reducing levels of meaty aroma compounds such as sul‐ fur-substituted thiophenes. Therefore, diets, feeding regimes, welfare and management of animals should be taken into account. Cooking conditions such as temperature, holding time and cooking methods play an important role in determining the formation volatile fla‐ vor compounds. In general, it has been demonstrated that cooking meat at high temperature (by roasting, grilling) will produce better aroma flavor characteristics due to the important Maillard products are formed. In addition to the cooking effect, it is suggested that a slow cooking and longer hold time can allow the undesirable volatile flavor compounds to dissi‐ pate, thus reduce warm-off flavor. Irradiation of meat can eliminate pathogens however, offflavor may result therefore, and to minimize the detrimental effect of this method we can lower temperature during irradiation of meat by freezing meat before irradiation. Modify‐ ing atmosphere packaging by using vacuum packaging (anaerobic packaging) or replace‐ ment with inert gases (i.e. nitrogen, helium, hydrogen, carbon dioxide) to eliminate oxygen in meat during irradiation are also the alternatives. Chiller ageing of meat should be applied to improve eating quality however should not age for a long time (3 week period in maxi‐ mum is encouraged) because chiller ageing meat for a too long period may result in flavor


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**Chapter 8**

**Novel Analytical Tools**

Christian W. Huck

**1. Introduction**

of analysis.

http://dx.doi.org/10.5772/51915

**for Quality Control in Food Science**

Additional information is available at the end of the chapter

Due to the fast technological and data treatment advancements new insights into food can be considered. The application of these novel analytical techniques belongs to the responsi‐ bility of food chemists and analysts. Thereby, an increase in efficiency is based on an im‐ proved lower limit of detection (LOD), selectivity to separate analytes of interest and speed

High-performance liquid chromatography (HPLC) belongs to the traditional separation techniques applied to a broad range of hydrophilic and hydrophobic ingredients in both the reversed-phase (RP) [1] as well as normal-phase (NP) [2] mode. In a conventional HPLC system the inner diameter of the separation column, which is the core of the sepa‐ ration unit, is 4.6 mm. During the last decade miniaturization down to 20 μm allowed to increase on one side the sensitivity and on the other side speed of analysis could be en‐ hanced dramatically. Therefore, novel stationary phases mainly based on polymers have been designed and brought to the market to enable both the separation of low and highmolecular weight analytes [3]. As an alternative separation technique capillary electropho‐ resis (CE), which separates analytes due to their different ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm can be applied [4]. Thereby, the appearance of the electroosmotic flow (EOF) can influence the separation efficiency by either speeding up the separation process or by improving the resolution. Capillary electrochromatography (CEC) is a hybrid technique of both HPLC and CE in which both pressure and an electri‐ cal field are applied and enables extreme high resolution. The drawback of this separa‐ tion method is the fact, that real samples can hardly be analysed due to the disturbance by the matrix [5]. In many cases the analyte of interest is only available in very low con‐

> © 2012 Huck; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Huck; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.


#### **Chapter 8**

## **Novel Analytical Tools for Quality Control in Food Science**

Christian W. Huck

[120] St, Angelo. A. J., Crippen, K. L., Dupuy, H. P., & James Jr, C., Jr. (1990). Chemical and sensory studies of antioxidant-treated beef. *Journal of Food Science*, 55, 1501-1539.

[121] James, J. M., & Calkins, C. R. (2005). Cooking rate and holding time effect on the fla‐ vor of muscles from the beef chuck and the round. In Proceedings of the internation‐

[122] Elmore, J. S. (2008). Aroma. In Nollet L. M. L and Toldra. F (eds.) Handbook of mus‐

[123] Madruga, M. S., Elmore, J. S., Dodson, A. T., & Mottram, D. S. (2009). Volatile flavor profile of goat meat extracted by three widely used techniques. *Food Chemistry*, 115,

[124] Moon, S. Y., Cliff, M. A., & Li-Chan, E. C. Y. (2006). Odour-active components of si‐ mulated beef flavor analyzed by solid phase microextraction and gas chromatogra‐ phy-mass spectrometry and-olfactometry. *Food Research International*, 39, 294-308.

[125] Xie, J. C., Sun, B. G., & Wang, S. B. (2008). Aromatic constituents from Chinese tradi‐ tional smoke-cured bacon of mini-pig. *Food science and technology international*, 14,

al congress of meat science and technology Baltimore, MD.

cle foods analysis Boca Raton CRC , 242-262.

1081-1087.

176 Latest Research into Quality Control

329-340.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51915

#### **1. Introduction**

Due to the fast technological and data treatment advancements new insights into food can be considered. The application of these novel analytical techniques belongs to the responsi‐ bility of food chemists and analysts. Thereby, an increase in efficiency is based on an im‐ proved lower limit of detection (LOD), selectivity to separate analytes of interest and speed of analysis.

High-performance liquid chromatography (HPLC) belongs to the traditional separation techniques applied to a broad range of hydrophilic and hydrophobic ingredients in both the reversed-phase (RP) [1] as well as normal-phase (NP) [2] mode. In a conventional HPLC system the inner diameter of the separation column, which is the core of the sepa‐ ration unit, is 4.6 mm. During the last decade miniaturization down to 20 μm allowed to increase on one side the sensitivity and on the other side speed of analysis could be en‐ hanced dramatically. Therefore, novel stationary phases mainly based on polymers have been designed and brought to the market to enable both the separation of low and highmolecular weight analytes [3]. As an alternative separation technique capillary electropho‐ resis (CE), which separates analytes due to their different ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm can be applied [4]. Thereby, the appearance of the electroosmotic flow (EOF) can influence the separation efficiency by either speeding up the separation process or by improving the resolution. Capillary electrochromatography (CEC) is a hybrid technique of both HPLC and CE in which both pressure and an electri‐ cal field are applied and enables extreme high resolution. The drawback of this separa‐ tion method is the fact, that real samples can hardly be analysed due to the disturbance by the matrix [5]. In many cases the analyte of interest is only available in very low con‐

© 2012 Huck; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Huck; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

centrations. Therefore, selective enrichment and purification steps are the method of choice, which can be accomplished by solid-phase extraction (SPE). Therefore, a material designed for a special analytical question is filled into a cartridge or pipette tip and the sample of interest is put onto the material in liquid form [6]. In the following, analytes of interest can interact with the functional groups of the stationary phase and compounds being not of interest can simply be washed away. In the final elution step, only some mi‐ cro liters of liquid are required to elute the analytes of interest from the stationary phase being available in relatively high concentrations for the following analytical steps. The fol‐ lowing analytical procedure can be either a separation or spectroscopic method. Spectro‐ scopic methods at this stage of the analytical procedure either include mass spectrometry (MS) and/or vibrational spectroscopy, respectively. In MS most of the samples are ana‐ lysed applying electrospray ionization (ESI) as an interface with different types of mass detectors including e.g. time of flight (TOF), ion trap, ion cyclotron and quadrupoles. As an alternative, matrix assisted laser desorption ionization time of flight mass spectrome‐ try (MALDI-TOF/MS) can be applied for the determination of high molecular weight com‐ pounds including proteins, peptides and lipids. For the analysis of low molecular ingredients < 1000 Da the so called matrix-free laser desorption ionization (mf-LDI) MS technique must be applied [7].Vibrational spectroscopy in the field of food analysis is mainly applied in the mid (400 – 4000 cm-1) as well as in the near infrared (4000 – 12000 cm-1) of the electromagnetic spectrum. In combination with chemo metrical algorithms these methods can be used for the authentication of the material on one hand, on the oth‐ er hand quantitative analysis allows to control selected quality parameters [8].

detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the spe‐ cific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical han‐ dling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the mid‐ dle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to be ana‐ lysed is carried out with only a few microliters causing a dramatic increase in concen‐ tration from which further analytical investigations can benefit due to the easier

Novel Analytical Tools for Quality Control in Food Science

http://dx.doi.org/10.5772/51915

179

4 *Figure 1. Multidimensional analytical approach* 

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of surface to size, which results in a high capacity and allows analyte detection with high sensitivity down to the femtomole range in the case when mass spectrometry is applied for detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the specific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical handling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the middle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to

6 *Sample enrichment/purification* 

3

handling of the systematic investigation.

1 2 3

5

**Figure 1.** Multidimensional analytical approach

Running Title

In the following a systematic analytical approach is introduced, which allows combining the different analytical techniques in a synergistic manner to get deeper insights into the compo‐ sition and origin of food samples.

#### **2. Systematic analytical approach**

The key technologies described in the above chapter can be combined according to the scheme depicted in Figure 1. In this approach extraction of the material for the further ana‐ lytical steps and individual procedures can be linked to sample enrichment/purification, separation, vibrational spectroscopy and mass spectroscopy followed by database analysis. The different parts are described in the following sub-chapters.

#### **2.1. Sample enrichment/purification**

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of sur‐ face to size, which results in a high capacity and allows analyte detection with high sen‐ sitivity down to the femtomole range in the case when mass spectrometry is applied for

3

detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the spe‐ cific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical han‐ dling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the mid‐ dle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to be ana‐ lysed is carried out with only a few microliters causing a dramatic increase in concen‐ tration from which further analytical investigations can benefit due to the easier handling of the systematic investigation.

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of surface to size, which results in a high capacity and allows analyte detection with high sensitivity down to the femtomole range in the case when mass spectrometry is applied for detection. Especially carbon nano materials can be easily further derivatised with a number of different functional groups including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), immobilized affinity (IMAC) and so on depending on the specific demand. As an alternative they can be incorporated into a polymer matrix for highly selective extraction by certain compound characteristics. For the practical handling pipette tips have been tested to be most suitable and this special type of SPE is called "hollow monolithic incorporated tip" as it has an open flow channel in the middle enabling an easy pipetting procedure. For the highly efficient pre-concentration of phosphopeptides nano particular TiO2, ZrO2 and mixtures thereof are incorporated into a polymer matrix as depicted in Figure 2 [9]. By this technique hundreds of microliters can be flushed over the system and finally elution of the desired compounds to

4 *Figure 1. Multidimensional analytical approach*  **Figure 1.** Multidimensional analytical approach

6 *Sample enrichment/purification* 

2 3

5

Running Title

centrations. Therefore, selective enrichment and purification steps are the method of choice, which can be accomplished by solid-phase extraction (SPE). Therefore, a material designed for a special analytical question is filled into a cartridge or pipette tip and the sample of interest is put onto the material in liquid form [6]. In the following, analytes of interest can interact with the functional groups of the stationary phase and compounds being not of interest can simply be washed away. In the final elution step, only some mi‐ cro liters of liquid are required to elute the analytes of interest from the stationary phase being available in relatively high concentrations for the following analytical steps. The fol‐ lowing analytical procedure can be either a separation or spectroscopic method. Spectro‐ scopic methods at this stage of the analytical procedure either include mass spectrometry (MS) and/or vibrational spectroscopy, respectively. In MS most of the samples are ana‐ lysed applying electrospray ionization (ESI) as an interface with different types of mass detectors including e.g. time of flight (TOF), ion trap, ion cyclotron and quadrupoles. As an alternative, matrix assisted laser desorption ionization time of flight mass spectrome‐ try (MALDI-TOF/MS) can be applied for the determination of high molecular weight com‐ pounds including proteins, peptides and lipids. For the analysis of low molecular ingredients < 1000 Da the so called matrix-free laser desorption ionization (mf-LDI) MS technique must be applied [7].Vibrational spectroscopy in the field of food analysis is mainly applied in the mid (400 – 4000 cm-1) as well as in the near infrared (4000 – 12000 cm-1) of the electromagnetic spectrum. In combination with chemo metrical algorithms these methods can be used for the authentication of the material on one hand, on the oth‐

er hand quantitative analysis allows to control selected quality parameters [8].

sition and origin of food samples.

178 Latest Research into Quality Control

**2. Systematic analytical approach**

**2.1. Sample enrichment/purification**

The different parts are described in the following sub-chapters.

In the following a systematic analytical approach is introduced, which allows combining the different analytical techniques in a synergistic manner to get deeper insights into the compo‐

The key technologies described in the above chapter can be combined according to the scheme depicted in Figure 1. In this approach extraction of the material for the further ana‐ lytical steps and individual procedures can be linked to sample enrichment/purification, separation, vibrational spectroscopy and mass spectroscopy followed by database analysis.

In many cases interesting analytes are only available in extremely low concentrations and/or in very complex matrices, respectively. Therefore, pre-concentration steps based on solid-phase extraction (SPE) can be very helpful. Nano-materials such as nanotubes, fullerenes, diamond offer excellent physiochemical properties due to a high ratio of sur‐ face to size, which results in a high capacity and allows analyte detection with high sen‐ sitivity down to the femtomole range in the case when mass spectrometry is applied for

**Figure 2.** TiO2, ZrO2 incorporated into a polymer matrix for phosphopeptide enrichment

Immobilisation of such polymers into pipette tips with trypsin can be used for fast digestion of peptides and proteins within only a few minutes ensuring high capacity and sequence coverage (Figure 3) even in the high-throughput mode using robotic pipetting systems [10]. In comparison to this quite young approach the conventional digestion procedure lasts ap‐ proximately 24 hours and doesn´t show in any case better results by higher sequence cover‐ ages. For this reason this approach is of high interest for the routine analysis and/or diagnostics, respectively. As a carrier glycidylmethacrylate-co-divinylbenzene (GMA/DVB) polymerized in pipette tips was chosen. The major advantages of in-tip digestion are easy handling and small sample amount required for analysis. Microwave-assisted digestion was applied for highly efficient and time saving proteolysis. Adaption to an automated robotic system allowed fast and reproducible sample treatment. Investigations with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography coupled to electrospray-ionization mass spectrometry (LC-ESI/MS) attest‐ ed high sequence coverages (SCs) for the three standard proteins, myoglobin (Myo, 89%), bovine serum albumin (BSA, 78%) and alpha-casein (α-Cas, 83%). Compared to commercial‐ ly available trypsin tips clear predominance concerning the digestion performance was ach‐ ieved. Storability was tested over a period of several weeks and results showed only less decrease (<5%) of protein sequence coverages. The application of microwave-assisted in-tip digestion (2 minutes) with full automation by a robotic system allows high-throughput anal‐ ysis (96 samples within 80 minutes) and highly effective proteolysis.

**Figure 3.** Trypsin immobilized pipette tips for high-throughput analysis of peptides

Material enhanced laser desorption ionisation (MELDI) is a method, which is based on the conventional matrix assisted laser desorption ionisation time of flight mass spectrometric (MALDI-TOF/MS) detection with the significant difference that before LDI MS step a selec‐ tive enrichment procedure is carried out for the distinct analysis of a certain compound class. Compared to other similar techniques in this field, this approach benefits from the physical properties of the material itself (pore size, surface area, capacity, etc.) and its chemi‐ cal derivatisation/functionalisation. In the past this technique was proven to be highly effi‐ cient for the analysis of biomarkers following an optimised strategy (Figure 4). In the first step a selected material including e.g., nanotubes, fullerenes, nano-crystalline diamond, pol‐ ymers, cellulose, etc., which are derivatised with functional groups (C18, IMAC (immobi‐ lised metal affinity chromatography), IEX and others) is activated and the serum sample of interest is incubated. During this step, selective binding of molecules according to their functional group is achieved and finally undesired components can be washed away apply‐ ing an optimised protocol. In the next step the incubated material is put onto a conventional

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**2.2. Selective MELDI-carriers**

**Figure 3.** Trypsin immobilized pipette tips for high-throughput analysis of peptides

#### **2.2. Selective MELDI-carriers**

**Figure 2.** TiO2, ZrO2 incorporated into a polymer matrix for phosphopeptide enrichment

180 Latest Research into Quality Control

ysis (96 samples within 80 minutes) and highly effective proteolysis.

Immobilisation of such polymers into pipette tips with trypsin can be used for fast digestion of peptides and proteins within only a few minutes ensuring high capacity and sequence coverage (Figure 3) even in the high-throughput mode using robotic pipetting systems [10]. In comparison to this quite young approach the conventional digestion procedure lasts ap‐ proximately 24 hours and doesn´t show in any case better results by higher sequence cover‐ ages. For this reason this approach is of high interest for the routine analysis and/or diagnostics, respectively. As a carrier glycidylmethacrylate-co-divinylbenzene (GMA/DVB) polymerized in pipette tips was chosen. The major advantages of in-tip digestion are easy handling and small sample amount required for analysis. Microwave-assisted digestion was applied for highly efficient and time saving proteolysis. Adaption to an automated robotic system allowed fast and reproducible sample treatment. Investigations with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography coupled to electrospray-ionization mass spectrometry (LC-ESI/MS) attest‐ ed high sequence coverages (SCs) for the three standard proteins, myoglobin (Myo, 89%), bovine serum albumin (BSA, 78%) and alpha-casein (α-Cas, 83%). Compared to commercial‐ ly available trypsin tips clear predominance concerning the digestion performance was ach‐ ieved. Storability was tested over a period of several weeks and results showed only less decrease (<5%) of protein sequence coverages. The application of microwave-assisted in-tip digestion (2 minutes) with full automation by a robotic system allows high-throughput anal‐

Material enhanced laser desorption ionisation (MELDI) is a method, which is based on the conventional matrix assisted laser desorption ionisation time of flight mass spectrometric (MALDI-TOF/MS) detection with the significant difference that before LDI MS step a selec‐ tive enrichment procedure is carried out for the distinct analysis of a certain compound class. Compared to other similar techniques in this field, this approach benefits from the physical properties of the material itself (pore size, surface area, capacity, etc.) and its chemi‐ cal derivatisation/functionalisation. In the past this technique was proven to be highly effi‐ cient for the analysis of biomarkers following an optimised strategy (Figure 4). In the first step a selected material including e.g., nanotubes, fullerenes, nano-crystalline diamond, pol‐ ymers, cellulose, etc., which are derivatised with functional groups (C18, IMAC (immobi‐ lised metal affinity chromatography), IEX and others) is activated and the serum sample of interest is incubated. During this step, selective binding of molecules according to their functional group is achieved and finally undesired components can be washed away apply‐ ing an optimised protocol. In the next step the incubated material is put onto a conventional steel target used in MALDI-TOF/MS, a matrix substance is added (e.g., sinapinic acid) and finally the mass spectrum is generated by the laser desorption ionisation process. The result is a mass spectrum being characteristic for a patient and/or the nutrition profile. Multivari‐ ate analysis (MVA) can be applied for further data analysis and interpretation, a clustering into certain stages of an illness can be achieved, respectively. From the mass spectrum po‐ tent biomarker molecules can be selected and identified by further analytical steps. The bio‐ marker itself and/or the profile of the corresponding mass spectrum can be used for the screening of certain diseases, stages therefrom, allergies, nutrition effects and so on [7].

rosity can be adjusted [12]. Extensive investigations on polymerisation time and tempera‐ ture have been carried out enabling a tailored design of micro-, meso- and macro-pore distribution [13, 14]. This results in the applicability of such capillaries with an inner diame‐ ter between 20 and 200 μm for even the separation of high- and low-molecular weight com‐ pounds. These capillaries can be highly successfully applied analysing peptides, proteins, oligonucleotides, DNA fragments as well as "small molecules" such as phenols, flavonoids, catechins, acids etc. Figure 5 shows as an example the separation of olive oil ingredients. This separation is characterised by a very high ratio of flow to back pressure, which is of high interest to perform extremely rapid Coupling to mass spectrometry enables a highly ef‐ ficient analysis even of crude samplesoffering all the possibilities of collision induced disso‐

0.0 2.0 4.0 6.0 8.0 10.0

Column, PA/PDA (Kap. 155, polymerised for 10 min), 80 x 0.2 mm; chromatographic conditions: mobile phase, A: 0.1 % TFA, B: ACN, 0.1 % TFA; gradient, 5-45 % B in 10 min; flow rate, 8 µl/min; temperature, RT; detection, UV 210 nm; Peak identification: (1) Hydroxytyrosol, (2) Tyrosol, (3) Caffeic acid, (4) Vanillin and

**Figure 5.** Separation of olive oil ingredients using a monolithic capillary column. Conditions: capillary 80 x 0.2 mm; mobile phase, A: 0.1% TFA; B: CAN; gradient, 5-45%B in 10 min; Flow rate 8 µl/min; temperature, RT; detection, UV

As an alternative separation method capillary electrophoresis (CE) and /or electrochroma‐ tography (CEC) can be applied. In CE separation of analytes is achieved due to their differ‐ ent ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm [4]. As has already been remarked the electroosmotic flow (EOF) has a main influence on the separation and can be used for speeding up. In CEC both an electrical field and high pressure are applied resulting in high resolution. This technique can be applied to check the identification and purity of standards compounds with very high efficiency. For the reproducible separation and analy‐ sis of food ingredients such as phenols, acids, peptides, lipids, coating of the capillary´s in‐ ner wall was shown being advantageous as irreversible analyte adsorption by free hydroxyl-groups from the silanole of the fused silica capillary can be avoided. Latex-diol

210 nm. Peak assignment, (1) hydroxytyrosol, (2) tyrosol, (3) caffeic acid, (4) vanillin, (5) oleuropein.

4 5

**time [min]**

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ciation (CID) and database search [15].

0

(5) Oleuropein.

2

4

6

8

10

12

1

2

3

**Figure 4.** Principle of material enhanced laser desorption ionization (MELDI)

For the analysis of low-molecular weight compounds (MW < 1000 Da) the conventional MELDI approach is replaced by the matrix-free (mf) MELDI approach for which the addi‐ tion of a matrix substance is not required so that no disturbing peaks appear. In this ap‐ proach a conventional steel target with a 50 nm thick titanium oxide layer can be applied fulfilling all requirements for a successful laser desorption ionization process [11].

As an alternative the incubated analytes of interest can be selectively eluted from the func‐ tinalised carrier material and further analysed by liquid chromatography (LC) or capillary electrophoresis (CE).

#### **2.3. Liquid chromatography, capillary electrophoresis and electrochromatography**

Novel materials used in miniaturised liquid chromatography (μ-LC) are mainly polymer based, e.g. poly(1,2-bis(p-vinylphenyl)ethane). These polymers possess the huge advantage that chemical (composition of the polymer) and physical parameters including mainly po‐ rosity can be adjusted [12]. Extensive investigations on polymerisation time and tempera‐ ture have been carried out enabling a tailored design of micro-, meso- and macro-pore distribution [13, 14]. This results in the applicability of such capillaries with an inner diame‐ ter between 20 and 200 μm for even the separation of high- and low-molecular weight com‐ pounds. These capillaries can be highly successfully applied analysing peptides, proteins, oligonucleotides, DNA fragments as well as "small molecules" such as phenols, flavonoids, catechins, acids etc. Figure 5 shows as an example the separation of olive oil ingredients. This separation is characterised by a very high ratio of flow to back pressure, which is of high interest to perform extremely rapid Coupling to mass spectrometry enables a highly ef‐ ficient analysis even of crude samplesoffering all the possibilities of collision induced disso‐ ciation (CID) and database search [15].

steel target used in MALDI-TOF/MS, a matrix substance is added (e.g., sinapinic acid) and finally the mass spectrum is generated by the laser desorption ionisation process. The result is a mass spectrum being characteristic for a patient and/or the nutrition profile. Multivari‐ ate analysis (MVA) can be applied for further data analysis and interpretation, a clustering into certain stages of an illness can be achieved, respectively. From the mass spectrum po‐ tent biomarker molecules can be selected and identified by further analytical steps. The bio‐ marker itself and/or the profile of the corresponding mass spectrum can be used for the screening of certain diseases, stages therefrom, allergies, nutrition effects and so on [7].

**Figure 4.** Principle of material enhanced laser desorption ionization (MELDI)

electrophoresis (CE).

182 Latest Research into Quality Control

For the analysis of low-molecular weight compounds (MW < 1000 Da) the conventional MELDI approach is replaced by the matrix-free (mf) MELDI approach for which the addi‐ tion of a matrix substance is not required so that no disturbing peaks appear. In this ap‐ proach a conventional steel target with a 50 nm thick titanium oxide layer can be applied

As an alternative the incubated analytes of interest can be selectively eluted from the func‐ tinalised carrier material and further analysed by liquid chromatography (LC) or capillary

Novel materials used in miniaturised liquid chromatography (μ-LC) are mainly polymer based, e.g. poly(1,2-bis(p-vinylphenyl)ethane). These polymers possess the huge advantage that chemical (composition of the polymer) and physical parameters including mainly po‐

fulfilling all requirements for a successful laser desorption ionization process [11].

**2.3. Liquid chromatography, capillary electrophoresis and electrochromatography**

**Figure 5.** Separation of olive oil ingredients using a monolithic capillary column. Conditions: capillary 80 x 0.2 mm; mobile phase, A: 0.1% TFA; B: CAN; gradient, 5-45%B in 10 min; Flow rate 8 µl/min; temperature, RT; detection, UV 210 nm. Peak assignment, (1) hydroxytyrosol, (2) tyrosol, (3) caffeic acid, (4) vanillin, (5) oleuropein.

(5) Oleuropein.

As an alternative separation method capillary electrophoresis (CE) and /or electrochroma‐ tography (CEC) can be applied. In CE separation of analytes is achieved due to their differ‐ ent ion mobility based on charge and molecular weight in an electric field within a fused silica capillary having an inner diameter of approximately 200 μm [4]. As has already been remarked the electroosmotic flow (EOF) has a main influence on the separation and can be used for speeding up. In CEC both an electrical field and high pressure are applied resulting in high resolution. This technique can be applied to check the identification and purity of standards compounds with very high efficiency. For the reproducible separation and analy‐ sis of food ingredients such as phenols, acids, peptides, lipids, coating of the capillary´s in‐ ner wall was shown being advantageous as irreversible analyte adsorption by free hydroxyl-groups from the silanole of the fused silica capillary can be avoided. Latex-diol and fullerene coated capillaries were successfully introduced and as a detection system online hyphenation to MALDI-TOF/MS was shown to be highly efficient not only for the in‐ vestigation of flavonoids but also for peptides, especially phosphorylated (Figure 6) [16, 17]. This system can be used for the investigation of the casein profile in milk offering the ad‐ vantage over all other more classical analysis tools that in this case also higher phosphory‐ lated species can be separated and detected. From the ratio of different phosphorylation degrees several interpretations concerning the quality but also the origin of the milk can be carried out.

analyse data. These are mainly multivariate (MVA) methods allowing to correct baseline, at‐ mospheric noise etc. For qualitative analysis in most cased principal component analysis

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An impressive example for the successful implementation in the food related production is the quality control of wine. It has been shown that NIR can be used to identify grapes, vines, age by qualitative (Figure 8) and its ingredients (acids, carbohydrates, pH etc.) simultane‐ ously, non-invasively within a few seconds by quantitative analysis [19]. Another big ad‐ vantage of this method can be found by the fact that the sample is not destroyed and can

Quantitative NIRS methods, which allow determining the carbohydrate, total acid, tartaric acid, malic acid, pH in grape variety and the polyphenol content in grapes were established [19]. The method can control the quality already at a very early stage during the wine pro‐ duction and allows improvement of its quality by this. Grapes of 12 different vines (*Weißburgunder, Chardonnay, Ruländer, Silvaner, Müller Thurgau, Gewürztraminer, Sauvignon, Lagrein, Grossvernatsch, Blauburgunder, Cabernet, Merlot*) were harvested in autumn 2000 and squeezed. The obtained grape variety was thermo stated at 23°C and analyzed quantitative‐ ly by NIRS in the transflection mode using an optical thin layer thickness of 1 mm. In order to establish a calibration model 252 spectra of samples with lower and upper concentration as a reference were recorded. 76 % of all spectra were randomly used for calibration, 24% for validation. Data preparation was carried out in order to minimize technical influences,

therefore be used for further purposes including following analytical steps.

(PCA), for quantitative partial least square regression (PLSR) are applied [18].

**Figure 7.** Sample measurement modes in NIR

**Figure 6.** Inner capillary wall coatings applied in CE and CEC

#### **2.4. Vibrational spectroscopy**

For quality control both mid- (MIR, 400 – 4000 cm-1) and near-infrared (NIR, 4000 – 12000 cm-1) can be conducted. In MIR fundamental stretching and bending vibrations occur, in NIR the corresponding overtones and combination vibrations are detected. This means that NIR-spectra can contain a lot of more vibrational information, which is an advantage for the analysis of highly complex samples. Therefore, during the last decade several applications in the field of food analysis were developed in the NIR region. Samples can be analysed ei‐ ther in transmission, reflectance and interactance mode (Figure 7) so that liquid as well as solid samples can be investigated. Due to the quite broad bands compared to MIR, chemo‐ metrical spectra treatment is required for establishing adequate calibration models and to analyse data. These are mainly multivariate (MVA) methods allowing to correct baseline, at‐ mospheric noise etc. For qualitative analysis in most cased principal component analysis (PCA), for quantitative partial least square regression (PLSR) are applied [18].

**Figure 7.** Sample measurement modes in NIR

and fullerene coated capillaries were successfully introduced and as a detection system online hyphenation to MALDI-TOF/MS was shown to be highly efficient not only for the in‐ vestigation of flavonoids but also for peptides, especially phosphorylated (Figure 6) [16, 17]. This system can be used for the investigation of the casein profile in milk offering the ad‐ vantage over all other more classical analysis tools that in this case also higher phosphory‐ lated species can be separated and detected. From the ratio of different phosphorylation degrees several interpretations concerning the quality but also the origin of the milk can be

For quality control both mid- (MIR, 400 – 4000 cm-1) and near-infrared (NIR, 4000 – 12000 cm-1) can be conducted. In MIR fundamental stretching and bending vibrations occur, in NIR the corresponding overtones and combination vibrations are detected. This means that NIR-spectra can contain a lot of more vibrational information, which is an advantage for the analysis of highly complex samples. Therefore, during the last decade several applications in the field of food analysis were developed in the NIR region. Samples can be analysed ei‐ ther in transmission, reflectance and interactance mode (Figure 7) so that liquid as well as solid samples can be investigated. Due to the quite broad bands compared to MIR, chemo‐ metrical spectra treatment is required for establishing adequate calibration models and to

carried out.

184 Latest Research into Quality Control

**Figure 6.** Inner capillary wall coatings applied in CE and CEC

**2.4. Vibrational spectroscopy**

An impressive example for the successful implementation in the food related production is the quality control of wine. It has been shown that NIR can be used to identify grapes, vines, age by qualitative (Figure 8) and its ingredients (acids, carbohydrates, pH etc.) simultane‐ ously, non-invasively within a few seconds by quantitative analysis [19]. Another big ad‐ vantage of this method can be found by the fact that the sample is not destroyed and can therefore be used for further purposes including following analytical steps.

Quantitative NIRS methods, which allow determining the carbohydrate, total acid, tartaric acid, malic acid, pH in grape variety and the polyphenol content in grapes were established [19]. The method can control the quality already at a very early stage during the wine pro‐ duction and allows improvement of its quality by this. Grapes of 12 different vines (*Weißburgunder, Chardonnay, Ruländer, Silvaner, Müller Thurgau, Gewürztraminer, Sauvignon, Lagrein, Grossvernatsch, Blauburgunder, Cabernet, Merlot*) were harvested in autumn 2000 and squeezed. The obtained grape variety was thermo stated at 23°C and analyzed quantitative‐ ly by NIRS in the transflection mode using an optical thin layer thickness of 1 mm. In order to establish a calibration model 252 spectra of samples with lower and upper concentration as a reference were recorded. 76 % of all spectra were randomly used for calibration, 24% for validation. Data preparation was carried out in order to minimize technical influences, which mainly cause a drift in baseline. Quantitative analysis was carried out by partial least square regression (PLSR).

ric acid content in grape variety with an absolute error of estimation of 0.40 g/l and predic‐

*Malic acid.* Malic acid often shows 2-5 times higher values compared to tartaric acid. Calibra‐ tion between 2.9 and 7.0 g/l after normalization between 0 and 1 and calculation of a second smoothened derivative was carried out using three factors, SEE and SEP showing acceptable agreement.Absolute values for SEE, and BIAS were 0.43 g/l and -4.25×10-15. Straight line for

*pH.* Normalization and calculation of the smoothed 2nd derivative between 4500 and 7308 cm-1 showed an optimum for BIAS at five factors. Despite the narrow calibration range of

In order to enable the determination of these parameters with only one single measurement, simultaneous analysis of the carbohydrate, total acid, tartaric acid, malic acid content and pH was achieved by performing normalization (between 0 and 1) and calculating its 2nd de‐ rivative (Taylor 3 points). Four factors over a wavenumber range from 4500 to 7308 cm-1 showed 73-100% agreement between SEE and SEP. Linear regression showed high linearity

described single analysis this method allows a quantitative analysis of all parameters at once

Polyphenols mainly influence taste, sensory properties and color of a wine. Therefore, a rap‐ id method to analyze its quantity is important. The method according to Folin - Ciocalteu was used as a reference method (see Materials and Methods). Gallic acid-1-hydrate was used as reference standard in a concentration range from 0 to 4.93 μg/ml with equidistant steps. 24 gallic acid-1-hydrate solutions in a concentration range between 0.442 and 7.08 mg/ml were measured in the transmission mode threefold and in random order by NIRS. Evaluation using PLSR was achieved by dividing 72-recorded spectra randomly into a cali‐ bration (54 spectra) and validation (18 spectra) set. Data pretreatment comprised normaliza‐ tion between 0 and 1 and calculation of the 1st derivative (Savitzky-Golay) between 4008-7512 cm-1. Using three factors, the PRESS function showed a minimum and a good agreement between SEE (0.45 mg/ml) and SEP (0.46 mg/ml). Linear regression between pre‐ dicted and true values allowed to predict the gallic acid-1-hydrate concentration between 0

In order to determine the total polyphenol concentration 30 must samples were measured in the transmission mode threefold and in random order. 90 spectra were divided into 72 cali‐ bration and 18 validation spectra. Normalization and performing of the 1st derivative al‐ lowed minimizing shifts in the baseline. 4 factors were necessary to obtain a minimum for the PRESS function and to get a maximum agreement of SEE and SEP. Linear regression al‐ lowed correlating true and predicted values with a R2 of 0.97. Compared to the traditionally used Folin - Ciocalteu method in a winery, which is very time-consuming and expensive due to the usage of different chemicals, the NIRS method is very simple, precise and incom‐

of 0.82.

=0.89 and allowed a prediction of the malic acid content

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. Compared to the above-

tion of 0.54 g/l.

calibration showed a linearity of R2

pH 3.09 - 3.74 the calibration equation shows a R2

for each investigated parameter with slightly lower values for R2

within a few seconds. Values for SEP are slightly increased (Table 1).

with an absolute error of 0.55 g/l.

and 7 mg/ml with R2

parably fast.

=0.98.

**Figure 8.** Factor plot of 141 specta of different wines (Lagrein, Chianti, Cabernet Sauvignon). Conditions: Normalisa‐ tion, 1. derivative; wavenumber range, 4500 - 10000 cm-1; thickness 3 mm; scans, 10; temperature, 23°C.

*Carbohydrates.* Data preparation comprised normalization between 0 and 1 and following calculation of the first derivative using a wavenumber range from 4500 - 7548 cm-1. The PRESS function showed that 3 factors were needed for the calculation of the model. Calcula‐ tion with 3 factors resulted in a good conformity between SEE and SEP. Linear regression between true and predicted values resulted in a value for the correlation coefficient of R2 =0.99 for calibration and R2 =0.99 for validation. Results for SEE and SEP: 0.13° KMW and 0.11 °KMW, the BIAS value is 2.30×10-15.

*Total acids.* PLSR in a concentration range between 5 and 11 g/l included normalization be‐ tween 0 and 1, full multiplicative scatter correction (MSC) and calculation of the 1st deriva‐ tive (Taylor 3 points) between 4500 and 7548 cm-1. 3 factors were necessary to obtain a minimum for PRESS and an agreement between SEE (0.60 g/l) and SEP (0.61 g/l) of nearly 100%.The highly linear model allows determining the total acid content with a prediction er‐ ror of 0.61 g/l.

*Tartaric acid.* After normalization, performing of the 1st derivative over between 4500 and 7308 cm-1, four factors were used for creation of the highly linear model depicted in Figure 6c with R2 =0.91 for calibration and R2 =0.87 for validation). Despite the small concentration range between 3.1 and 6.7 g/l used for calibration this system allows to determine the tarta‐ ric acid content in grape variety with an absolute error of estimation of 0.40 g/l and predic‐ tion of 0.54 g/l.

which mainly cause a drift in baseline. Quantitative analysis was carried out by partial least

**Figure 8.** Factor plot of 141 specta of different wines (Lagrein, Chianti, Cabernet Sauvignon). Conditions: Normalisa‐

*Carbohydrates.* Data preparation comprised normalization between 0 and 1 and following calculation of the first derivative using a wavenumber range from 4500 - 7548 cm-1. The PRESS function showed that 3 factors were needed for the calculation of the model. Calcula‐ tion with 3 factors resulted in a good conformity between SEE and SEP. Linear regression between true and predicted values resulted in a value for the correlation coefficient of

*Total acids.* PLSR in a concentration range between 5 and 11 g/l included normalization be‐ tween 0 and 1, full multiplicative scatter correction (MSC) and calculation of the 1st deriva‐ tive (Taylor 3 points) between 4500 and 7548 cm-1. 3 factors were necessary to obtain a minimum for PRESS and an agreement between SEE (0.60 g/l) and SEP (0.61 g/l) of nearly 100%.The highly linear model allows determining the total acid content with a prediction er‐

*Tartaric acid.* After normalization, performing of the 1st derivative over between 4500 and 7308 cm-1, four factors were used for creation of the highly linear model depicted in Figure

range between 3.1 and 6.7 g/l used for calibration this system allows to determine the tarta‐

tion, 1. derivative; wavenumber range, 4500 - 10000 cm-1; thickness 3 mm; scans, 10; temperature, 23°C.

Cabernet Sauvignon

=0.99 for validation. Results for SEE and SEP: 0.13° KMW and

=0.87 for validation). Despite the small concentration

Chianti

square regression (PLSR).

186 Latest Research into Quality Control

Lagrein

=0.99 for calibration and R2

0.11 °KMW, the BIAS value is 2.30×10-15.

=0.91 for calibration and R2

R2

ror of 0.61 g/l.

6c with R2

*Malic acid.* Malic acid often shows 2-5 times higher values compared to tartaric acid. Calibra‐ tion between 2.9 and 7.0 g/l after normalization between 0 and 1 and calculation of a second smoothened derivative was carried out using three factors, SEE and SEP showing acceptable agreement.Absolute values for SEE, and BIAS were 0.43 g/l and -4.25×10-15. Straight line for calibration showed a linearity of R2 =0.89 and allowed a prediction of the malic acid content with an absolute error of 0.55 g/l.

*pH.* Normalization and calculation of the smoothed 2nd derivative between 4500 and 7308 cm-1 showed an optimum for BIAS at five factors. Despite the narrow calibration range of pH 3.09 - 3.74 the calibration equation shows a R2 of 0.82.

In order to enable the determination of these parameters with only one single measurement, simultaneous analysis of the carbohydrate, total acid, tartaric acid, malic acid content and pH was achieved by performing normalization (between 0 and 1) and calculating its 2nd de‐ rivative (Taylor 3 points). Four factors over a wavenumber range from 4500 to 7308 cm-1 showed 73-100% agreement between SEE and SEP. Linear regression showed high linearity for each investigated parameter with slightly lower values for R2 . Compared to the abovedescribed single analysis this method allows a quantitative analysis of all parameters at once within a few seconds. Values for SEP are slightly increased (Table 1).

Polyphenols mainly influence taste, sensory properties and color of a wine. Therefore, a rap‐ id method to analyze its quantity is important. The method according to Folin - Ciocalteu was used as a reference method (see Materials and Methods). Gallic acid-1-hydrate was used as reference standard in a concentration range from 0 to 4.93 μg/ml with equidistant steps. 24 gallic acid-1-hydrate solutions in a concentration range between 0.442 and 7.08 mg/ml were measured in the transmission mode threefold and in random order by NIRS. Evaluation using PLSR was achieved by dividing 72-recorded spectra randomly into a cali‐ bration (54 spectra) and validation (18 spectra) set. Data pretreatment comprised normaliza‐ tion between 0 and 1 and calculation of the 1st derivative (Savitzky-Golay) between 4008-7512 cm-1. Using three factors, the PRESS function showed a minimum and a good agreement between SEE (0.45 mg/ml) and SEP (0.46 mg/ml). Linear regression between pre‐ dicted and true values allowed to predict the gallic acid-1-hydrate concentration between 0 and 7 mg/ml with R2 =0.98.

In order to determine the total polyphenol concentration 30 must samples were measured in the transmission mode threefold and in random order. 90 spectra were divided into 72 cali‐ bration and 18 validation spectra. Normalization and performing of the 1st derivative al‐ lowed minimizing shifts in the baseline. 4 factors were necessary to obtain a minimum for the PRESS function and to get a maximum agreement of SEE and SEP. Linear regression al‐ lowed correlating true and predicted values with a R2 of 0.97. Compared to the traditionally used Folin - Ciocalteu method in a winery, which is very time-consuming and expensive due to the usage of different chemicals, the NIRS method is very simple, precise and incom‐ parably fast.


biological tissues and the modern analytical techniques enabling molecular imaging of com‐ plex samples. These techniques are based on the absorption of IR radiations by vibrational transitions in covalent bonds and their major advantage is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue by avoiding time-consuming extraction, purification and separation steps. These new techniques enable global analysis of biological samples with high spatial resolution and provide unique chemi‐ cal-morphological information about the tissue status. With these non-destructive examina‐ tion methods it is possible to get qualitative and quantitative information of heterogeneous

Novel Analytical Tools for Quality Control in Food Science

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189

Additionally, MALDI-TOF/MS imaging can be applied from the same sample of interest to get knowledge concerning the molecular weight distribution. This method is also suitable for studying the effect of nutrition onto different kinds of diseases, e.g. prostate cancer.

The techniques described can be applied according to the scheme depicted in Figure 1. This systematic analytical strategy allows getting multifacial knowledge and insights into food

samples.

**Figure 9.** Principle of hyperspectral cube

and samples derived therefrom.

**3. Conclusions**

**Table 1.** Prediction results for the determination of the carbohydrate, total acid, tartaric acid, malic acid content and pH

Quality control of coffee ingredients including caffeine, theobromine and theophylline [20] and of food additives deriving from the highly interesting field of Traditional Chinese Medi‐ cine (TCM) [21] can be carried out in a similar way. Thereby, emphasis must be put onto the calibration method for which the above mentioned techniques can be applied as a reference. A new analytical method based on near infrared spectroscopy (NIRS) for the quantitation of the three main alkaloids caffeine (Caf), theobromine (Tbr) and theophylline (Tph) in roasted coffee after discrimination of the rough green beans into Arabic and Robusta was establish‐ ed. This validated method was compared to the most commonly used liquid chromatogra‐ phy (LC) connected to UV and mass spectrometric (MS) detection. As analysis time plays an important role in choosing a reference method for the calibration of the NIR-spectrometer, the non-porous silica-C18 phase offers a very fast method. Coupling of the optimised LC method to a mass spectrometer (MS) via an electrospray ionisation (ESI) interface not only allowed to identify Caf, Tbr and Tph by their characteristic fragmentation pattern using col‐ lisionally induced dissociation (CID), but also to quantitate the content of the three analytes, which was found to be 6% higher compared to UV-detection. The validated LC–UV method was chosen as a reference method for the calibration of the NIRS system. Analysis of 83 liq‐ uid coffee extracts in random order resulted for Caf and Tbr in values for S.E.E. (standard error of estimation) of 0.34, 0.40 g/100 g, S.E.P. (standard error of prediction) of 0.07 and 0.10 g/100 g with correlation coefficients of 0.86 and 0.85 in a concentration range between 0.10 and 4.13 g/100 g. Compared to LC the lower limit of detection (LOD) of the NIRS-method is found at 0.05 g/100 g compared to 0.244–0.60 ng/100 g in LC, which makes it impossible to analyse Tph by NIRS.

The possibility to hyphenate a MIR/NIR spectrometer to a microscope unit allows determining the distribution of active ingredients within a tissue sample down to a resolution of 1.2 μm [22]. A "hyperspectral cube" is recorded with the dimensions of the sample on the x- and y-axis and the absorbance on the z-axis from which the image can be extracted (Figure 9).

Fourier Transform Infrared (FTIR) spectroscopic imaging and mapping techniques have be‐ come essential tools for the detection and characterization of the molecular components of biological tissues and the modern analytical techniques enabling molecular imaging of com‐ plex samples. These techniques are based on the absorption of IR radiations by vibrational transitions in covalent bonds and their major advantage is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue by avoiding time-consuming extraction, purification and separation steps. These new techniques enable global analysis of biological samples with high spatial resolution and provide unique chemi‐ cal-morphological information about the tissue status. With these non-destructive examina‐ tion methods it is possible to get qualitative and quantitative information of heterogeneous samples.

**Figure 9.** Principle of hyperspectral cube

Additionally, MALDI-TOF/MS imaging can be applied from the same sample of interest to get knowledge concerning the molecular weight distribution. This method is also suitable for studying the effect of nutrition onto different kinds of diseases, e.g. prostate cancer.

#### **3. Conclusions**

**Parameter Unit SEE SEP BIAS**

Note. a Single analysis; b Simultaneous analysis

188 Latest Research into Quality Control

pH

analyse Tph by NIRS.

Carbohydrates KMW 0.13 0.21 0.11 0.19 2.30 × 10-15 3.33 × 10-16 Total acids g/l 0.60 0.43 0.61 0.53 7.17 × 10-15 -1.08 × 10-14 Tartaric acid g/l 0.40 0.41 0.54 0.55 -1.08 × 10-14 -3.43 × 10-15 Malic acid g/l 0.43 0.49 0.55 0.65 -4.25 × 10-15 -2.44 × 10-15 pH 0.07 0.09 0.06 0.09 -1.26 × 10-15 -7.15 × 10-15

**Table 1.** Prediction results for the determination of the carbohydrate, total acid, tartaric acid, malic acid content and

Quality control of coffee ingredients including caffeine, theobromine and theophylline [20] and of food additives deriving from the highly interesting field of Traditional Chinese Medi‐ cine (TCM) [21] can be carried out in a similar way. Thereby, emphasis must be put onto the calibration method for which the above mentioned techniques can be applied as a reference. A new analytical method based on near infrared spectroscopy (NIRS) for the quantitation of the three main alkaloids caffeine (Caf), theobromine (Tbr) and theophylline (Tph) in roasted coffee after discrimination of the rough green beans into Arabic and Robusta was establish‐ ed. This validated method was compared to the most commonly used liquid chromatogra‐ phy (LC) connected to UV and mass spectrometric (MS) detection. As analysis time plays an important role in choosing a reference method for the calibration of the NIR-spectrometer, the non-porous silica-C18 phase offers a very fast method. Coupling of the optimised LC method to a mass spectrometer (MS) via an electrospray ionisation (ESI) interface not only allowed to identify Caf, Tbr and Tph by their characteristic fragmentation pattern using col‐ lisionally induced dissociation (CID), but also to quantitate the content of the three analytes, which was found to be 6% higher compared to UV-detection. The validated LC–UV method was chosen as a reference method for the calibration of the NIRS system. Analysis of 83 liq‐ uid coffee extracts in random order resulted for Caf and Tbr in values for S.E.E. (standard error of estimation) of 0.34, 0.40 g/100 g, S.E.P. (standard error of prediction) of 0.07 and 0.10 g/100 g with correlation coefficients of 0.86 and 0.85 in a concentration range between 0.10 and 4.13 g/100 g. Compared to LC the lower limit of detection (LOD) of the NIRS-method is found at 0.05 g/100 g compared to 0.244–0.60 ng/100 g in LC, which makes it impossible to

The possibility to hyphenate a MIR/NIR spectrometer to a microscope unit allows determining the distribution of active ingredients within a tissue sample down to a resolution of 1.2 μm [22]. A "hyperspectral cube" is recorded with the dimensions of the sample on the x- and y-axis and

Fourier Transform Infrared (FTIR) spectroscopic imaging and mapping techniques have be‐ come essential tools for the detection and characterization of the molecular components of

the absorbance on the z-axis from which the image can be extracted (Figure 9).

**a b a b a b**

The techniques described can be applied according to the scheme depicted in Figure 1. This systematic analytical strategy allows getting multifacial knowledge and insights into food and samples derived therefrom.

#### **Acknowledgements**

This work was financially supported by the Efre-project "Originalp" (EU) and Agrarmarket‐ ing Austria (AMA, Vienna, Austria).

[9] Rainer, M., Sonderegger, H., Bakry, R., Huck, C. W., Morandell, S., Huber, L. A., Gjerde, D. T., & Bonn, G. K. Analysis of protein phosphorylation by monolithic ex‐ traction columns based on poly(divinylbenzene) containing embedded titanium di‐ oxide and zirconium dioxide nano-powders. Proteomics (2008). , 8(21), 4593-4602. [10] Hahn, H., Rainer, M., Ringer, T., Gjerde, D., Huck, C. W., & Bonn, G. K. Ultra-fast Microwave-Assisted In-Tip Digestion of Proteins. J. Proteom. Res. (2009). , 8(9),

Novel Analytical Tools for Quality Control in Food Science

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191

[11] Bonn, G. K., Bakry, R., Huck, C. W., Vallant, R., & Szabo, Z. Analysis of low molecu‐ lar weight molecules by maldi-ms. Eur. Pat. Appl. ((2008). pp. CODEN: EPXXDW EP

[12] Greiderer A., Clark Ligon S.Jr., Huck C.W., Bonn G.K.Monolithic poly( -bis(p-vinyl‐ phenyl)ethane capillary columns for simultaneous separation of low- and high-mo‐

[13] Greiderer A., Trojer L., Huck C.W., Bonn G.K.Influence of the Polymerisation time on the Porous and Chromatographic Properties of Monolithic Poly(1,2-bis(p-vinyl‐ phenyl)ethane Capillary Columns.J.Chromatogr. A (2009). , 1216(45), 7747-7754. [14] Heigl, N., Greiderer, A., Petter, C. H., Siesler, H. W., Bonn, G. K., & Huck, C. W. Si‐ multaneous Determination of Physical and Chemical Parameters of Monolithic Po‐ rous Polymers with a Combined Use of Fourier-Transform Near Infrared Diffuse Reflection Spectroscopy and Multivariate Techniques. Anal. Chem. (2008). , 80(22),

[15] Trojer, L., Greiderer, A., Bisjak, C. P., Wieder, W., Heigl, N., Huck, C. W., Bonn, G. K., in, Handbook., of, H. P. L. C., Second, Edition., edited, by., Corradini, D., &

[16] Bachmann S., Vallant R., Bakry R., Huck C.W., Corradini D., Bonn G.K.Capillary electrophoresis coupled to matrix assisted laser desorption ionization with novel co‐

[17] Stöggl W.M., Huck C.W., Stecher G., Bonn G.K.Capillary Electrochromatography of

[18] Petter C.H., Heigl N., Bachmann S., Huck-Pezzei V.A.C., Najam-ul-Haq M., Bakry R., Bernkop-Schnürch A., Bonn G.K., Huck C.W.Near Infrared Spectroscopy Compared to Liquid Chromatography Coupled to Mass Spectrometry and Capillary Electropho‐ resis as a Detection Tool for Peptide Reaction Monitoring.Amino Acids, (2008). ,

[19] Guggenbichler, W., Huck, C. W., Kobler, A., Bonn, G. K., Near, Infrared., Reflectance, Spectroscopy., Tool, A., for, Quality., Control, In., & Wine, Production. J. Food Agric.

[20] Huck C.W., Guggenbichler W., Bonn G.K., Analysis of Caffeine, Theobromine and Theophylline in Coffee by Near Infrared Reflectance Spectroscopy (NIRS) Compared

Press, . (2010). Print 978-1-57444-554-1eBook ISBN: 978-1-4200-1694-9, 3-45.

valently coated capillaries.Electrophoresis, (2010). , 31, 618-629.

Biologically Relevant Flavonoids.Electrophoresis, (2006). , 27-787.

4225-4230.

8493-8500.

34(4), 605-616.

Environm., (2006). , 4(2), 98-106.

1973142 A1 20080924 AN 2008:1151584

lecular weight compounds.J.Sep. Sci. (2009).

#### **Author details**

Christian W. Huck

Institute of Analytical Chemistry and Radiochemistry, CCB – Center for Chemistry and Bio‐ medicine, Innsbruck, Austria

#### **References**


[9] Rainer, M., Sonderegger, H., Bakry, R., Huck, C. W., Morandell, S., Huber, L. A., Gjerde, D. T., & Bonn, G. K. Analysis of protein phosphorylation by monolithic ex‐ traction columns based on poly(divinylbenzene) containing embedded titanium di‐ oxide and zirconium dioxide nano-powders. Proteomics (2008). , 8(21), 4593-4602.

**Acknowledgements**

190 Latest Research into Quality Control

**Author details**

Christian W. Huck

**References**

medicine, Innsbruck, Austria

ing Austria (AMA, Vienna, Austria).

This work was financially supported by the Efre-project "Originalp" (EU) and Agrarmarket‐

Institute of Analytical Chemistry and Radiochemistry, CCB – Center for Chemistry and Bio‐

[1] Huck C.W., Popp M., Scherz H., Bonn G.K.Development and Evaluation of a New Method for the Determination of the Carotenoid Content in Selected Vegetables by

[2] Stöggl, W. M., Huck, C. W., Scherz, H., Bonn, G. K., Analysis, of., Vitamin, E., in, Food., Phytopharmaceutical, Preparations., by, H. P. L. C., -A, H. P. L. C., -M, P. C. I.,

[3] JakschitzT., HuckC.W., LubbadS., Bonn G.K.Monolithic Poly(TMSM-DMSBMS) Sta‐ tionary Phases for the fast Separation of Oligonucleotides and Proteins.J. Chroma‐

[4] Huck C.W., Bakry R., Bonn G.K.Progress in capillary electrophoresis of biomarkers

[5] Stöggl W.M., Huck C.W., Stecher G., Bonn G.K., Capillary Electrochromatography of

[6] Huck C.W., Bonn G.K.Review: Polymer based sorbents for solid-phase extraction.J.

[7] Feuerstein I., Najam-ul-Haq M., Rainer M., Trojer L., Bakry R., Hidayat Aprilita N., Stecher G., Huck C.W., Klocker H., Bartsch G., Guttman A., BonnG.K.Material En‐ hanced Laser Desorption/Ionization (MELDI)- a new protein profiling tool utilizing specific carrier materials for TOF-MS Analysis.J. Am. Soc. Mass Spectrom. (2006). ,

[8] Petter C.H., Heigl N., Bachmann S., Huck-Pezzei V.A.C., Najam-ul-Haq M., Bakry R., Bernkop-Schnürch A., Bonn G.K., Huck\*C.W.Near Infrared Spectroscopy Compared to Liquid Chromatography Coupled to Mass Spectrometry and Capillary Electropho‐

resis as a Detection Tool for Peptide Reaction Monitoring.Amino Acids (2008).

and metabolites between 2002 and 2005.Electrophoresis (2006). , 27-111.

Biologically Relevant Flavonoids. (2006). *Electrophoresis*, 27-787.

HPLC and HPLC-MS/MS.J. Chromatogr. Sci. (2000). , 38-441.

& , S. M. S. Chromatographia (2001). , 54-179.

togr. A (2007). , 1147-53.

Chromatogr. A (2000). , 885-51.

17-1203.


to High Performance Liquid Chromatography (HPLC) Coupled to Mass Spectrome‐ try.Anal. Chim. Acta, (2005). , 538-195.

**Section 3**

**Quality Control in Pharmaceutics**


**Quality Control in Pharmaceutics**

to High Performance Liquid Chromatography (HPLC) Coupled to Mass Spectrome‐

[21] Mattle, C., Heigl, N., Bonn, G. K., & Huck, C. W. Near Infrared Diffuse Reflection Spectroscopy and Multivariate Calibration Hyphenated to Thin Layer Chromatogra‐ phy for Quality Control and Simultaneous Quantification of Methoxylated Flavones

[22] Pezzei, C., Pallua, J. D., Schaefer, G., Seifarth, C., Huck-Pezzei, V., Bittner, L. K., Klocker, H., Bartsch, G., Bonn, G. K., Huck, C. W., Prostate, cancer., characterization, by., Fourier, Transform., & infrared, microspectroscopy. Mol. Biosys., (2010). , 6-2287.

in a Phytomedicine. J. Planar Chromatogr., (2010). , 23(5), 348-352.

try.Anal. Chim. Acta, (2005). , 538-195.

192 Latest Research into Quality Control

**Chapter 9**

**Microbial Quality**

http://dx.doi.org/10.5772/52114

**1. Introduction**

and biological product.

pharmaceutical product.

packaging in its final container and labeling." [3]

properly cited.

**Concerns for Biopharmaceuticals**

Farzaneh Lotfipour and Somayeh Hallaj-Nezhadi

Finding an appropriate definition or a clear classification for biologically occurring pharma‐ ceutical products is a complicated task because of overlapping borders and consequent mis‐ conceptions in this area. Indeed, numerous definitions and classifications for this category of products have been proposed so far, and different points of view for this concept can be

To obtain a better view of biopharmaceutical concept, first, it is necessary to know the present definitions for the main constituents of the word, that is, pharmaceutical product

According to the WHO, a finished pharmaceutical product (FPP) is "A finished dosage form of a pharmaceutical product, which has undergone all stages of manufacture, including

An active pharmaceutical ingredient can be defined as "A substance used in a finished phar‐ maceutical product (FPP), intended to furnish pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings." [3] Hence, in brief, it can be said that any material, regardless of its origin or structure, with treatment, diagnosis, or prevention applications and passing regulatory requirements, is a

On the other hand, the FDA definition for biological products is as follows: "Biological products or biologics are medical products made from a variety of natural sources (human, animal or microorganism). Like drugs, some biologics are intended to treat diseases and

> © 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

found in research literature, business, industry, and even the general public [1, 2].

Additional information is available at the end of the chapter

### **Chapter 9**

## **Microbial Quality Concerns for Biopharmaceuticals**

Farzaneh Lotfipour and Somayeh Hallaj-Nezhadi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52114

#### **1. Introduction**

Finding an appropriate definition or a clear classification for biologically occurring pharma‐ ceutical products is a complicated task because of overlapping borders and consequent mis‐ conceptions in this area. Indeed, numerous definitions and classifications for this category of products have been proposed so far, and different points of view for this concept can be found in research literature, business, industry, and even the general public [1, 2].

To obtain a better view of biopharmaceutical concept, first, it is necessary to know the present definitions for the main constituents of the word, that is, pharmaceutical product and biological product.

According to the WHO, a finished pharmaceutical product (FPP) is "A finished dosage form of a pharmaceutical product, which has undergone all stages of manufacture, including packaging in its final container and labeling." [3]

An active pharmaceutical ingredient can be defined as "A substance used in a finished phar‐ maceutical product (FPP), intended to furnish pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings." [3] Hence, in brief, it can be said that any material, regardless of its origin or structure, with treatment, diagnosis, or prevention applications and passing regulatory requirements, is a pharmaceutical product.

On the other hand, the FDA definition for biological products is as follows: "Biological products or biologics are medical products made from a variety of natural sources (human, animal or microorganism). Like drugs, some biologics are intended to treat diseases and

properly cited.

© 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2012 Lotfipour and Hallaj-Nezhadi; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

medical conditions or to prevent or diagnose diseases." [4] Consequently, any product of bi‐ ological origin with treatment, diagnosis, or prevention applications is a biological product.

A biotechnology-derived product is another concept that should be taken into consideration. It is defined by Walsh as "any pharmaceutical product used for a therapeutic or in vivo di‐ agnostic purpose, which is produced in full or in part by either traditional or modern bio‐ technological means." [5]

On comparing the definitions for a biotechnology-derived product and a biological product, both of which should be of biological origin, it is obvious that the key element in the former definition is the application of biotechnological means for production.

In practice, the regulatory requirements needed for a biotechnology-derived product and a biological product are methodologically different from the pharmaceutical product due to their biological essence. For example, the determination of adventitious agents such as vi‐ ruses, transmitting spongiform encephalopathy (TSE), and mycoplasma are included in most of the related guidelines and pharmacopeias for a biotechnology-derived product and a biological product.

**Figure 1.** The schematic representation for biotechnology-derived products and biopharmaceutical categories and

Microbial Quality Concerns for Biopharmaceuticals

http://dx.doi.org/10.5772/52114

197

The main category of biopharmaceuticals is manufactured via recombinant DNA technolo‐ gy. Indeed, recombinant DNA technologies are enabling techniques that manipulate and en‐ gineer different gene fragments and which have been introduced less than 50 years ago by

DNA and RNA extraction from different cell types, cutting DNA fragments using restriction endonucleases, joining DNA fragments by DNA ligases, PCR to amplify gene fragments, cloning of the gene fragments into different vectors, introduction of recombinant constructs into proper hosts, protein expression, extraction, and purification are some of the most

Figure 2 schematically represents the summarized process of production of a recombinant protein. As can be seen from the chart, first, the gene of interest should be isolated and am‐ plified from the original cell. According to the type of the cell, it can be done through direct total DNA extraction followed by a PCR using proper primers to obtain the gene in prokar‐ yotes. On the other hand, in eukaryotes, due to the existence of introns and some modifica‐ tions that occur in the transcribed mRNA, the process is considerably complicated. Introns are non-coding sequences which are removed after transcription versus coding sequences

the revolutionary invention of Polymerase Chain Reaction (PCR) by Kary Mullis [7].

the overlapping areas between them.

*2.1.1. Recombinant DNA technology*

**2. Methods for biopharmaceutical production**

**2.1. Production of an original recombinant system**

widely used means in recombinant protein production.

Finally, a biopharmaceutical is defined by Walsh as "A protein or nucleic acid based phar‐ maceutical substance used for therapeutic or in vivo diagnostic purposes, which is produced by means other than direct extraction from a native (non-engineered) biological source." This definition that will be used in the present chapter for biopharmaceuticals includes all pharmaceutical products produced by modern biotechnology techniques as well as nucleic acid (DNA or RNA) based pharmaceutical products for gene therapy. Hence, the overlap‐ ping area between biotechnology-derived products and biopharmaceuticals is the applica‐ tion of modern biotechnological means in their production. However, the differentiating area can be the application of traditional biotechnological means for the production of bio‐ technology-derived products. In addition, nucleic acid-based pharmaceutical products that are categorized as biopharmaceuticals are not biotechnology-derived products [6]. Figure 1 illustrates these overlapping and differentiating areas. Moreover, some examples of prod‐ ucts in these categories are shown in Table1.


**Table 1.** Some examples of products related to biological, biotechnology-derived, and biopharmaceutical products.

**Figure 1.** The schematic representation for biotechnology-derived products and biopharmaceutical categories and the overlapping areas between them.

### **2. Methods for biopharmaceutical production**

#### **2.1. Production of an original recombinant system**

#### *2.1.1. Recombinant DNA technology*

medical conditions or to prevent or diagnose diseases." [4] Consequently, any product of bi‐ ological origin with treatment, diagnosis, or prevention applications is a biological product.

A biotechnology-derived product is another concept that should be taken into consideration. It is defined by Walsh as "any pharmaceutical product used for a therapeutic or in vivo di‐ agnostic purpose, which is produced in full or in part by either traditional or modern bio‐

On comparing the definitions for a biotechnology-derived product and a biological product, both of which should be of biological origin, it is obvious that the key element in the former

In practice, the regulatory requirements needed for a biotechnology-derived product and a biological product are methodologically different from the pharmaceutical product due to their biological essence. For example, the determination of adventitious agents such as vi‐ ruses, transmitting spongiform encephalopathy (TSE), and mycoplasma are included in most of the related guidelines and pharmacopeias for a biotechnology-derived product and

Finally, a biopharmaceutical is defined by Walsh as "A protein or nucleic acid based phar‐ maceutical substance used for therapeutic or in vivo diagnostic purposes, which is produced by means other than direct extraction from a native (non-engineered) biological source." This definition that will be used in the present chapter for biopharmaceuticals includes all pharmaceutical products produced by modern biotechnology techniques as well as nucleic acid (DNA or RNA) based pharmaceutical products for gene therapy. Hence, the overlap‐ ping area between biotechnology-derived products and biopharmaceuticals is the applica‐ tion of modern biotechnological means in their production. However, the differentiating area can be the application of traditional biotechnological means for the production of bio‐ technology-derived products. In addition, nucleic acid-based pharmaceutical products that are categorized as biopharmaceuticals are not biotechnology-derived products [6]. Figure 1 illustrates these overlapping and differentiating areas. Moreover, some examples of prod‐

**Biotechnology-derived product by:**

**Biological products Traditional technology Modern technology Biopharmaceuticals**

**Table 1.** Some examples of products related to biological, biotechnology-derived, and biopharmaceutical products.

natural sources Recombinant proteins Recombinant proteins

Nucleic acid-based pharmaceutical products for gene therapy

Monoclonal antibody produced by hybridoma technology

definition is the application of biotechnological means for production.

technological means." [5]

196 Latest Research into Quality Control

a biological product.

ucts in these categories are shown in Table1.

Blood and blood products Therapeutic proteins from

Human cells and tissues Antibiotics fully or partially

from microorganisms

The main category of biopharmaceuticals is manufactured via recombinant DNA technolo‐ gy. Indeed, recombinant DNA technologies are enabling techniques that manipulate and en‐ gineer different gene fragments and which have been introduced less than 50 years ago by the revolutionary invention of Polymerase Chain Reaction (PCR) by Kary Mullis [7].

DNA and RNA extraction from different cell types, cutting DNA fragments using restriction endonucleases, joining DNA fragments by DNA ligases, PCR to amplify gene fragments, cloning of the gene fragments into different vectors, introduction of recombinant constructs into proper hosts, protein expression, extraction, and purification are some of the most widely used means in recombinant protein production.

Figure 2 schematically represents the summarized process of production of a recombinant protein. As can be seen from the chart, first, the gene of interest should be isolated and am‐ plified from the original cell. According to the type of the cell, it can be done through direct total DNA extraction followed by a PCR using proper primers to obtain the gene in prokar‐ yotes. On the other hand, in eukaryotes, due to the existence of introns and some modifica‐ tions that occur in the transcribed mRNA, the process is considerably complicated. Introns are non-coding sequences which are removed after transcription versus coding sequences that are called *exons*. In addition, mRNA is more modified by the addition of a methylated guanine (CAP) on its 5'end and a poly-adenine tail on its 3'end. After these modifications, mature mRNA is exported to the cytoplasm in order to start the translation process. Conse‐ quently, to obtain a gene of interest in eukaryotes, the mature mRNA should be extracted from the cell, and the complementary DNA should be synthesized followed by amplifica‐ tion of the gene by PCR using proper primers. However, in both cases (prokaryotes and eu‐ karyotes), the short genes can be obtained by a solid-phase synthesis process.

fragment that can be inserted into them. Ultimately, the new recombinant construct should be introduced into an expression system for production. Different classes of expression sys‐ tems with their certain merits and disadvantages are available and range from cell systems such as bacterial, animal, fungal, and yeast cells to transgenic systems such as transgenic

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Each specific antibody is secreted by a specific B cell and could recognize a specific region on the antigen that is called *epitope*. Each antibody-secreting B cell could be used as a source of an antibody of interest if it is isolated and cultured in vitro. Nevertheless, B cells are not considered a satisfactory source, as they are not immortalized and cannot survive for a long time. The main approach for overcoming this problem is cell hybridization, which includes the fusion of antibody-secreting B cells with tumor cells (such as mouse myeloma cells) pro‐ liferating ever more. The classical hybridoma technology was first introduced by Georges Kohler and Cesar Milstein in the mid 1970s for the generation of immortalized hybridoma cells that could grow in cell culture for a long time and produce the desired monoclonal an‐ tibodies [9, 10]. The basic process (Figure 3) includes the immunization of a mouse with the desired antigen. The mouse was then sacrificed, and B lymphocytes secreting antibodies that were selective for the specific epitope on the antigen were isolated from the spleen. The spleen is considered the most ready source for antigen-specific lymphocytes that provides access to a large number of antibody-secreting cells [1]. The isolated B cells were subse‐ quently fused with immortal mouse myeloma cells. The resultant hybridoma cells were then separated from the unfused cells by culturing in specific cell culture media. The cell culture media for the hybridoma growth and production of monoclonal antibodies have been re‐

In general, for the successful fusion of hybridomas, the cells are grown in HAT selection medi‐ um. The selection medium is called *HAT*, as it has Hypoxanthine, Aminopterin, and Thymi‐ dine. This is because a mutation in either the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or the thymidine kinase (TK) gene of the cells would cause their death in the HAT medium. Generally, a TK-deficient cell (TK-negative mutant) is resistant to bromodeoxyuridine (BrdU), and an HGPRT-deficient cell (HGPRT-negative mu‐ tant) is resistant to 6-thioguanine (6-TG) and 8-azaguanine. Hence, in order to make myeloma cells sensitive to HAT (unable to grow in HAT media), they are treated with one of these drugs

Normal cells can synthesize the required nucleotides in two pathways: (1) the main one or de novo biosynthetic pathway, and (2) the alternative one or the salvage pathway (when the

Aminopterin (a folic acid analog that inhibits dihydrofolate reductase) blocks the activation of tetrahydrofolate, which is required for the synthesis of nucleotides via the de novo syn‐ thetic pathway, and, therefore, the main pathway is blocked. Thus, in aminopterin-treated

plants and animals [7, 8].

*2.1.2. Monoclonal antibody production*

*2.1.2.1. Classical hybridoma technology*

viewed in detail by Bols et al. [11].

before their passage to HAT media.

main pathway is blocked).

**Figure 2.** Schematic representation of recombinant protein production process

Based on their sizes, the obtained genes can now be introduced into a proper vector for maintenance, replication, or expression purposes. Plasmids, cosmids, and bacteriophages are the most important cloning vectors that are classified according to the size of the DNA fragment that can be inserted into them. Ultimately, the new recombinant construct should be introduced into an expression system for production. Different classes of expression sys‐ tems with their certain merits and disadvantages are available and range from cell systems such as bacterial, animal, fungal, and yeast cells to transgenic systems such as transgenic plants and animals [7, 8].

#### *2.1.2. Monoclonal antibody production*

that are called *exons*. In addition, mRNA is more modified by the addition of a methylated guanine (CAP) on its 5'end and a poly-adenine tail on its 3'end. After these modifications, mature mRNA is exported to the cytoplasm in order to start the translation process. Conse‐ quently, to obtain a gene of interest in eukaryotes, the mature mRNA should be extracted from the cell, and the complementary DNA should be synthesized followed by amplifica‐ tion of the gene by PCR using proper primers. However, in both cases (prokaryotes and eu‐

karyotes), the short genes can be obtained by a solid-phase synthesis process.

198 Latest Research into Quality Control

**Figure 2.** Schematic representation of recombinant protein production process

Based on their sizes, the obtained genes can now be introduced into a proper vector for maintenance, replication, or expression purposes. Plasmids, cosmids, and bacteriophages are the most important cloning vectors that are classified according to the size of the DNA

#### *2.1.2.1. Classical hybridoma technology*

Each specific antibody is secreted by a specific B cell and could recognize a specific region on the antigen that is called *epitope*. Each antibody-secreting B cell could be used as a source of an antibody of interest if it is isolated and cultured in vitro. Nevertheless, B cells are not considered a satisfactory source, as they are not immortalized and cannot survive for a long time. The main approach for overcoming this problem is cell hybridization, which includes the fusion of antibody-secreting B cells with tumor cells (such as mouse myeloma cells) pro‐ liferating ever more. The classical hybridoma technology was first introduced by Georges Kohler and Cesar Milstein in the mid 1970s for the generation of immortalized hybridoma cells that could grow in cell culture for a long time and produce the desired monoclonal an‐ tibodies [9, 10]. The basic process (Figure 3) includes the immunization of a mouse with the desired antigen. The mouse was then sacrificed, and B lymphocytes secreting antibodies that were selective for the specific epitope on the antigen were isolated from the spleen. The spleen is considered the most ready source for antigen-specific lymphocytes that provides access to a large number of antibody-secreting cells [1]. The isolated B cells were subse‐ quently fused with immortal mouse myeloma cells. The resultant hybridoma cells were then separated from the unfused cells by culturing in specific cell culture media. The cell culture media for the hybridoma growth and production of monoclonal antibodies have been re‐ viewed in detail by Bols et al. [11].

In general, for the successful fusion of hybridomas, the cells are grown in HAT selection medi‐ um. The selection medium is called *HAT*, as it has Hypoxanthine, Aminopterin, and Thymi‐ dine. This is because a mutation in either the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or the thymidine kinase (TK) gene of the cells would cause their death in the HAT medium. Generally, a TK-deficient cell (TK-negative mutant) is resistant to bromodeoxyuridine (BrdU), and an HGPRT-deficient cell (HGPRT-negative mu‐ tant) is resistant to 6-thioguanine (6-TG) and 8-azaguanine. Hence, in order to make myeloma cells sensitive to HAT (unable to grow in HAT media), they are treated with one of these drugs before their passage to HAT media.

Normal cells can synthesize the required nucleotides in two pathways: (1) the main one or de novo biosynthetic pathway, and (2) the alternative one or the salvage pathway (when the main pathway is blocked).

Aminopterin (a folic acid analog that inhibits dihydrofolate reductase) blocks the activation of tetrahydrofolate, which is required for the synthesis of nucleotides via the de novo syn‐ thetic pathway, and, therefore, the main pathway is blocked. Thus, in aminopterin-treated cells (HGPRT+ and TK+ ), the synthesis of nucleotides shifts to the salvage pathway only if hypoxanthine and thymidine are supplied in the medium. HGPRT and TK, the two en‐ zymes, are required for the salvage pathway, and they catalyze the synthesis of purine and thymidylate from hypoxanthine and thymidine substrates, respectively.

**5.** When in vitro methods result in monoclonal antibodies that are glycosylated at posi‐ tions different from those harvested from mouse ascites, they affect antigen-binding ca‐

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Taken together, the cell culture technique is a method of choice for large-scale monoclonal antibody production due to the simplicity of the cell culture and financial considerations

**Figure 3.** The diagram of the monoclonal antibody production via classical hybridoma technology

In 1986, about 10 years after the conception of monoclonal antibody technology, Orthoclone OKT3 was approved by the FDA for use in patients with acute rejection of a transplanted kidney [14]. Unfortunately, early clinical applications of murine monoclonal antibodies were disappointing. This was due to the fact that monoclonal antibodies produced via the classi‐ cal method are of murine origin and are, therefore, immunogenic to human subjects. In gen‐ eral, patients receiving an antibody exhibit HAMA responses (human anti-mouse antibodies) within two weeks. Multiple infusions of murine monoclonal antibodies signifi‐ cantly enhance the HAMA reactions [6]. In addition, the immune system eliminates the murine monoclonal antibody molecule. Thus, murine monoclonal antibodies demonstrate short serum half lives after administration to humans. Furthermore, the other main difficul‐ ty related to murine monoclonal antibodies is the poor recognition of the Fc region by hu‐

Thus, new strategies that are used for producing humanized mouse antibodies that are less immunogenic have been discovered. The first strategy includes the production of functional specific recombinant IgG molecules consisting of mouse variable regions and human con‐ stant regions; these are known as chimeric antibodies. Taken together, in the chimeric anti‐ body, 8 out of 12 domains are of human origin (constant regions of the heavy and light chains) (CH and CL) [15]. Chimeric antibodies exhibit reduced HAMA responses compared

pacity as well as biological functions [12].

without ethical concerns that are related to animal use.

*2.1.2.2. Chimeric and humanized antibodies*

man effector systems of complement and Fc receptors.

Since unfused myeloma cells lack HGPRT or TK, they cannot use the salvage pathway. Thus, the unfused myeloma cells get killed in the HAT medium, as both biosynthetic path‐ ways are blocked. Normal unfused B cells die in the HAT medium, as they are not immor‐ talized and cannot grow for a long time. Nevertheless, the fusion of normal B cells with the HGPRTor TKmyeloma cells allows the hybridoma cells to grow in HAT medium, as the B cells provide the necessary enzymes for growth of the hybridoma cells.

Hence, the HAT selection medium offers an ideal environment for the isolation of fused myeloma and B cells (hybridoma cells) from unfused myeloma cells and unfused B cells, as this medium allows only the hybridoma cells to survive in the culture.

The production of monoclonal antibodies could be accomplished by ascites (ascitic fluid) production (in vivo) or by cell culture (in vitro) methods. In the in vivo method, hybridoma cells are injected intraperitoneally into mice. The peritoneum serves as a growth chamber for the injected cells. These cells could secrete a high-titered solution of desired antibodies as they grow in the cavity. Finally, the produced antibodies are extracted from the ascitic fluid accumulated in the peritoneal cavity [6]. The antibody concentrations typically range be‐ tween 1 and 15 mg/ml. The in vivo method offers a very high concentration of monoclonal antibody that often does not need more concentration procedures. Nevertheless, monoclonal antibodies produced by this technique may be contaminated by considerable levels of mouse proteins and other contaminants that might require more complicated, subsequent downstream purifications. The other disadvantage of the ascites production is related to ani‐ mal welfare issues, as these could cause distress in mice.

Currently, more than 90% of monoclonal antibodies are produced by in vitro techniques [12] that use large-scale manufacturing plants containing several 10,000-L or larger culture bio‐ reactors [13]. The in vitro method of monoclonal antibody production decreases the use of mice and also avoids the need for experienced personnel for animal handling. Regardless of the privileges and importance of the in vitro methods of antibody production, there are some situations in which this method is not applicable; for instance:


**5.** When in vitro methods result in monoclonal antibodies that are glycosylated at posi‐ tions different from those harvested from mouse ascites, they affect antigen-binding ca‐ pacity as well as biological functions [12].

Taken together, the cell culture technique is a method of choice for large-scale monoclonal antibody production due to the simplicity of the cell culture and financial considerations without ethical concerns that are related to animal use.

**Figure 3.** The diagram of the monoclonal antibody production via classical hybridoma technology

#### *2.1.2.2. Chimeric and humanized antibodies*

cells (HGPRT+

200 Latest Research into Quality Control

HGPRT-

or TK-

activity.

valuable hybridoma.

and TK+

), the synthesis of nucleotides shifts to the salvage pathway only if

hypoxanthine and thymidine are supplied in the medium. HGPRT and TK, the two en‐ zymes, are required for the salvage pathway, and they catalyze the synthesis of purine and

Since unfused myeloma cells lack HGPRT or TK, they cannot use the salvage pathway. Thus, the unfused myeloma cells get killed in the HAT medium, as both biosynthetic path‐ ways are blocked. Normal unfused B cells die in the HAT medium, as they are not immor‐ talized and cannot grow for a long time. Nevertheless, the fusion of normal B cells with the

Hence, the HAT selection medium offers an ideal environment for the isolation of fused myeloma and B cells (hybridoma cells) from unfused myeloma cells and unfused B cells, as

The production of monoclonal antibodies could be accomplished by ascites (ascitic fluid) production (in vivo) or by cell culture (in vitro) methods. In the in vivo method, hybridoma cells are injected intraperitoneally into mice. The peritoneum serves as a growth chamber for the injected cells. These cells could secrete a high-titered solution of desired antibodies as they grow in the cavity. Finally, the produced antibodies are extracted from the ascitic fluid accumulated in the peritoneal cavity [6]. The antibody concentrations typically range be‐ tween 1 and 15 mg/ml. The in vivo method offers a very high concentration of monoclonal antibody that often does not need more concentration procedures. Nevertheless, monoclonal antibodies produced by this technique may be contaminated by considerable levels of mouse proteins and other contaminants that might require more complicated, subsequent downstream purifications. The other disadvantage of the ascites production is related to ani‐

Currently, more than 90% of monoclonal antibodies are produced by in vitro techniques [12] that use large-scale manufacturing plants containing several 10,000-L or larger culture bio‐ reactors [13]. The in vitro method of monoclonal antibody production decreases the use of mice and also avoids the need for experienced personnel for animal handling. Regardless of the privileges and importance of the in vitro methods of antibody production, there are

**2.** Downstream purification methods cause protein denaturation or decreased antibody

**4.** When hybridoma cells are contaminated with infectious agents (such as yeasts or fun‐ gi), the cells must often be passed through mice. Since removal of the organisms cannot be accomplished by current antimicrobial drugs, thus the in vivo method may save a

myeloma cells allows the hybridoma cells to grow in HAT medium, as the B

thymidylate from hypoxanthine and thymidine substrates, respectively.

cells provide the necessary enzymes for growth of the hybridoma cells.

this medium allows only the hybridoma cells to survive in the culture.

mal welfare issues, as these could cause distress in mice.

some situations in which this method is not applicable; for instance:

**3.** The cell line cannot maintain the production of monoclonal antibodies.

**1.** Hybridoma cells do not adapt well to in vitro conditions.

In 1986, about 10 years after the conception of monoclonal antibody technology, Orthoclone OKT3 was approved by the FDA for use in patients with acute rejection of a transplanted kidney [14]. Unfortunately, early clinical applications of murine monoclonal antibodies were disappointing. This was due to the fact that monoclonal antibodies produced via the classi‐ cal method are of murine origin and are, therefore, immunogenic to human subjects. In gen‐ eral, patients receiving an antibody exhibit HAMA responses (human anti-mouse antibodies) within two weeks. Multiple infusions of murine monoclonal antibodies signifi‐ cantly enhance the HAMA reactions [6]. In addition, the immune system eliminates the murine monoclonal antibody molecule. Thus, murine monoclonal antibodies demonstrate short serum half lives after administration to humans. Furthermore, the other main difficul‐ ty related to murine monoclonal antibodies is the poor recognition of the Fc region by hu‐ man effector systems of complement and Fc receptors.

Thus, new strategies that are used for producing humanized mouse antibodies that are less immunogenic have been discovered. The first strategy includes the production of functional specific recombinant IgG molecules consisting of mouse variable regions and human con‐ stant regions; these are known as chimeric antibodies. Taken together, in the chimeric anti‐ body, 8 out of 12 domains are of human origin (constant regions of the heavy and light chains) (CH and CL) [15]. Chimeric antibodies exhibit reduced HAMA responses compared with mouse antibodies, but the affinity and the selectivity are the same. Furthermore, since the Fc region contains human sequences, the activation of Fc-mediated immune effector functions is allowed.

**2.2. Mass production of a recombinant product**

**Figure 5.** Mass production of a recombinant product.

Figure 5.

Mass production of recombinant products can be achieved in a process that is divided into two main sections called *upstream* and *downstream* processing, as schematically depicted in

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The first step, the upstream processing step, is the mass production of a recombinant protein via the fermentation process. The original recombinant system that is used for the expres‐ sion of the recombinant protein (i.e. in the form of a recombinant plasmid maintained in a suitable host cell) forms the cell deposit in a cell banking system. There are two levels of cell deposits in the cell banking system: The first line is called *master cell bank*, which is prepared

To further minimize the antigenicity of murine antibodies, humanized antibodies were devel‐ oped. For their generation, hyper-variable complementarity-determining regions (CDRs) of the specific murine antibody are transferred to a fully human framework. In comparison with the mouse antibodies, humanized antibodies suggest a lower occurrence of HAMA responses.

Further efforts have been invested in the development of technologies that generate fully hu‐ man monoclonal antibodies. One of the approaches entails the development of transgenic mice, in which a repertoire of human immunoglobulin germline gene segments is inserted into the mouse genome. After the immunization of these mice, they produce fully human antibodies, which can subsequently be separated with the classical hybridoma technology [15].

Figure 4 illustrates the schematic structures of mouse, chimeric, humanized, and human antibodies.

**Figure 4.** The structure of mouse (shown in red color), chimeric, humanized (shown in black color), and human anti‐ bodies. Chimeric antibodies comprise mouse variable regions and human constant regions. Humanized antibodies consist of murine hyper-variable complementarity-determining regions (CDRs) that are grafted to fully human frame‐ work.

#### **2.2. Mass production of a recombinant product**

with mouse antibodies, but the affinity and the selectivity are the same. Furthermore, since the Fc region contains human sequences, the activation of Fc-mediated immune effector

To further minimize the antigenicity of murine antibodies, humanized antibodies were devel‐ oped. For their generation, hyper-variable complementarity-determining regions (CDRs) of the specific murine antibody are transferred to a fully human framework. In comparison with the mouse antibodies, humanized antibodies suggest a lower occurrence of HAMA responses.

Further efforts have been invested in the development of technologies that generate fully hu‐ man monoclonal antibodies. One of the approaches entails the development of transgenic mice, in which a repertoire of human immunoglobulin germline gene segments is inserted into the mouse genome. After the immunization of these mice, they produce fully human antibodies,

Figure 4 illustrates the schematic structures of mouse, chimeric, humanized, and human

**Figure 4.** The structure of mouse (shown in red color), chimeric, humanized (shown in black color), and human anti‐ bodies. Chimeric antibodies comprise mouse variable regions and human constant regions. Humanized antibodies consist of murine hyper-variable complementarity-determining regions (CDRs) that are grafted to fully human frame‐

which can subsequently be separated with the classical hybridoma technology [15].

functions is allowed.

202 Latest Research into Quality Control

antibodies.

work.

Mass production of recombinant products can be achieved in a process that is divided into two main sections called *upstream* and *downstream* processing, as schematically depicted in Figure 5.

**Figure 5.** Mass production of a recombinant product.

The first step, the upstream processing step, is the mass production of a recombinant protein via the fermentation process. The original recombinant system that is used for the expres‐ sion of the recombinant protein (i.e. in the form of a recombinant plasmid maintained in a suitable host cell) forms the cell deposit in a cell banking system. There are two levels of cell deposits in the cell banking system: The first line is called *master cell bank*, which is prepared directly from a culture of the original recombinant system that includes several hundred stored ampoules.

Furthermore, high-temperature, short-time treatment strategies are sometimes employed for the elimination of biological contaminants from small solutes such as vitamins and amino

Another important raw material that is used in the production of any pharmaceutical prod‐ uct, including biopharmaceuticals, is water, which can be considered an important source for contamination, with water-borne bacteria such as Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacter spp., and Serratia spp. Water for pharmaceutical pur‐ poses is discussed in detail in the USP [17]. Due to the fact that the intended administration of biopharmaceuticals in the majority of cases is via injection, Water for Injection (WFI)

Pharmaceutical products' contamination may occur from the transformation of microorgan‐ isms from the production environment to the product. The production environment in‐

The main groups of microorganisms that are isolated from air are the spore-forming bacteria (Bacillus spp. and Clostridium spp., the non-sporing bacteria Staphylococcus spp., Streptococ‐ cus spp., and Corynebacterium spp.), the molds (Penicillium spp., Cladosporium spp., Asper‐ gillus spp., and Mucor spp.), and the yeast (Rhodotorula spp.). These contaminants may be air borne or can be initiated from process equipment or personnel [16]. Consequently, environ‐ mental monitoring programs in a production environment are essential actions. Furthermore, the critical operations in biopharmaceutical production should be performed in controlled en‐ vironments or clean rooms. A clean room is a place with high control of the entrance of parti‐ cles via the establishment of some air filters called *high-efficiency particulate air (HEPA) filters*. HEPA filters made from a microglass material with a pleated construction system provide a large surface area that efficiently filters the incoming air and generates a constant air motion. Based on the permitted quantity of viable microorganisms and particulates, various classes of

According to the *EC Guide to Good Manufacturing Practice for Medicinal Products* (EC GGMP), four grades for clean rooms are available, such as grade A, B, C, or D, based on the number

> **Maximum permitted CFU of viable microorganisms/m** <sup>3</sup> **in air sample**

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which is sterile and apyrogen is routinely used in this area.

cludes air, surfaces, instruments, equipments, and personnel.

clean rooms can be established using HEPA filters with required efficiencies.

**particles/m** <sup>3</sup>

A 3500 <1 B 3500 10 C 350000 100 D 3500000 200

**Table 2.** Clean room grades according to the number of viable microorganisms and the number of particulates

of viable microorganisms and particulates (Table 2).

**Clean room grade Maximum permitted number of**

acids [16].

**3.2. Production environment**

The second line that is referred to as the *working cell bank* is produced from a single master cell bank ampoule. Each ampoule from the working cell bank is thawed and used to seed the fermentor for the production of a batch. Obviously, the fermentation process for various recombinant systems, such as bacterial fermenting systems or animal cell systems, is quite different and needs its own certain requirements.

On the other hand, *downstream processing*, which is the next step, refers to the purification of the mass produced protein. The first step in downstream processing is the initial extraction and concentration of the product, which depends on the situation of the expressed protein; that is, it should be extracted from the pellet cells for intracellular proteins or from the su‐ pernatant for extracellular proteins after centrifugation.

The second stage entails (1) several chromatographic steps that complete the purification of the product; (2) the potency test; (3) the addition of suitable excipients; (4) sterilization; (5) filling of the product in its final form (liquid or solid) into the final container before sealing; and labeling [6].

#### **3. Sources of biological contamination of biopharmaceuticals**

In line with conventional pharmaceutical products, the main sources of biological contami‐ nation in biopharmaceuticals can be related to raw materials and the production environ‐ ment. Indeed, the biological contaminant content of any pharmaceutical product is a representative of their starting materials and the production environment flora.

#### **3.1. Raw materials**

Animal origin materials, such as cell culture media, sera, and supplements that are exten‐ sively used in biopharmaceutical production, are of high contamination risk. These materi‐ als can be considered the main source for the contamination of biopharmaceuticals with adventitious agents such as TSEs, viruses, and mycoplasmas. Therefore, they should be sup‐ plied from reliable resources, and special attention should be paid to their quality control procedure. It should be ensured that all raw materials, especially those of high risk, gain quality specifications for current good manufacturing practice.

Standard methods for sterilization of cell culture media, sera, and supplements should be established according to the properties of the materials. Due to the heat-labile nature of the majority of materials used in biopharmaceutical production, autoclaving is usually replaced with alternative strategies such as filter-sterilization or less frequently high-temperature, short-time treatment strategies. In spite of the routine filter-sterilization procedure that uses 0.22 μm, it is usually performed with 0.1-μm membrane filters due to the risk of contamina‐ tion with adventitious agents.

Furthermore, high-temperature, short-time treatment strategies are sometimes employed for the elimination of biological contaminants from small solutes such as vitamins and amino acids [16].

Another important raw material that is used in the production of any pharmaceutical prod‐ uct, including biopharmaceuticals, is water, which can be considered an important source for contamination, with water-borne bacteria such as Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacter spp., and Serratia spp. Water for pharmaceutical pur‐ poses is discussed in detail in the USP [17]. Due to the fact that the intended administration of biopharmaceuticals in the majority of cases is via injection, Water for Injection (WFI) which is sterile and apyrogen is routinely used in this area.

#### **3.2. Production environment**

directly from a culture of the original recombinant system that includes several hundred

The second line that is referred to as the *working cell bank* is produced from a single master cell bank ampoule. Each ampoule from the working cell bank is thawed and used to seed the fermentor for the production of a batch. Obviously, the fermentation process for various recombinant systems, such as bacterial fermenting systems or animal cell systems, is quite

On the other hand, *downstream processing*, which is the next step, refers to the purification of the mass produced protein. The first step in downstream processing is the initial extraction and concentration of the product, which depends on the situation of the expressed protein; that is, it should be extracted from the pellet cells for intracellular proteins or from the su‐

The second stage entails (1) several chromatographic steps that complete the purification of the product; (2) the potency test; (3) the addition of suitable excipients; (4) sterilization; (5) filling of the product in its final form (liquid or solid) into the final container before sealing;

In line with conventional pharmaceutical products, the main sources of biological contami‐ nation in biopharmaceuticals can be related to raw materials and the production environ‐ ment. Indeed, the biological contaminant content of any pharmaceutical product is a

Animal origin materials, such as cell culture media, sera, and supplements that are exten‐ sively used in biopharmaceutical production, are of high contamination risk. These materi‐ als can be considered the main source for the contamination of biopharmaceuticals with adventitious agents such as TSEs, viruses, and mycoplasmas. Therefore, they should be sup‐ plied from reliable resources, and special attention should be paid to their quality control procedure. It should be ensured that all raw materials, especially those of high risk, gain

Standard methods for sterilization of cell culture media, sera, and supplements should be established according to the properties of the materials. Due to the heat-labile nature of the majority of materials used in biopharmaceutical production, autoclaving is usually replaced with alternative strategies such as filter-sterilization or less frequently high-temperature, short-time treatment strategies. In spite of the routine filter-sterilization procedure that uses 0.22 μm, it is usually performed with 0.1-μm membrane filters due to the risk of contamina‐

**3. Sources of biological contamination of biopharmaceuticals**

representative of their starting materials and the production environment flora.

quality specifications for current good manufacturing practice.

stored ampoules.

204 Latest Research into Quality Control

and labeling [6].

**3.1. Raw materials**

tion with adventitious agents.

different and needs its own certain requirements.

pernatant for extracellular proteins after centrifugation.

Pharmaceutical products' contamination may occur from the transformation of microorgan‐ isms from the production environment to the product. The production environment in‐ cludes air, surfaces, instruments, equipments, and personnel.

The main groups of microorganisms that are isolated from air are the spore-forming bacteria (Bacillus spp. and Clostridium spp., the non-sporing bacteria Staphylococcus spp., Streptococ‐ cus spp., and Corynebacterium spp.), the molds (Penicillium spp., Cladosporium spp., Asper‐ gillus spp., and Mucor spp.), and the yeast (Rhodotorula spp.). These contaminants may be air borne or can be initiated from process equipment or personnel [16]. Consequently, environ‐ mental monitoring programs in a production environment are essential actions. Furthermore, the critical operations in biopharmaceutical production should be performed in controlled en‐ vironments or clean rooms. A clean room is a place with high control of the entrance of parti‐ cles via the establishment of some air filters called *high-efficiency particulate air (HEPA) filters*. HEPA filters made from a microglass material with a pleated construction system provide a large surface area that efficiently filters the incoming air and generates a constant air motion. Based on the permitted quantity of viable microorganisms and particulates, various classes of clean rooms can be established using HEPA filters with required efficiencies.

According to the *EC Guide to Good Manufacturing Practice for Medicinal Products* (EC GGMP), four grades for clean rooms are available, such as grade A, B, C, or D, based on the number of viable microorganisms and particulates (Table 2).


**Table 2.** Clean room grades according to the number of viable microorganisms and the number of particulates

Critical operations such as inoculum preparation and aseptic filling are generally performed in the highest air grade (A); however, less critical operations can be performed in lower grades or even non-classified air.

The eradication of bacteria and fungi from the products is generally carried out via inactiva‐

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Since most of the biopharmaceuticals are administered intravenously, finished-product bio‐ pharmaceuticals must be sterile and free from pyrogenic substances. The endotoxin limit for the intravenous administration of pharmaceutical and biological products is 5 endotoxin units (EU)/kg of body weight/hour by all pharmacopoeias [19]. Hence, the detection and re‐ moval of pyrogenic substances, especially endotoxins (lipopolysaccharides in the cell wall of gram-negative bacteria), are necessary to ensure safety of biopharmaceutical products. Cur‐ rently available methods for endotoxin detection include the U.S. Pharmacopeia rabbit test

The rabbit pyrogen test entails measurements of the rise in body temperature of rabbits after an intravenous injection of a test substance. The presence of pyrogens of all kinds can be tested using this method. However, this method suffers from a number of disadvantages and limitations: (1) Endotoxin tolerance occurs after repeated use of rabbits; (2) variations in the response depending on sex, age, and species; (3) differences between the responses of rabbits and humans to various pyrogen types; and (4) the rabbit pyrogen test is inadequate for sera, radiopharmaceuticals, chemotherapeutics, analgesics, cytokines, immunosuppres‐

Accordingly, the use of the rabbit pyrogen test has been reduced. Nowadays, the most wide‐ ly used endotoxin detection systems are based on the highly sensitive LAL test. It is based on the coagulation cascade of the blood of a horseshoe crab, Limulus polyphemus, which is induced by lipopolysaccharide. The currently known methods for lipopolysaccharide detec‐ tion entail (1) gel-clot assay, (2) turbidimetric LAL technique, and (3) the chromogenic LAL

The gel-clot assay is a limit test that provides simple positive or negative results. The LAL reagent is introduced to a sample, and the test material is considered endotoxin positive if a

The turbidimetric and the chromogenic LAL techniques are quantitative tests. The former is based on the fact that turbidity increases as a result of the precipitation of the clottable pro‐ tein that is related to endotoxin concentration in the sample. The optical density is read by a spectrophotometer at either a fixed time (for the end-point method) or progressively (for the

The chromogenic LAL technique makes use of a synthetic substrate which contains an amino acid sequence similar to that of the clottable protein, coagulogen, in order to detect endotoxin. The enzyme cleaves a yellow-colored substance from the chromogenic sub‐ strate, and the color intensity produced is proportional to the amount of endotoxin

tion and sterile filtration.

sive agents, and others [20].

gel is formed via a clotting reaction.

kinetic assay) as turbidity develops.

present in the sample.

technique.

and the Limulus amebocyte lysate (LAL) test [7].

**5.2. Endotoxins**

In addition to the establishment of suitable filters in the clean rooms, special attention should be paid to the position, type, and texture of surfaces, floors, and fixtures. They should be made from smooth and chemically stable materials. In addition, a distinct transfer lock area should exist before entry to the clean room for sanitization of materials and per‐ sonnel or garment changing. Furthermore, all doors should be interlocking [6].

### **4. Hazards of biological contamination of biopharmaceuticals**

Similar to other pharmaceuticals, biological contamination of biopharmaceuticals may per‐ haps cause product spoilage. It may result in product metabolization by microorganisms, and, therefore, lead to a decrease in biopharmaceutical potency. The product spoilage may also provide a potential health hazard to patients and lead to outbreaks of infections that may cause additional complications. In addition, microbial-derived agents secreted in prod‐ ucts such as endotoxins can be hazardous to a patient's health.

#### **5. Determination of biological contaminants**

#### **5.1. Bacteria and fungi**

Bacteria and fungi can be considered important contamination sources for all kinds ofphar‐ maceutical products, including biopharmaceuticals; hence, the control of them is of critical importance. The control of both bacteria and fungi is considered to be worthy of mandatory tests for nearly all kinds of pharmaceuticals in pharmacopoeias. All the related tests and procedures are covered in detail in the major pharmacopoeias such as USP and EP [17, 18].

Since almost all the biopharmaceuticals are administered intravenously, general sterility testing must be carried out for these products. Basically, sterility testing can be defined as "a test that evaluates whether a sterilized pharmaceutical product is free of contaminating microorganisms." The European Pharmacopoeia (2002) proposes two media for sterility testing:

(1) fluid mercaptoacetate medium (also known as *fluid thioglycollate medium*), which is main‐ ly appropriate for the culture of anaerobic organisms at 30–35°C; and (2) soyabean casein di‐ gest medium, which is used for the culture of both aerobic bacteria at 30–35°C and fungi at 20–25°C.

Two main methods are used for sterility tests: (1) direct inoculation of the test samples in the media mentioned earlier; or (2) filtration of the test material through a sterile membrane fil‐ ter with a pore size of 0.45 μm; then, the filter containing any microorganism present in the fluids is divided aseptically, and portions are transferred to the media.

The eradication of bacteria and fungi from the products is generally carried out via inactiva‐ tion and sterile filtration.

#### **5.2. Endotoxins**

Critical operations such as inoculum preparation and aseptic filling are generally performed in the highest air grade (A); however, less critical operations can be performed in lower

In addition to the establishment of suitable filters in the clean rooms, special attention should be paid to the position, type, and texture of surfaces, floors, and fixtures. They should be made from smooth and chemically stable materials. In addition, a distinct transfer lock area should exist before entry to the clean room for sanitization of materials and per‐

Similar to other pharmaceuticals, biological contamination of biopharmaceuticals may per‐ haps cause product spoilage. It may result in product metabolization by microorganisms, and, therefore, lead to a decrease in biopharmaceutical potency. The product spoilage may also provide a potential health hazard to patients and lead to outbreaks of infections that may cause additional complications. In addition, microbial-derived agents secreted in prod‐

Bacteria and fungi can be considered important contamination sources for all kinds ofphar‐ maceutical products, including biopharmaceuticals; hence, the control of them is of critical importance. The control of both bacteria and fungi is considered to be worthy of mandatory tests for nearly all kinds of pharmaceuticals in pharmacopoeias. All the related tests and procedures are covered in detail in the major pharmacopoeias such as USP and EP [17, 18]. Since almost all the biopharmaceuticals are administered intravenously, general sterility testing must be carried out for these products. Basically, sterility testing can be defined as "a test that evaluates whether a sterilized pharmaceutical product is free of contaminating microorganisms." The European Pharmacopoeia (2002) proposes two media for sterility

(1) fluid mercaptoacetate medium (also known as *fluid thioglycollate medium*), which is main‐ ly appropriate for the culture of anaerobic organisms at 30–35°C; and (2) soyabean casein di‐ gest medium, which is used for the culture of both aerobic bacteria at 30–35°C and fungi at

Two main methods are used for sterility tests: (1) direct inoculation of the test samples in the media mentioned earlier; or (2) filtration of the test material through a sterile membrane fil‐ ter with a pore size of 0.45 μm; then, the filter containing any microorganism present in the

fluids is divided aseptically, and portions are transferred to the media.

sonnel or garment changing. Furthermore, all doors should be interlocking [6].

**4. Hazards of biological contamination of biopharmaceuticals**

ucts such as endotoxins can be hazardous to a patient's health.

**5. Determination of biological contaminants**

**5.1. Bacteria and fungi**

testing:

20–25°C.

grades or even non-classified air.

206 Latest Research into Quality Control

Since most of the biopharmaceuticals are administered intravenously, finished-product bio‐ pharmaceuticals must be sterile and free from pyrogenic substances. The endotoxin limit for the intravenous administration of pharmaceutical and biological products is 5 endotoxin units (EU)/kg of body weight/hour by all pharmacopoeias [19]. Hence, the detection and re‐ moval of pyrogenic substances, especially endotoxins (lipopolysaccharides in the cell wall of gram-negative bacteria), are necessary to ensure safety of biopharmaceutical products. Cur‐ rently available methods for endotoxin detection include the U.S. Pharmacopeia rabbit test and the Limulus amebocyte lysate (LAL) test [7].

The rabbit pyrogen test entails measurements of the rise in body temperature of rabbits after an intravenous injection of a test substance. The presence of pyrogens of all kinds can be tested using this method. However, this method suffers from a number of disadvantages and limitations: (1) Endotoxin tolerance occurs after repeated use of rabbits; (2) variations in the response depending on sex, age, and species; (3) differences between the responses of rabbits and humans to various pyrogen types; and (4) the rabbit pyrogen test is inadequate for sera, radiopharmaceuticals, chemotherapeutics, analgesics, cytokines, immunosuppres‐ sive agents, and others [20].

Accordingly, the use of the rabbit pyrogen test has been reduced. Nowadays, the most wide‐ ly used endotoxin detection systems are based on the highly sensitive LAL test. It is based on the coagulation cascade of the blood of a horseshoe crab, Limulus polyphemus, which is induced by lipopolysaccharide. The currently known methods for lipopolysaccharide detec‐ tion entail (1) gel-clot assay, (2) turbidimetric LAL technique, and (3) the chromogenic LAL technique.

The gel-clot assay is a limit test that provides simple positive or negative results. The LAL reagent is introduced to a sample, and the test material is considered endotoxin positive if a gel is formed via a clotting reaction.

The turbidimetric and the chromogenic LAL techniques are quantitative tests. The former is based on the fact that turbidity increases as a result of the precipitation of the clottable pro‐ tein that is related to endotoxin concentration in the sample. The optical density is read by a spectrophotometer at either a fixed time (for the end-point method) or progressively (for the kinetic assay) as turbidity develops.

The chromogenic LAL technique makes use of a synthetic substrate which contains an amino acid sequence similar to that of the clottable protein, coagulogen, in order to detect endotoxin. The enzyme cleaves a yellow-colored substance from the chromogenic sub‐ strate, and the color intensity produced is proportional to the amount of endotoxin present in the sample.

Endotoxins are temperature and pH stable, and, therefore, their removal is one of the most challenging issues. Numerous techniques are used to reduce endotoxin contamination of bi‐ opharmaceuticals, including ion-exchange chromatography, sucrose gradient centrifugation, gel filtration chromatography [19], affinity adsorption [21], charged membrane/depth filtra‐ tion, and ultrafiltration [22].

An immunoassay for viral-specific proteins can be undertaken through production of the relevant antibodies after an injection of a virus of interest into animals. Currently commer‐

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209

Another method that is used for virus detection is TEM (Transmission Electron Microscopy). TEM is a quantitative assay that is based on the visualization and morphological identifica‐

Nevertheless, more sensitive methods, such as the PCR identification methods, can be em‐

Since the biopharmaceuticals can be originated from mammalian cell lines with a high risk of endogenous retroviruses, on one hand, and these products may be infected with adventi‐ tious viruses through processing, on the other hand, virus inactivation and removal steps in the purification process are required [24]. These entail gamma irradiation, low pH treat‐

Indeed, ensuring the absence of virus contamination in biopharmaceuticals is challenging. For instance, a limited number of commercial poultry vaccines were contaminated by avian leukosis virus even after routine quality assurance procedures. In addition, reovirus was found as a contaminant in urokinase. On the whole, sourcing and testing alone cannot guar‐ antee the virological safety of biopharmaceuticals owing to some limitations: the limit of sensitivity for cell culture and PCR tests and also due to the fact that cell culture or in vivo tests are not able to detect all known kinds of potential contaminants [25]. Thus, practical methods are required for the virological safety of biopharmaceuticals, which involve the in‐

Mycoplasmas are the smallest free-living and self-replicating organisms in nature that are sized between 50 and 500nm. They lack a rigid cell wall and, consequently, are highly pleo‐ morphic from round to filamentous. They are filterable and penicillin-resistant forms. Fur‐ thermore, their membrane contains sterol and due to this, mycoplasmas require the addition of serum or cholesterol to the growth medium. They grow on special media in aerobic or anaerobic conditions with optimum growth at 37°C and pH 7.0 and form with a "fried egg"

Mycoplasma contamination of cell culture systems for the production of mycoplasmas is a critical problem due to its effect on various parameters within the cell culture system. Myco‐ plasma contaminates cell cultures approximately without any sign, and it persists for a long time. Indeed, mycoplasma-positive cell cultures can be considered the major source of bio‐ pharmaceutical infection, and they should be discarded or effectively decontaminated. Tak‐ en together, mycoplasma-positive cell cultures pose a serious problem and should be

cially available immunoassays are able to detect various viruses.

ployed for the detection of sequences of the viruses [16].

clusion of risk assessment as well as management policies.

tion of virus particles in samples [23].

ment, or virus filtration.

**5.4. Mycoplasma**

morphology on agar media (Figure 6).

effectively detected and eradicated [26].

#### **5.3. Viruses**

Owing to the risks of transmission of adventitious agents to patients, the different cell levels should be studied for the absence of these agents. Among the adventitious agents, special attention should be paid to viruses that are capable of contaminating the original species. Generally, the virological safety of biopharmaceuticals includes several levels of control at various manufacturing stages, including 1 - rigorous screening of cell banks (both master cell bank and working cell bank) for viruses; 2 - screening of each cell culture harvest for adventitious agents; and 3 - a demonstration that the purification process can clear potential adventitious agents [15].

The detection of viruses in cell lines can be carried out via various techniques. The common‐ ly used methods of detecting viral infections include


For the co-cultivation assays (specific in vitro tests), the cells used for production, or cul‐ ture supernatant, or the final product are incubated with the detector cells. The detector cell lines are susceptible to different viruses and are used to detect desired viruses via monitoring subsequent cytopathic effects, hemadsorption, morphological changes, or oth‐ er signs of viral infection. The detector cells usually contain humans, primates, and cells from the same species.

The in vivo assay can be performed by the inoculation of cells or cell lysates into animals, including newborn and adult mice, guinea pigs, rabbits, or embryonated chicken eggs to de‐ tect viruses. The animals are consequently monitored for any abnormality.

Species-specific viruses potentially present in rodent cell lines can be examined using assays for antibody production in the animals. The MAP, RAP, and HAP (mouse, rat, and hamster antibody production assays, respectively) tests involve an injection of the test article into the animals. The inoculated animals are bled after four weeks, and the sera are tested for the presence of the antibodies against the specific viral antigens. For instance, Hantaan virus, Lactic Dehydrogenase virus, and Sendai virus have been screened using MAP.

An immunoassay for viral-specific proteins can be undertaken through production of the relevant antibodies after an injection of a virus of interest into animals. Currently commer‐ cially available immunoassays are able to detect various viruses.

Another method that is used for virus detection is TEM (Transmission Electron Microscopy). TEM is a quantitative assay that is based on the visualization and morphological identifica‐ tion of virus particles in samples [23].

Nevertheless, more sensitive methods, such as the PCR identification methods, can be em‐ ployed for the detection of sequences of the viruses [16].

Since the biopharmaceuticals can be originated from mammalian cell lines with a high risk of endogenous retroviruses, on one hand, and these products may be infected with adventi‐ tious viruses through processing, on the other hand, virus inactivation and removal steps in the purification process are required [24]. These entail gamma irradiation, low pH treat‐ ment, or virus filtration.

Indeed, ensuring the absence of virus contamination in biopharmaceuticals is challenging. For instance, a limited number of commercial poultry vaccines were contaminated by avian leukosis virus even after routine quality assurance procedures. In addition, reovirus was found as a contaminant in urokinase. On the whole, sourcing and testing alone cannot guar‐ antee the virological safety of biopharmaceuticals owing to some limitations: the limit of sensitivity for cell culture and PCR tests and also due to the fact that cell culture or in vivo tests are not able to detect all known kinds of potential contaminants [25]. Thus, practical methods are required for the virological safety of biopharmaceuticals, which involve the in‐ clusion of risk assessment as well as management policies.

#### **5.4. Mycoplasma**

Endotoxins are temperature and pH stable, and, therefore, their removal is one of the most challenging issues. Numerous techniques are used to reduce endotoxin contamination of bi‐ opharmaceuticals, including ion-exchange chromatography, sucrose gradient centrifugation, gel filtration chromatography [19], affinity adsorption [21], charged membrane/depth filtra‐

Owing to the risks of transmission of adventitious agents to patients, the different cell levels should be studied for the absence of these agents. Among the adventitious agents, special attention should be paid to viruses that are capable of contaminating the original species. Generally, the virological safety of biopharmaceuticals includes several levels of control at various manufacturing stages, including 1 - rigorous screening of cell banks (both master cell bank and working cell bank) for viruses; 2 - screening of each cell culture harvest for adventitious agents; and 3 - a demonstration that the purification process can clear potential

The detection of viruses in cell lines can be carried out via various techniques. The common‐

For the co-cultivation assays (specific in vitro tests), the cells used for production, or cul‐ ture supernatant, or the final product are incubated with the detector cells. The detector cell lines are susceptible to different viruses and are used to detect desired viruses via monitoring subsequent cytopathic effects, hemadsorption, morphological changes, or oth‐ er signs of viral infection. The detector cells usually contain humans, primates, and cells

The in vivo assay can be performed by the inoculation of cells or cell lysates into animals, including newborn and adult mice, guinea pigs, rabbits, or embryonated chicken eggs to de‐

Species-specific viruses potentially present in rodent cell lines can be examined using assays for antibody production in the animals. The MAP, RAP, and HAP (mouse, rat, and hamster antibody production assays, respectively) tests involve an injection of the test article into the animals. The inoculated animals are bled after four weeks, and the sera are tested for the presence of the antibodies against the specific viral antigens. For instance, Hantaan virus,

tect viruses. The animals are consequently monitored for any abnormality.

Lactic Dehydrogenase virus, and Sendai virus have been screened using MAP.

tion, and ultrafiltration [22].

208 Latest Research into Quality Control

adventitious agents [15].

**•** in vivo assays,

from the same species.

ly used methods of detecting viral infections include

**•** antibody production in animals (MAPs, RAPs, or HAPs),

**•** co-cultivation assays (specific in vitro tests),

**•** immunoassays for viral specific proteins,

**•** Transmission Electron Microscopy (TEM),

**•** Polymerase Chain Reaction (PCR).

**5.3. Viruses**

Mycoplasmas are the smallest free-living and self-replicating organisms in nature that are sized between 50 and 500nm. They lack a rigid cell wall and, consequently, are highly pleo‐ morphic from round to filamentous. They are filterable and penicillin-resistant forms. Fur‐ thermore, their membrane contains sterol and due to this, mycoplasmas require the addition of serum or cholesterol to the growth medium. They grow on special media in aerobic or anaerobic conditions with optimum growth at 37°C and pH 7.0 and form with a "fried egg" morphology on agar media (Figure 6).

Mycoplasma contamination of cell culture systems for the production of mycoplasmas is a critical problem due to its effect on various parameters within the cell culture system. Myco‐ plasma contaminates cell cultures approximately without any sign, and it persists for a long time. Indeed, mycoplasma-positive cell cultures can be considered the major source of bio‐ pharmaceutical infection, and they should be discarded or effectively decontaminated. Tak‐ en together, mycoplasma-positive cell cultures pose a serious problem and should be effectively detected and eradicated [26].

and expressed in human cells and result in the initiation of cancer cells. According to guide‐ lines, the acceptable level of residual DNA in recombinant products is 10 pg per therapeutic dose. DNA hybridization studies that use radiolabeled DNA probes with a specific nucleic acid sequence constitute one of the most widely used methods for the detection of DNA contaminants in the product to a nanogram (ng) range [6]. The important steps involved in

Microbial Quality Concerns for Biopharmaceuticals

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211

Cell cultures may be infected with other cell types due to the use of contaminated items or operator mistakes. Also fail of the sterilization process can be another reason. The detection of cross-contamination is very challenging due to the fact that macroscopic and microscopic properties of the original and contaminant cells are commonly the same. Cross-contamina‐ tion in the production of biopharmaceuticals would prove to be disastrous and terrible.

Various tests for detecting cross-contaminations can be applied; however, a product-specific

DNA hybridization are shown in Figure 7.

**Figure 7.** The main steps of the DNA hybridization procedure

identity test will be the best choice [16].

**5.6. Cross-contamination**

**Figure 6.** Mycoplasma colonies with fried egg morphology on mycoplasma agar medium. Picture was taken at Myco‐ plasma Reference laboratory, Razi vaccine and serum research institute, Iran

Different methods are used in international pharmacopoeias and guidance for detecting my‐ coplasma in biological test samples, mainly categorizing them as direct assay by microbio‐ logical culture, indirect assay by indicator mammalian cell culture, and PCR.

Direct assay by microbiological culture: The principle of detection is based on the growth of mycoplasma on supporting agar and liquid media (broth). First, the test sample is intro‐ duced into a special broth culture, is incubated for an appropriate time, and, consequently, it is sub-passaged to plate agar. After the required incubation period, the presence of myco‐ plasma colonies is observed microscopically in the agar plates (Figure 6).

Indirect assay by indicator mammalian cell culture: The indirect method requires the co-cul‐ tivation of the test sample with an indicator cell line for two to three days. Typically, VERO cells with a large cytoplasm area around the nucleus were used. Consequently, the cells were stained using a DNA binding stain (such as Hoechst stain) that binds specifically to DNA and is observed via fluorescent microscopy. Due to the affinity of mycoplasmas for the mammalian cell membrane, mycoplasmas appear as granules surrounding the nucleus.

Mycoplasma PCR: In this method, detection is carried out using specific oligonucleotide pri‐ mers for the amplification of mycoplasma DNA. This method is specially recommended for detecting contamination with the non-cultivable strains of *M. hyorhinis* [27].

On the whole, it is advisable to use two different methods in the detection of mycoplasmas in order to allow for the differentiation between false-positive and false-negative results.

#### **5.5. DNA**

The importance of DNA contamination detection in biopharmaceuticals is related to the fact that the DNA from some sources such as hybridoma cell lines in monoclonal antibody pro‐ duction may act as active oncogenes. These kinds of DNA contaminants can be introduced and expressed in human cells and result in the initiation of cancer cells. According to guide‐ lines, the acceptable level of residual DNA in recombinant products is 10 pg per therapeutic dose. DNA hybridization studies that use radiolabeled DNA probes with a specific nucleic acid sequence constitute one of the most widely used methods for the detection of DNA contaminants in the product to a nanogram (ng) range [6]. The important steps involved in DNA hybridization are shown in Figure 7.

**Figure 7.** The main steps of the DNA hybridization procedure

#### **5.6. Cross-contamination**

**Figure 6.** Mycoplasma colonies with fried egg morphology on mycoplasma agar medium. Picture was taken at Myco‐

Different methods are used in international pharmacopoeias and guidance for detecting my‐ coplasma in biological test samples, mainly categorizing them as direct assay by microbio‐

Direct assay by microbiological culture: The principle of detection is based on the growth of mycoplasma on supporting agar and liquid media (broth). First, the test sample is intro‐ duced into a special broth culture, is incubated for an appropriate time, and, consequently, it is sub-passaged to plate agar. After the required incubation period, the presence of myco‐

Indirect assay by indicator mammalian cell culture: The indirect method requires the co-cul‐ tivation of the test sample with an indicator cell line for two to three days. Typically, VERO cells with a large cytoplasm area around the nucleus were used. Consequently, the cells were stained using a DNA binding stain (such as Hoechst stain) that binds specifically to DNA and is observed via fluorescent microscopy. Due to the affinity of mycoplasmas for the mammalian cell membrane, mycoplasmas appear as granules surrounding the nucleus.

Mycoplasma PCR: In this method, detection is carried out using specific oligonucleotide pri‐ mers for the amplification of mycoplasma DNA. This method is specially recommended for

On the whole, it is advisable to use two different methods in the detection of mycoplasmas in order to allow for the differentiation between false-positive and false-negative results.

The importance of DNA contamination detection in biopharmaceuticals is related to the fact that the DNA from some sources such as hybridoma cell lines in monoclonal antibody pro‐ duction may act as active oncogenes. These kinds of DNA contaminants can be introduced

logical culture, indirect assay by indicator mammalian cell culture, and PCR.

plasma colonies is observed microscopically in the agar plates (Figure 6).

detecting contamination with the non-cultivable strains of *M. hyorhinis* [27].

**5.5. DNA**

plasma Reference laboratory, Razi vaccine and serum research institute, Iran

210 Latest Research into Quality Control

Cell cultures may be infected with other cell types due to the use of contaminated items or operator mistakes. Also fail of the sterilization process can be another reason. The detection of cross-contamination is very challenging due to the fact that macroscopic and microscopic properties of the original and contaminant cells are commonly the same. Cross-contamina‐ tion in the production of biopharmaceuticals would prove to be disastrous and terrible.

Various tests for detecting cross-contaminations can be applied; however, a product-specific identity test will be the best choice [16].

#### **6. Summary**

Microbial quality control plays a prominent role in the manufacture of safe and effective bio‐ pharmaceuticals. The main sources of microbial contamination can be related to raw materi‐ als and the production environment. The main categories of raw materials that are involved in the manufacturing of biopharmaceuticals with a high risk of contamination are those of animal origin such as cell culture media, sera, and supplements. The production environ‐ ment includes air, surfaces, instruments, equipments, and personnel. All these can be con‐ sidered the main source for the contamination of biopharmaceuticals with adventitious agents such as viruses, bacteria, fungi, transmitting spongiform encephalopathy, and myco‐ plasma. The use of contaminated biopharmaceuticals causes product spoilage, which may lead to (i) metabolization of the therapeutic agents by microorganisms, thus bringing about a decrease in the potency of the therapeutic agent; (ii) a potential health hazard to patients as a result of either infectious diseases or microbial-derived agents such as endotoxins that are secreted into products.

**References**

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initions. BioExecutive. March and May

DA/Transparency/Basics/ucm194516.htm.

menclature. Eur J Pharm Sci. 15(2):, 135-138.

ceutical Microbiology. SEVENTH ed: Blackwell Science.

England: John Wiley & Sons, Ltd.

duction. Biotechnol Adv. 6(2):, 169-182.

Blackwell.

2(5):, 466-479.

PI-QAS Rev1-08082011.pdf.

[1] Rader, R. A. (2005). What is a Biopharmaceutical, Part 1: (Bio)Technology-Based Defi‐

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[2] Rader, R. A. (2005). What is a Biopharmaceutical, Part 2: Company and Industry Def‐

[3] WHO. Definition of active pharmaceutical ingredient.(2011). July Available from: http://www.who.int/medicines/areas/quality\_safety/quality\_assurance/DefinitionA‐

[4] FDA. What is a biological product? ; Available from: http://www.fda.gov/AboutF‐

[5] Walsh, G. (2002). Biopharmaceuticals and biotechnology medicines: an issue of no‐

[6] Walsh, G. (2003). Biopharmaceuticals Biochemistry And Biotechnology. Second ed,

[7] Denyer, S. P., Hodges, N. A., & Gorman, S. P. (2004). Hugo and Russell's Pharma‐

[8] Brown, T.A., Gene Cloning and DNA Analysis: An Introduction.th ed(2010). Wiley

[9] Kohler, G., & Milstein, C. (2005). Continuous cultures of fused cells secreting anti‐

[10] Kohler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting anti‐

[11] Bols, N. C., et al. (1988). Media for hybridoma growth and monoclonal antibody pro‐

[12] ANTIBODIES, C.O.M.O.P.M., *Monoclonal Antibody Production A Report of the Commit‐ tee on Methods of Producing Monoclonal Antibodies Institute for Laboratory Animal Re‐*

[13] Li, F., et al. (2010). Cell culture processes for monoclonal antibody production. MAbs.

[14] Ho, R. J. Y., & Gibaldi, M. (2003). Biotechnology and Biopharmaceuticals: Transform‐ ing Proteins and Genes into Drugs, New Jersey.: A John Wiley & Sons, Inc.

[15] Knäblein, J. (2005). Modern Biopharmaceuticals: Design, Development and Optimi‐

[16] Denyer, S. P., & Baird, R. M. (2007). Guide to Microbiological Control in Pharmaceut‐

body of predefined specificity. 1975. J Immunol. 174(5):, 2453-2455.

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*search National Research Council*., (1999). : Washington, DC.

zation: WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

icals and Medical Devices. second ed: CRC Press.

Various methods are used for detecting and eliminating different biological contaminants that are used in the manufacturing of biopharmaceuticals. Generally, bacteria and fungi can be detected by standard sterility testing or macroscopic and microscopic characteristics, as well as biochemical tests. In addition, viruses can be detected via a number of methods such as co-cultivation assays (specific in vitro tests), in vivo assays, antibody production in ani‐ mals (MAPs, RAPs, or HAPs), immunoassays for viral specific proteins, TEM, or PCR. The detection of endotoxin can be carried out using the pharmacopeial rabbit test or LAL test. The available approaches for the detection of mycoplasma include direct assay using special culture media, indirect assay by mammalian cells, and DNA staining, as well as PCR. Fur‐ thermore, DNA hybridization is a widely used approach for the detection of DNA contami‐ nants in biopharmaceuticals. The best method for cross-contamination detection includes a product-specific identity test.

With regard to the collection of tests for biological quality control of biopharmaceuticals summarized in this chapter, it is obvious that various sets of methods are available in differ‐ ent guidelines and pharmacopeias which are complicated and problematic. The develop‐ ment and compilation of harmonized guidelines for biological quality control of biopharmaceuticals is a critical necessity that can facilitate the control of the safety of these ever-increasing products.

#### **Author details**

Farzaneh Lotfipour and Somayeh Hallaj-Nezhadi

Faculty of Pharmacy, Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

#### **References**

**6. Summary**

212 Latest Research into Quality Control

are secreted into products.

product-specific identity test.

ever-increasing products.

Farzaneh Lotfipour and Somayeh Hallaj-Nezhadi

**Author details**

Tabriz, Iran

Microbial quality control plays a prominent role in the manufacture of safe and effective bio‐ pharmaceuticals. The main sources of microbial contamination can be related to raw materi‐ als and the production environment. The main categories of raw materials that are involved in the manufacturing of biopharmaceuticals with a high risk of contamination are those of animal origin such as cell culture media, sera, and supplements. The production environ‐ ment includes air, surfaces, instruments, equipments, and personnel. All these can be con‐ sidered the main source for the contamination of biopharmaceuticals with adventitious agents such as viruses, bacteria, fungi, transmitting spongiform encephalopathy, and myco‐ plasma. The use of contaminated biopharmaceuticals causes product spoilage, which may lead to (i) metabolization of the therapeutic agents by microorganisms, thus bringing about a decrease in the potency of the therapeutic agent; (ii) a potential health hazard to patients as a result of either infectious diseases or microbial-derived agents such as endotoxins that

Various methods are used for detecting and eliminating different biological contaminants that are used in the manufacturing of biopharmaceuticals. Generally, bacteria and fungi can be detected by standard sterility testing or macroscopic and microscopic characteristics, as well as biochemical tests. In addition, viruses can be detected via a number of methods such as co-cultivation assays (specific in vitro tests), in vivo assays, antibody production in ani‐ mals (MAPs, RAPs, or HAPs), immunoassays for viral specific proteins, TEM, or PCR. The detection of endotoxin can be carried out using the pharmacopeial rabbit test or LAL test. The available approaches for the detection of mycoplasma include direct assay using special culture media, indirect assay by mammalian cells, and DNA staining, as well as PCR. Fur‐ thermore, DNA hybridization is a widely used approach for the detection of DNA contami‐ nants in biopharmaceuticals. The best method for cross-contamination detection includes a

With regard to the collection of tests for biological quality control of biopharmaceuticals summarized in this chapter, it is obvious that various sets of methods are available in differ‐ ent guidelines and pharmacopeias which are complicated and problematic. The develop‐ ment and compilation of harmonized guidelines for biological quality control of biopharmaceuticals is a critical necessity that can facilitate the control of the safety of these

Faculty of Pharmacy, Immunology Research Center, Tabriz University of Medical Sciences,


[17] United States Pharmacopocia 29, National Formulary 24. USP Convention,Rockville (2006).

**Chapter 10**

**New Approachs in Drug Quality Control: Matrices and**

Quality control refers to the process of quality evaluation that focuses on the internal meas‐ urement of the quality of a process, institution, product, service, or other. Often used inter‐

Quality Assurance plays a very important role in making sure that the GMP standards are met and products comply with the international quality standards. The main functions car‐

Manufacturing processes are monitored and controlled by testing of raw materials, in-process parameters. Final active pharmaceutical ingredients and dosage forms are tested for specified parameters before release. Analytical testing is carried out with highly sophisticated instru‐ ments: viz. HPLC, GC, IR, UV spectrophotometer mettler titrators, particle size analyzer etc.

> © 2012 Mennickent et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2012 Mennickent et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Sigrid Mennickent, M. de Diego, B. Schulz, M. Vega

changeably with *quality management* and *quality assurance* [1-3].

Additional information is available at the end of the chapter

**Chemometrics**

and C. G. Godoy

**1. Quality control**

**2. Drug quality control**

**•** Approval of raw materials

**•** Implementation of cGMP

**•** Approval of finished products

ried out by drug quality control are:

**•** Monitoring of manufacturing processes

**•** Documentation of technical information

http://dx.doi.org/10.5772/50825


## **New Approachs in Drug Quality Control: Matrices and Chemometrics**

Sigrid Mennickent, M. de Diego, B. Schulz, M. Vega

and C. G. Godoy

[17] United States Pharmacopocia 29, National Formulary 24. USP Convention,Rockville

[19] Magalhaes, P. O., et al. (2007). Methods of endotoxin removal from biological prepa‐

[20] Daneshian, M., et al. (2006). In vitro pyrogen test for toxic or immunomodulatory

[21] Petsch, D., & Anspach, F. B. (2000). Endotoxin removal from protein solutions. J Bio‐

[22] Salema, V., Saxena, L., & Pattnaik, P. (2009). Removing endotoxin from biopharma‐

[23] Vale, F. F., et al. (2010). Applications of transmission electron microscopy to virus de‐ tection and identification Microscopy: Science, Technology, Applications and Educa‐

[24] Liu, H. F., et al. (2010). Recovery and purification process development for monoclo‐

[25] Bailey, A. (2011). Learning from Experience European Biopharmaceutical Review,

[27] Edition, T. J. P. F. Mycoplasma Testing for Cell Substrates used for the Production of Biotechnological/Biological Products. Available from: http://db.yaozh.com/foreign/

ceutical solutions. Pharmaceutical Technology Europe. 21(10):, 36.

[18] European Pharmacopoeia, 4th ed. EPSecretariat, Strasbourg.(2002).

rations: a review. J Pharm Pharm Sci. 10(3):, 388-404.

drugs. J Immunol Methods. 313(1-2):, 169-175.

nal antibody production. MAbs,. 2(5):, 480-499.

[26] Fleckenstein, E., & Drexler, H.G. Iochemica n 1996 1.

technol. 76(2-3):, 97-119.

(2006).

214 Latest Research into Quality Control

tion.

Spring.

JP15/jp5/F5\_14.pdf.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50825

### **1. Quality control**

Quality control refers to the process of quality evaluation that focuses on the internal meas‐ urement of the quality of a process, institution, product, service, or other. Often used inter‐ changeably with *quality management* and *quality assurance* [1-3].

### **2. Drug quality control**

Quality Assurance plays a very important role in making sure that the GMP standards are met and products comply with the international quality standards. The main functions car‐ ried out by drug quality control are:


Manufacturing processes are monitored and controlled by testing of raw materials, in-process parameters. Final active pharmaceutical ingredients and dosage forms are tested for specified parameters before release. Analytical testing is carried out with highly sophisticated instru‐ ments: viz. HPLC, GC, IR, UV spectrophotometer mettler titrators, particle size analyzer etc.

All the analytical test procedures and manufacturing procedures are well documented and revision is undertaken as per specified protocol. Analytical methods are validated to give the reproducible results. Stability study as per stability protocol is considered to be very im‐ portant area of Quality Assurance.

Traces of such compounds have been detected in surface water samples from all countries

New Approachs in Drug Quality Control: Matrices and Chemometrics

http://dx.doi.org/10.5772/50825

217

Pharmaceutically active compounds have captured the attention of the scientific community because such pollutants result not primarily from manufacturing but from widespread, con‐ tinual use in human and veterinary clinical practice. The biological activity of these com‐ pounds can lead to adverse effects in aquatic ecosystems and potentially have an impact on

In the human body, pharmaceuticals can be transformed to one or more metabolites and ex‐ creted as a mixture of parent compound and metabolites, in which the parent compound is often the minor component. However, some drugs are poorly metabolized and are excreted unchanged. The degree of metabolism depends on a number of parameters, including age, gender and ethnicity, the constitution of the patient and the time of administration. Drugdrug interactions caused by enzyme induction or inhibition, as well as enhanced metabo‐

Both the parent compound and the metabolites enter the aquatic environment once they are excreted from the human body. Monitoring studies in the environment have demonstrated the discharge of pharmaceuticals and their metabolites through municipal wastewater-treat‐ ment plants (WWTPs). Although unchanged drugs can undergo biochemical transforma‐ tions during sewage treatment, some studies indicate that the absence of pharmaceutical compounds in treated water does not necessarily imply their complete removal. In most in‐ stances, human drugs are metabolized in the body to more polar compounds that are more likely to pass through the WWTP. In some cases, pharmaceuticals and their human metabo‐

Knowledge of the formation of stable metabolites in WWTPs is also important in order to understand the environmental fate of the parent compound. Once in the environment, these compounds can be transported and distributed in rivers, streams, and possibly further bio‐ degraded. For most pharmaceuticals and their biotransformation products, these pathways in the aquatic environment are largely unknown, and investigations into their occurrence in

Studies have been carried out to investigate their fate not only in surface waters, but also in sediment and soil environments. By nature, most pharmaceuticals are designed to be at least moderately water-soluble and to possess half-lives in the human body in the range of hours. Because human and microbial degradates will generally coexist with their parent com‐ pounds in the environment, indicators that summarize all the information on parent substan‐ ces and degradates would be important instruments for decision-making and assessment [6].

Progress in instrumental analytical chemistry has resulted in the availability of methods that

Improvements in detection limits over the past years have mainly been due to sophisticated mass spectrometric detection techniques. Furthermore, robust sample preparation and preconcentration protocols have contributed significantly to the achievements observed so far.

lism due to previous exposure, can also influence the pharmacokinetics of drugs [5].

lites can be microbially degraded in the activated sludge treatment.

environmental compartments are still rare.

allow a monitoring of these pollutants at ng levels.

where pharmaceuticals are widely in use.

drinking-water supplies [4].

Automated systems are becoming increasingly important tools for appropriate monitoring and controlling of the pharmaceutical packaging process. Solutions for comprehensive qual‐ ity assurance or production data acquisition and evaluation are just as important as applica‐ tions that meet the legislative requirements of different countries in terms of serial numbering and the unique marking of products.

Quality control involves many phases, such as sample collection, measuring, analysis of re‐ sults, and the approval/rejection of the batch. Nonetheless, the most important thing is the continuity and systematization of the quality control.

Effective process validation contributes significantly to assuring drug quality. The basic principle of quality assurance is that a drug should be produced that is fit for its intended use. This principle incorporates the understanding that the following conditions exist: Qual‐ ity, safety, and efficacy are designed or built into the product.

Quality cannot be adequately assured merely by in-process and finished-product inspec‐ tion or testing.

Process validation is defined as the collection and evaluation of data, from the process de‐ sign stage through commercial production, which establishes scientific evidence that a proc‐ ess is capable of consistently delivering quality product. Process validation involves a series of activities taking place over the lifecycle of the product and process. Usually, process vali‐ dation includes three stages:


#### **3. New approaches in drug quality control**

#### **3.1. Matrices**

#### *3.1.1. Residue analysis of pharmaceuticals in the aquatic environment*

Residue analysis of pharmaceuticals in the aquatic environment has attracted considerable interest during the last few years.

Traces of such compounds have been detected in surface water samples from all countries where pharmaceuticals are widely in use.

All the analytical test procedures and manufacturing procedures are well documented and revision is undertaken as per specified protocol. Analytical methods are validated to give the reproducible results. Stability study as per stability protocol is considered to be very im‐

Automated systems are becoming increasingly important tools for appropriate monitoring and controlling of the pharmaceutical packaging process. Solutions for comprehensive qual‐ ity assurance or production data acquisition and evaluation are just as important as applica‐ tions that meet the legislative requirements of different countries in terms of serial

Quality control involves many phases, such as sample collection, measuring, analysis of re‐ sults, and the approval/rejection of the batch. Nonetheless, the most important thing is the

Effective process validation contributes significantly to assuring drug quality. The basic principle of quality assurance is that a drug should be produced that is fit for its intended use. This principle incorporates the understanding that the following conditions exist: Qual‐

Quality cannot be adequately assured merely by in-process and finished-product inspec‐

Process validation is defined as the collection and evaluation of data, from the process de‐ sign stage through commercial production, which establishes scientific evidence that a proc‐ ess is capable of consistently delivering quality product. Process validation involves a series of activities taking place over the lifecycle of the product and process. Usually, process vali‐

**•** Process Design: The commercial manufacturing process is defined during this stage based

**•** Process Qualification: During this stage, the process design is evaluated to determine if

**•** Process Verification: Ongoing assurance is gained during routine production that the

Residue analysis of pharmaceuticals in the aquatic environment has attracted considerable

on knowledge gained through development and scale-up activities.

the process is capable of reproducible commercial manufacturing.

portant area of Quality Assurance.

216 Latest Research into Quality Control

tion or testing.

**3.1. Matrices**

dation includes three stages:

process remains in a state of control [1-3].

interest during the last few years.

**3. New approaches in drug quality control**

*3.1.1. Residue analysis of pharmaceuticals in the aquatic environment*

numbering and the unique marking of products.

continuity and systematization of the quality control.

ity, safety, and efficacy are designed or built into the product.

Pharmaceutically active compounds have captured the attention of the scientific community because such pollutants result not primarily from manufacturing but from widespread, con‐ tinual use in human and veterinary clinical practice. The biological activity of these com‐ pounds can lead to adverse effects in aquatic ecosystems and potentially have an impact on drinking-water supplies [4].

In the human body, pharmaceuticals can be transformed to one or more metabolites and ex‐ creted as a mixture of parent compound and metabolites, in which the parent compound is often the minor component. However, some drugs are poorly metabolized and are excreted unchanged. The degree of metabolism depends on a number of parameters, including age, gender and ethnicity, the constitution of the patient and the time of administration. Drugdrug interactions caused by enzyme induction or inhibition, as well as enhanced metabo‐ lism due to previous exposure, can also influence the pharmacokinetics of drugs [5].

Both the parent compound and the metabolites enter the aquatic environment once they are excreted from the human body. Monitoring studies in the environment have demonstrated the discharge of pharmaceuticals and their metabolites through municipal wastewater-treat‐ ment plants (WWTPs). Although unchanged drugs can undergo biochemical transforma‐ tions during sewage treatment, some studies indicate that the absence of pharmaceutical compounds in treated water does not necessarily imply their complete removal. In most in‐ stances, human drugs are metabolized in the body to more polar compounds that are more likely to pass through the WWTP. In some cases, pharmaceuticals and their human metabo‐ lites can be microbially degraded in the activated sludge treatment.

Knowledge of the formation of stable metabolites in WWTPs is also important in order to understand the environmental fate of the parent compound. Once in the environment, these compounds can be transported and distributed in rivers, streams, and possibly further bio‐ degraded. For most pharmaceuticals and their biotransformation products, these pathways in the aquatic environment are largely unknown, and investigations into their occurrence in environmental compartments are still rare.

Studies have been carried out to investigate their fate not only in surface waters, but also in sediment and soil environments. By nature, most pharmaceuticals are designed to be at least moderately water-soluble and to possess half-lives in the human body in the range of hours. Because human and microbial degradates will generally coexist with their parent com‐ pounds in the environment, indicators that summarize all the information on parent substan‐ ces and degradates would be important instruments for decision-making and assessment [6].

Progress in instrumental analytical chemistry has resulted in the availability of methods that allow a monitoring of these pollutants at ng levels.

Improvements in detection limits over the past years have mainly been due to sophisticated mass spectrometric detection techniques. Furthermore, robust sample preparation and preconcentration protocols have contributed significantly to the achievements observed so far.

Nowadays it is a well-established fact that pharmaceutical drugs used during medical treat‐ ment may partly be excreted in an un-metabolized form, enter municipal sewage systems, and can even survive the passage through the sewage treatment plant. Therefore, sewage treatment plant effluents are the major source for introduction of pharmaceuticals into the aquatic environment. Furthermore, pharmaceuticals employed in veterinary medicine may be introduced into soil (and eventually into water) via manure, or may find a direct way in‐ to the aquatic system when used in fish farms.

hydrophilic–hydrophobic balance material) or by considerably increased surface area. Some of these new materials have turned out to be well suited for multi-class analysis of pharma‐ ceuticals in water samples. Nowadays, one of the most widely used sorbent is a copolymer

New Approachs in Drug Quality Control: Matrices and Chemometrics

http://dx.doi.org/10.5772/50825

219

Sorptive extraction based on a single partitioning equilibrium of analytes between the aque‐ ous sample and a solid sorbent includes solid-phase microextraction (SPME), stir-bar sorp‐ tive extraction (SBSE), and several related variants. Originally, these techniques were based on polydimethylsiloxane (PDMS) as material for trapping trace analytes from a water sam‐ ple due to partitioning between the aqueous matrix and the PDMS phase. Besides PDMS, some alternative sorptive materials have become commercially available recently, such as polyacrylates, copolymers of PDMS with divinylbenzene, copolymers of polyethylene glycol with divinylbenzene, and mixtures of carboxen (an inorganic adsorbent) with PDMS or di‐

Extraction of pharmaceuticals from sediment and sludge is generally done by blending the sample with an organic solvent or with mixtures of aqueous buffers and organic solvents.

Additional clean-up steps for the extract may be necessary employing SPE or liquid–liq‐ uid extraction. Somewhat more advanced procedures are based on pressurized liquid ex‐ traction (accelerated solvent extraction) which may need less time and less solvent

Various groups of pharmaceuticals can be derivatized to make them suited for GC analysis. Typical derivatization reagents for acidic pharmaceuticals include pentafluorobenzylbro‐ mide, methyl chloromethanoate, methanol/BF3, or tetrabutylammonium salts (for derivati‐ zation during injection). Phenazone-type drugs have been derivatized by silylation using *Ntert-*butyldimethylsilyl-*N-*methyltrifluoroacetamide (MTBSTFA). Silylation procedures are

*3.2.1.1. Pharmaceuticals in the aquatic environment: a critical review of the evidence for health effects*

The authors review the current data on the presence and reported biological effects in fish of some of the most commonly detected pharmaceuticals in the aquatic environment; namely nonsteroidal anti-inflammatory drugs (NSAIDs), fibrates, beta-blockers, selective serotonin

*3.1.1.3. Sample pre-concentration procedures for sediment and sludge samples*

Ultrasonication is frequently applied to assist the extraction process.

of divinylbenzene and vinylpyrrolidone [7-23].

*3.1.1.2. Sorptive extraction*

vinylbenzene [7-23].

consumption [7-23].

*in fish*

*3.1.1.4. Derivatization of the compounds*

*3.2.1.Some latest researches in this area*

also commonly used for synthetic estrogens [7-23].

Unfortunately, the consequences of continuous presence of low concentrations of pharma‐ ceuticals for the ecosystem are still not fully known.

In many cases, the analytical procedures for residue analysis of pharmaceutical drugs nowa‐ days available includes a pre-concentration and clean-up step by solid-phase extraction or related techniques, followed by chromatography in combination with mass spectrometry (MS) as detector.

Although GC–MS may still be the perfect technique for certain classes of pharmaceuticals, high-performance liquid chromatography (HPLC) hyphenated with atmospheric pressure ionization-MS has established itself as the better choice for simultaneous determination of pharmaceuticals of widely differing structures.

The concentration levels of pharmaceuticals found in environmental water samples are gen‐ erally too low to allow a direct injection into a chromatographic system. Therefore, efficient pre-concentration steps are necessary which should also result in some sample clean-up. One of the most widely used sample treatment technique for residue analysis of pharma‐ ceuticals in water is the extraction of the analytes by means of a solid sorbent.

This extraction procedure can be based on multiple equilibria between the liquid phase and the sorbent filled into a small cartridge (solid-phase extraction, SPE), or on a single equilibri‐ um (sorptive extraction) [7-23].

#### *3.1.1.1. Solid-phase extraction*

Pharmaceuticals of adequate hydrophobicity can easily be pre-concentrated using any re‐ versed-phase material such as alkyl-modified silica or polymer-based materials. Deprotona‐ tion of acidic compounds and protonation of basic compounds should be suppressed to ensure sufficient hydrophobicity of the analytes. Therefore, acidic pharmaceuticals should be pre-concentrated under acidic conditions, whereas basic analytes should be pre-concen‐ trated at an alkaline pH. Alternatively, mixed-mode SPE materials can be used which exhib‐ it both reversed-phase and cation-exchange properties due to the presence of sulfonic acid groups on the hydrophobic surface of the particles. Using acidified sample solutions, acidic and neutral analytes would be extracted by hydrophobic interactions, whereas protonated basic analytes would interact via ion exchange mechanisms.

A recent review has summarized new SPE materials that can improve the recoveries for po‐ lar analytes. These materials are mainly polymeric sorbents that improve the retention of po‐ lar compounds either by novel functional groups in the polymeric structure (resulting in a hydrophilic–hydrophobic balance material) or by considerably increased surface area. Some of these new materials have turned out to be well suited for multi-class analysis of pharma‐ ceuticals in water samples. Nowadays, one of the most widely used sorbent is a copolymer of divinylbenzene and vinylpyrrolidone [7-23].

#### *3.1.1.2. Sorptive extraction*

Nowadays it is a well-established fact that pharmaceutical drugs used during medical treat‐ ment may partly be excreted in an un-metabolized form, enter municipal sewage systems, and can even survive the passage through the sewage treatment plant. Therefore, sewage treatment plant effluents are the major source for introduction of pharmaceuticals into the aquatic environment. Furthermore, pharmaceuticals employed in veterinary medicine may be introduced into soil (and eventually into water) via manure, or may find a direct way in‐

Unfortunately, the consequences of continuous presence of low concentrations of pharma‐

In many cases, the analytical procedures for residue analysis of pharmaceutical drugs nowa‐ days available includes a pre-concentration and clean-up step by solid-phase extraction or related techniques, followed by chromatography in combination with mass spectrometry

Although GC–MS may still be the perfect technique for certain classes of pharmaceuticals, high-performance liquid chromatography (HPLC) hyphenated with atmospheric pressure ionization-MS has established itself as the better choice for simultaneous determination of

The concentration levels of pharmaceuticals found in environmental water samples are gen‐ erally too low to allow a direct injection into a chromatographic system. Therefore, efficient pre-concentration steps are necessary which should also result in some sample clean-up. One of the most widely used sample treatment technique for residue analysis of pharma‐

This extraction procedure can be based on multiple equilibria between the liquid phase and the sorbent filled into a small cartridge (solid-phase extraction, SPE), or on a single equilibri‐

Pharmaceuticals of adequate hydrophobicity can easily be pre-concentrated using any re‐ versed-phase material such as alkyl-modified silica or polymer-based materials. Deprotona‐ tion of acidic compounds and protonation of basic compounds should be suppressed to ensure sufficient hydrophobicity of the analytes. Therefore, acidic pharmaceuticals should be pre-concentrated under acidic conditions, whereas basic analytes should be pre-concen‐ trated at an alkaline pH. Alternatively, mixed-mode SPE materials can be used which exhib‐ it both reversed-phase and cation-exchange properties due to the presence of sulfonic acid groups on the hydrophobic surface of the particles. Using acidified sample solutions, acidic and neutral analytes would be extracted by hydrophobic interactions, whereas protonated

A recent review has summarized new SPE materials that can improve the recoveries for po‐ lar analytes. These materials are mainly polymeric sorbents that improve the retention of po‐ lar compounds either by novel functional groups in the polymeric structure (resulting in a

ceuticals in water is the extraction of the analytes by means of a solid sorbent.

basic analytes would interact via ion exchange mechanisms.

to the aquatic system when used in fish farms.

pharmaceuticals of widely differing structures.

um (sorptive extraction) [7-23].

*3.1.1.1. Solid-phase extraction*

(MS) as detector.

218 Latest Research into Quality Control

ceuticals for the ecosystem are still not fully known.

Sorptive extraction based on a single partitioning equilibrium of analytes between the aque‐ ous sample and a solid sorbent includes solid-phase microextraction (SPME), stir-bar sorp‐ tive extraction (SBSE), and several related variants. Originally, these techniques were based on polydimethylsiloxane (PDMS) as material for trapping trace analytes from a water sam‐ ple due to partitioning between the aqueous matrix and the PDMS phase. Besides PDMS, some alternative sorptive materials have become commercially available recently, such as polyacrylates, copolymers of PDMS with divinylbenzene, copolymers of polyethylene glycol with divinylbenzene, and mixtures of carboxen (an inorganic adsorbent) with PDMS or di‐ vinylbenzene [7-23].

#### *3.1.1.3. Sample pre-concentration procedures for sediment and sludge samples*

Extraction of pharmaceuticals from sediment and sludge is generally done by blending the sample with an organic solvent or with mixtures of aqueous buffers and organic solvents.

Ultrasonication is frequently applied to assist the extraction process.

Additional clean-up steps for the extract may be necessary employing SPE or liquid–liq‐ uid extraction. Somewhat more advanced procedures are based on pressurized liquid ex‐ traction (accelerated solvent extraction) which may need less time and less solvent consumption [7-23].

#### *3.1.1.4. Derivatization of the compounds*

Various groups of pharmaceuticals can be derivatized to make them suited for GC analysis. Typical derivatization reagents for acidic pharmaceuticals include pentafluorobenzylbro‐ mide, methyl chloromethanoate, methanol/BF3, or tetrabutylammonium salts (for derivati‐ zation during injection). Phenazone-type drugs have been derivatized by silylation using *Ntert-*butyldimethylsilyl-*N-*methyltrifluoroacetamide (MTBSTFA). Silylation procedures are also commonly used for synthetic estrogens [7-23].

#### *3.2.1.Some latest researches in this area*

#### *3.2.1.1. Pharmaceuticals in the aquatic environment: a critical review of the evidence for health effects in fish*

The authors review the current data on the presence and reported biological effects in fish of some of the most commonly detected pharmaceuticals in the aquatic environment; namely nonsteroidal anti-inflammatory drugs (NSAIDs), fibrates, beta-blockers, selective serotonin reuptake inhibitors (SSRIs), azoles, and antibiotics. Reported biological effects in fish in the laboratory have often been shown to be in accordance with known effects of pharmaceuti‐ cals in mammals. Water concentrations at which such effects have been reported, however, are generally, between microg L(-1) and mg L(-1), typically at least 1 order of magnitude higher than concentrations normally found in surface waters (ng L(-1)). There are exceptions to this, however, as for the case of synthetic oestrogens, which can induce biological effects in the low ng L(-1) range. Although generally effect levels for pharmaceuticals are higher than those found in the environment, the risks to wild fish populations have not been thor‐ oughly characterised, and there has been a lack of consideration given to the likely chronic nature of the exposures, or the potential for mixture effects. As global consumption of phar‐ maceuticals rises, an inevitable consequence is an increased level of contamination of sur‐ face and ground waters with these biologically active drugs, and thus in turn a greater potential for adverse effects in aquatic wildlife [24].

tions ranging from approximately 10- 3,000 ng/L for high use pharmaceuticals such as beta‐ blockers (*e.g.,* metoprolol, propranolol) and acidic drugs (*e.g.,* gemfibrozil, ibuprofen). The concentration of pharmaceuticals in effluent from conventional wastewater treatment plants is similar. Advanced wastewater treatment plants equipped with reverse osmosis systems reduce concentrations of pharmaceuticals below detection limits. In addition to removal during biological wastewater treatment, pharmaceuticals also are attenuated in engineered natural systems (*i.e.,* treatment wetlands, ground water infiltration basins). Preliminary evi‐ dence suggests limited removal of pharmaceuticals in engineered treatment wetlands and

New Approachs in Drug Quality Control: Matrices and Chemometrics

http://dx.doi.org/10.5772/50825

221

Environmental fates and effects of pharmaceuticals in the aquatic environment have been the focus of recent research in environmental ecotoxicology. Worldwide studies of common over-the-counter pharmaceuticals have reported detectable levels in the aquatic environ‐ ment, but there are few studies examining impacts on marine habitats. These drugs can af‐ fect the functions of various vertebrates and invertebrates. The stability of two pharmaceuticals, cyclizine (CYC) and prochlorperazine (PCZ), in seawater was examined under light and dark conditions, as well as the toxicity of these compounds to larvae of the barnacle Balanus amphitrite, which is a cosmopolitan marine organism found in most of the world's oceans. CYC was very stable under all the tested conditions. On the other hand, PCZ degraded in light but not in the dark, and was more stable in seawater than fresh wa‐ ter. For the barnacle larvae, the LC50 of prochlorperazine was 0.93 microg/mL and the LC50

In the last years pharmaceuticals have aroused great interest as environmental pollu‐ tants for their toxic effects towards non target organisms. This study wants to draw atten‐ tion to a further adverse effect of drugs, the endocrine interference. The most representative drugs of the widespread classes in environment were investigated. The YES-test and the E-screen assay were performed to detect the capability of these substances to bind the human estrogenic receptor alpha (hER alpha) in comparison with 17beta-estradiol. Out of 14 tested pharmaceuticals, 9 were positive to YES-assay and 11 were positive to Escreen assay; in particular, Furosemide and the fibrates (Bezafibrate, Fenofibrate and Gem‐ fibrozil) gave the maximal estrogenic response. Tamoxifen showed its dual activity as

The occurrence and fate of pharmaceuticals in the aquatic environment is recognized as one of the emerging issues in environmental chemistry and as a matter of public concern. Exist‐ ing data tend to focus on the concentrations of pharmaceuticals in the aqueous phase, with limited studies on their concentrations in particulate phase such as sediments. Furthermore, current water quality monitoring does not differentiate between soluble and colloidal phas‐

nearly complete removal of pharmaceuticals during ground water infiltration [26].

*3.2.1.3. A preliminary ecotoxicity study of pharmaceuticals in the marine environment*

for CYC was approximately 0.04 microg/mL [27].

agonist and antagonist of hER alpha [28].

*3.2.1.4. Estrogenic activity of pharmaceuticals in the aquatic environment*

*3.2.1.5. Colloids as a sink for certain pharmaceuticals in the aquatic environment*

#### *3.1.1.2. Human Pharmaceuticals, Hormones and Fragrances: The Challenge of Micropollutants in Urban Water Management*

The observed concentrations of pharmaceuticals and personal care products (PPCPs) in raw wastewater confirm that municipal wastewater represents the main disposal pathway for the PPCPs consumed in households, hospitals and industry. In sewage treatment plant ef‐ fluents most PPCPs are still present, since many of these polar and persistent compounds are being removed only partially or, in some cases, not at all. Treated wastewater therefore represents an important point source for PPCPs into the environment. After passing a sew‐ age treatment plant the treated wastewater is mostly discharged into rivers and streams or sometimes used to irrigate fields. If drinking water is produced using resources containing a substantial proportion of treated wastewater (e.g. from river water downstream of commun‐ ities) the water cycle is closed and indirect potable reuse occurs. Human Pharmaceuticals, Hormones and Fragrances provides an overview of the occurrence, analytics, removal and environmental risk of pharmaceuticals and personal care products in wastewater, surface water and drinking water. [25].

#### *3.2.1.2. Factors affecting the concentrations of pharmaceuticals released to the aquatic environment*

Although recent research has demonstrated that pharmaceuticals are widely distributed in the aquatic environment, it is difficult to assess the threat that they

pose to drinking water supplies or their rate of attenuation in natural systems without an adequate understanding of the sources of contamination. To identify pharmaceutical com‐ pounds of significance to water supplies in the United States, the authors have reviewed available data on the use of prescription drugs. Results of our analysis indicate that approxi‐ mately 40 compounds could be present in municipal wastewater effluent at concentrations above 1,000 ng/L and at least 120 compounds could be present at concentrations above 1 ng/L. Important classes of prescription drugs include analgesics, beta-blockers, and antibiot‐ ics. Analysis of a group of the most commonly used pharmaceuticals in the United States indicates that they are ubiquitous in wastewater effluents. Authors have detected concentra‐ tions ranging from approximately 10- 3,000 ng/L for high use pharmaceuticals such as beta‐ blockers (*e.g.,* metoprolol, propranolol) and acidic drugs (*e.g.,* gemfibrozil, ibuprofen). The concentration of pharmaceuticals in effluent from conventional wastewater treatment plants is similar. Advanced wastewater treatment plants equipped with reverse osmosis systems reduce concentrations of pharmaceuticals below detection limits. In addition to removal during biological wastewater treatment, pharmaceuticals also are attenuated in engineered natural systems (*i.e.,* treatment wetlands, ground water infiltration basins). Preliminary evi‐ dence suggests limited removal of pharmaceuticals in engineered treatment wetlands and nearly complete removal of pharmaceuticals during ground water infiltration [26].

#### *3.2.1.3. A preliminary ecotoxicity study of pharmaceuticals in the marine environment*

reuptake inhibitors (SSRIs), azoles, and antibiotics. Reported biological effects in fish in the laboratory have often been shown to be in accordance with known effects of pharmaceuti‐ cals in mammals. Water concentrations at which such effects have been reported, however, are generally, between microg L(-1) and mg L(-1), typically at least 1 order of magnitude higher than concentrations normally found in surface waters (ng L(-1)). There are exceptions to this, however, as for the case of synthetic oestrogens, which can induce biological effects in the low ng L(-1) range. Although generally effect levels for pharmaceuticals are higher than those found in the environment, the risks to wild fish populations have not been thor‐ oughly characterised, and there has been a lack of consideration given to the likely chronic nature of the exposures, or the potential for mixture effects. As global consumption of phar‐ maceuticals rises, an inevitable consequence is an increased level of contamination of sur‐ face and ground waters with these biologically active drugs, and thus in turn a greater

*3.1.1.2. Human Pharmaceuticals, Hormones and Fragrances: The Challenge of Micropollutants in*

The observed concentrations of pharmaceuticals and personal care products (PPCPs) in raw wastewater confirm that municipal wastewater represents the main disposal pathway for the PPCPs consumed in households, hospitals and industry. In sewage treatment plant ef‐ fluents most PPCPs are still present, since many of these polar and persistent compounds are being removed only partially or, in some cases, not at all. Treated wastewater therefore represents an important point source for PPCPs into the environment. After passing a sew‐ age treatment plant the treated wastewater is mostly discharged into rivers and streams or sometimes used to irrigate fields. If drinking water is produced using resources containing a substantial proportion of treated wastewater (e.g. from river water downstream of commun‐ ities) the water cycle is closed and indirect potable reuse occurs. Human Pharmaceuticals, Hormones and Fragrances provides an overview of the occurrence, analytics, removal and environmental risk of pharmaceuticals and personal care products in wastewater, surface

*3.2.1.2. Factors affecting the concentrations of pharmaceuticals released to the aquatic environment*

the aquatic environment, it is difficult to assess the threat that they

Although recent research has demonstrated that pharmaceuticals are widely distributed in

pose to drinking water supplies or their rate of attenuation in natural systems without an adequate understanding of the sources of contamination. To identify pharmaceutical com‐ pounds of significance to water supplies in the United States, the authors have reviewed available data on the use of prescription drugs. Results of our analysis indicate that approxi‐ mately 40 compounds could be present in municipal wastewater effluent at concentrations above 1,000 ng/L and at least 120 compounds could be present at concentrations above 1 ng/L. Important classes of prescription drugs include analgesics, beta-blockers, and antibiot‐ ics. Analysis of a group of the most commonly used pharmaceuticals in the United States indicates that they are ubiquitous in wastewater effluents. Authors have detected concentra‐

potential for adverse effects in aquatic wildlife [24].

*Urban Water Management*

220 Latest Research into Quality Control

water and drinking water. [25].

Environmental fates and effects of pharmaceuticals in the aquatic environment have been the focus of recent research in environmental ecotoxicology. Worldwide studies of common over-the-counter pharmaceuticals have reported detectable levels in the aquatic environ‐ ment, but there are few studies examining impacts on marine habitats. These drugs can af‐ fect the functions of various vertebrates and invertebrates. The stability of two pharmaceuticals, cyclizine (CYC) and prochlorperazine (PCZ), in seawater was examined under light and dark conditions, as well as the toxicity of these compounds to larvae of the barnacle Balanus amphitrite, which is a cosmopolitan marine organism found in most of the world's oceans. CYC was very stable under all the tested conditions. On the other hand, PCZ degraded in light but not in the dark, and was more stable in seawater than fresh wa‐ ter. For the barnacle larvae, the LC50 of prochlorperazine was 0.93 microg/mL and the LC50 for CYC was approximately 0.04 microg/mL [27].

#### *3.2.1.4. Estrogenic activity of pharmaceuticals in the aquatic environment*

In the last years pharmaceuticals have aroused great interest as environmental pollu‐ tants for their toxic effects towards non target organisms. This study wants to draw atten‐ tion to a further adverse effect of drugs, the endocrine interference. The most representative drugs of the widespread classes in environment were investigated. The YES-test and the E-screen assay were performed to detect the capability of these substances to bind the human estrogenic receptor alpha (hER alpha) in comparison with 17beta-estradiol. Out of 14 tested pharmaceuticals, 9 were positive to YES-assay and 11 were positive to Escreen assay; in particular, Furosemide and the fibrates (Bezafibrate, Fenofibrate and Gem‐ fibrozil) gave the maximal estrogenic response. Tamoxifen showed its dual activity as agonist and antagonist of hER alpha [28].

#### *3.2.1.5. Colloids as a sink for certain pharmaceuticals in the aquatic environment*

The occurrence and fate of pharmaceuticals in the aquatic environment is recognized as one of the emerging issues in environmental chemistry and as a matter of public concern. Exist‐ ing data tend to focus on the concentrations of pharmaceuticals in the aqueous phase, with limited studies on their concentrations in particulate phase such as sediments. Furthermore, current water quality monitoring does not differentiate between soluble and colloidal phas‐ es in water samples, hindering our understanding of the bioavailability and bioaccumula‐ tion of pharmaceuticals in aquatic organisms. In this study, an investigation was conducted into the concentrations and phase association (soluble, colloidal, suspended particulate mat‐ ter or SPM) of selected pharmaceuticals (propranolol, sulfamethoxazole, meberverine, thio‐ ridazine, carbamazepine, tamoxifen, indomethacine, diclofenac, and meclofenamic acid) in river water, effluents from sewage treatment works (STW), and groundwater in the UK. Colloids were isolated by cross-flow ultrafiltration (CFUF). Water samples were extracted by solid-phase extraction (SPE), while SPM was extracted by microwave. All sample extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring.

**4. Chemometrics**

chemical engineering.

or observations.

**Author details**

Chile

**References**

Sigrid Mennickent1\*, M. de Diego1

Concepción, Concepción, , Chile

\*Address all correspondence to: smennick@udec.cl

Chemometrics is the science of extracting information from chemical systems by data-driven means. It is a highly interfacial discipline, using methods frequently employed in core dataanalytic disciplines such as multivariate statistics, applied mathematics, and computer sci‐ ence, in order to address problems in chemistry, biochemistry, medicine, biology and

Chemometrics is applied to solve both descriptive and predictive problems in experimental life sciences, especially in chemistry. In descriptive applications, properties of chemical sys‐ tems are modeled with the intent of learning the underlying relationships and structure of the system (i.e., model understanding and identification). In predictive applications, proper‐ ties of chemical systems are modeled with the intent of predicting new properties or behav‐ ior of interest. In both cases, the datasets can be small but are often very large and highly complex, involving hundreds to thousands of variables, and hundreds to thousands of cases

Chemometric techniques are particularly heavily used in analytical chemistry and metabolo‐ mics, and the development of improved chemometric methods of analysis also continues to advance the state of the art in analytical instrumentation and methodology. It is an applica‐ tion driven discipline, and thus while the standard chemometric methodologies are very widely used industrially, academic groups are dedicated to the continued development of

, M. Vega2

1 Department of Pharmacy, Faculty of Pharmacy, University of Concepción, Concepción,,

2 Department of Bromatology, Nutrition and Dietetic, Faculty of Pharmacy, University of

[1] USP (1999). The United States Pharmacopeia/ The National Formulary (USP 24/NF

[2] ICH. (2003). *The Sixth ICH International Conference on Armonization of Technical Re‐*

and C. G. Godoy1

New Approachs in Drug Quality Control: Matrices and Chemometrics

http://dx.doi.org/10.5772/50825

223

chemometric theory, method and application development [30-33].

, B. Schulz1

19), United States Pharmacopeial Convection, Inc., Rockville

*quirements for Registration of Pharmaceuticals for Human Use, Osaka,*.

Five compounds propranolol, sulfamethoxazole, carbamazepine, indomethacine, and diclo‐ fenac were detected in all samples, with carbamazepine showing the highest concentra‐ tions in all phases. The highest concentrations of these compounds were detected in STW effluents, confirming STW as a key source of these compounds in the aquatic environ‐ ments. The calculation of partition coefficients of pharmaceuticals between SPM and fil‐ trate, between SPM and soluble phase, and between colloids and soluble phase showed that intrinsic partition coefficients are between 25% and 96%, and between 18% and 82% higher than relevant observed partition coefficients values, and are much less variable. Secondly, *K* coc values are 3–4 orders of magnitude greater than Kocint values, indicat‐ ing that aquatic colloids are substantially more powerful sorbents for accumulating phar‐ maceuticals than sediments. Furthermore, mass balance calculations of pharmaceutical concentrations demonstrate that between 23% and 70% of propranolol, 17–62% of sulfame‐ thoxazole, 7–58% of carbamazepine, 19–84% of indomethacine, and 9–74% of diclofenac are present in the colloidal phase.

The results provide direct evidence that sorption to colloids provides an important sink for the pharmaceuticals in the aquatic environment. Such strong pharmaceutical/colloid interac‐ tions may provide a long-term storage of pharmaceuticals, hence, increasing their persis‐ tence while reducing their bioavailability in the environment.

Recommendations and perspectives from this study:

Pharmaceutical compounds have been detected not only in the aqueous phase but also in suspended particles; it is important, therefore, to have a holistic approach in future envi‐ ronmental fate investigation of pharmaceuticals. For example, more research is needed to assess the storage and long-term record of pharmaceutical residues in aquatic sediments by which benthic organisms will be most affected. Aquatic colloids have been shown to account for the accumulation of major fractions of total pharmaceutical concentrations in the aquatic environment, demonstrating unequivocally the importance of aquatic col‐ loids as a sink for such residues in the aquatic systems. As aquatic colloids are abun‐ dant, ubiquitous, and highly powerful sorbents, they are expected to influence the bioavailability and bioaccumulation of such chemicals by aquatic organisms. It is there‐ fore critical for colloids to be incorporated into water quality models for prediction and risk assessment purposes [29].

#### **4. Chemometrics**

es in water samples, hindering our understanding of the bioavailability and bioaccumula‐ tion of pharmaceuticals in aquatic organisms. In this study, an investigation was conducted into the concentrations and phase association (soluble, colloidal, suspended particulate mat‐ ter or SPM) of selected pharmaceuticals (propranolol, sulfamethoxazole, meberverine, thio‐ ridazine, carbamazepine, tamoxifen, indomethacine, diclofenac, and meclofenamic acid) in river water, effluents from sewage treatment works (STW), and groundwater in the UK. Colloids were isolated by cross-flow ultrafiltration (CFUF). Water samples were extracted by solid-phase extraction (SPE), while SPM was extracted by microwave. All sample extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the

Five compounds propranolol, sulfamethoxazole, carbamazepine, indomethacine, and diclo‐ fenac were detected in all samples, with carbamazepine showing the highest concentra‐ tions in all phases. The highest concentrations of these compounds were detected in STW effluents, confirming STW as a key source of these compounds in the aquatic environ‐ ments. The calculation of partition coefficients of pharmaceuticals between SPM and fil‐ trate, between SPM and soluble phase, and between colloids and soluble phase showed that intrinsic partition coefficients are between 25% and 96%, and between 18% and 82% higher than relevant observed partition coefficients values, and are much less variable. Secondly, *K* coc values are 3–4 orders of magnitude greater than Kocint values, indicat‐ ing that aquatic colloids are substantially more powerful sorbents for accumulating phar‐ maceuticals than sediments. Furthermore, mass balance calculations of pharmaceutical concentrations demonstrate that between 23% and 70% of propranolol, 17–62% of sulfame‐ thoxazole, 7–58% of carbamazepine, 19–84% of indomethacine, and 9–74% of diclofenac

The results provide direct evidence that sorption to colloids provides an important sink for the pharmaceuticals in the aquatic environment. Such strong pharmaceutical/colloid interac‐ tions may provide a long-term storage of pharmaceuticals, hence, increasing their persis‐

Pharmaceutical compounds have been detected not only in the aqueous phase but also in suspended particles; it is important, therefore, to have a holistic approach in future envi‐ ronmental fate investigation of pharmaceuticals. For example, more research is needed to assess the storage and long-term record of pharmaceutical residues in aquatic sediments by which benthic organisms will be most affected. Aquatic colloids have been shown to account for the accumulation of major fractions of total pharmaceutical concentrations in the aquatic environment, demonstrating unequivocally the importance of aquatic col‐ loids as a sink for such residues in the aquatic systems. As aquatic colloids are abun‐ dant, ubiquitous, and highly powerful sorbents, they are expected to influence the bioavailability and bioaccumulation of such chemicals by aquatic organisms. It is there‐ fore critical for colloids to be incorporated into water quality models for prediction and

multiple reaction monitoring.

222 Latest Research into Quality Control

are present in the colloidal phase.

risk assessment purposes [29].

tence while reducing their bioavailability in the environment.

Recommendations and perspectives from this study:

Chemometrics is the science of extracting information from chemical systems by data-driven means. It is a highly interfacial discipline, using methods frequently employed in core dataanalytic disciplines such as multivariate statistics, applied mathematics, and computer sci‐ ence, in order to address problems in chemistry, biochemistry, medicine, biology and chemical engineering.

Chemometrics is applied to solve both descriptive and predictive problems in experimental life sciences, especially in chemistry. In descriptive applications, properties of chemical sys‐ tems are modeled with the intent of learning the underlying relationships and structure of the system (i.e., model understanding and identification). In predictive applications, proper‐ ties of chemical systems are modeled with the intent of predicting new properties or behav‐ ior of interest. In both cases, the datasets can be small but are often very large and highly complex, involving hundreds to thousands of variables, and hundreds to thousands of cases or observations.

Chemometric techniques are particularly heavily used in analytical chemistry and metabolo‐ mics, and the development of improved chemometric methods of analysis also continues to advance the state of the art in analytical instrumentation and methodology. It is an applica‐ tion driven discipline, and thus while the standard chemometric methodologies are very widely used industrially, academic groups are dedicated to the continued development of chemometric theory, method and application development [30-33].

### **Author details**

Sigrid Mennickent1\*, M. de Diego1 , B. Schulz1 , M. Vega2 and C. G. Godoy1

\*Address all correspondence to: smennick@udec.cl

1 Department of Pharmacy, Faculty of Pharmacy, University of Concepción, Concepción,, Chile

2 Department of Bromatology, Nutrition and Dietetic, Faculty of Pharmacy, University of Concepción, Concepción, , Chile

#### **References**


[25] Ternes, T. (2006). Human Pharmaceuticals, Hormones and Fragrances: The Chal‐

New Approachs in Drug Quality Control: Matrices and Chemometrics

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[26] Sedlak, D., & Pinkston, K. (2011). *Factors affecting the concentrations of pharmaceuticals*

[27] Choong, A. M., Teo, S. L., Leow, J. L., & Ho, P. C. (2006). *J. Toxicol. Environ. Health A.*,

[28] Isidori, M., Bellota, M., Cangiano, M., & Parrella, A. (2009). *Environ. Int*, 35(5),

[29] Maskaoui, K., & Zhou, J. (2010). *Environmental Science and Pollution Research*, 17(4),

[31] Gemperline, P. J. (2006). Practical guide to chemometrics. 2nd Edition, CRC Press

[32] Mark, H., & Workman, J. (2007). Chemometrics in spectroscopy. Academic Press-

[33] Maeder, M., & Neuhold, Y. M. (2007). Practical Data Analysis in Chemistry. Elsevier.

[30] Verenitch, S. S., Lowe, C. J., & Mazumder, A. (2006). *J. Chromatogr. A*, 1116.

lenge of Micropollutants in Urban Water Management. Iwa Publishing.

*released to the aquatic environment*, University of California.

69(21), 1959-1970.

10.1201/9781420018301

826-829.

898-907.

Elsevier.


[25] Ternes, T. (2006). Human Pharmaceuticals, Hormones and Fragrances: The Chal‐ lenge of Micropollutants in Urban Water Management. Iwa Publishing.

[3] Buchberger, W. W. (2007). *Analytica Chimica Acta*, 593, 129-139.

163-167.

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369A.

69, 334.

*A*, 1114, 224.

97.

*Chemosphere*, 65, 1990.

(2006). *Chemosphere*, 64, 1318.

khäuser,, New York, USA.

[7] Moldovan, Z. (2006). *Chemosphere*, 64, 1808.

(2006). *Env. Sci. Technol*, 40, 3457.

(2006). *Anal. Chim. Acta*, 556, 415.

[4] Jones, O., Lester, J. N., & Voulvoulis, N. (2005). *Trends in Biotechonology*, 23(4),

[5] Lemmer, B. (1996). Chronopharmacology- Cellular and Biochemical Interactions. Bir‐

[6] Boxall, A., Fenner, K., Kolpin, D. W., & Maund, S. (2004). *Environ. Sci. Technol.*, 38,

[8] Cunningham, V. L., Buzby, M., Hutchinson, T., Mastrocco, F., Parke, N., & Roden, N.

[11] Hernando, M. D., Mezcua, M., Fernandez-Alba, A. R., & Barcelo, D. (2006). *Talanta*,

[12] Benito-Peña, E., Partal-Rodera, A. I., Leon-Gonzalez, M. E., & Moreno-Bondi, M. C.

[14] Gomez, M. J., Petrovic, M., Fernandez-Alba, A. R., & Barcelo, D. (2006). *J. Chromatogr.*

[17] Trenholm, R. A., Vanderford, B. J., Holady, J. C., Rexing, D. J., & Snyder, S. A. (2006).

[19] Himmelsbach, M., Buchberger, W., & Klampfl, C. (2006). *Electrophoresis*, 27, 1220.

[20] Seitz, W., Weber, W. H., Jiang, J. Q., Lloyd, B. J., Maier, M., Maier, D., & Schulz, W.

[21] Pozo, O. J., Guerrero, C., Sancho, J. V., Ibañez, M., Pitarch, E., Hogendoorn, E., &

[22] Rodriguez-Mozaz, S., Lopez de Alda, D., & Barcelo, D. (2007). *J. Chromatogr. A*, 1152,

[23] Quintana, J. B., Miro, M., Estela, J. M., & Cerda, V. (2006). *Anal. Chem.*, 78, 2832.

[24] Corcoran, J., Winter, M. J., & Tyler, C. R. (2010). *Crit, Rev. Toxicol*, 40(4), 287-304.

[13] Fontanals, N., Marce, R. M., & Borrull, F. (2005). Trends Anal. Chem ., 24, 394.

[9] Fent, K., Weston, A. A., & Caminada, D. (2006). *Aquatic Toxicol*, 76, 122.

[10] Crane, M., Watts, C., & Boucard, T. (2006). *Sci. Tot. Environ*, 367, 23.

[15] Gros, M., Petrovic, M., & Barcelo, D. (2006). *Talanta*, 70, 678.

[16] Petrovic, M., Gros, M., & Barcelo, D. (2006). *J. Chromatogr. A*, 1124, 68.

[18] Roberts, P. H., & Bersuder, P. (2006). *J. Chromatogr. A*, 1134, 143.

Hernandez, F. (2006). *J. Chromatogr. A*, 1103, 83.


**Chapter 11**

**Quality Control of Formulated Medicines**

A pharmaceutical drug is technically obtained or prepared for prophylactic, curative, pallia‐ tive or diagnostic purposes. The final product must meet quality standard, be safe and effec‐ tive. In Brazil, there is a high demand for formulated drugs. This is mainly due to their lower price compared to manufactured drugs, evidenced by the rapid growth – an increase

Even after the ANVISA's (Agency National Health Surveillance) establishment of the new handling standards to be followed by the magistral pharmacies, several serious cases, in‐ cluding death reports, caused by the consumption of formulated drugs have recently be‐

Other problems related to this subject occurred in 2004, when deaths caused by manipulated medicines of low therapeutic index (clonidine and levotiroxine) led ANVISA to modify the regulation for manipulated medicines. The concentration of each compound was not totally assured and the contamination by impurities, not included in the original formula, were

In a previous work, we have also observed the presence of impurities – such as metals - in different kinds of medicines. The ingestion of metals, even at low levels, can be very harm‐ ful to humans. Besides this, the long-term uptake of some drugs is also risky. This should

The quality and safety of drugs must follow the specifications described in the official compendia - among them, the pharmacopoeias. Medicines cannot contain impurities or

> © 2012 Leal et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Leal et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

require attention and surveillance from the public health-related agencies [6-10].

Alexandre S. Leal, Maria Ângela de B. C. Menezes,

Ilza Dalmázio, Fernanda P. Sepe,

http://dx.doi.org/10.5772/51459

**1. Introduction**

come public [1-5].

of 350% from 1998 to 2010.

present in the final product.

Tatiana C. B. Gomes, Amalia S. Santana, Luzia H. da Cunha and Radojko Jaćimović

Additional information is available at the end of the chapter

### **Chapter 11**

## **Quality Control of Formulated Medicines**

Alexandre S. Leal, Maria Ângela de B. C. Menezes, Ilza Dalmázio, Fernanda P. Sepe, Tatiana C. B. Gomes, Amalia S. Santana, Luzia H. da Cunha and Radojko Jaćimović

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51459

#### **1. Introduction**

A pharmaceutical drug is technically obtained or prepared for prophylactic, curative, pallia‐ tive or diagnostic purposes. The final product must meet quality standard, be safe and effec‐ tive. In Brazil, there is a high demand for formulated drugs. This is mainly due to their lower price compared to manufactured drugs, evidenced by the rapid growth – an increase of 350% from 1998 to 2010.

Even after the ANVISA's (Agency National Health Surveillance) establishment of the new handling standards to be followed by the magistral pharmacies, several serious cases, in‐ cluding death reports, caused by the consumption of formulated drugs have recently be‐ come public [1-5].

Other problems related to this subject occurred in 2004, when deaths caused by manipulated medicines of low therapeutic index (clonidine and levotiroxine) led ANVISA to modify the regulation for manipulated medicines. The concentration of each compound was not totally assured and the contamination by impurities, not included in the original formula, were present in the final product.

In a previous work, we have also observed the presence of impurities – such as metals - in different kinds of medicines. The ingestion of metals, even at low levels, can be very harm‐ ful to humans. Besides this, the long-term uptake of some drugs is also risky. This should require attention and surveillance from the public health-related agencies [6-10].

The quality and safety of drugs must follow the specifications described in the official compendia - among them, the pharmacopoeias. Medicines cannot contain impurities or

other substances that endanger the patient's health. According to the second edition of the Brazilian Pharmacopoeia National Formulary [1], to ensure safety, efficacy and quali‐ ty of the handled products it is necessary correct calculations, exact measurements, and adequate conditions and procedures of preparation. The prudent judgment of the phar‐ macist, who must be a qualified professional for this purpose, is another fundamental as‐ pect. Additionally, an appropriate profile with a proven stability must be sought in the literature [11-14].

In this study, the quality of medicines Omeprazole and Enalapril Maleate from five (5) dif‐ ferent magistral pharmacies was evaluated according to the methodology described in phar‐ macopoeia. The analyses for mass determination, identification of active principle, content,

Quality Control of Formulated Medicines http://dx.doi.org/10.5772/51459 229

The target drugs, Omeprazole and Enalapril Maleate, were chosen because of their represen‐ tativeness of consumption and availability of related reference data in the pharmacopoeias.

In order to evaluate the presence and concentration of chemical elements, the technique used in this study was neutron activation analysis (NAA), applying the k0-standardization method [23-25]. The neutron activation analysis is a very sensitive and reliable multielemen‐ tal technique, suitable for determination of the elements such as: As, Ba, Br, Ca, Ce, Cl, Co, Cr, Eu, Fe, Hf, Mg, Mn, Na, Sb, Sc, Sm, Ti and Zn, in different drugs [14]. The technique is based on the principle that when the material is irradiated by neutrons, some elements with suitable nuclear characteristics become radioactive isotopes. Thus, the concentration of each element can be determined by counting the respective radiation emitted by the correspond‐

The results described here are part of a wider project which also includes the analyses of

The 5th edition of the Brazilian Pharmacopoeia [27] defines quality control as: "The set of measures to ensure, at any time, the batch production of medicines and other products that meet the standards of identity, activity, content, purity, efficacy and safety." According to Resolution RDC Nº. 67, October 8, 2007 [15], which provides the Technical Regulation estab‐ lishing the Good Handling Practices in Pharmacies (Good Compounding Practices) quality control of magistral and officinal preparations, is given by the completion of at minimum, the tests described in Table 1, according to the Brazilian Pharmacopoeia or other Official

Results of tests must be recorded in the same order of handling, in addition to other relevant information. The pharmacist must evaluate the results to approve or not the preparation for dispensing. Each pharmacy is responsible for the quality of magistral preparations that han‐ dles, keeps, transports and dispenses. Raw materials should be checked in its receipt and moved to quarantine soon after, until the release of the reports of quality control. In the ab‐ sence of pharmacopoeia monograph, the scientific literature should be used as a reference, and only with the lack of literature, the specification provided by the supplier may be used.

Compendium recognized by the National Health Surveillance Agency (ANVISA).

content uniformity and related compounds were performed.

Fluoxetin and Sinvastatin medicines and will be published briefly.

**2. Quality control of formulated drugs**

All results must be written and stored [15].

ing radionuclide [26].

**2.1. The pharmacopeia**

The requirements of sanitary legislation and quality control of raw materials for magistral solid preparations are:


The legislation also determines that all pharmacies must perform analyzes every two months of at least one of the formulas containing drug(s) ≤ 25 mg of drugs. The priority is to those that contain ≤ 5 mg of drugs. The legislation establishes special quality con‐ trol requirements for preparations of substances with low therapeutic index, like hor‐ mones, antibiotics and cytotoxic drugs, homeopathic products, and sterile products. The raw materials used in sterile preparations must also be analyzed [15-17].

The Legislation on Good Practices for Handling does not require impurity tests for the raw materials received by the pharmacies. It is only necessary to check the certificate of a qualified supplier – issued in accordance with methods described in the pharmaco‐ poeia, which are only suitable for the detection of some elements (Ag, As, Bi, Cd, Hg, Mo, Pb, Sb, and Sn) [18].

Quality control tests for the products handled do not include detection and quantifica‐ tion of impurities. Moreover, the analysis required for formulated preparations allow limited conclusions about the quality of the process, since they do not testify the homo‐ geneity of the active principle directly, but only as to the uniformity of filling of the cap‐ sules. So, a particular formulation can have the acceptance criteria for average mass, standard deviation and coefficient of variation but not the uniformity of this active con‐ tent in the capsules [19].

Periodic reviews performed every two months for formulated drugs do not statistically have significant value, so that a reliable conclusion about the quality of formulated drugs can not be reached [20]. The analysis of thirty batches of 20 mg of Sinvastatin medicine manipulated in pharmacies of Belo Horizonte, showed that only fourteen of them, met the quality stand‐ ard required by pharmacopeia. Thus, the therapeutic efficacy of 53% of the analyzed prod‐ ucts can not be totally dependable [21].

In this study, the quality of medicines Omeprazole and Enalapril Maleate from five (5) dif‐ ferent magistral pharmacies was evaluated according to the methodology described in phar‐ macopoeia. The analyses for mass determination, identification of active principle, content, content uniformity and related compounds were performed.

The target drugs, Omeprazole and Enalapril Maleate, were chosen because of their represen‐ tativeness of consumption and availability of related reference data in the pharmacopoeias.

In order to evaluate the presence and concentration of chemical elements, the technique used in this study was neutron activation analysis (NAA), applying the k0-standardization method [23-25]. The neutron activation analysis is a very sensitive and reliable multielemen‐ tal technique, suitable for determination of the elements such as: As, Ba, Br, Ca, Ce, Cl, Co, Cr, Eu, Fe, Hf, Mg, Mn, Na, Sb, Sc, Sm, Ti and Zn, in different drugs [14]. The technique is based on the principle that when the material is irradiated by neutrons, some elements with suitable nuclear characteristics become radioactive isotopes. Thus, the concentration of each element can be determined by counting the respective radiation emitted by the correspond‐ ing radionuclide [26].

The results described here are part of a wider project which also includes the analyses of Fluoxetin and Sinvastatin medicines and will be published briefly.

#### **2. Quality control of formulated drugs**

#### **2.1. The pharmacopeia**

other substances that endanger the patient's health. According to the second edition of the Brazilian Pharmacopoeia National Formulary [1], to ensure safety, efficacy and quali‐ ty of the handled products it is necessary correct calculations, exact measurements, and adequate conditions and procedures of preparation. The prudent judgment of the phar‐ macist, who must be a qualified professional for this purpose, is another fundamental as‐ pect. Additionally, an appropriate profile with a proven stability must be sought in the

The requirements of sanitary legislation and quality control of raw materials for magistral

**•** raw material: character sensory, solubility, pH determination, melting point, density,

**•** raw material of vegetal origin: organoleptic characters, solubility, pH determination, melting point, density, weight and volume, evaluation of vendor analysis report;

**•** manipulated product: solid dosage forms: description, appearance and organoleptic char‐

The legislation also determines that all pharmacies must perform analyzes every two months of at least one of the formulas containing drug(s) ≤ 25 mg of drugs. The priority is to those that contain ≤ 5 mg of drugs. The legislation establishes special quality con‐ trol requirements for preparations of substances with low therapeutic index, like hor‐ mones, antibiotics and cytotoxic drugs, homeopathic products, and sterile products. The

The Legislation on Good Practices for Handling does not require impurity tests for the raw materials received by the pharmacies. It is only necessary to check the certificate of a qualified supplier – issued in accordance with methods described in the pharmaco‐ poeia, which are only suitable for the detection of some elements (Ag, As, Bi, Cd, Hg,

Quality control tests for the products handled do not include detection and quantifica‐ tion of impurities. Moreover, the analysis required for formulated preparations allow limited conclusions about the quality of the process, since they do not testify the homo‐ geneity of the active principle directly, but only as to the uniformity of filling of the cap‐ sules. So, a particular formulation can have the acceptance criteria for average mass, standard deviation and coefficient of variation but not the uniformity of this active con‐

Periodic reviews performed every two months for formulated drugs do not statistically have significant value, so that a reliable conclusion about the quality of formulated drugs can not be reached [20]. The analysis of thirty batches of 20 mg of Sinvastatin medicine manipulated in pharmacies of Belo Horizonte, showed that only fourteen of them, met the quality stand‐ ard required by pharmacopeia. Thus, the therapeutic efficacy of 53% of the analyzed prod‐

weight and volume; analysis report of manufacturer/supplier;

raw materials used in sterile preparations must also be analyzed [15-17].

acteristics, determination of average weight

literature [11-14].

solid preparations are:

228 Latest Research into Quality Control

Mo, Pb, Sb, and Sn) [18].

tent in the capsules [19].

ucts can not be totally dependable [21].

The 5th edition of the Brazilian Pharmacopoeia [27] defines quality control as: "The set of measures to ensure, at any time, the batch production of medicines and other products that meet the standards of identity, activity, content, purity, efficacy and safety." According to Resolution RDC Nº. 67, October 8, 2007 [15], which provides the Technical Regulation estab‐ lishing the Good Handling Practices in Pharmacies (Good Compounding Practices) quality control of magistral and officinal preparations, is given by the completion of at minimum, the tests described in Table 1, according to the Brazilian Pharmacopoeia or other Official Compendium recognized by the National Health Surveillance Agency (ANVISA).

Results of tests must be recorded in the same order of handling, in addition to other relevant information. The pharmacist must evaluate the results to approve or not the preparation for dispensing. Each pharmacy is responsible for the quality of magistral preparations that han‐ dles, keeps, transports and dispenses. Raw materials should be checked in its receipt and moved to quarantine soon after, until the release of the reports of quality control. In the ab‐ sence of pharmacopoeia monograph, the scientific literature should be used as a reference, and only with the lack of literature, the specification provided by the supplier may be used. All results must be written and stored [15].


obtaining the substance which can be normally found within a specified limit. This test is

Quality Control of Formulated Medicines http://dx.doi.org/10.5772/51459 231

The variation of the mass allows checking the uniformity of mass between units within a batch. For products in hard capsules should be weighed individually, twenty units, the con‐ tents of each one should be removed, properly cleaned and reweighed. The mass content of each capsule is determined by mass difference between the full and the empty capsule. Then the average mass of the contents can be determined. For hard capsules, the limit of variation is ± 10% of the mass corresponding to less than 300 mg. If the mass corresponds to 300 mg or more, the maximum range is ± 7.5%. It cannot be tolerated more than two units outside the limits specified in the official compendia, but none can be above or below twice the percen‐

The uniformity of dosage units evaluate the uniformity of distribution of active component units in a single batch can be determined by two methods: mass variation and content uni‐ formity. The mass variation test is only applicable in specific cases. The test for content uni‐ formity is based on the content of each active ingredients in a number of unit doses in order to determine whether the content is within specified limits, being applicable in all cases [28].

In this study the identification tests, content, related compounds and content uniformity was performed by high performance liquid chromatography (HPLC) according to the speci‐ fications of literature [28]. The chromatograph Shimadzu detector was coupled to molecular absorption spectrophotometry in the ultraviolet-visible Perkin Elmer Lambda 25 model, Class-VP software. All chemical reference substances (SQR) were purchased from USP (The

To analyze the Enalapril Maleate, L7 C8 column (4.6 mm x 25 cm x 5 mm) was used. Isocratic elution was performed with a buffer monobasic sodium phosphate pH 2.2 /acetonitrile at a ratio of 75:25. Solvents and solutions were degassed in ultrasonic bath (Elma Transsonic Digitals) and filtered through a Millipore membrane of 0.45 micrometers. Chromatography was performed at 50°C, flow rate of 2 mL.min-1, with injections of 50 μL, detection at 215 nm and running time of 30 min. The calculations were based on the content of the samples ob‐ tained areas of the areas of the SQR of Enalapril Maleate. For related compounds the content

For omeprazole, L7 C8 column (4.6 mm x 15 cm x 5 mm) was used. Elution was performed by mixing two solutions – solution A (6 g of glycine in 1500 mL water, pH 9) and solution B (acetonitrile and methanol, 85:15 ratio) – as shown in Table 3. Solvents and solutions were degassed in ultrasonic bath (Elma Transsonic Digitals) and filtered through a Millipore membrane of 0.45 micrometers. Chromatography was performed with a flow of 1,2 mL.min-1, with injections of 10 μL, detection at 305 nm and running time of 30 min. The cal‐

of diketopiperazine compounds and enalaprilat was also calculated.

done only when specified in the pharmacopoeia.

**3.1. High performance liquid chromatography**

tages indicated [27].

**3. Methodology**

United States Pharmacopeia).

**Table 1.** Tests for quality control of magistral drugs

However, some studies also show that the rule of Good Practices on Handling does not an‐ swer and does not guarantee the quality of compounded drugs [20].

#### **2.2. Analyses performed**

The analyses of quality control were performed at the Laboratory for Quality Control of Chemical Physics Drug, and Cosmetic Sanitizing of the Ezequiel Dias Foundation (FUNED). The following tests were performed [15,27,28]:


The test of aspect is just a visual description of the product to be analyzed, coloration of the capsule and its content.

The test of identification allows determining the presence of the active principle in the product analyzed. It is performed through the high performance liquid chromatography (HPLC) [28].

The analysis of content aims to verify whether the drug has a dose of active ingredient on the label provided and used to quantify the active ingredient in the product analyzed. This test is performed according to the pharmacopoeia for each product, and may be performed in the ultraviolet and visible spectrophotometry, by high performance liquid chromatogra‐ phy, among other methods. The test uses usually ten to twenty capsules and each capsule analyzed separately, but the "pool" of these. There are limits specified in the monograph, which should be within the active drug, usually 90 to 110%. Results below the limit can re‐ sult in ineffective therapy and above, intoxication, depending on the drug analized [28].

The analysis of related compounds determines the amount of by-products of synthesis of the substance and / or its degradation products and / or contaminants from the process of obtaining the substance which can be normally found within a specified limit. This test is done only when specified in the pharmacopoeia.

The variation of the mass allows checking the uniformity of mass between units within a batch. For products in hard capsules should be weighed individually, twenty units, the con‐ tents of each one should be removed, properly cleaned and reweighed. The mass content of each capsule is determined by mass difference between the full and the empty capsule. Then the average mass of the contents can be determined. For hard capsules, the limit of variation is ± 10% of the mass corresponding to less than 300 mg. If the mass corresponds to 300 mg or more, the maximum range is ± 7.5%. It cannot be tolerated more than two units outside the limits specified in the official compendia, but none can be above or below twice the percen‐ tages indicated [27].

The uniformity of dosage units evaluate the uniformity of distribution of active component units in a single batch can be determined by two methods: mass variation and content uni‐ formity. The mass variation test is only applicable in specific cases. The test for content uni‐ formity is based on the content of each active ingredients in a number of unit doses in order to determine whether the content is within specified limits, being applicable in all cases [28].

#### **3. Methodology**

**Preparation Test**

swer and does not guarantee the quality of compounded drugs [20].

**Table 1.** Tests for quality control of magistral drugs

The following tests were performed [15,27,28]:

**2.2. Analyses performed**

230 Latest Research into Quality Control

**•** Aspect;

**•** Labeling;

**•** Content;

**•** Identification ;

**•** Related compounds;

**•** Dosage uniformity;

**•** Unit Change in mass;

capsule and its content.

(HPLC) [28].

Solid Description, appearance, organoleptic characteristics, average mass Semi-solid Description, appearance, organoleptic characteristics, pH (where applicable), mass Non-sterile liquid Description, appearance, organoleptic characteristics, pH, mass or volume before filling

However, some studies also show that the rule of Good Practices on Handling does not an‐

The analyses of quality control were performed at the Laboratory for Quality Control of Chemical Physics Drug, and Cosmetic Sanitizing of the Ezequiel Dias Foundation (FUNED).

The test of aspect is just a visual description of the product to be analyzed, coloration of the

The test of identification allows determining the presence of the active principle in the product analyzed. It is performed through the high performance liquid chromatography

The analysis of content aims to verify whether the drug has a dose of active ingredient on the label provided and used to quantify the active ingredient in the product analyzed. This test is performed according to the pharmacopoeia for each product, and may be performed in the ultraviolet and visible spectrophotometry, by high performance liquid chromatogra‐ phy, among other methods. The test uses usually ten to twenty capsules and each capsule analyzed separately, but the "pool" of these. There are limits specified in the monograph, which should be within the active drug, usually 90 to 110%. Results below the limit can re‐ sult in ineffective therapy and above, intoxication, depending on the drug analized [28].

The analysis of related compounds determines the amount of by-products of synthesis of the substance and / or its degradation products and / or contaminants from the process of

#### **3.1. High performance liquid chromatography**

In this study the identification tests, content, related compounds and content uniformity was performed by high performance liquid chromatography (HPLC) according to the speci‐ fications of literature [28]. The chromatograph Shimadzu detector was coupled to molecular absorption spectrophotometry in the ultraviolet-visible Perkin Elmer Lambda 25 model, Class-VP software. All chemical reference substances (SQR) were purchased from USP (The United States Pharmacopeia).

To analyze the Enalapril Maleate, L7 C8 column (4.6 mm x 25 cm x 5 mm) was used. Isocratic elution was performed with a buffer monobasic sodium phosphate pH 2.2 /acetonitrile at a ratio of 75:25. Solvents and solutions were degassed in ultrasonic bath (Elma Transsonic Digitals) and filtered through a Millipore membrane of 0.45 micrometers. Chromatography was performed at 50°C, flow rate of 2 mL.min-1, with injections of 50 μL, detection at 215 nm and running time of 30 min. The calculations were based on the content of the samples ob‐ tained areas of the areas of the SQR of Enalapril Maleate. For related compounds the content of diketopiperazine compounds and enalaprilat was also calculated.

For omeprazole, L7 C8 column (4.6 mm x 15 cm x 5 mm) was used. Elution was performed by mixing two solutions – solution A (6 g of glycine in 1500 mL water, pH 9) and solution B (acetonitrile and methanol, 85:15 ratio) – as shown in Table 3. Solvents and solutions were degassed in ultrasonic bath (Elma Transsonic Digitals) and filtered through a Millipore membrane of 0.45 micrometers. Chromatography was performed with a flow of 1,2 mL.min-1, with injections of 10 μL, detection at 305 nm and running time of 30 min. The cal‐ culations were based on the content of the samples obtained areas of the areas of the SQR of omeprazole.

**Pharmacy Enalaprilat (%) Diketopiperazine (%) Enalaprilat +**

A 0.66 0.82 1.48 B 0.16 0.07 0.23 C 0.53 0.87 1.40 D 0.05 0.001 0.055 E 1.18 2.77 3.95

The reference values for the content of both drugs should not be less than 90% nor exceed 110% of the declared value, 10 mg and 20 mg for omeprazole and enalapril, respectively.

It can be observed that four from the five samples of omeprazole were unsatisfactory; two of them with content above the permissible and the other two with the content below. For the samples of enalapril, two were unsatisfactory, one exceeding the limit and the other with

**Pharmacy Omeprazole (10 mg/caps) Enalapril Maleate (20 mg/caps)**

All Omeprazole samples were considered unsatisfactory for uniformity of the dosage unit. Three samples were satisfactory for Enalapril Maleate. The results for uniformity of dosage unit are described in Table 7 as contained in the final analysis report issued by FUNED. Var‐

**Table 3.** Content of related compounds of Enalapril Maleate

The results for content of active ingredient are described in Table 6.

recommended content lower than expected, as showed in Table 6.

112.7% declared

84.4% declared

68.7% declared

111.5% declared

109.6% declared

<sup>A</sup> (11.3 ± 0.4) mg/caps or

<sup>B</sup> (8.4 ± 2.2) mg/caps or

<sup>C</sup> (6.9 ± 2.6) mg/caps or

<sup>D</sup> (11.2 ± 0.1) mg/caps or

<sup>E</sup> (11.0 ± 0.2) mg/caps or

iations in dose uniformity should not exceed 15% [28].

**Table 4.** Final content of Omeprazole and Enalapril Maleate

**d.** Uniformity of the dosage unit

**c.** Content

**Diketopiperazine (%)**

233

Quality Control of Formulated Medicines http://dx.doi.org/10.5772/51459

> (18.5 ± 0.2) mg/caps or 92.6% of declared

> (20.5 ± 0.1) mg/caps or 102.7% declared

> (18.5 ± 0.1) mg/caps or 92.5% declared

> (11.2 ± 4.3) mg/caps or 56.0% declared

(16.9 ±1.2) mg/caps or 84.7% do declared


**Table 2.** Parameters of elution of the HPLC analysis of Omeprazole

#### *3.1.1. Results and discussion*

**a.** Appearance, Identification and Labeling

Both, Omeprazole and Enalapril Maleate samples, showed similar aspects as their samples: hard capsule containing white pellets for Omeprazole, and hard capsule containing white powder varying only the color of the hard capsule used by each pharmacy. All samples, Omeprazole and Enalapril Maleate, were satisfactory for labeling and identification, con‐ firming that the identity of the material was in accordance with the label from its packaging. In addition, all labels contain information provided by RDC Resolution Nº 67, October 8, 2007 [15]:


The test for related substances is performed only when described in the pharmacopeia of the compound to be analyzed, or another official compendium regulated by ANVISA [28]. For the Enalapril Maleate it is specified that no more than 5% of diketopiperazine and enalapri‐ lat can be found in the final product. All samples of Enalapril Maleate were satisfactory for this analysis and the results are shown in Table 5.


**Table 3.** Content of related compounds of Enalapril Maleate

#### **c.** Content

culations were based on the content of the samples obtained areas of the areas of the SQR of

**Time (minutes) Solution A (%) Solution B (%) Elution**

**Table 2.** Parameters of elution of the HPLC analysis of Omeprazole

**a.** Appearance, Identification and Labeling

**3.** Registration Number of the formulation;

**b.** Related substances (Enalapril Maleate)

this analysis and the results are shown in Table 5.

**5.** Formulation components and their quantities;

**8.** Identification of pharmacy, full address and federal registration;

**9.** Name and professional register of the responsible person.

0 – 20 88 → 40 12 → 60 Linear gradient 20 – 21 40 → 88 60 → 12 Linear gradient 21 – 25 88 12 Isocratic

Both, Omeprazole and Enalapril Maleate samples, showed similar aspects as their samples: hard capsule containing white pellets for Omeprazole, and hard capsule containing white powder varying only the color of the hard capsule used by each pharmacy. All samples, Omeprazole and Enalapril Maleate, were satisfactory for labeling and identification, con‐ firming that the identity of the material was in accordance with the label from its packaging. In addition, all labels contain information provided by RDC Resolution Nº 67, October 8,

The test for related substances is performed only when described in the pharmacopeia of the compound to be analyzed, or another official compendium regulated by ANVISA [28]. For the Enalapril Maleate it is specified that no more than 5% of diketopiperazine and enalapri‐ lat can be found in the final product. All samples of Enalapril Maleate were satisfactory for

omeprazole.

232 Latest Research into Quality Control

2007 [15]:

*3.1.1. Results and discussion*

**1.** Name of the prescriber;

**4.** Data handling and shelf life;

**2.** Name of the patient;

**6.** Number of units;

**7.** Dosage;

The reference values for the content of both drugs should not be less than 90% nor exceed 110% of the declared value, 10 mg and 20 mg for omeprazole and enalapril, respectively. The results for content of active ingredient are described in Table 6.

It can be observed that four from the five samples of omeprazole were unsatisfactory; two of them with content above the permissible and the other two with the content below. For the samples of enalapril, two were unsatisfactory, one exceeding the limit and the other with recommended content lower than expected, as showed in Table 6.


**Table 4.** Final content of Omeprazole and Enalapril Maleate

#### **d.** Uniformity of the dosage unit

All Omeprazole samples were considered unsatisfactory for uniformity of the dosage unit. Three samples were satisfactory for Enalapril Maleate. The results for uniformity of dosage unit are described in Table 7 as contained in the final analysis report issued by FUNED. Var‐ iations in dose uniformity should not exceed 15% [28].

#### **e.** Mass Variation

The acceptable limit for the analysis of variation in mass of capsules, weighing less than 300 mg is ± 10% above the average mass, and it is tolerable no more than two units outside the specified limit and any unit may be above or below twice the percentages indicated. Thus, only a sample of Omeprazole was considered unsatisfactory. The results for the samples of Omeprazole and Enalapril Maleate are presented in Tables 8 and 9, respectively [27].

From the ten samples analyzed, seven were rated as *unsatisfactory*, considering the analysis of aspect, mass variation, identification, related substances, uniformity of dosage units, con‐

Quality Control of Formulated Medicines http://dx.doi.org/10.5772/51459 235

It was observed that, if only the official established procedures (description, appearance, or‐ ganoleptic characteristics and average mass) were considered from the seven samples rated as unsatisfactory, just one would be classified in this status. The remaining six samples would erroneously be rated satisfactory, meaning that would be approved for human con‐

Some factors may cause deviations, inherent to the handling process of drugs in capsules, such as the loss of substance during the grinding, mixing and filling the capsules. Miscalcu‐ lations and weight of the formulation components, errors inherent to the operator and the use of damaged equipment may also compromise the process and therefore the quality of

The results of Omerazole and Enalapril Maleate were analyzed by ANOVA followed by Tu‐ key's test for uniformity of content and unit dose. Results were considered significantly dif‐

All samples of Omeprazole and Enalapril Maleate were purchased in the market of the Belo

Due to operational reasons, Jožef Stefan Institute (JSI) performed analyses only from three (3) different pharmacies from the sampling group. Due to same operational reasons, the JSI did not analyze the short half-lives radionuclides of elements like Al, Cl, Mg, Mn and Ti.

The samples of Omeprazole and Enalapril Maleate performed by the JSI were packed in polyethylene capsules in plastic bottle containing 20 capsules each. Whole powder mass from 20 capsules was taken to prepare homogenized samples, which was transferred in clean polyethylene bottle. In the samples of Omeprazole performed by CDTN, just one the mass of one capsule taken randomly was considered by each sample. The difference of pro‐

Both institutes CDTN/CNEN and JSI followed the same procedure to prepare the samples. The aliquots of each sample were manually crushed or ground using an agate mortar with pestle, whenever necessary, to avoid any contamination. In most cases, unless the amount of material did not allow it, two replicates were taken and weighed in polyethylene vials. It is relevant to emphasize that no additional chemical sample preparation was performed. At CDTN/CNEN and IJS, the samples were irradiated together with several Al–0.1% Au disks as neutron flux monitors, according to the k0-standardisation method procedure [23,24,30]. Table 10 shows the characteristics of the applied technique such as the parameters *f* (thermal to epithermal fluxes ratio) and the *α* (parameter which measures the epithermal flux devia‐

Horizonte, state of Minas Gerais, Brazil from five (5) different magistral pharmacies.

cedures carried out by both Institutes was due to operational reasons.

tent and labeling.

sumption [18].

the final product [29].

**3.2. Neutron activation analysis**

*3.2.1. Material and methods*

ferent at p < 0.05.


**Table 5.** Dose uniformity of the capsules of Omeprazole and Enalapril Maleate


\*One unit above the limit. \*\* Two units above the limit. Caps, capsules

**Table 6.** Variation (%) in mass of the capsules of Enalapril Maleate (20 mg/caps)


\*Two units above the limit. \*\* Four units above the limit. Unsatisfactory. \*\*\* One unit above the limit.

**Table 7.** Variation (%) in mass of the capsules of Enalapril Maleate (20 mg/caps)

From the ten samples analyzed, seven were rated as *unsatisfactory*, considering the analysis of aspect, mass variation, identification, related substances, uniformity of dosage units, con‐ tent and labeling.

It was observed that, if only the official established procedures (description, appearance, or‐ ganoleptic characteristics and average mass) were considered from the seven samples rated as unsatisfactory, just one would be classified in this status. The remaining six samples would erroneously be rated satisfactory, meaning that would be approved for human con‐ sumption [18].

Some factors may cause deviations, inherent to the handling process of drugs in capsules, such as the loss of substance during the grinding, mixing and filling the capsules. Miscalcu‐ lations and weight of the formulation components, errors inherent to the operator and the use of damaged equipment may also compromise the process and therefore the quality of the final product [29].

The results of Omerazole and Enalapril Maleate were analyzed by ANOVA followed by Tu‐ key's test for uniformity of content and unit dose. Results were considered significantly dif‐ ferent at p < 0.05.

#### **3.2. Neutron activation analysis**

#### *3.2.1. Material and methods*

**e.** Mass Variation

234 Latest Research into Quality Control

The acceptable limit for the analysis of variation in mass of capsules, weighing less than 300 mg is ± 10% above the average mass, and it is tolerable no more than two units outside the specified limit and any unit may be above or below twice the percentages indicated. Thus, only a sample of Omeprazole was considered unsatisfactory. The results for the samples of

Omeprazole and Enalapril Maleate are presented in Tables 8 and 9, respectively [27].

**Pharmacy Omeprazole (10 mg/caps) Enalapril Maleate**

A 18.7% 7.8% B 24.0% 14.2% C 47.9% 10.6% D 15.6% 14.0% E 15.8% 46.5%

**Pharmacy average weight (mg/caps.) Lower Higher** \*A 220.4 ± 1.2 2.4 1.6 B 226.8 ± 5.8 9.5 8.7 C 210.0 ± 4.9 9.7 7.5 \*D 119.0 ± 2.1 6.5 10.9 \*\*E 120.7 ± 2.9 10.9 7.9

**Pharmacy average mass (mg/caps.) Lower Higher** \*A 108.3 ± 3.0 7.1 15.1 B 195.4 ± 1.8 4.6 3.0 C 175.9 ± 3.0 9.1 4.7 \*\*D 107.0 ± 7.0 15.3 40.8 \*\*\*E 105.0 ± 2.1 10.6 7.2

\*Two units above the limit. \*\* Four units above the limit. Unsatisfactory. \*\*\* One unit above the limit.

**Table 5.** Dose uniformity of the capsules of Omeprazole and Enalapril Maleate

\*One unit above the limit. \*\* Two units above the limit. Caps, capsules

**Table 6.** Variation (%) in mass of the capsules of Enalapril Maleate (20 mg/caps)

**Table 7.** Variation (%) in mass of the capsules of Enalapril Maleate (20 mg/caps)

**(20 mg/caps)**

All samples of Omeprazole and Enalapril Maleate were purchased in the market of the Belo Horizonte, state of Minas Gerais, Brazil from five (5) different magistral pharmacies.

Due to operational reasons, Jožef Stefan Institute (JSI) performed analyses only from three (3) different pharmacies from the sampling group. Due to same operational reasons, the JSI did not analyze the short half-lives radionuclides of elements like Al, Cl, Mg, Mn and Ti.

The samples of Omeprazole and Enalapril Maleate performed by the JSI were packed in polyethylene capsules in plastic bottle containing 20 capsules each. Whole powder mass from 20 capsules was taken to prepare homogenized samples, which was transferred in clean polyethylene bottle. In the samples of Omeprazole performed by CDTN, just one the mass of one capsule taken randomly was considered by each sample. The difference of pro‐ cedures carried out by both Institutes was due to operational reasons.

Both institutes CDTN/CNEN and JSI followed the same procedure to prepare the samples. The aliquots of each sample were manually crushed or ground using an agate mortar with pestle, whenever necessary, to avoid any contamination. In most cases, unless the amount of material did not allow it, two replicates were taken and weighed in polyethylene vials. It is relevant to emphasize that no additional chemical sample preparation was performed. At CDTN/CNEN and IJS, the samples were irradiated together with several Al–0.1% Au disks as neutron flux monitors, according to the k0-standardisation method procedure [23,24,30].

Table 10 shows the characteristics of the applied technique such as the parameters *f* (thermal to epithermal fluxes ratio) and the *α* (parameter which measures the epithermal flux devia‐ tion from the ideal (1/E) distribution), needed for the k0-method, the irradiation times and gamma spectrometry systems at each Institute.

The data presented in Tables 12, 13 and 14 cannot be compared directly because the samples analyzed are not from the batch, but the results are, in general, very similar. Most results determined by the CDTN in one capsule of Omeprazole taken randomly were also deter‐ mined by the JSI in the homogenized samples, except for the elements Br, Cr and La. The concentrations of the elements determined by both institutes have, in general, the same

Quality Control of Formulated Medicines http://dx.doi.org/10.5772/51459 237

The discussion about toxicity levels and possible consequences for humans being is very dif‐ ficult, due to the low concentration of the elements and limitations on the studies available in the literature. For most trace elements, there are just some available data on acute and chronic toxicity in experimental animals, not sufficient data to assess the risks to the human

The obtained results of samples of omeprazole and enalapril from five different magistral pharmacies of Belo Horizonte, Brazil, confirm the concern about the quality and safety for consumption of formulated medicines. They represent a preliminary part of a more com‐

From the ten samples analyzed, seven were considered unsatisfactory. Most of the problems found through analyses Omeprazole and Enalapril Maleate medicines, like the variation of active principle mass, mass variation and dosage unit, come from the inadequacy of proce‐

Problems can also be caused by the quality of the raw material used and inefficient or inexis‐ tence of test for checking the material. Diversified impurities reinforce the hypothesis that these elements are not controlled by the quality system. It also suggests that quality control over the purity of medicines in general should be established, as well as the concentration limits for the impurities, at least for some elements like As, Cd, Cu, Hg, Pb and Sn, already

The possible harmful and/or toxicological effects for the human health as a consequence of long term use of the formulated medicines represent an important concern for the authori‐ ties of the public health system. Recent cases of contamination and death in Brazil due to the consumption of inadequate formulated medicines has been enhancing the debate about the

In conclusion, the results point out the necessity of prompt and efficient actions by the au‐ thorities of the health public system to assure the quality of formulated medicines. The aim

of this work is just to provide evidences in order to contribute with this initiative.

magnitude.

**4. Conclusion**

health on a long term daily intake [22].

plete investigation, still under way.

dures for handling the ingredients in the pharmacy.

foreseen for food in the Brazilian legislation.

quality of the magistral pharmacy.

#### *3.2.2. Results and discussion*

The obtained results of NAA from the medicines Omeprazole and Enalapril Maleate are showed in the Tables 11 to 13.

The technique applied was suitable for determining 20 chemical elements – Al, Br, Ca, Cl, Co, Cr, Fe, Mg, Mn, Na, Sb, Sc, Sm, Sr, Ta, Th, Ti, U and Zn – in a large range of concentra‐ tion, without any chemical process. The elements Cl, Fe, K, Mg, Mn, Na, and Zn could be expected in this kind of samples. Other elements, not considered essential, for the human being such as As and Sr, found in lower concentration compared to Cl, Fe, K, Mg, Mn, Na, and Zn can also represent a health problem in a long term consumption. Even essential ele‐ ments were determined but in high concentrations, like Fe may be toxic.

High concentration of elements such as Cl, Ca, Mg, Na and Ti are expected because they are frequently components of excipients in the preparation of pellets. The presence of Mg is due to the excipients usually used: magnesium is a component of magnesium estearate (Mg[C18H35O2]), a lubricant for tablets and capsules and opadry, coloring agent, respectively [26]. Mg also is present in magnesium silicate (Mg3SiO4(OH)2] Na is a component of sodium laurilsulfate, ([CH3(CH2)10(CH2O)(SO3)Na] and sodium bicarbonate NaHCO3. Ca is added as excipient as calcium phosphate and Ti as titanium dioxide, TiO2. Fe comes from red iron ox‐ ide, used as excipient as well [10,13]. The impurities such as Br, Co, Cr, Hf, La, Sb, Sc, Sm, Sr, Ta, Th and U, are probably original from the raw material and/or from the process of pro‐ duction and manipulation of the medicine. All elements determined not foreseen in the orig‐ inal formula can be considered as impurities.


**Table 8.** Experimental information of neutron activation analysis

The data presented in Tables 12, 13 and 14 cannot be compared directly because the samples analyzed are not from the batch, but the results are, in general, very similar. Most results determined by the CDTN in one capsule of Omeprazole taken randomly were also deter‐ mined by the JSI in the homogenized samples, except for the elements Br, Cr and La. The concentrations of the elements determined by both institutes have, in general, the same magnitude.

The discussion about toxicity levels and possible consequences for humans being is very dif‐ ficult, due to the low concentration of the elements and limitations on the studies available in the literature. For most trace elements, there are just some available data on acute and chronic toxicity in experimental animals, not sufficient data to assess the risks to the human health on a long term daily intake [22].

#### **4. Conclusion**

tion from the ideal (1/E) distribution), needed for the k0-method, the irradiation times and

The obtained results of NAA from the medicines Omeprazole and Enalapril Maleate are

The technique applied was suitable for determining 20 chemical elements – Al, Br, Ca, Cl, Co, Cr, Fe, Mg, Mn, Na, Sb, Sc, Sm, Sr, Ta, Th, Ti, U and Zn – in a large range of concentra‐ tion, without any chemical process. The elements Cl, Fe, K, Mg, Mn, Na, and Zn could be expected in this kind of samples. Other elements, not considered essential, for the human being such as As and Sr, found in lower concentration compared to Cl, Fe, K, Mg, Mn, Na, and Zn can also represent a health problem in a long term consumption. Even essential ele‐

High concentration of elements such as Cl, Ca, Mg, Na and Ti are expected because they are frequently components of excipients in the preparation of pellets. The presence of Mg is due to the excipients usually used: magnesium is a component of magnesium estearate (Mg[C18H35O2]), a lubricant for tablets and capsules and opadry, coloring agent, respectively [26]. Mg also is present in magnesium silicate (Mg3SiO4(OH)2] Na is a component of sodium laurilsulfate, ([CH3(CH2)10(CH2O)(SO3)Na] and sodium bicarbonate NaHCO3. Ca is added as excipient as calcium phosphate and Ti as titanium dioxide, TiO2. Fe comes from red iron ox‐ ide, used as excipient as well [10,13]. The impurities such as Br, Co, Cr, Hf, La, Sb, Sc, Sm, Sr, Ta, Th and U, are probably original from the raw material and/or from the process of pro‐ duction and manipulation of the medicine. All elements determined not foreseen in the orig‐

> 20.4 0.197

Genie 2000 (CANBERRA) HyperLab Kayzero for Windows, V.2.42

Sample mass (mg) 200-250 240-250

Irradiation time (h) 8 13 Detector nominal efficiency (%) 50 40

**INSTITUTE CDTN/CNEN JSI**

6.4x 1011 1.1x 1012

28.6 - 0.011

Genie 2000 (CANBERRA) HyperLab Kayzero for Windows, V.2.42

ments were determined but in high concentrations, like Fe may be toxic.

gamma spectrometry systems at each Institute.

inal formula can be considered as impurities.

Thermal Flux (neutrons cm-2 s-1)

k0-standardisation parameters f α

Software used for: Acquisition spectra Spectra Analysis Concentration calculation

**Table 8.** Experimental information of neutron activation analysis

*3.2.2. Results and discussion*

236 Latest Research into Quality Control

showed in the Tables 11 to 13.

The obtained results of samples of omeprazole and enalapril from five different magistral pharmacies of Belo Horizonte, Brazil, confirm the concern about the quality and safety for consumption of formulated medicines. They represent a preliminary part of a more com‐ plete investigation, still under way.

From the ten samples analyzed, seven were considered unsatisfactory. Most of the problems found through analyses Omeprazole and Enalapril Maleate medicines, like the variation of active principle mass, mass variation and dosage unit, come from the inadequacy of proce‐ dures for handling the ingredients in the pharmacy.

Problems can also be caused by the quality of the raw material used and inefficient or inexis‐ tence of test for checking the material. Diversified impurities reinforce the hypothesis that these elements are not controlled by the quality system. It also suggests that quality control over the purity of medicines in general should be established, as well as the concentration limits for the impurities, at least for some elements like As, Cd, Cu, Hg, Pb and Sn, already foreseen for food in the Brazilian legislation.

The possible harmful and/or toxicological effects for the human health as a consequence of long term use of the formulated medicines represent an important concern for the authori‐ ties of the public health system. Recent cases of contamination and death in Brazil due to the consumption of inadequate formulated medicines has been enhancing the debate about the quality of the magistral pharmacy.

In conclusion, the results point out the necessity of prompt and efficient actions by the au‐ thorities of the health public system to assure the quality of formulated medicines. The aim of this work is just to provide evidences in order to contribute with this initiative.


**Pharmacy**

Quality Control of Formulated Medicines http://dx.doi.org/10.5772/51459 239

**Element A B C**

Mo 0.34 ± 0.05 DL ± DL

\* DL – Lower than the Detection Limit

\* DL – Lower than the Detection Limit

**Table 11.** Elemental concentration (mg.kg-1) for Omeprazole (JSI)

**Table 12.** Elemental concentration (mg.kg-1) for Enalapril Maleate (JSI)

Co 0.18 ± 0.01 0.016 ± 0.001 0.51 ± 0.02 Cr 0.52 ± 0.03 0.26 ± 0.02 0.59 ± 0.04 Fe 102 ± 4 53 ± 2 265 ± 9 Hf 0.020 ± 0.001 0.009 ± 0.001 0.021 ± 0.002 La 0.019 ± 0.002 0.030 ± 0.004 0.040 ± 0.002

Na 6616 ± 232 5988 ± 210 4836 ± 169 Sb 0.014 ± 0.001 0.012 ± 0.001 0.011 ± 0.001 Sc 0.013 ± 0.005 0.010 ± 0.001 0.022 ± 0.001 Sm DL DL 0.0052 ± 0.0003 Sr 22.3 ± 1.1 17.4 ± 1.0 9.0 ± 1.0 Ta 0.34 ± 0.01 0.076 ± 0.003 0.42 ± 0.02 Th 0.012 ± 0.002 DL 0.027 ± 0.002 U 0.09 ± 0.01 0.14 ± 0.01 0.053 ± 0.004 Zn 0.60 ± 0.1 0.4 ± 0.1 0.7 ± 0.1

**Pharmacy**

**Element A B C**

Br 0.13 ± 0.01 0.47 ± 0.02 0.24 ± 0.02 Cr 0.07 ± 0.01 0.15 ± 0.01 0.10 ± 0.01 Na 12540 ± 439 271 ± 10 72480 ± 2538 Sc 0.0009 ± 0.0001 0.0007 ± 0.0001 0.0054 ± 0.0002 Sb 0.09 ± 0.01 0.41 ± 0.02 0.04 ± 0.01 Th DL DL 0.015 ± 0.001 Zn 0.49 ± 0.04 DL 0.015 ± 0.001

**Table 9.** Elemental concentration (mg.kg-1) for Omeprazole (CDTN/CNEN)


\* DL – Lower than the Detection Limit

**Table 10.** Elemental concentration (mg.kg-1) for Omeprazole (CDTN/CNEN)



\* DL – Lower than the Detection Limit

**Pharmacy**

**Pharmacy**

Sc 0.03 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 0.02 ± 0.01 0.02 ± 0.01

Ta 0.02 ± 0.01 0.14 ± 0.01 DL 0.15 ± 0.01 0.09 ± 0.01 Ti 2748 ± 105 1483 ± 55 897 ± 34 2124 ± 79 1907 ± 71 U DL 0.22 ± 0.01 DL DL 0.4 ± 0.1 Zn 3.4 ± 0.4 1.8 ± 0.3 DL 2.9 ± 0.4 2.5 ± 0.3

**Pharmacy**

**Element A B C D E**

Sm DL 0.02 ± DL 0.02 0.01 DL Sr DL DL DL 21 ± 4 DL

**Element A B C**

Br 0.19 ± 0.01 0.17 ± 0.01 0.22 ± 0.01 Ca 16453 ± 592 14908 ± 540 12817 ± 467 Ce DL DL ± 0.09 ± 0.01

**Element A B C D E**

Br DL 0.60 ± 0.03 DL DL DL

**Table 9.** Elemental concentration (mg.kg-1) for Omeprazole (CDTN/CNEN)

**Table 10.** Elemental concentration (mg.kg-1) for Omeprazole (CDTN/CNEN)

\* DL – Lower than the Detection Limit

238 Latest Research into Quality Control

Al 532 ± 20 260 ± 10 305 ± 11 452 ± 17 335 ± 12

Ca 17740 ± 793 9379 ± 446 11230 ± 512 19600 ± 880 14260 ± 190 Cl 806 ± 48 313 ± 18 262 ± 18 490 ± 30 514 ± 31 Co 0.5 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 DL ± 0.10 ± 0.01 Cr 11.7 ± 0.5 7.9 ± 0.3 6.7 ± 0.3 11.6 ± 0.5 13 ± 1 Fe 65 ± 5 64 ± 11 47 ± 10 49 ± 11 51 ± 5 Mg 4643 ± 192 449 ± 28 390 ± 28 998 ± 57 697 ± 47 Mn 2.8 ± 0.3 1.4 ± 0.1 DL 1.8 0.2 2.2 0.2 Na 8134 ± 326 4003 ± 144 3918 ± 140 6838 ± 241 6718 ± 247 Sb 0.09 ± 0.01 0.41 ± 0.02 0.04 ± 0.01 0.06 ± 0.01 0.13 ± 0.01

**Table 11.** Elemental concentration (mg.kg-1) for Omeprazole (JSI)


\* DL – Lower than the Detection Limit

**Table 12.** Elemental concentration (mg.kg-1) for Enalapril Maleate (JSI)

#### **Acknowledgements**

The authors would like to thank Dr. Adailton Pereira de Paiva and Dr. Marco Antonio Fran‐ zero for their kind and helpful collaboration.

[8] Notícia, A. Remédio pode ter matado duas pessoas em Corupá, Norte de SC. http:// www.clicrbs.com.br/anoticia/jsp/default.jsp?uf=2&local=18&section=Geral&news‐

Quality Control of Formulated Medicines http://dx.doi.org/10.5772/51459 241

[9] G1MG. Análise parcial comprova erro em produção de remédio, diz secretaria. http://g1.globo.com/minas-gerais/noticia/2011/12/analise-parcial-comprova-erro-em-

[10] Remington, J. P, & Gennaro, A. R. The Science and Practice of Pharmacy. Philadel‐ phia College of Pharmacy and Science, Philadelphia, ed. 20, (2000). , 2077.

[11] Fergusson, J. E. The Heavy Elements: Chemistry, Environmental Impact and Health

[13] Leal, A. S, Menezes, M. Â. B. C, Vermaercke, R. R, & Rodrigues, O. Andonie; Sneyers, L. Investigation of chemical impurities in formulations, phytotherapics and polyvita‐ minic medicines by k0-instrumental neutron activation analysis. Nucl. Instrum.

[14] Leal, A. S, Menezes, M. Â. B. C, Vermaercke, P, Sneyers, L, & Jensen, C. A compara‐ tive neutron activation analysis study of common generic manipulated and reference

[15] BrasilResolução RDC n° 67, de 8 de outubro de (2007). Diário Oficial da União, Poder

[16] BrasilResolução RDC n° 87, de 21 de novembro de (2008). Diário Oficial da União,

[17] BrasilResolução RDC n° 21, de 20 de maio de (2009). Diário Oficial da União, Poder

[18] Wang, T, Wu, J, Hartman, R, Jia, X, & Egan, R. S. A multi-element ICP-MS survey method as an alternative to the heavy metals limit test for pharmaceutical materials.

[19] Buurma, H, Smet, P. A. G. M, Hoff, O. P, Sysling, H, Storimans, M, & Egberts, A. C. G. Frequency, nature and determinants of pharmacy compounded medicines in Dutch community pharmacies. Pharmacy World & Science, Netherlands, n. 6,

[20] Almeida, M. L, & Filho, A. P. N. Análise das cápsulas manipuladas segundo a RDC 67/2007 da ANVISA/MS para a garantia da qualidade. Rev. Bras. Farm., n. 3, (2010). ,

[21] Marinho, F. D. M. Zanon, J.C.C; Sakurai, E. Reis, I.A.; Lima, A.A.; Soares, C.D.V. Quality evaluation of simvastatin compounded capsules. Brazilian Journal of Phar‐

Journal of Pharmaceutical and Biomedical Analysis. 23, (2000). , 867-890.

medicines commercialized in Brazil. App. Rad. Isotop. 66, (2008). , 1307-1312.

producao-de-remedio-diz-secretaria.html (Acessed 23 Dec. 2011).

[12] World Health Organization (WHO)www.who.intAcessed 03 Mar. (2010).

ID=a3497429.xmlAcessed 10 Oct. (2011).

Effects. Pergamon, Oxford, (1990). , 614.

Methods Phys. Res. A 564, (2006). , 729-732.

Executivo, Brasília, DF, 09 Oct. 2007. Seção I, , 29-58.

Executivo, Brasília, DF, 21 May 2009, Seção I, , 53.

maceutical Sciences, n. 3, (2011). , 47, 495-502.

(2003). , 25, 280-287.

91, 119-125.

Poder Executivo, Brasília, DF, 24 Nov. 2008, Seção I, , 58-59.

#### **Author details**

Alexandre S. Leal1\*, Maria Ângela de B. C. Menezes1 , Ilza Dalmázio1 , Fernanda P. Sepe1 , Tatiana C. B. Gomes1 , Amalia S. Santana2 , Luzia H. da Cunha2 and Radojko Jaćimović<sup>3</sup>

\*Address all correspondence to: asleal@cdtn.br

1 Centro de Desenvolvimento da Tecnologia Nuclear/Comissão Nacional de Energia Nucle‐ ar (CDTN/CNEN), Belo Horizonte, Brazil

2 Fundação Estadual Ezequiel Dias (FUNED), Belo Horizonte, Brazil

3 Jožef Stefan Institute (JSI), Ljubljana, Slovenia

#### **References**


[8] Notícia, A. Remédio pode ter matado duas pessoas em Corupá, Norte de SC. http:// www.clicrbs.com.br/anoticia/jsp/default.jsp?uf=2&local=18&section=Geral&news‐ ID=a3497429.xmlAcessed 10 Oct. (2011).

**Acknowledgements**

240 Latest Research into Quality Control

**Author details**

Tatiana C. B. Gomes1

**References**

2011).

zero for their kind and helpful collaboration.

Alexandre S. Leal1\*, Maria Ângela de B. C. Menezes1

\*Address all correspondence to: asleal@cdtn.br

3 Jožef Stefan Institute (JSI), Ljubljana, Slovenia

ar (CDTN/CNEN), Belo Horizonte, Brazil

, Amalia S. Santana2

2 Fundação Estadual Ezequiel Dias (FUNED), Belo Horizonte, Brazil

%2009%20Dez%202012.pdfAcessed 23 May 2012)., 37.

cípio de Toledo. Infarma, Brasília, DF, n. 1-2, (2004). , 16, 77-80.

The authors would like to thank Dr. Adailton Pereira de Paiva and Dr. Marco Antonio Fran‐

1 Centro de Desenvolvimento da Tecnologia Nuclear/Comissão Nacional de Energia Nucle‐

[1] Formulário Nacional Da Farmacopeia BrasileiraEd. 2. (2011). http://www.anvi‐ sa.gov.br/farmacopeiabrasileira/arquivos/FNFB%202%20Vers%C3%A3o%20DICOL

[2] BrasilLei nº 5991, de 17 de dezembro de (1973). Dispõe sobre o controle sanitário do comércio de drogas, medicamentos, insumos farmacêuticos e correlatos, e dá outras providências. Diário Oficial da República Federativa do Brasil, Brasília, 19 Dec. 1973.

[3] Szatkowski, L. T. D, & Oliveira, C. L. O uso de medicamentos manipulados no muni‐

[5] Agência Nacional De Vigilância Sanitária (ANVISA)Boletim Informativo. Ed. 56. Jun. 2005. http://www.anvisa.gov.br/divulga/public/boletim/56\_05.pdfAcessed (26 Sep.

[6] Gisele, H. Comunicação na Oficina: "Medicamentos Manipulados: um Desafio para o Sistema Nacional de Vigilância Sanitária". Rio de Janeiro, 08 and 09 May (2006).

[7] UOLFarmácia assume culpa por contaminação que causou doping de Cielo. http:// esporte.uol.com.br/natacao/ultimas-noticias/2011/07/01/farmacia-assume-culpa-por-

contaminacao-que-causou-doping-de-cesar-cielo.htmAcessed (10 Oct. 2011).

[4] Conselho Federal De FarmáciaHttp://Www.Cff.Org.Br/Acessed 01 Oct. (2011).

, Ilza Dalmázio1

, Luzia H. da Cunha2

, Fernanda P. Sepe1

and Radojko Jaćimović<sup>3</sup>

,


[22] Oskarsson, A, & Sandstrom, B. A Nordic Project-risk evaluation of essencial trace ele‐ ments: esencial versus toxic levels of intake. Analyst., (1995). , 120, 911-912.

**Section 4**

**Quality Control in Radiology and Clinical**

**Imaging**


**Quality Control in Radiology and Clinical Imaging**

[22] Oskarsson, A, & Sandstrom, B. A Nordic Project-risk evaluation of essencial trace ele‐ ments: esencial versus toxic levels of intake. Analyst., (1995). , 120, 911-912.

[23] De Corte, F, & The, k. standardisation method; a move to the optimisation of neutron activation analysis. Ed. Ryksuniversiteit Gent, Faculteit Van de Wetenschappen,

[24] De Wispelaere, F, De Corte, F, Bossus, D. A. W, Swagten, J. J. M. G, & Vermaercke, P. Re-determination and re-evaluation of the f and α parameters in channels Y4 and S84 of the BR1 reactor for use in K0- NAA at DSM research. Nuclear Instruments and

[25] Vermaercke, P, Robouch, P, Eguskiza, M, De Corte, F, Kennedy, G, Smodis, B, Yone‐ zawa, C, Matsue, H, Lin, X, Blaauw, M, & Kucêra, J. Characterization of synthetic multi-element standards SMELS used for validation of K0- NAA. Nuclear Instru‐

[26] Nordberg, G. F, et al. Handbook on the Toxicology of Metals. (2007). http:// pt.scribd.com/doc/59673422/Toxic-Metals-HandbookAcessed 01 Oct. 2011).

[27] Farmacopéia BrasileiraAgência Nacional de Vigilância Sanitária, Ed. 5, Brasília,

[28] USP 32 (The United States Pharmacopeia 32)United States Pharmacopeial Conven‐

[29] Futuro, D. O, & Silva, R. F. Uso de ferramentas de controle estatístico para o entendi‐ mento do processo de produção de cápsulas em farmácias magistrais. XXVIII Encon‐ tro Nacional de Engenharia de Produção Foz do Iguaçu, Paraná, Brasil, Oct. (2007). ,

[30] Menezes, M. Â. B. C, Jacimovic, R, & Optimised, k. instrumental neutron activation method using the TRIGA MARK I IPR-R1 reactor at CDTN/CNEN, Belo Horizonte, Brazil. Nuclear Instruments and Methods in Physics Research Section A, (2006). , 564,

ments and Methods in Physics Research Section A, (2006). , 564, 675-682.

Methods in Physics Research Section A, (2005). , 564, 636-640.

(1986). , 464.

242 Latest Research into Quality Control

(2010).

09-11.

707-715.

tion, (2009).

**Chapter 12**

**Quality Assurance in Diagnostic Medical Exposures in**

It is well known that medical expsoure procedures such as diagnostic radiology, nuclear medicine and radiotherapy remains the largest source of man made exposure to ionising ra‐ diation and continues to grow substantially. This makes the role of quality assurance (QA), an important tool in medical exposure procedures. This paper reviews the future of quality assurance in diagnostic medical exposures in Ghana from the perspective of a Medical Phys‐ icist, since a viable QA programme must be developed under the guidance and supervision of a medical physicist who is qualified in this area of expertise by education, training and experience. The Medical Physicist is expected to give guidance and supervision to the Tech‐ nologists and other staff to execute the programme but should be prepared to perform high‐ er level QA procedures as required. The focus of this review is on diagnostic radiology since it is the dominant mode of medical exposure as compared to nuclear medicine and radio‐ therapy procedures in Ghana as per the database of the Regulatory Authority Information System "(RAIS)" of the Radiation Protection Institute. It is also worth noting that most of the issues under consideration for discussion mirrors similar conditions in many developing countries. The main goal of a diagnostic quality assurance programme is to make sure that radiation doses to patients, staff and public are as low as reasonable achievable (ALARA) consistent with high quality diagnostic images of patients. An adequate diagnostic QA pro‐ gram involves periodic checks of all major components in the respective diagnostic imaging modalities. On the other hand, an optimum QA programme for any individual diagnostic facility will depend on some items such as the type of procedures performed, type of equip‐ ment utilized, patient workload, etc. The current scope of diagnostic imaging procedures in Ghana covers conventional, fluoroscopy, dental, computed tomography, interventional pro‐ cedures and nuclear medicine scans. Interventional radiology procedures performed are

> © 2012 Inkoom; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Inkoom; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Ghana - A Medical Physicist's Perspective**

Additional information is available at the end of the chapter

Stephen Inkoom

**1. Introduction**

http://dx.doi.org/10.5772/51445

## **Quality Assurance in Diagnostic Medical Exposures in Ghana - A Medical Physicist's Perspective**

Stephen Inkoom

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51445

#### **1. Introduction**

It is well known that medical expsoure procedures such as diagnostic radiology, nuclear medicine and radiotherapy remains the largest source of man made exposure to ionising ra‐ diation and continues to grow substantially. This makes the role of quality assurance (QA), an important tool in medical exposure procedures. This paper reviews the future of quality assurance in diagnostic medical exposures in Ghana from the perspective of a Medical Phys‐ icist, since a viable QA programme must be developed under the guidance and supervision of a medical physicist who is qualified in this area of expertise by education, training and experience. The Medical Physicist is expected to give guidance and supervision to the Tech‐ nologists and other staff to execute the programme but should be prepared to perform high‐ er level QA procedures as required. The focus of this review is on diagnostic radiology since it is the dominant mode of medical exposure as compared to nuclear medicine and radio‐ therapy procedures in Ghana as per the database of the Regulatory Authority Information System "(RAIS)" of the Radiation Protection Institute. It is also worth noting that most of the issues under consideration for discussion mirrors similar conditions in many developing countries. The main goal of a diagnostic quality assurance programme is to make sure that radiation doses to patients, staff and public are as low as reasonable achievable (ALARA) consistent with high quality diagnostic images of patients. An adequate diagnostic QA pro‐ gram involves periodic checks of all major components in the respective diagnostic imaging modalities. On the other hand, an optimum QA programme for any individual diagnostic facility will depend on some items such as the type of procedures performed, type of equip‐ ment utilized, patient workload, etc. The current scope of diagnostic imaging procedures in Ghana covers conventional, fluoroscopy, dental, computed tomography, interventional pro‐ cedures and nuclear medicine scans. Interventional radiology procedures performed are

© 2012 Inkoom; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Inkoom; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

quite few but the future looks promising in this field. The performance of QA practices are done on three fronts; namely at the hospital, equipment engineers and the Regulatory Au‐ thority (RA). The hospital based QA are done mainly by the Radiographic Technologist through their routine equipment warm ups and minor quality checks. Equipment Engineers perform engineer related QA checks through installation and acceptance testing, perform‐ ance tests and periodic preventative maintenance procedures. On the other hand, the RA is largely in charge of major QA procedures through it's on site safety assessment inspections by assessing the compliance of the equipment within regulatory requirements. This is large‐ ly so because the RA has the technical expertise and equipment. Due to the expansion of di‐ agnostic imaging procedures in medicine coupled with rapid technological advances, the availability of qualified and trained personnel is crucial if the desired quality is to be ach‐ ieved. Some measures have been put in place for human resource development, but there is room for improvement. On the way forward, there is a strong need for the establishment of National Quality Control Centre for Diagnostic Radiology. This body must be equipped with the requisite state of the art equipment, highly qualified and trained personnel in order to coordinate all QA activities in the country. Such a body can initiate some guidelines on the minimum instrumentation requirements for all imaging modalities. Nevertheless, a good QA programmme is not a guarantee for the assurance of the radiation safety of pa‐ tients, staff and public. What is also needed is a separate radiation safety programme, which is very essential in every diagnostic imaging facility and must also be under the direction of a qualified expert in radiation protection.

involves the use of intense radiation beams and high-activity sources for the treatment of

Quality Assurance in Diagnostic Medical Exposures in Ghana - A Medical Physicist's Perspective

http://dx.doi.org/10.5772/51445

247

Source Annual per caput effective dose (mSv) Contribution (%)

**Table 1.** Sources of ionizing radiation and the annual per caput effective dose to the global population.

**Figure 1.** An illustration of (a) diagnostic radiology, (b) nuclear medicine and (c) radiation therapy procedures.

This makes the role of (QA), an important tool in medical exposure procedures. Quality as‐ surance procedures must aim at to produce images of optimal diagnostic quality while en‐ suring that the radiation exposures to patients, staff and the public are kept as low as practicable. In achieving this goal, QA procedures if well implemented would ensure that any problem in the imaging chain will be dealt without a compromise in the diagnostic quality of the images. The World Health Organization (WHO), (WHO, 1982), indicates that achieving adequate diagnostic information and least possible exposure of the patient to radi‐ ation should be done at the lowest possible cost. An adequate diagnostic QA program in‐ volves periodic checks of all major components in the respective diagnostic imaging modalities. On the other hand, an optimum QA programme for any individual diagnostic facility will depend on some items such as the type of procedures performed, type of equip‐ ment utilized, patient workload, etc. Any QA procedure must be in agreement with the rele‐ vant national and international legislation or regulations on the protection and safe uses of ionising radiation. Another form of QA is to establish clinical audit, which is an essential

Natural background 2.4 79 Diagnostic medical radiology 0.62 20 Diagnostic dental radiology 0.0018 <0.1 Nuclear medicine 0.031 1.1 Fallout 0.005 <0.2 Total 3.1 100

Source: UNSCEAR 2008 report on medical radiation exposures. (UNSCEAR, 2008).

many types of cancer.

#### **2. Overview of quality assurance**

The 2008 United Nations Scientific Committee on the Effects of Atomic Radiation (UN‐ SCEAR) report on medical exposures from the assessment of the global population dose from medical exposures for the period 1997-2007 indicates that medical exposure remains the largest source of man made exposure to ionizing radiation and continues to grow sub‐ stantially. (UNSCEAR, 2008). A summary of the annual per caput effective dose to the glob‐ al population due to all sources of ionizing radiation is illustrated in Table 1.

It is evident that diagnostic examinations result in a per caput effective dose of 0.66 mSv, while medical exposures now contribute around 20% of the average annual per caput dose to the global population. Medical exposures are defined as; (i) exposure of patients as part of their medical diagnosis or treatment; (ii) exposure of individuals as part of health screening programmes; and (iii) exposure of healthy individuals or patients voluntarily participating in medical, biomedical, diagnostic or therapeutic research programmes. These exposures in‐ clude diagnostic radiology, nuclear medicine and radiation therapy (Fig. 1), out of which di‐ agnostic radiology accounts for the largest contribution. Diagnostic radiology generally refers to the analysis of images obtained using x-rays. In nuclear medicine, a radiopharma‐ ceutical is administered to the patient and concentrates primarily in a specific region of the body which allows: (i) external imaging of the body to evaluate structure and/or function, and (ii) or delivery of a large radiation dose to control a specific disease. Radiation therapy involves the use of intense radiation beams and high-activity sources for the treatment of many types of cancer.

quite few but the future looks promising in this field. The performance of QA practices are done on three fronts; namely at the hospital, equipment engineers and the Regulatory Au‐ thority (RA). The hospital based QA are done mainly by the Radiographic Technologist through their routine equipment warm ups and minor quality checks. Equipment Engineers perform engineer related QA checks through installation and acceptance testing, perform‐ ance tests and periodic preventative maintenance procedures. On the other hand, the RA is largely in charge of major QA procedures through it's on site safety assessment inspections by assessing the compliance of the equipment within regulatory requirements. This is large‐ ly so because the RA has the technical expertise and equipment. Due to the expansion of di‐ agnostic imaging procedures in medicine coupled with rapid technological advances, the availability of qualified and trained personnel is crucial if the desired quality is to be ach‐ ieved. Some measures have been put in place for human resource development, but there is room for improvement. On the way forward, there is a strong need for the establishment of National Quality Control Centre for Diagnostic Radiology. This body must be equipped with the requisite state of the art equipment, highly qualified and trained personnel in order to coordinate all QA activities in the country. Such a body can initiate some guidelines on the minimum instrumentation requirements for all imaging modalities. Nevertheless, a good QA programmme is not a guarantee for the assurance of the radiation safety of pa‐ tients, staff and public. What is also needed is a separate radiation safety programme, which is very essential in every diagnostic imaging facility and must also be under the direction of

The 2008 United Nations Scientific Committee on the Effects of Atomic Radiation (UN‐ SCEAR) report on medical exposures from the assessment of the global population dose from medical exposures for the period 1997-2007 indicates that medical exposure remains the largest source of man made exposure to ionizing radiation and continues to grow sub‐ stantially. (UNSCEAR, 2008). A summary of the annual per caput effective dose to the glob‐

It is evident that diagnostic examinations result in a per caput effective dose of 0.66 mSv, while medical exposures now contribute around 20% of the average annual per caput dose to the global population. Medical exposures are defined as; (i) exposure of patients as part of their medical diagnosis or treatment; (ii) exposure of individuals as part of health screening programmes; and (iii) exposure of healthy individuals or patients voluntarily participating in medical, biomedical, diagnostic or therapeutic research programmes. These exposures in‐ clude diagnostic radiology, nuclear medicine and radiation therapy (Fig. 1), out of which di‐ agnostic radiology accounts for the largest contribution. Diagnostic radiology generally refers to the analysis of images obtained using x-rays. In nuclear medicine, a radiopharma‐ ceutical is administered to the patient and concentrates primarily in a specific region of the body which allows: (i) external imaging of the body to evaluate structure and/or function, and (ii) or delivery of a large radiation dose to control a specific disease. Radiation therapy

al population due to all sources of ionizing radiation is illustrated in Table 1.

a qualified expert in radiation protection.

246 Latest Research into Quality Control

**2. Overview of quality assurance**


**Table 1.** Sources of ionizing radiation and the annual per caput effective dose to the global population.

**Figure 1.** An illustration of (a) diagnostic radiology, (b) nuclear medicine and (c) radiation therapy procedures.

This makes the role of (QA), an important tool in medical exposure procedures. Quality as‐ surance procedures must aim at to produce images of optimal diagnostic quality while en‐ suring that the radiation exposures to patients, staff and the public are kept as low as practicable. In achieving this goal, QA procedures if well implemented would ensure that any problem in the imaging chain will be dealt without a compromise in the diagnostic quality of the images. The World Health Organization (WHO), (WHO, 1982), indicates that achieving adequate diagnostic information and least possible exposure of the patient to radi‐ ation should be done at the lowest possible cost. An adequate diagnostic QA program in‐ volves periodic checks of all major components in the respective diagnostic imaging modalities. On the other hand, an optimum QA programme for any individual diagnostic facility will depend on some items such as the type of procedures performed, type of equip‐ ment utilized, patient workload, etc. Any QA procedure must be in agreement with the rele‐ vant national and international legislation or regulations on the protection and safe uses of ionising radiation. Another form of QA is to establish clinical audit, which is an essential tool for quality improvement in any diagnostic centre. For instance, the European Council directive (Commission of the European Communities. 1997) defines clinical audit as: "a sys‐ tematic examination or review of medical radiological procedures which seeks to improve the quality and the outcome of patient care, through structured review whereby radiological practices, procedures, and results are examined against agreed standards for good medical radiological procedures, with modifications of the practices where indicated and the appli‐ cation of new standards if necessary."

equipment within regulatory requirements. This is largely so because the RA has the requi‐

Quality Assurance in Diagnostic Medical Exposures in Ghana - A Medical Physicist's Perspective

http://dx.doi.org/10.5772/51445

249

In Ghana, Medical Physicists are engaged in service, teaching, research and administration activities. They perform some of the tasks outlined by the International Organisation for Medical Physics (IOMP) in its definition of who a Medical Physicist is and the roles and re‐

**•** teaching principles of medical physics to physicians, residents, graduate students, medi‐ cal students, technologists, and other health care professionals by means of lectures, prob‐

**•** conducting research into various human disorders, illnesses and disabilities, develop in‐ strumentation, mathematical analysis and applications of computers in medicine; investi‐ gating biophysical techniques associated with any branch of medicine. Research is very

**•** responsible for ensuring the quality, safety testing and correct maintenance of all radia‐ tion emitting devices in order to get an accurate diagnosis of illnesses. Medical Physicists also involved in the formulation of radiation protection guides and procedures specific to clinical environment and producing protocols to minimize radiation exposure of patients,

**•** in administration, they supervise and manage radiation workers and other health profes‐

**•** participating in and contributing to the development and implementation of national and **•** prepares guidance on education and training drawing-up standards and guidance relat‐

Ghana is involved in several International Atomic Energy Agency (IAEA) Technical and Re‐

**•** RAF/9/033 - Strengthening Radiological Protection of Patients and Medical Exposure Con‐

**•** RAF/9/034 - Establishment of National Capabilities for Response to a Radiological and

**•** RAF/9/032 - Development of Technical Capabilities for the Protection of Health and Safe‐

**•** RAF/9/027 - National Regulatory Control and Occupational Radiation Protection Pro‐

**•** RAF/9/031 - Strengthening National Regulatory Infrastructure for the Control of Radia‐

**•** RAF/9/035 - Education and Training in Support of Radiation Protection Infrastructure.

search Projects. Some of the Projects in which Medical Physicists are involved are:

sponsibilities (International Organisation for Medical Physics, 2010) such as:

important for advancement of medical physics as a profession and science.

**•** preparing, publishing and presenting scientific papers and reports

site technical expertise and equipment.

lem solving, and laboratory sessions.

staff and the general public.

sional workers.

trol.

grammes.

tion Sources.

ing to medical devices.

Nuclear Emergency.

ty of Workers Exposed to Ionizing Radiation.

This paper reviews the future of quality assurance in diagnostic medical exposures in Ghana from the perspective of a Medical Physicist, since a viable QA programme must be devel‐ oped under the guidance and supervision of a medical physicist who is qualified in this area of expertise by education, training and experience. The Medical Physicist is expected to give guidance and supervision to the Technologists and other staff to executive the programme but should be prepared to perform higher level QA procedures as required.

#### **3. Current status of quality assurance in diagnostic medical exposures**

The types of diagnostic medical exposure procedures in Ghana are;


In addition to the above procedures for diagnosis, some hospitals perform interventional or in‐ vasive procedures on a limited scale. On the other hand, the types of medical exposure in nu‐ clear medicine procedures are the use of gamma camera and single photon emission computed tomography (SPECT/CT) for imaging various organs. Plain radiography (screen-film and dig‐ ital systems) is the dominant mode of all the diagnostic medical procedures, accounting for more than 80% of the total contribution of all the imaging modalities. (RAIS, 2011).

The performance of QA practices are done on three fronts; namely at the hospital, equip‐ ment/service engineers and the (RA). The hospital based QA are done mainly by the Radiog‐ raphers/Radiologic Technologist through their routine equipment warm ups and minor quality checks. In this scenario, a qualified Medical Physicist with the requisite expertise must supervise such QA procedures. Unfortunately, there are not many Medical Physicists in diagnostic departments as compared to radiation therapy centres. Equipment/service en‐ gineers perform engineer related QA checks through installation and acceptance testing, performance tests and periodic preventative maintenance procedures as well as when there is equipment down time. On the other hand, the RA is largely in charge of major QA proce‐ dures through it's on site safety assessment inspections by assessing the compliance of the equipment within regulatory requirements. This is largely so because the RA has the requi‐ site technical expertise and equipment.

tool for quality improvement in any diagnostic centre. For instance, the European Council directive (Commission of the European Communities. 1997) defines clinical audit as: "a sys‐ tematic examination or review of medical radiological procedures which seeks to improve the quality and the outcome of patient care, through structured review whereby radiological practices, procedures, and results are examined against agreed standards for good medical radiological procedures, with modifications of the practices where indicated and the appli‐

This paper reviews the future of quality assurance in diagnostic medical exposures in Ghana from the perspective of a Medical Physicist, since a viable QA programme must be devel‐ oped under the guidance and supervision of a medical physicist who is qualified in this area of expertise by education, training and experience. The Medical Physicist is expected to give guidance and supervision to the Technologists and other staff to executive the programme

**3. Current status of quality assurance in diagnostic medical exposures**

In addition to the above procedures for diagnosis, some hospitals perform interventional or in‐ vasive procedures on a limited scale. On the other hand, the types of medical exposure in nu‐ clear medicine procedures are the use of gamma camera and single photon emission computed tomography (SPECT/CT) for imaging various organs. Plain radiography (screen-film and dig‐ ital systems) is the dominant mode of all the diagnostic medical procedures, accounting for

The performance of QA practices are done on three fronts; namely at the hospital, equip‐ ment/service engineers and the (RA). The hospital based QA are done mainly by the Radiog‐ raphers/Radiologic Technologist through their routine equipment warm ups and minor quality checks. In this scenario, a qualified Medical Physicist with the requisite expertise must supervise such QA procedures. Unfortunately, there are not many Medical Physicists in diagnostic departments as compared to radiation therapy centres. Equipment/service en‐ gineers perform engineer related QA checks through installation and acceptance testing, performance tests and periodic preventative maintenance procedures as well as when there is equipment down time. On the other hand, the RA is largely in charge of major QA proce‐ dures through it's on site safety assessment inspections by assessing the compliance of the

more than 80% of the total contribution of all the imaging modalities. (RAIS, 2011).

but should be prepared to perform higher level QA procedures as required.

The types of diagnostic medical exposure procedures in Ghana are;

cation of new standards if necessary."

248 Latest Research into Quality Control

**•** Plain radiography

**•** Computed Tomography

**•** Mammography

**•** Fluoroscopy

**•** Dental

In Ghana, Medical Physicists are engaged in service, teaching, research and administration activities. They perform some of the tasks outlined by the International Organisation for Medical Physics (IOMP) in its definition of who a Medical Physicist is and the roles and re‐ sponsibilities (International Organisation for Medical Physics, 2010) such as:


Ghana is involved in several International Atomic Energy Agency (IAEA) Technical and Re‐ search Projects. Some of the Projects in which Medical Physicists are involved are:


**•** GHA/6/015 - Upgrading and Expansion of Radiotherapy and Nuclear Medicine Services

surance/quality control (QA/QC) programmes, including the promotion of safety culture

Quality Assurance in Diagnostic Medical Exposures in Ghana - A Medical Physicist's Perspective

http://dx.doi.org/10.5772/51445

251

**•** RAF/6/045 - To establish national and regional networks in clinical radiation oncology. To interact with National Organizations with the aim to promote the comprehensive man‐ agement of commonest cancers. Support academic education, training and accreditation, patients and personnel safety in radiotherapy improvement of documentation of clinical

The National Competent/Regulatory Authority in Ghana charged with the responsibility for authorization and inspection of practices using ionizing radiation sources and radioactive materials is the Radiation Protection Board (RPB) (Radiation Protection Instrument LI 1559, 1993). However, the operational functions of the RPB are carried out by RPI, which was es‐ tablished in 2000 to provide scientific and technical support for the enforcement of the pro‐ visions in LI 1559. Details about how the RA was established and the main activities have been described elsewhere (Inkoom et al, 2011). There are plans to establish a new Regulatory Body to regulate the peaceful uses of nuclear energy and technology which will be inde‐ pendent of any governmental agency. Currently, the RA is answerable to the Ghana Atomic Energy Commission (GAEC) which is a promoter for the peaceful uses of nuclear energy and technology and also plays the role of a regulator. However, the new RA is expected to

and innovative practices in dosimetry.

**5. Human resource development**

and Sub-Regional levels.

outcomes through regular patient assessment.

**4. Regulatory guidelines for quality assurance procedures**

be only a regulator and not a promoter of the application of nuclear technology.

The categories of Radiographic Staff available in Ghana are Radiologists, Medical Physicists, Biomedical Engineers and Radiographers/X-ray Technicians. Most of our Radiologists were trained overseas until the last few years when local training of Radiologists started and the accreditation is given by either the Ghana College of Surgeons or the West African College of Physicians and Surgeons. Similarly, the other professionals were also trained overseas. Currently, the School of Allied Health Sciences (SAHS), College of Health Sciences (CHS) of the University of Ghana (UG) is responsible for churning out medical and dental technical graduates in physiotherapy, medical laboratory science and radiography. There are plans to establish another Allied Health University and some private institutions are also running some of the programmes. A Post-Graduate School of Nuclear and Allied Sciences which was established jointly by the GAEC and UG, in co-operation with the IAEA is training the Med‐ ical Physicists, Radiation Protection Professionals, Nuclear Engineers, etc. at the National


The main objectives of some of the projects are discussed. For instance in RAF/9/033, the ob‐ jectives are to upgrade / strengthen radiological protection of the patient in medical expo‐ sures due to:


The objectives of other projects are as follows:


surance/quality control (QA/QC) programmes, including the promotion of safety culture and innovative practices in dosimetry.

**•** RAF/6/045 - To establish national and regional networks in clinical radiation oncology. To interact with National Organizations with the aim to promote the comprehensive man‐ agement of commonest cancers. Support academic education, training and accreditation, patients and personnel safety in radiotherapy improvement of documentation of clinical outcomes through regular patient assessment.

#### **4. Regulatory guidelines for quality assurance procedures**

The National Competent/Regulatory Authority in Ghana charged with the responsibility for authorization and inspection of practices using ionizing radiation sources and radioactive materials is the Radiation Protection Board (RPB) (Radiation Protection Instrument LI 1559, 1993). However, the operational functions of the RPB are carried out by RPI, which was es‐ tablished in 2000 to provide scientific and technical support for the enforcement of the pro‐ visions in LI 1559. Details about how the RA was established and the main activities have been described elsewhere (Inkoom et al, 2011). There are plans to establish a new Regulatory Body to regulate the peaceful uses of nuclear energy and technology which will be inde‐ pendent of any governmental agency. Currently, the RA is answerable to the Ghana Atomic Energy Commission (GAEC) which is a promoter for the peaceful uses of nuclear energy and technology and also plays the role of a regulator. However, the new RA is expected to be only a regulator and not a promoter of the application of nuclear technology.

#### **5. Human resource development**

**•** GHA/6/015 - Upgrading and Expansion of Radiotherapy and Nuclear Medicine Services

**•** RAF/2/008 - Strengthening and Expanding Radiopharmacy Services in Africa (AFRA)

**•** RAF/6/032 - Promoting Regional and National Quality Assurance Programmes for Medi‐

**•** RAF/6/041 - Supporting the Development of Comprehensive National Cancer Control

**•** RAF/6/045 - Enhancing Accessibility and Quality in the Care of Cancer Patients (AFRA

The main objectives of some of the projects are discussed. For instance in RAF/9/033, the ob‐ jectives are to upgrade / strengthen radiological protection of the patient in medical expo‐

**•** GHA/6/015 - To consolidate existing radiotherapy and nuclear medicine facilities at two leading Teaching Hospitals located in the southern part of the country, and establish a third one in the northern part to cater for the diagnosis, curative and palliative treatment

**•** INT/6/054 - To promote the recognition of medical physics in radiation medicine and to harmonize educational material in order to ensure safe and effective diagnosis and treat‐

**•** RAF/2/008 - To strengthen radiopharmacy in support of in vivo and in vitro nuclear medi‐

**•** RAF/6/032 - To improve the effectiveness and safety of nuclear medicine procedures by providing support for design and implementation of quality assurance (QA) programmes and by establishing training and education programmes in medical radiation physics, fo‐

**•** RAF/6/041 - To assist Member States in performing comprehensive cancer capacity need assessments and national cancer strategic planning via collaboration with IAEA, WHO, and other partners under the Programme of Action for Cancer Therapy (PACT) umbrella.

**•** RAF/6/044 - To strengthen national and regional medical physics capabilities to ensure ef‐ ficient support of cancer management in AFRA Member States and to sustain quality as‐

cusing on aspects related to the application of nuclear medicine techniques.

of cancer patients and the efficient diagnosis and management of other diseases.

**•** RAF/6/044 - Medical Physics in Support of Cancer Management (AFRA II-8)

**i.** Diagnostic Radiology and Interventional Radiological procedures

**•** INT/6/054 - Strengthening Medical Physics in Radiation Medicine

cal Physics in Nuclear Medicine (AFRA II-7)

**ii.** Nuclear Medicine procedures

The objectives of other projects are as follows:

**iii.** Radiotherapy practice

ment of patients.

cine in Africa.

Programmes

250 Latest Research into Quality Control

II-10)

sures due to:

The categories of Radiographic Staff available in Ghana are Radiologists, Medical Physicists, Biomedical Engineers and Radiographers/X-ray Technicians. Most of our Radiologists were trained overseas until the last few years when local training of Radiologists started and the accreditation is given by either the Ghana College of Surgeons or the West African College of Physicians and Surgeons. Similarly, the other professionals were also trained overseas. Currently, the School of Allied Health Sciences (SAHS), College of Health Sciences (CHS) of the University of Ghana (UG) is responsible for churning out medical and dental technical graduates in physiotherapy, medical laboratory science and radiography. There are plans to establish another Allied Health University and some private institutions are also running some of the programmes. A Post-Graduate School of Nuclear and Allied Sciences which was established jointly by the GAEC and UG, in co-operation with the IAEA is training the Med‐ ical Physicists, Radiation Protection Professionals, Nuclear Engineers, etc. at the National and Sub-Regional levels.

#### **6. Recent trends in quality assurance**

The increasing expansion of diagnostic imaging procedures in medicine coupled with rap‐ id technological advances makes the availability of qualified and trained personnel to be very crucial if the desired quality is to be achieved. This come with a lot challenges to the medical imaging community. This offers practitioners the opportunity to continually un‐ dergo retraining and other continuous professional development programmes in their re‐ spective fields. Also with the emergence of picture archiving and communication system (PACS) in many hospitals, there is the need for the development of appropriate on line QA procedures and in corporating them into hospital PACS systems. Special attention must also be given to the emergence of digital technology over the last decade as one of the greatest technological advances in medical imaging. This new technolgy poses a great challenge in medical imaging, requiring re-training of staff on the safe use of equipment and radiation protection issues. In Ghana for instance, the RPI of GAEC, in collaboration with the IAEA, has in the previous years developed a lot of expertise in the training of occupationally exposed workers in Ghana and the rest of Africa, spanning a period of al‐ most two decades (Boadu et al. 2011). This local expertise in training can be tapped. In this regard, a critical review of all QA procedures that were developed for screen-film systems needs special attention.

in all major hospitals which have a myriad of imaging modalities. The Ghana Society of Medical Physics, RA, Ministry of Health and other stakeholders must initiate procedures for the establishment of Medical Physics Departments in such hospitals. This would give the necessary recognition to the profession of Medical Physics in Ghana, which has been given recognition by the International Labour Organization (ILO) in its International Standard Classification of Occupations (ISCO) (ILO, 2008). With this recognition, Medical Physics has been accepted as modern applied branch of physics. Clinical audit should al‐

Quality Assurance in Diagnostic Medical Exposures in Ghana - A Medical Physicist's Perspective

http://dx.doi.org/10.5772/51445

253

As the uses of ionizing radiation continue to increase in medicine, it is also expected that the services of Medical Physicist would increase. As such, more physicists would be re‐ quired to be trained in subsequent years. Appropriate accreditation bodies charged with issuing accreditation certificates, for a period of years must be put in place to regulate the

The role of an effective QA programme in any diagnostic department cannot be overem‐ phasized especially if the desired quality of producing good diagnostic images and the least radiation exposure are to be achieved. Nevertheless, a good QA programmme is not a guarantee for the assurance of the radiation safety of patients, staff and public. What is also needed is a separate radiation safety programme, which is very essential in every di‐ agnostic imaging facility and must also be under the direction of a qualified expert in ra‐ diation protection or a Medical Physicist expert. With significant contributions in clinical service, education, and research, Medical Physics continues to grow in importance both as a profession and as science, driven by the technological developments of societies in gen‐

The support received from the Radiation Protection Institute of Ghana Atomic Energy Com‐

Radiation Protection Institute, Ghana Atomic Energy Commission, Legon, Accra, Ghana

profession of Medical Physics and maintain international standards of practice.

so be incorporated in the overall QA procedures in the country.

**8. Conclusion**

eral and medicine in particular.

**Acknowledgements**

mission is appreciated.

**Author details**

Stephen Inkoom

#### **7. The way forward**

Various practitioners in the medical imaging community must brace themselves in order to face challenges of technological developments. With the advent of digital radiography: advances in computed radiography, direct digital radiography, digital subtraction angiog‐ raphy, new digital receivers, image processing techniques, computer applications in radi‐ ology and PACS offers enormous challenges. The advantages of digital technology:postprocessing capabilities, decreased costs, multiple viewing options, electronic transfer, possibilities of archiving, wide dynamic range of flat panel detectors and increased detec‐ tion quantum efficiency has led to a high demand of this technology by the medical imag‐ ing community. Therefore, the development of the requisite human resource must be continued and sustained in order to deal with the challenges.

There is a strong need for the establishment of National Quality Control Centre for Diag‐ nostic Radiology. This body must be equipped with the requisite state of the art equip‐ ment, highly qualified and trained personnel in order to coordinate all QA activities in the country. Such a body can initiate some guidelines on the minimum instrumentation requirements for all imaging modalities. With the training of more Medical Physicists sand Radiation Protection Professionals, it is expected that they would take up positions in all major hospitals which have a myriad of imaging modalities. The Ghana Society of Medical Physics, RA, Ministry of Health and other stakeholders must initiate procedures for the establishment of Medical Physics Departments in such hospitals. This would give the necessary recognition to the profession of Medical Physics in Ghana, which has been given recognition by the International Labour Organization (ILO) in its International Standard Classification of Occupations (ISCO) (ILO, 2008). With this recognition, Medical Physics has been accepted as modern applied branch of physics. Clinical audit should al‐ so be incorporated in the overall QA procedures in the country.

As the uses of ionizing radiation continue to increase in medicine, it is also expected that the services of Medical Physicist would increase. As such, more physicists would be re‐ quired to be trained in subsequent years. Appropriate accreditation bodies charged with issuing accreditation certificates, for a period of years must be put in place to regulate the profession of Medical Physics and maintain international standards of practice.

#### **8. Conclusion**

**6. Recent trends in quality assurance**

252 Latest Research into Quality Control

systems needs special attention.

**7. The way forward**

The increasing expansion of diagnostic imaging procedures in medicine coupled with rap‐ id technological advances makes the availability of qualified and trained personnel to be very crucial if the desired quality is to be achieved. This come with a lot challenges to the medical imaging community. This offers practitioners the opportunity to continually un‐ dergo retraining and other continuous professional development programmes in their re‐ spective fields. Also with the emergence of picture archiving and communication system (PACS) in many hospitals, there is the need for the development of appropriate on line QA procedures and in corporating them into hospital PACS systems. Special attention must also be given to the emergence of digital technology over the last decade as one of the greatest technological advances in medical imaging. This new technolgy poses a great challenge in medical imaging, requiring re-training of staff on the safe use of equipment and radiation protection issues. In Ghana for instance, the RPI of GAEC, in collaboration with the IAEA, has in the previous years developed a lot of expertise in the training of occupationally exposed workers in Ghana and the rest of Africa, spanning a period of al‐ most two decades (Boadu et al. 2011). This local expertise in training can be tapped. In this regard, a critical review of all QA procedures that were developed for screen-film

Various practitioners in the medical imaging community must brace themselves in order to face challenges of technological developments. With the advent of digital radiography: advances in computed radiography, direct digital radiography, digital subtraction angiog‐ raphy, new digital receivers, image processing techniques, computer applications in radi‐ ology and PACS offers enormous challenges. The advantages of digital technology:postprocessing capabilities, decreased costs, multiple viewing options, electronic transfer, possibilities of archiving, wide dynamic range of flat panel detectors and increased detec‐ tion quantum efficiency has led to a high demand of this technology by the medical imag‐ ing community. Therefore, the development of the requisite human resource must be

There is a strong need for the establishment of National Quality Control Centre for Diag‐ nostic Radiology. This body must be equipped with the requisite state of the art equip‐ ment, highly qualified and trained personnel in order to coordinate all QA activities in the country. Such a body can initiate some guidelines on the minimum instrumentation requirements for all imaging modalities. With the training of more Medical Physicists sand Radiation Protection Professionals, it is expected that they would take up positions

continued and sustained in order to deal with the challenges.

The role of an effective QA programme in any diagnostic department cannot be overem‐ phasized especially if the desired quality of producing good diagnostic images and the least radiation exposure are to be achieved. Nevertheless, a good QA programmme is not a guarantee for the assurance of the radiation safety of patients, staff and public. What is also needed is a separate radiation safety programme, which is very essential in every di‐ agnostic imaging facility and must also be under the direction of a qualified expert in ra‐ diation protection or a Medical Physicist expert. With significant contributions in clinical service, education, and research, Medical Physics continues to grow in importance both as a profession and as science, driven by the technological developments of societies in gen‐ eral and medicine in particular.

### **Acknowledgements**

The support received from the Radiation Protection Institute of Ghana Atomic Energy Com‐ mission is appreciated.

#### **Author details**

#### Stephen Inkoom

Radiation Protection Institute, Ghana Atomic Energy Commission, Legon, Accra, Ghana

#### **References**

[1] Boadu, M., Schandorf, C., Emi-Reynolds, G., Faanu, A., Inkoom, S., Kwabena, Gye‐ kye. P., & Kaikor, M.C. (2011). Systematic approach to training of occupationally ex‐ posed workers in Ghana andthe rest of Africa. *Health Phys*, 101(2: S116YS120).

**Chapter 13**

**Quality by Design and Risk Assessment for**

Kung-Tien Liu, Jian-Hua Zhao, Lee-Chung Men and

Additional information is available at the end of the chapter

opment can improve the product and process knowledge.

**Imaging**

Chien-Hsin Chen

**1. Introduction**

oped in many countries.

http://dx.doi.org/10.5772/51112

**Radiopharmaceutical Manufacturing and Clinical**

Radiopharmaceuticals have been widely used in many clinical and nonclinical applications, such as *in vivo* and non-invasive diagnosis or treatment of human diseases. The quality of radiopharmaceuticals administered for a patient is primarily related for the radiation dose delivered to achieve optimizing diagnostic imaging or therapeutic efficacy. Radiopharmaceut‐ icals with different half-lives (short, medium, and long), decay modes (alpha, beta, gamma, and electron capture), and biochemical properties (of ligands) can determine their utilities in medicine. Moreover, chemical and radiochemical impurities in a radiopharmaceutical can produce a serious trouble of diagnosis or treatment. Therefore, different requirements, regu‐ lations, and instrumentations for ensuring their high quality and high safety have been devel‐

There are only few years for the progress of "Quality by Design (QbD)" in International Conference on Harmonisation (ICH) Guidelines, e.g. ICH Q8, ICH Q9, and ICH Q10 [1-3]. According to the requirement of ICH Q8, quality can not be tested into products; i.e., quali‐ ty should be built in by design, i.e. QbD. Enhanced QbD approach to pharmaceutical devel‐

In this chapter, we provide a harmonized framework of QbD for manufacturing and clini‐ cal applications of radiopharmaceuticals in accordance with the requirements and guide‐ lines of U.S. Food and Drug Administration (FDA), International Atomic Energy Agency

> © 2012 Liu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Liu et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.


## **Quality by Design and Risk Assessment for Radiopharmaceutical Manufacturing and Clinical Imaging**

Kung-Tien Liu, Jian-Hua Zhao, Lee-Chung Men and Chien-Hsin Chen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51112

#### **1. Introduction**

**References**

254 Latest Research into Quality Control

[1] Boadu, M., Schandorf, C., Emi-Reynolds, G., Faanu, A., Inkoom, S., Kwabena, Gye‐ kye. P., & Kaikor, M.C. (2011). Systematic approach to training of occupationally ex‐

[2] Commission of the European Communities. (1997). Council Directive 97/43/ Euratom of 30 Juneon health protection of individuals against the dangers of ionizing radia‐ tion in relation to medical exposure, and repealing Directive 84/466 Euratom. *Off. J.*

[3] International Labour Organization (ILO). *International Standard Classification of Occu‐*

[4] International Organisation for Medical Physics (IOMP). (2010). IOMP Policy State‐ ment No.1. *The Medical Physicist: Role and Responsibilities, IOMP Working Group on Pol‐*

[5] Radiation Protection Instrument LI 1559. (1993). *Provisional National Defence Council*

[6] Regulatory Authority Information System [RAIS]. (2011). *Radiation Protection Insti‐*

[7] Inkoom, S., Schandorf, C., Reynolds, G. E., & Fletcher, J. J. (2011). Quality Assurance and Quality Control of Equipment in Diagnostic Radiology Practice-The Ghanaian Experience. *Wide Spectra of Quality Control, Isin Akyar (Ed.)*, 291-308, 978-9-53307-683-6, InTech, http://www.intechopen.com/articles/show/title/quality-as‐ surance-and-quality-control-of-equipment-in-diagnostic-radiology-practice-the-gha‐

[8] United Nations Scientific Committee on the Effects of Atomic Radiation. (2008). *Sour‐ ces and effects of ionizing radiation. Report to the General Assembly with scientific annexes*

[9] World Health Organization (1982). Quality Assurance in Diagnostic Radiology, Mac‐

*Law 308, Accra, Ghana. Date of Gazette Notification: 2nd April, 1993.*

posed workers in Ghana andthe rest of Africa. *Health Phys*, 101(2: S116YS120).

*Eur. Commun. Rep. L.*, 180(1997), 22-27.

*icy Statement* [1].

naian-exp.

*(UNSCEAR)*.

millan Procrom,9-24154-164-4

*pations (ISCO), Geneva, Switzerland, (ISCO-08), 2008.*

*tute, Ghana Atomic Energy Commission, Accra, Ghana.*

Radiopharmaceuticals have been widely used in many clinical and nonclinical applications, such as *in vivo* and non-invasive diagnosis or treatment of human diseases. The quality of radiopharmaceuticals administered for a patient is primarily related for the radiation dose delivered to achieve optimizing diagnostic imaging or therapeutic efficacy. Radiopharmaceut‐ icals with different half-lives (short, medium, and long), decay modes (alpha, beta, gamma, and electron capture), and biochemical properties (of ligands) can determine their utilities in medicine. Moreover, chemical and radiochemical impurities in a radiopharmaceutical can produce a serious trouble of diagnosis or treatment. Therefore, different requirements, regu‐ lations, and instrumentations for ensuring their high quality and high safety have been devel‐ oped in many countries.

There are only few years for the progress of "Quality by Design (QbD)" in International Conference on Harmonisation (ICH) Guidelines, e.g. ICH Q8, ICH Q9, and ICH Q10 [1-3]. According to the requirement of ICH Q8, quality can not be tested into products; i.e., quali‐ ty should be built in by design, i.e. QbD. Enhanced QbD approach to pharmaceutical devel‐ opment can improve the product and process knowledge.

In this chapter, we provide a harmonized framework of QbD for manufacturing and clini‐ cal applications of radiopharmaceuticals in accordance with the requirements and guide‐ lines of U.S. Food and Drug Administration (FDA), International Atomic Energy Agency

© 2012 Liu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Liu et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(IAEA), World Health Organization (WHO) and European Association of Nuclear Medi‐ cine (EANM). The attributes of the components in the quality system (QA/QC), including organization, staffing and personnel, facilities, instrumentation and equipment, operation procedure, radiopharmaceuticals, protocol and conduct of a study or a treatment, records and reports, and audit framework were further characterized. Assessments and compari‐ sons of critical quality attributes (CQAs) for assuring accurate radioactive dosimetry calcu‐ lation in the efficiency tracing of absolute activity measurement and patient- and technologistrelated risks for nuclear medicine imaging including Positron Emission Tomography (PET), Computed Tomography (CT), PET/CT, and Single Photon Emission Computed Tomogra‐ phy (SPECT) were identified.

#### **2. Quality system design based on the Requirements and Guidelines**

#### **2.1. Quality policy and system**

The quality system by design for radiopharmaceuticals and clinical imaging techniques is aimed to maintain and improve the qualified service for the patients, fulfill the regulatory requirements, optimize the safety and efficacy for patient care, demonstrate a proper equip‐ ment operating condition, and obtain a reliable quantitative performance in both diagnos‐ tic and therapeutic nuclear medicine procedures [4,5]. The pursuit of excellence in quality system is not a single action over a short period, instead, it is achieved through the whole life cycle of instruments, analytical methods or education for example, from planning and procurement to decommissioning based on advanced technology [6]. Continuous quality improvement implies a commitment to continuously struggle to advance based on state-ofthe-art information and techniques developed by the nuclear medicine and metrology com‐ munity at large [5].

**Figure 1.** Quality policy and system for the radiopharmaceuticals [7].

The organization of quality system could be grouped into two categories: (a) synthesis and preparation of nuclear medicine and (b) clinical imaging as shown in Figure 2 [8-10]. For synthesis and preparation of nuclear medicine, three important guidelines were considered [11-13]. Basically, preparation of "classical" radiopharmaceuticals in "kit" procedures and in a "distinct chemical" procedures for PET radiopharmaceuticals are distinguished as two dif‐

Quality by Design and Risk Assessment for Radiopharmaceutical Manufacturing and Clinical Imaging

http://dx.doi.org/10.5772/51112

257

For the clinical imaging, the major differences PET and SPECT in QbD are related to the properties and applications of a radiotracer. The most commonly used nuclides for PET imaging, such as carbon-11, oxygen-15, nitrogen-13, and fluorine-18, exhibit shorter half-life and more complicated labelling technology than that for SPECT imaging (Table 1)[14-31]. For example, the short half-lives of radionuclides used in PET modality allow for better de‐

**2.2. Quality plan and key factors**

*2.2.1. Organization*

ferent parts [11].

Implementation of a quality system must be in accordance with the quality police, i.e. the overall quality intentions and direction of an organization, as formally expressed by top management. And quality system includes the structure, responsibilities, and proce‐ dures for implementing quality management. An integrated infrastructure of quality pol‐ icy and system design is demonstrated as in Figure 1, which is mainly developed from the European Standard EN 28402 proposed by Bergmann *et al.* [7]. The attributes of the components in the quality sub-system (QA/QC), e.g. organization, personnel, facilities, instrumentation, operation procedures, preparation of radiopharmaceuticals, protocol and conduct, records and reports, and audit or inspection, were further integrated and classi‐ fied in this article.

Quality by Design and Risk Assessment for Radiopharmaceutical Manufacturing and Clinical Imaging http://dx.doi.org/10.5772/51112 257

**Figure 1.** Quality policy and system for the radiopharmaceuticals [7].

#### **2.2. Quality plan and key factors**

#### *2.2.1. Organization*

(IAEA), World Health Organization (WHO) and European Association of Nuclear Medi‐ cine (EANM). The attributes of the components in the quality system (QA/QC), including organization, staffing and personnel, facilities, instrumentation and equipment, operation procedure, radiopharmaceuticals, protocol and conduct of a study or a treatment, records and reports, and audit framework were further characterized. Assessments and compari‐ sons of critical quality attributes (CQAs) for assuring accurate radioactive dosimetry calcu‐ lation in the efficiency tracing of absolute activity measurement and patient- and technologistrelated risks for nuclear medicine imaging including Positron Emission Tomography (PET), Computed Tomography (CT), PET/CT, and Single Photon Emission Computed Tomogra‐

**2. Quality system design based on the Requirements and Guidelines**

The quality system by design for radiopharmaceuticals and clinical imaging techniques is aimed to maintain and improve the qualified service for the patients, fulfill the regulatory requirements, optimize the safety and efficacy for patient care, demonstrate a proper equip‐ ment operating condition, and obtain a reliable quantitative performance in both diagnos‐ tic and therapeutic nuclear medicine procedures [4,5]. The pursuit of excellence in quality system is not a single action over a short period, instead, it is achieved through the whole life cycle of instruments, analytical methods or education for example, from planning and procurement to decommissioning based on advanced technology [6]. Continuous quality improvement implies a commitment to continuously struggle to advance based on state-ofthe-art information and techniques developed by the nuclear medicine and metrology com‐

Implementation of a quality system must be in accordance with the quality police, i.e. the overall quality intentions and direction of an organization, as formally expressed by top management. And quality system includes the structure, responsibilities, and proce‐ dures for implementing quality management. An integrated infrastructure of quality pol‐ icy and system design is demonstrated as in Figure 1, which is mainly developed from the European Standard EN 28402 proposed by Bergmann *et al.* [7]. The attributes of the components in the quality sub-system (QA/QC), e.g. organization, personnel, facilities, instrumentation, operation procedures, preparation of radiopharmaceuticals, protocol and conduct, records and reports, and audit or inspection, were further integrated and classi‐

phy (SPECT) were identified.

256 Latest Research into Quality Control

**2.1. Quality policy and system**

munity at large [5].

fied in this article.

The organization of quality system could be grouped into two categories: (a) synthesis and preparation of nuclear medicine and (b) clinical imaging as shown in Figure 2 [8-10]. For synthesis and preparation of nuclear medicine, three important guidelines were considered [11-13]. Basically, preparation of "classical" radiopharmaceuticals in "kit" procedures and in a "distinct chemical" procedures for PET radiopharmaceuticals are distinguished as two dif‐ ferent parts [11].

For the clinical imaging, the major differences PET and SPECT in QbD are related to the properties and applications of a radiotracer. The most commonly used nuclides for PET imaging, such as carbon-11, oxygen-15, nitrogen-13, and fluorine-18, exhibit shorter half-life and more complicated labelling technology than that for SPECT imaging (Table 1)[14-31]. For example, the short half-lives of radionuclides used in PET modality allow for better de‐ tection sensitivity over a given period of time. This is because radiotracers with shorter halflives can be injected in higher activities to the patient without posing any additional radiation damage to the patient (since overall accumulation over time remains the same) leading to the increased detectable radiation over a shorter time. Moreover, arguments that the natural occurrence of PET isotopes in biologically active molecules (as opposed to heavy isotopes used in SPECT) results in a less challenging task of synthesizing physiologically useful tracers in PET modality [32,33]. In general, PET generally has a higher resolution, higher sensitivity, and a better quantitation capability than SPECT. However, SPECT is more practical as a routine procedure [18] and is more cost-effective for the system setting or maintain than a PET facility [8].

F-18 109.8 min F-18-fallypride D2/D3 dopamine receptor

Cu-64 12.7 h Cu-64-ATSM Tumor hypoxia I-124 4.12 d I-124-FIAU HSV1-tk expresssion

Tc-99m 6 hr Tc-99m-HMPAO Brain perfusion

SPECT imaging agents

SPECT imaging agents

F-18-FDG Oncology imaging, metabolism of

F-18-NaF Osseous metastasis

Quality by Design and Risk Assessment for Radiopharmaceutical Manufacturing and Clinical Imaging

I-124-HMFGI (IgG1) Breast ductal carcinoma

Tc-99m-TRODAT-1 Dopamine transporter

Tc-99m-Annexin-V Acute myocardial infarction,

tumors

acid) receptor

I-123-IBZM D2/D3 dopamine receptor I-123-iodobenzofuran D2/D3 dopamine receptor I-123-epidepride D2/D3 dopamine receptor I-123-FP-β-CIT Dopamine-transporter I-123-ADAM Serotonin transporter

Tc-99m-sestabmbi Myocardium perfusion Tc-99m-MAG3 Kidney perfusion Tc-99m-DTPA Kidney perfusion Tc-99m-DMSA Kidney perfusion

Tc-99m pertechnetate Thyroid Tc-99m sulfur colloid Lymph nodes

I-123 13 hr I-123-Iomazenil Benzodiazepine (γ-aminobutyric

I-123-IMP Brain perfusion

I-123-NaI Thyroid In-111 2.8 d In-111-Zevalin Non-Hodgkin's lymphoma

chemotherapy response monitoring, apoptosis of lung

Tc-99m-ECD Brain perfusion

Tc-99m-Prostascint Prostate cancer Tc-99m-CEA Colon cancer Tc-99m-Depreotide Lung cancer

myocardium

glucose in tumors, brain and

http://dx.doi.org/10.5772/51112

259

**Figure 2.** Quality system of organization [8-10].


tection sensitivity over a given period of time. This is because radiotracers with shorter halflives can be injected in higher activities to the patient without posing any additional radiation damage to the patient (since overall accumulation over time remains the same) leading to the increased detectable radiation over a shorter time. Moreover, arguments that the natural occurrence of PET isotopes in biologically active molecules (as opposed to heavy isotopes used in SPECT) results in a less challenging task of synthesizing physiologically useful tracers in PET modality [32,33]. In general, PET generally has a higher resolution, higher sensitivity, and a better quantitation capability than SPECT. However, SPECT is more practical as a routine procedure [18] and is more cost-effective for the system setting or

maintain than a PET facility [8].

258 Latest Research into Quality Control

**Figure 2.** Quality system of organization [8-10].

PET imaging agents

**Agents Isotope Half-life**

**(t1/2)**

**Radiopharmaceutical Applications**

C-11-MADAM Serotonin transporter

perfusion

C-11 20.4 min C-11-raclopride D2/D3 dopamine receptor

O-15 2.07 min 0-15 water Myocardium perfusion, brain

Ga-68 68 min Ga-68-DOTA Neuroendocrine tumours

N-13 9.96 min N-13-ammonia Blood flow (ventricle)



**Table 1.** Some examples of radiopharmaceutical classification and applications [14-31]. ADAM: 2-((2- ((dimethylamino)-methyl) phenyl)thio)-5- iodophenylamine; DTPA: diethylenetriaminepentaacetic acid; ECD: ethyl cysteinate dimer; FDG: fluoro-deoxy-glucose; FIAU: 1-(2-fluoro-2-deoxy-ß-D-arabinofuranosyl)-5-[I-124]iodouracil; FPβ-CIT: N-propyl-2-beta-carboxy-methoxy-3-beta(4-iodophenyl)-nortropane; HMPAO: hexamethyl propylene amine oxime; IBZM: iodobenzamide.

**Figure 3.** Quality system of staffing and personnel [9,34,35].

for the management of the archive.

scheduling studies.

[9,34,35] and briefly introduced below [4,6,7,36-38]:

The responsibilities for staffing and personnel in a quality system are classified in Figure 3

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261

**a.** Facility management: ensure the requirements, guidelines, and practices are complied within facility, sufficient qualified personnel, appropriate facilities, equipment, and ma‐ terials are available, ensure that personnel clearly understand the functions they are to perform and appropriate and technically valid Standard Operating Procedures (SOPs) are established and followed, ensure that there is a QA manager or QAU with designat‐ ed personnel and their responsibility is being performed, ensure that for each study an individual with the appropriate qualifications, training, and experience is designated by the management as the SD and PI, ensure that an individual is identified as responsible

**b.** Administrative staff: represent the first encounter a patient has with the centre. They re‐ ceive the patients according to the established protocols. In collaboration with the medi‐ cal and technical staff, they are responsible of the application of the procedures for

#### *2.2.2. Staffing and personnel*

Facilities should have written staff and personnel responsibilities and requirements. Two types of staff in the requirements for synthesis and preparation of nuclear medicine and clinical imaging are necessary [4]:


Quality by Design and Risk Assessment for Radiopharmaceutical Manufacturing and Clinical Imaging http://dx.doi.org/10.5772/51112 261

**Figure 3.** Quality system of staffing and personnel [9,34,35].

In-111-Octreotide Somatostatin receptor

Tl-201 3.04 d Tl-201 Myocardium perfusion Ga-67 3.3 d Ga-67 citrate Non-Hodgkin's lymphoma

Sm-153 1.95 d Sm-153 EDTMP Metastatic bone pain palliation Sr-89 50.5 d SrCl2 Palliative treatment of bone

P-32 14.28 d Orthophosphate Metastatic bone pain palliation Re-186 3.78 d Re-186-HEDP Metastatic bone pain palliation Re-188 17 h Re-188-bisphosphonate Metastatic bone pain palliation

I-131 8 d I-131 Tositumomab B-cell non-Hodgkin's lymphoma

Sn-117m 13.6 d Sn-117m-DTPA Metastatic bone pain palliation At-211 7.2 h At-211-81C6 Glioblastoma multiforme tumors

Y-90 64.14 h Y-90 Ibritumomab

Lu-177 6.7 d Lu-177-DOTA-Tyr3-

Tiuxetan

Octreotate

**Table 1.** Some examples of radiopharmaceutical classification and applications [14-31]. ADAM: 2-((2-

Ho-166 1.1 d Ho-166-DOTMP Multiple myeloma

((dimethylamino)-methyl) phenyl)thio)-5- iodophenylamine; DTPA: diethylenetriaminepentaacetic acid; ECD: ethyl cysteinate dimer; FDG: fluoro-deoxy-glucose; FIAU: 1-(2-fluoro-2-deoxy-ß-D-arabinofuranosyl)-5-[I-124]iodouracil; FPβ-CIT: N-propyl-2-beta-carboxy-methoxy-3-beta(4-iodophenyl)-nortropane; HMPAO: hexamethyl propylene amine

Facilities should have written staff and personnel responsibilities and requirements. Two types of staff in the requirements for synthesis and preparation of nuclear medicine and

**a.** Personnel for synthesis and preparation of nuclear medicine may include such as facili‐ ty management, administrative staff, study director (SD), principal investigator (PI), production chemists, QA manager or quality assurance unit (QAU), radiochemists, QC

**b.** Personnel for PET and SPECT imaging examination may include such as facility man‐ agement, administrative staff, medical physicists, nurses, referring physicians, nuclear medicine physicians, radiopharmacist, radiochemists, radiation protection officer, en‐

Therapy agents

260 Latest Research into Quality Control

oxime; IBZM: iodobenzamide.

*2.2.2. Staffing and personnel*

clinical imaging are necessary [4]:

chemists, cyclotron operators, and technologists.

gineers, QA manager or QAU, and technologists.

(Neuroendocrine tumors)

cancers and for prostate cancer

B-cell non-Hodgkin's lymphoma

Small cell lung cancer

The responsibilities for staffing and personnel in a quality system are classified in Figure 3 [9,34,35] and briefly introduced below [4,6,7,36-38]:


**c.** SD and PI: they are responsible for approving, conducting, documenting, recording and archiving the overall of the study and for its final report.

**m.** Nurses: manage and care of the patient, collaborate in preparing protocols of patient management and information material as well as in checking the operation of other in‐

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263

In IAEA, quality manager is responsible for the entire quality management system supervi‐ sion, the authority to enforce it and act on its findings, and should be involved in the evalua‐ tion and periodic review of the results [5,6]. But, in EANM, the responsibility for overseeing

In a PET facility, it should include the facility for (a) PET/CT scanner, (b) cyclotron, and (c) radiopharmacy. The location of the facility is a very important issue for the flow of patients, materials, and radiation protection. According to the risk of radiation exposure, two areas

**a.** low risk area, cold area or uncontrolled area is the area of offices, reception, waiting

**b.** high risk area, hot area or controlled area is the area of hot laboratory, preparation, in‐ jection and uptake room, toilet, control and scanning room, post-examination waiting

More considerations for the requirements of radiation protection and cleanliness are sum‐

room, consulting room, cleaning utilities room or store, and

room, reporting room, and waste disposal room.

the preparation operations of a qualified radiopharmaceutical is called QAU [11,12].

stitutional services.

*2.2.3. Facility*

are planned [4]:

**Figure 4.** Quality system of facilities [34].

marized in Figure 4 [34].


**m.** Nurses: manage and care of the patient, collaborate in preparing protocols of patient management and information material as well as in checking the operation of other in‐ stitutional services.

In IAEA, quality manager is responsible for the entire quality management system supervi‐ sion, the authority to enforce it and act on its findings, and should be involved in the evalua‐ tion and periodic review of the results [5,6]. But, in EANM, the responsibility for overseeing the preparation operations of a qualified radiopharmaceutical is called QAU [11,12].

#### *2.2.3. Facility*

**c.** SD and PI: they are responsible for approving, conducting, documenting, recording and

**d.** Nuclear medicine physicians: responsible for quality encompasses the general services of the centre. In particular, supervises all patient care and management procedures and all clinical protocols. In addition, he/she supports and enforces the QA/QC of equip‐

**e.** QA manager or QAU: all those planned and systematic actions necessary to provide ad‐ equate confidence that a product or service will satisfy given requirements for quality, express the closeness with which the outcome of a given procedure approaches some ideal, free from all errors and artefacts. Quality assurance embraces all efforts made to

**f.** Radiopharmacist and Radiochemists (Nuclear pharmacy): they are responsible for compounding, dispensing, quality assessment, patient monitoring, drug use review, new drug development and evaluation, product selection and performance evalua‐ tion, pharmacokinetic modeling, drug information and educational services. They are also responsible for the performance of acceptance testing and organization/supervi‐ sion of routine calibration and QC of all radiopharmacy equipment; QC of chemi‐ cals, enriched materials, precursors, and kits; QC of radiopharmaceuticals products

**g.** Cyclotron operators: they are in charge of the daily operations, take part in the accept‐ ance test of the cyclotron and related equipment, and are responsible for calibration and

**i.** QC chemists: the restriction of QC persons is independent of the production opera‐ tions or must have independent oversight of these duties. The operational techniques and activities that are used to fulfill requirements for quality and are used in refer‐ ence to the specific measures taken to ensure that one particular aspect of the proce‐

**j.** Medical physicists: specialized in nuclear medicine and responsible for the perform‐ ance of acceptance testing and organization/supervision of routine calibration and QC of imaging and radiation measurement equipment, including radiation protec‐

**k.** Radiation protection officer: ensure the radiation safety for patient, staffing, and environ‐

**l.** Engineers and Technologists: contribute to the preparation of clinical examination pro‐ tocols and the performance of patient examinations according to the established proto‐

cols, involved in the performance of routine calibration and QC of scanners.

**h.** Production chemists: synthesis and preparation of nuclear medicine.

archiving the overall of the study and for its final report.

ment, establish clinical review and auditing.

this end.

262 Latest Research into Quality Control

and batch release.

dure is satisfactory.

tion instrumentation.

mental.

QC procedures for equipment.

In a PET facility, it should include the facility for (a) PET/CT scanner, (b) cyclotron, and (c) radiopharmacy. The location of the facility is a very important issue for the flow of patients, materials, and radiation protection. According to the risk of radiation exposure, two areas are planned [4]:


**Figure 4.** Quality system of facilities [34].

More considerations for the requirements of radiation protection and cleanliness are sum‐ marized in Figure 4 [34].

#### *2.2.4. Instrumentation and equipment*

The instrumentation and equipment in the quality system are summarized in Figure 5 [34,38]. Apparatus and equipment for the purposes of manufacturing QC, diagnosis, and therapy, including validated computerized systems, used for the generation, storage and re‐ trieval of data, and for controlling environmental factors relevant to the study should be suitably located and of appropriate design and adequate capacity. Apparatus used in a study should be periodically inspected, cleaned, maintained, and calibrated according to SOPs. Records of these activities should be maintained. Calibration should be traceable to national or international standards of measurement [39].

*2.2.5. Operation procedures*

*2.2.6. Radiopharmaceuticals*

(a) Manufacturing of radiopharmaceuticals

Current Good Radiopharmacy Practice (cGRPP) [11-13,34].

imaging are summarized in Figures 6 and 7 [11,12,40-44].

**Figure 6.** Quality system of clinical operation procedures for nuclear medicine [11,12,40].

Manufacturing and quality control plans for radiopharmaceuticals are indicated in Figure 8 [34,40,45]. Radiopharmaceuticals might be manufactured or prepared in hospital radiophar‐ macies, centralized radiopharmacies, nuclear centers, institutes, industrial manufacturers, or PET centers in accordance with the requirements of good manufacturing practices (GMP) or

A test facility should have written SOPs approved by facility management for ensuring the quality and integrity of the data generation. Deviations from SOPs related to the manufac‐ turing, study, or treatment should be documented and should be acknowledged by the study director, the principal investigator, the medical physician, quality assurance personnel and/or radiopharmacist. The historical file of different version of all SOPs should be well re‐ corded and stored. The requirements of SOPs for nuclear medicine manufacturing and

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**Figure 5.** Quality system of instrumentation and equipment [34,38].

Performance tests and operation verification for the nuclear medicine units are achieved dai‐ ly, weekly, monthly, quarterly, or annually by a qualified medical physicist, a qualified nu‐ clear medicine technologist, or a medical physicist in training, with management by a qualified medical physicist. The tests results of intrinsic or system spatial resolution, uni‐ formity, center of rotation, sensitivity, energy resolution, counting rate parameters, multi‐ ple-window spatial registration, formatter and video display, linearity, leak test, overall system performance for imaging systems, interlocks, dose calibrators, thyroid uptake and counting systems must be reviewed and documented in an annual survey report in accord‐ ance with the ACR Technical Standard for Medical Nuclear Physics Performance Monitor‐ ing of Nuclear Medicine Imaging Equipment [37].

#### *2.2.5. Operation procedures*

*2.2.4. Instrumentation and equipment*

264 Latest Research into Quality Control

national or international standards of measurement [39].

**Figure 5.** Quality system of instrumentation and equipment [34,38].

ing of Nuclear Medicine Imaging Equipment [37].

The instrumentation and equipment in the quality system are summarized in Figure 5 [34,38]. Apparatus and equipment for the purposes of manufacturing QC, diagnosis, and therapy, including validated computerized systems, used for the generation, storage and re‐ trieval of data, and for controlling environmental factors relevant to the study should be suitably located and of appropriate design and adequate capacity. Apparatus used in a study should be periodically inspected, cleaned, maintained, and calibrated according to SOPs. Records of these activities should be maintained. Calibration should be traceable to

Performance tests and operation verification for the nuclear medicine units are achieved dai‐ ly, weekly, monthly, quarterly, or annually by a qualified medical physicist, a qualified nu‐ clear medicine technologist, or a medical physicist in training, with management by a qualified medical physicist. The tests results of intrinsic or system spatial resolution, uni‐ formity, center of rotation, sensitivity, energy resolution, counting rate parameters, multi‐ ple-window spatial registration, formatter and video display, linearity, leak test, overall system performance for imaging systems, interlocks, dose calibrators, thyroid uptake and counting systems must be reviewed and documented in an annual survey report in accord‐ ance with the ACR Technical Standard for Medical Nuclear Physics Performance Monitor‐ A test facility should have written SOPs approved by facility management for ensuring the quality and integrity of the data generation. Deviations from SOPs related to the manufac‐ turing, study, or treatment should be documented and should be acknowledged by the study director, the principal investigator, the medical physician, quality assurance personnel and/or radiopharmacist. The historical file of different version of all SOPs should be well re‐ corded and stored. The requirements of SOPs for nuclear medicine manufacturing and imaging are summarized in Figures 6 and 7 [11,12,40-44].

**Figure 6.** Quality system of clinical operation procedures for nuclear medicine [11,12,40].

#### *2.2.6. Radiopharmaceuticals*

#### (a) Manufacturing of radiopharmaceuticals

Manufacturing and quality control plans for radiopharmaceuticals are indicated in Figure 8 [34,40,45]. Radiopharmaceuticals might be manufactured or prepared in hospital radiophar‐ macies, centralized radiopharmacies, nuclear centers, institutes, industrial manufacturers, or PET centers in accordance with the requirements of good manufacturing practices (GMP) or Current Good Radiopharmacy Practice (cGRPP) [11-13,34].

Two categories of radiopharmaceuticals are classified in EANM Radiopharmacy Committee according to the significant difference of preparation procedures, i.e. "kit" and PET radio‐ pharmaceuticals. Also, significant consideration in the "Guidelines on Current Good Radio‐ pharmacy Practice (cGRPP) in the Preparation of Radiopharmaceuticals" is proposed by EANM Radiopharmacy Committee. Two types of preparation methods, i.e. in "classical" procedure and in "synthetical" procedure, have been distinguished in cGRPP [11]. Accord‐ ing to WHO guideline, radiopharmaceuticals are divided into four categories including ready-to-use, radionuclide generators, "kits" for the labelled with a radioactive component, and precursors used for radiolabelling other substances before administration (e.g. samples from patients) [13].

pensing and destruction of investigational drugs within the hospital [9]. Manufacturing or preparation of radiopharmaceuticals must follow the FDA 21CFR Part 212 "Current Good Manufacturing (cGMP) for PET drugs," USP Chapter <797> "Pharmaceutical Compound‐ ing-Sterile Preparations," USP Chapter <823> "Radiopharmaceuticals for Positron Emission Tomography - Compounding," and U.S. FDA Guidance: PET Drugs - Current Good Manu‐

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267

Three essential parts i.e. chemical, inventory, and radiochemical QC diagrams for radio‐

facturing Practice (CGMP) [10].

(b) Quality control of radiopharmaceuticals

pharmaceuticals are also indicated in Figure 8 [30,50,45].

**Figure 8.** Quality system for radiopharmaceutical manufacturing and quality control [34,40,45].

Method developments for the chemical and radiochemical analysis of starting material, intermediates, precursor used for the radiolabelling, active pharmaceutical ingredient (APIs or drug substance), and finished product (drug product or finished dosage form) are essen‐ tial requirements of Chemistry, and Manufacturing and Controls (CMC). In the applica‐ tions of investigational new drug (IND), New Drug Application (NDA), and Abbreviated New Drug Application (ANDA), information on the CMC has to be filed as per 21 CFR 312.23(a) for a drug substance and drug product. The contents for the CMC sections of the

**Figure 7.** Quality system of clinical operation procedures for imaging [41-44].

Clinical investigations of radiopharmaceuticals can be approved by FDA as "legend drugs." The investigational radiopharmaceutical drug service (IRDS) is responsible for establishing study-specific procedures for radiopharmaceutical drug, including preparation, storage, dis‐ pensing and destruction of investigational drugs within the hospital [9]. Manufacturing or preparation of radiopharmaceuticals must follow the FDA 21CFR Part 212 "Current Good Manufacturing (cGMP) for PET drugs," USP Chapter <797> "Pharmaceutical Compound‐ ing-Sterile Preparations," USP Chapter <823> "Radiopharmaceuticals for Positron Emission Tomography - Compounding," and U.S. FDA Guidance: PET Drugs - Current Good Manu‐ facturing Practice (CGMP) [10].

```
(b) Quality control of radiopharmaceuticals
```
Two categories of radiopharmaceuticals are classified in EANM Radiopharmacy Committee according to the significant difference of preparation procedures, i.e. "kit" and PET radio‐ pharmaceuticals. Also, significant consideration in the "Guidelines on Current Good Radio‐ pharmacy Practice (cGRPP) in the Preparation of Radiopharmaceuticals" is proposed by EANM Radiopharmacy Committee. Two types of preparation methods, i.e. in "classical" procedure and in "synthetical" procedure, have been distinguished in cGRPP [11]. Accord‐ ing to WHO guideline, radiopharmaceuticals are divided into four categories including ready-to-use, radionuclide generators, "kits" for the labelled with a radioactive component, and precursors used for radiolabelling other substances before administration (e.g. samples

from patients) [13].

266 Latest Research into Quality Control

**Figure 7.** Quality system of clinical operation procedures for imaging [41-44].

Clinical investigations of radiopharmaceuticals can be approved by FDA as "legend drugs." The investigational radiopharmaceutical drug service (IRDS) is responsible for establishing study-specific procedures for radiopharmaceutical drug, including preparation, storage, dis‐

Three essential parts i.e. chemical, inventory, and radiochemical QC diagrams for radio‐ pharmaceuticals are also indicated in Figure 8 [30,50,45].

**Figure 8.** Quality system for radiopharmaceutical manufacturing and quality control [34,40,45].

Method developments for the chemical and radiochemical analysis of starting material, intermediates, precursor used for the radiolabelling, active pharmaceutical ingredient (APIs or drug substance), and finished product (drug product or finished dosage form) are essen‐ tial requirements of Chemistry, and Manufacturing and Controls (CMC). In the applica‐ tions of investigational new drug (IND), New Drug Application (NDA), and Abbreviated New Drug Application (ANDA), information on the CMC has to be filed as per 21 CFR 312.23(a) for a drug substance and drug product. The contents for the CMC sections of the EU and U.S. are very much the same. However, the sequence and titles of the sections are quite different [46,47].

dioactive Drug Research Committee (RDRC) and IND. For an investigational medical prod‐ uct (IMP, investigation only), if there are adequate data from literature or original assessments that no pharmacologic effects are likely in humans, and the chosen radioactivity is small enough to result in the total radiation absorbed dose, clinical trial can be approved by National Competent Authority (NCA) and Ethical Committee (EC) in EU or approved by RDRC in U.S.. Otherwise, it is approved by EC in EU or approved by FDA in U.S., depend‐

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The FDA allows certain unique applications by the local RDRC, consisting of at least five individuals and three individual specialists in nuclear medicine, in formulate radioactive drugs, and in radiation safety, to approve and monitor for the use of radiopharmaceuticals in humans without IND approval. This is due to the low potential for toxicity of radiophar‐ maceuticals that are typically administered in tracer quantities. Requirements to establish a local RDRC at one's institution is outlined in regulation 21 CFR 361.1. And RDRC has to submit an annual report to the FDA as part of the procedures for maintaining an active and

ing on the phase of drug development [47].

**Figure 9.** Quality system of protocol and conduct for a study or a treatment [10,47].

Records and reports for the manufacturing of radiopharmaceuticals and imaging trial or testing are summarized in Figure 10 [7,9,40-44]. All records and reports should be main‐ tained at the radiopharmaceutical laboratory or another location that is accessible to respon‐

approved RDRC program [48].

*2.2.8. Records and reports*

According to International Conference on Harmonization (ICH) guidelines, the parameters for the validation of analytical methods should basically include specificity (selectivity), lin‐ earity, precision (repeatability, reproducibility and intermediate), accuracy, recovery, limit of detection (LOD), limit of quantification (LOQ), robustness, and stability. However, instru‐ ment validation parameters for the radioactivity measurement or isotopic analysis, such as dose calibrator or liquid scintillation spectrometry, are partially different.

Radiopharmaceuticals are usually used before all quality control testing has been complet‐ ed. The implementation of and compliance with the quality assurance program are therefore essential. Principal responsibilities of QA/QC are detailed by WHO and De vos et al., includ‐ ing preparation of detailed instructions for each test and analysis, ensuring the adequate identification, ensuring equipment and process validation, release or rejection of materials, evaluation of the quality and stability of the finished products, expiry dates, storage condi‐ tions, control procedures, specifications, and records keeping [13,34].

#### *2.2.7. Protocol and conduct*

Protocol for a medicine manufacturing study or imaging examination should be evaluated according to the purposes of a study, a treatment, or a clinical trial. Safety issue, such as al‐ gorithm proposed by ASNC for maximal benefit in patient radiation exposure must be in‐ cluded [33].

For each study and treatment, a written plan or protocol should exist prior to the initiation of the study. The protocol should be approved by dated signature of the study director, principal investigator or medical physician, facility management, sponsor and verified by quality assurance personnel and/or radiopharmacist. The study and treatment should be conducted in accordance with the study plan or protocol by using a unique identification to each study.

Clinical protocol should be evaluated based on the patient characteristics (e.g. patient histo‐ ry of disease or ability to complete the examination) and complexity of clinical situation in accordance with the current statements and guidelines [33]. For instance, advantages and disadvantages of assessing myocardial perfusion with PET, as compared to SPECT imag‐ ing, was reported and concluded that use of very short half-life tracers injected at very high activities, as well as the introduction of increasingly fast scintillators technology, which in turn has allowed reduction of random coincidences and introduced the possibility of timeof-flight (TOF) PET are expected to further contribute to high sensitivity imaging capabili‐ ties of PET [32].

An example for approving of protocol design for a clinical trial is shown in Figure. 9 [10,47]. Two pathways for the clinical studies of investigational radiopharmaceuticals are called Ra‐ dioactive Drug Research Committee (RDRC) and IND. For an investigational medical prod‐ uct (IMP, investigation only), if there are adequate data from literature or original assessments that no pharmacologic effects are likely in humans, and the chosen radioactivity is small enough to result in the total radiation absorbed dose, clinical trial can be approved by National Competent Authority (NCA) and Ethical Committee (EC) in EU or approved by RDRC in U.S.. Otherwise, it is approved by EC in EU or approved by FDA in U.S., depend‐ ing on the phase of drug development [47].

The FDA allows certain unique applications by the local RDRC, consisting of at least five individuals and three individual specialists in nuclear medicine, in formulate radioactive drugs, and in radiation safety, to approve and monitor for the use of radiopharmaceuticals in humans without IND approval. This is due to the low potential for toxicity of radiophar‐ maceuticals that are typically administered in tracer quantities. Requirements to establish a local RDRC at one's institution is outlined in regulation 21 CFR 361.1. And RDRC has to submit an annual report to the FDA as part of the procedures for maintaining an active and approved RDRC program [48].

**Figure 9.** Quality system of protocol and conduct for a study or a treatment [10,47].

#### *2.2.8. Records and reports*

EU and U.S. are very much the same. However, the sequence and titles of the sections are

According to International Conference on Harmonization (ICH) guidelines, the parameters for the validation of analytical methods should basically include specificity (selectivity), lin‐ earity, precision (repeatability, reproducibility and intermediate), accuracy, recovery, limit of detection (LOD), limit of quantification (LOQ), robustness, and stability. However, instru‐ ment validation parameters for the radioactivity measurement or isotopic analysis, such as

Radiopharmaceuticals are usually used before all quality control testing has been complet‐ ed. The implementation of and compliance with the quality assurance program are therefore essential. Principal responsibilities of QA/QC are detailed by WHO and De vos et al., includ‐ ing preparation of detailed instructions for each test and analysis, ensuring the adequate identification, ensuring equipment and process validation, release or rejection of materials, evaluation of the quality and stability of the finished products, expiry dates, storage condi‐

Protocol for a medicine manufacturing study or imaging examination should be evaluated according to the purposes of a study, a treatment, or a clinical trial. Safety issue, such as al‐ gorithm proposed by ASNC for maximal benefit in patient radiation exposure must be in‐

For each study and treatment, a written plan or protocol should exist prior to the initiation of the study. The protocol should be approved by dated signature of the study director, principal investigator or medical physician, facility management, sponsor and verified by quality assurance personnel and/or radiopharmacist. The study and treatment should be conducted in accordance with the study plan or protocol by using a unique identification to

Clinical protocol should be evaluated based on the patient characteristics (e.g. patient histo‐ ry of disease or ability to complete the examination) and complexity of clinical situation in accordance with the current statements and guidelines [33]. For instance, advantages and disadvantages of assessing myocardial perfusion with PET, as compared to SPECT imag‐ ing, was reported and concluded that use of very short half-life tracers injected at very high activities, as well as the introduction of increasingly fast scintillators technology, which in turn has allowed reduction of random coincidences and introduced the possibility of timeof-flight (TOF) PET are expected to further contribute to high sensitivity imaging capabili‐

An example for approving of protocol design for a clinical trial is shown in Figure. 9 [10,47]. Two pathways for the clinical studies of investigational radiopharmaceuticals are called Ra‐

dose calibrator or liquid scintillation spectrometry, are partially different.

tions, control procedures, specifications, and records keeping [13,34].

quite different [46,47].

268 Latest Research into Quality Control

*2.2.7. Protocol and conduct*

cluded [33].

each study.

ties of PET [32].

Records and reports for the manufacturing of radiopharmaceuticals and imaging trial or testing are summarized in Figure 10 [7,9,40-44]. All records and reports should be main‐ tained at the radiopharmaceutical laboratory or another location that is accessible to respon‐ sible officials and to government employees designated to perform inspections [11,12]. Storage of records must ensure safekeeping for many years. Archive facilities of independ‐ ent locations should be provided for the secure storage and retrieval of study plans, raw da‐ ta, final reports, samples of test items and specimens. Archive conditions, e.g. fireproof, waterproof, and insect prevention are designed for protecting contents from untimely dete‐ rioration [38].

#### *2.2.9. Audit framework*

Laboratory inspections and study audits should be established for periodical monitoring compliance with GLP, GCP, or GMP principles, study protocol, and SOPs [9,38]. Audits for radiopharmaceutical drug products typically begin by confirming the clinical site is appro‐ priately licensed and authorized to receive, possess, store, handle, prepare and administer radiopharmaceuticals. The audit framework of quality system for radiopharmaceuticals, imaging equipment, laboratory equipment, safety, computer systems, data handling, and ra‐ diation protection are displayed in Figure 11 [9,39].

**Figure 11.** Quality system of audit framework. Audit (a) for radiopharmaceuticals manufacturing, and (b) for imaging equipment, laboratory equipment, safety, computer systems, data handling, and radiation protection. [9,39]

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sible officials and to government employees designated to perform inspections [11,12]. Storage of records must ensure safekeeping for many years. Archive facilities of independ‐ ent locations should be provided for the secure storage and retrieval of study plans, raw da‐ ta, final reports, samples of test items and specimens. Archive conditions, e.g. fireproof, waterproof, and insect prevention are designed for protecting contents from untimely dete‐

Laboratory inspections and study audits should be established for periodical monitoring compliance with GLP, GCP, or GMP principles, study protocol, and SOPs [9,38]. Audits for radiopharmaceutical drug products typically begin by confirming the clinical site is appro‐ priately licensed and authorized to receive, possess, store, handle, prepare and administer radiopharmaceuticals. The audit framework of quality system for radiopharmaceuticals, imaging equipment, laboratory equipment, safety, computer systems, data handling, and ra‐

rioration [38].

270 Latest Research into Quality Control

**Figure 10.** Quality system of records and reports [7,9,40-44].

diation protection are displayed in Figure 11 [9,39].

*2.2.9. Audit framework*


**Figure 11.** Quality system of audit framework. Audit (a) for radiopharmaceuticals manufacturing, and (b) for imaging equipment, laboratory equipment, safety, computer systems, data handling, and radiation protection. [9,39]

#### **3. Quality evaluation and sources of uncertainty**

#### **3.1. Radiopharmaceuticals**

#### *3.1.1. Standardization: principle and applications*

Quality control for the quantification of radiopharmaceutical activity is critical for accurate dosimetry calculations, from whole body to cell microscopy. Tumor uptake of radiopharma‐ ceutical need to be correlated with tumor response and to be related to the tumor radiation absorbed dose. [14]

4πβ-γ coincidence

ε: 65-87% 3194 ± 12 kBq/g

4πγ method ε: ~83% 3174 ± 25 kBq/g

Quality by Design and Risk Assessment for Radiopharmaceutical Manufacturing and Clinical Imaging

γ ray (167.4 keV) probability: 0.1000

± 0.0006

εβ: 71 - 91% εEC (~ εAEs): 50 - 100%

**Table 2.** Some examples of absolute standardization of radiopharmaceuicals and related radioisotopes [17,50-58]

The theoretical counting efficiency, i.e. counts/disintegration or counts per minute/disinte‐ gration per minute (cpm/dpm), for a radionuclide can be used to examine the absolute activ‐ ity, in disintegration or disintegration per minute (dpm) of the radionuclide. Different efficiency tracing methods has been developed for more than six decade by characterizing the effects of sample volume, medium composition (matrix), pulse discrimination condi‐ tions, photomultiplier voltage, amplifier gain, and luminophor concentration on counting efficiency of a radioactive species [49]. The use of 4πβ scintillation counting and 4πβ-γ coin‐ cidence counting for the standardization of certain electron capture (EC) nuclides with sim‐

Some examples of absolute standardization of radiopharmaceuicals and related radioiso‐ topes are shown in Table 2 [17,50-58]. Below, we introduce different tracing methods, in‐ cluding (a) efficiency tracing (and extrapolation) method using a non-H-3 standard solution, (b) CIEMAT-NIST (C/N) efficiency tracing method, (c) non-extrapolation tracer method, (d) coincidence method by a 4πβ-γ system, (e) triple to double coincidence ratio (TDCR) meth‐

(a) The efficiency tracing (and extrapolation) method using a non-H-3 standard solution

(0.88%)

(2.09%)

7.207 ± 0.033 (NIST) 7.197 ± 0.027 (NPL) 7.116 ± 0.050 (PTB)

UC: 0.76% 57

UC: 2.7% 58

55

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56

method

C/N method

High-pressure IC 4πγ coincidence method

Windowless 4π-CsI(Tl) -sandwich spectrometer, LSC,

4πβ-γ coincidence method and Cs-134

4πβ-γ coincidence

PPC

tracer

method Germanium γ spectrometry

ple decay schemes is established since 1952 [49] and 1957 [50].

Tl-201 γ decays to Hg-201

Pb-210 β decay to

od, and (f) 4πγ counting method.

Bi-210 (t1/2 5.103 d), Po-210 (t1/2 138.4 d), and α decay to Pb-206

 decay (97.4%) to Pb-204 and EC decay (2.6%) to Hg-204

Tl-204 β-



**3. Quality evaluation and sources of uncertainty**

Quality control for the quantification of radiopharmaceutical activity is critical for accurate dosimetry calculations, from whole body to cell microscopy. Tumor uptake of radiopharma‐ ceutical need to be correlated with tumor response and to be related to the tumor radiation

> **Detection efficiency (ε)**

To ground state: β<sup>+</sup> (87.85%), EC (8.92%) To 1077 keV: β<sup>+</sup> (1.29%), EC (1.93%) Annihilation radiation: 178.29% γ-ray to 1077 keV:

3.22%

LSC ε: ~95% 3191 ± 8 kBq/g

εpure γ: ~100% εpure EC: < 75% εEC-γ: ~100% εα: ~100%

**Activity accuracy & uncertainty (U)**

U: 0.2-0.5% (pure β) U: 0.2-0.5% (β-γ)

U: 1% 52

UC: 0.60% (for DTPA

UC: 2.43% (for DTPA by NaI(T1))

by LS)


(0.54%)

ε: ~75% U: 2% 50

**Ref.**

51

17

54

**Method for standardization**

4πβ-γ coincidence

x-ray spectrometer 4πβ-γ coincidence

method

method LSC

Calibrated IC

4πβ LS and 4πγ methods

Coincidence methods: L Auger electrons plus L Xrays and K X-rays

LSC and C/N method

**3.1. Radiopharmaceuticals**

272 Latest Research into Quality Control

absorbed dose. [14]

*3.1.1. Standardization: principle and applications*

**Isotope Major decay**


Co-57 EC decay to Fe-57

1. Ge-68 EC decay to Zn-68 2. Ga-68 β<sup>+</sup>, EC, γ decay to Zn-68

Sn-117m-DTPA

Cs-134 β-

Decay to Sn-117

 and γ decay to Ba-134

Cs-131 EC decay to Xe-131

**modes**

Ge-68/Ga-68 γ spectrometer

**Table 2.** Some examples of absolute standardization of radiopharmaceuicals and related radioisotopes [17,50-58]

The theoretical counting efficiency, i.e. counts/disintegration or counts per minute/disinte‐ gration per minute (cpm/dpm), for a radionuclide can be used to examine the absolute activ‐ ity, in disintegration or disintegration per minute (dpm) of the radionuclide. Different efficiency tracing methods has been developed for more than six decade by characterizing the effects of sample volume, medium composition (matrix), pulse discrimination condi‐ tions, photomultiplier voltage, amplifier gain, and luminophor concentration on counting efficiency of a radioactive species [49]. The use of 4πβ scintillation counting and 4πβ-γ coin‐ cidence counting for the standardization of certain electron capture (EC) nuclides with sim‐ ple decay schemes is established since 1952 [49] and 1957 [50].

Some examples of absolute standardization of radiopharmaceuicals and related radioiso‐ topes are shown in Table 2 [17,50-58]. Below, we introduce different tracing methods, in‐ cluding (a) efficiency tracing (and extrapolation) method using a non-H-3 standard solution, (b) CIEMAT-NIST (C/N) efficiency tracing method, (c) non-extrapolation tracer method, (d) coincidence method by a 4πβ-γ system, (e) triple to double coincidence ratio (TDCR) meth‐ od, and (f) 4πγ counting method.

(a) The efficiency tracing (and extrapolation) method using a non-H-3 standard solution

The efficiency tracer techniques, using Co-60, Cs-134, C-14, Cr-51, Mn-54 or Am-241 stand‐ ard solution for the standardization of the β-γ nucides were developed. The 4π liquid scin‐ tillation (LS) consisted of the extrapolation of the 4π counting rate to the zero discrimination level for the standardization of the Tl-204 (97.6% β emission and 2.4% electron capture) solu‐ tion was carried out for efficiency tracing using a Co-60 standard solution received in the framework of the 1997 BIPM comparison was carried out by Sahagia et al. [59]. A germani‐ um spectrometer was calibrated for the standardization of Pb-210 using Am-241 as a nor‐ malizing agent has been proposed [58]. Instead, Dias et al. chose Cs-134 as an efficiency tracer to standardize Tl-204 as well as a 4πβ-γ coincidence system for the calibration [57]. This method can be also successfully used for the standardization of radionuclides such as Ir-192, Zn-65, Mn-54, with the detection of the β rays, Auger electrons, X rays, in the propor‐ tional counter (PC) [60]. Efficiency tracing with C-14 and zero detection threshold techni‐ ques with H-3 as tracers was applied for standardization of various β-emitting radionuclides, e.g. C-14, Cl-36, and Tl-204 using LS spectrometer [61].

**•** This relation is used in conjunction with the measured data to calculate the efficiency for

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The parameters of emitters in different decay modes used for the C/N calculations are sum‐

**•** Pure β emitters (Sr-89, Sr-90, Y-90, and K-40): atomic number Z of the radionuclide, the

**•** β+γ emitters, if the radionuclide has significant levels with half-lives in the order of the coincidence resolving time or the dead time of the equipment, a C/N calculation is not

**•** Pure EC emitters: the input parameters are the capture probabilities, PK; PL; PM, and the atomic parameters for the rearrangement: the fluorescence yields ωK and ωL (averaged), the probabilities of the X-rays (PKL, PKX, and PLX) and their average energies (EKL, EKX, and ELX), the emission probabilities of the Auger electrons (PKLL, PKLX, PKXY,

**•** EC+γ emitters (Co-57, Se-75, Sr-85, and Ba-133): the calculation method is the same as for

**•** The efficiency of LSC systems with respect to alpha radiation is in each case very close to

An alternative called "non-extrapolation tracer method" was proposed by Steyn et al. in 1979, where Fe-55 was used as a tracer to establish the figure-of-merit (FOM) of the detec‐ tion system for the calculation of counting efficiency [65]. The liquid scintillation method, for the determination of absolute activity of Mn-54 and Zn-65 from 4π(LS)e-γ data by direct calculation without efficiency extrapolation was performed. The non-extrapolation LS meth‐ od relies on determining the probability of the γ-ray interacting with the scintillator solu‐

Coincidence method comes from the additional coincidence channel, which records a disin‐ tegration event when it is detected in both β- and γ-channels. Typically, the system for abso‐ lute standardization is usually consisted of a gas-flow or pressurized proportional counter with 4π geometry as the α, β, electrons or X-ray detector and coupled to a pair of NaI(Tl) scintillation counters or a semiconductor detector, as γ detectors. The 4πβ-γ coincidence technique has been considered a primary standardization method due to its high accuracy and because it can obtain the radionuclide activity depending only on observables quantities

Alternatively, solid or liquid scintillation counters (LSC) are used in place of gas-flow pro‐ portional counters. Advantages of using LSC counting in the 4π channel are that self-ab‐ sorption does not occur, leading to Auger electrons being detected with relatively high

the nuclide and an activity value in dpm for each single measurement.

mass number A, the endpoint energy EMax, and the shape parameters.

and PLXY) and their average energies (EKLL, EKLX, EKXY, and ELXY).

tion, is described and validated by measurements made on Co-60 [66].

**•** Pure γ emitters (Nb-93m): the efficiency is nearly 100%.

marized as follows [51]:

possible.

β+γ nuclides.

[57,67].

unity. A tracer method is not necessary.

(d) The coincidence method by a 4πβ-γ system

(c) The non-extrapolation tracer method

Recently, different methodologies were proposed. Koskinas et al. developed a "dual-trac‐ ers", e.g. Cr-51 and Mn-54 procedure followed by the Laboratório de Metrologia Nuclear (LMN) for the standardization of EC nuclide, i.e. Fe-55. The efficiency was obtained by se‐ lecting a γ-ray window set at 320 keV (Cr-51) and at 834 keV (Mn-54) [62]. The activity of EC radionuclides is usually determined by 4π (proportional counter, PC)-γ coincidence count‐ ing and by an efficiency extrapolation method. However, an alternative method, called "wet extrapolation method", utilizes an absorption change during the drying of a water droplet added onto the source surface, variation of the PC detection efficiency can be achieved. Slopes of extrapolation curves and resulting activity values obtained are compared for sev‐ eral radionuclides (Mn-54, Ce-139, Y-88, and Co-57) [63].

#### (b) The CIEMAT-NIST (C/N) efficiency tracing method

CIEMAT/NIST (C/N) method, developed by Centro de Investigationes Energéticas, Medio‐ ambientales y Tecnologicas (CIEMAT), Spain and the National Institute of Standards and Technology (NIST), U.S. is used for standardization of radionuclides with Liquid Scintilla‐ tion (LS) Spectrometry by calculating the counting efficiency of the radionuclide to be as‐ sayed and using H-3 as a tracer [61]. C/N program is suitable used for the calculation of the efficiency of nuclides decayed by β, β-γ, EC, EC-γ and nuclides with mixed decay [51]. The basic principle of C/N LS efficiency tracing method is a combination of a theoretical calcula‐ tion of the counting efficiency and an experimental determination of correction factors in three steps [61,64]:


**•** This relation is used in conjunction with the measured data to calculate the efficiency for the nuclide and an activity value in dpm for each single measurement.

The parameters of emitters in different decay modes used for the C/N calculations are sum‐ marized as follows [51]:


The efficiency tracer techniques, using Co-60, Cs-134, C-14, Cr-51, Mn-54 or Am-241 stand‐ ard solution for the standardization of the β-γ nucides were developed. The 4π liquid scin‐ tillation (LS) consisted of the extrapolation of the 4π counting rate to the zero discrimination level for the standardization of the Tl-204 (97.6% β emission and 2.4% electron capture) solu‐ tion was carried out for efficiency tracing using a Co-60 standard solution received in the framework of the 1997 BIPM comparison was carried out by Sahagia et al. [59]. A germani‐ um spectrometer was calibrated for the standardization of Pb-210 using Am-241 as a nor‐ malizing agent has been proposed [58]. Instead, Dias et al. chose Cs-134 as an efficiency tracer to standardize Tl-204 as well as a 4πβ-γ coincidence system for the calibration [57]. This method can be also successfully used for the standardization of radionuclides such as Ir-192, Zn-65, Mn-54, with the detection of the β rays, Auger electrons, X rays, in the propor‐ tional counter (PC) [60]. Efficiency tracing with C-14 and zero detection threshold techni‐ ques with H-3 as tracers was applied for standardization of various β-emitting

Recently, different methodologies were proposed. Koskinas et al. developed a "dual-trac‐ ers", e.g. Cr-51 and Mn-54 procedure followed by the Laboratório de Metrologia Nuclear (LMN) for the standardization of EC nuclide, i.e. Fe-55. The efficiency was obtained by se‐ lecting a γ-ray window set at 320 keV (Cr-51) and at 834 keV (Mn-54) [62]. The activity of EC radionuclides is usually determined by 4π (proportional counter, PC)-γ coincidence count‐ ing and by an efficiency extrapolation method. However, an alternative method, called "wet extrapolation method", utilizes an absorption change during the drying of a water droplet added onto the source surface, variation of the PC detection efficiency can be achieved. Slopes of extrapolation curves and resulting activity values obtained are compared for sev‐

CIEMAT/NIST (C/N) method, developed by Centro de Investigationes Energéticas, Medio‐ ambientales y Tecnologicas (CIEMAT), Spain and the National Institute of Standards and Technology (NIST), U.S. is used for standardization of radionuclides with Liquid Scintilla‐ tion (LS) Spectrometry by calculating the counting efficiency of the radionuclide to be as‐ sayed and using H-3 as a tracer [61]. C/N program is suitable used for the calculation of the efficiency of nuclides decayed by β, β-γ, EC, EC-γ and nuclides with mixed decay [51]. The basic principle of C/N LS efficiency tracing method is a combination of a theoretical calcula‐ tion of the counting efficiency and an experimental determination of correction factors in

**•** Count rates (cpm) and the quench-indicating parameters (QIPs, i.e. tSIE) are determined for a set of samples of the nuclide to be measured, and for a set of H-3 standard samples, with a different quench. The tSIE values were calculated using the Ba-133 source inside of the instrument. By combining these data, a corresponding H-3 efficiency is obtained for

**•** The universal curve of Figure of Merit (FOM) as a function of tSIE was plotted. The effi‐ ciency of the nuclide is theoretically calculated as a function of the efficiency of the tracer

radionuclides, e.g. C-14, Cl-36, and Tl-204 using LS spectrometer [61].

eral radionuclides (Mn-54, Ce-139, Y-88, and Co-57) [63].

(b) The CIEMAT-NIST (C/N) efficiency tracing method

three steps [61,64]:

274 Latest Research into Quality Control

nuclide H-3.

each sample of the nuclide.

An alternative called "non-extrapolation tracer method" was proposed by Steyn et al. in 1979, where Fe-55 was used as a tracer to establish the figure-of-merit (FOM) of the detec‐ tion system for the calculation of counting efficiency [65]. The liquid scintillation method, for the determination of absolute activity of Mn-54 and Zn-65 from 4π(LS)e-γ data by direct calculation without efficiency extrapolation was performed. The non-extrapolation LS meth‐ od relies on determining the probability of the γ-ray interacting with the scintillator solu‐ tion, is described and validated by measurements made on Co-60 [66].

(d) The coincidence method by a 4πβ-γ system

Coincidence method comes from the additional coincidence channel, which records a disin‐ tegration event when it is detected in both β- and γ-channels. Typically, the system for abso‐ lute standardization is usually consisted of a gas-flow or pressurized proportional counter with 4π geometry as the α, β, electrons or X-ray detector and coupled to a pair of NaI(Tl) scintillation counters or a semiconductor detector, as γ detectors. The 4πβ-γ coincidence technique has been considered a primary standardization method due to its high accuracy and because it can obtain the radionuclide activity depending only on observables quantities [57,67].

Alternatively, solid or liquid scintillation counters (LSC) are used in place of gas-flow pro‐ portional counters. Advantages of using LSC counting in the 4π channel are that self-ab‐ sorption does not occur, leading to Auger electrons being detected with relatively high efficiency; source preparation is easy; and the source geometry is highly reproducible. The latter leads to good reproducibility of the counting efficiency of the X-rays and Auger elec‐ trons, which in turn gives rise to consistent results amongst the counting sources. The effi‐ ciency data can generally be fitted with a linear function, particularly in the high-efficiency region, or by a low-order polynomial expression, giving rise to reliable extrapolated activity values [68].

calculate the Čerenkov counting efficiencies. Since Čerenkov counting is more sensitive to changes in the computed β spectra, the method was extensively used to investigate β

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An ionization chamber system referring to a long living and stable standard source is very adequate for the comparison of γ-ray emitting radio-nuclides. In most cases Ra-226 sealed sources have been used as the reference because the Ra-226 sources were widely used in ra‐ diotherapy [69]. Zimmerman et al. standardized and compared solution of Sn-117m by 4πβ liquid scintillation (LS) spectrometry and 4π γ-ray spectrometry (NaI(Tl) and high-purity germanium detectors). Massic activities were measured for determining the dose calibrator

Examples for the evaluation of detection efficiency (ε), activity accuracy, and measure‐ ment uncertainty (U) of absolute activity of radiopharmaceuicals and related radioisotopes are shown in Table 2. Components of combined uncertainty were further summarized in

(a) Uncertainty for the efficiency tracing (and extrapolation) method using a non-H-3 stand‐

Components of combined uncertainty in the activity determination include counting statis‐ tics, background, dead time, weighing, decay scheme parameter, half-life, and extrapolation of efficiency curve [57]. Source of uncertainty evaluated by Woods et al. in the absolute standardization of low energy β emitter, i.e. Pb-210 are counting, background, half life, β dead time, γ dead time, resolving times, choice of fit, count rate dependence, dead time formula, weighing, separation time, extrapolation range, contaminants, and reproducibility [58].

Component of uncertainty in the standardization of Re-186 by the C/N method of LS effi‐ ciency tracing with H-3 include source preparation, scintillator stability, dead time, liquidscintillation measurements, uncertainty due to H-3 reference standard, EC/β- branching

branches. [74]

The contributions to the uncertainty of the value of the specific activity are volatility of H2 [GeCl6] during the preparation of solid sources for coincidence measurements, drop mass‐ es, counting statistics, background variation, accidental coincidences and dead time losses, Compton continuum of the 1077 keV peak included in the γ window around the 511 keV peak, decay scheme correct ion factor, correction factor for non-vanishing εEC, impurities and halflife uncertainty, and detection of 511 keV quanta in the β detector due to its γ sensitivity [52].

The components contributing to the uncertainty of 4πβ-γ coincidence method were estimat‐ ed as follows: counting statistics and background variation, instrumental corrections, impur‐ ities, half-life uncertainty, decay scheme correction factor, and mass of droplet. Standard

shape factor functions [73]. (f) The 4πγ counting method.

*3.1.2. Uncertainty of measurement*

(b) Uncertainty for the C/N efficiency tracing method

ratio, spectral distributions for EC and β-

factor settings [17].

this section.

ard solution

Several examples for the applications of the coincidence method by a 4πβ-γ system are such as standardization of Ho-166m using the normal gas flow 4πβ-γ coincidence method [69], standardization of Tl-204 using Cs-134 as tracer and a 4πβ-γ coincidence system was used for the calibration [57], directly measured of radionuclides with EC decay schemes, e.g. I-125, Ir-192, Zn-65, and Ce-139 by a LS coincidence extrapolation technique [68], and stand‐ ardization of Fe-55 using a "dual-tracers" method coupled with a 4πβ-γ coincidence calibra‐ tion system [62].

#### (e) The triple to double coincidence ratio (TDCR) method

The TDCR method was first developed at the R.C., Poland and at the LNHB, France. The equipment consists in a detection unit, provided with three photomultipliers (PMs), acted by the light emitted in the vial containing the radioactive solution dissolved in a liquid scin‐ tillator, and the electronic unit [60]. TDCR, allowing the observation of three kind of double coincidences (2-photodetectors) and triple coincidence (3-photodetectors) method in LSC, is a fundamental measurement method suitable to the standardization of pure-beta emitters, i.e. H-3, C-14, P-32, Ni-63, Tc-99, Tl-204 and some low energy electron-capture emitters, i.e. Fe-55 [59,60,70,71]. Detection efficiency variation can be achieved using techniques of chemi‐ cal quenching, coaxial grey filters and PM tubes defocusing. The two former processes re‐ duce the mean quantity of light emitted and the later reduces the detection probability [71]. Basically, the specific experimental parameter (K) is equal to the ratio of the triple coinciden‐ ces counting rate (NT) to the sum of double coincidences counting rate (ND). Determination of a counting efficiency (εD) for each counting point (ND) leads to the activity of the source (N0). The efficiency functions εT and εD are nonlinear functions for a particular emitter and counting system [70].

Two innovative TDCR instrumentations were developed:


calculate the Čerenkov counting efficiencies. Since Čerenkov counting is more sensitive to changes in the computed β spectra, the method was extensively used to investigate β shape factor functions [73].

(f) The 4πγ counting method.

efficiency; source preparation is easy; and the source geometry is highly reproducible. The latter leads to good reproducibility of the counting efficiency of the X-rays and Auger elec‐ trons, which in turn gives rise to consistent results amongst the counting sources. The effi‐ ciency data can generally be fitted with a linear function, particularly in the high-efficiency region, or by a low-order polynomial expression, giving rise to reliable extrapolated activity

Several examples for the applications of the coincidence method by a 4πβ-γ system are such as standardization of Ho-166m using the normal gas flow 4πβ-γ coincidence method [69], standardization of Tl-204 using Cs-134 as tracer and a 4πβ-γ coincidence system was used for the calibration [57], directly measured of radionuclides with EC decay schemes, e.g. I-125, Ir-192, Zn-65, and Ce-139 by a LS coincidence extrapolation technique [68], and stand‐ ardization of Fe-55 using a "dual-tracers" method coupled with a 4πβ-γ coincidence calibra‐

The TDCR method was first developed at the R.C., Poland and at the LNHB, France. The equipment consists in a detection unit, provided with three photomultipliers (PMs), acted by the light emitted in the vial containing the radioactive solution dissolved in a liquid scin‐ tillator, and the electronic unit [60]. TDCR, allowing the observation of three kind of double coincidences (2-photodetectors) and triple coincidence (3-photodetectors) method in LSC, is a fundamental measurement method suitable to the standardization of pure-beta emitters, i.e. H-3, C-14, P-32, Ni-63, Tc-99, Tl-204 and some low energy electron-capture emitters, i.e. Fe-55 [59,60,70,71]. Detection efficiency variation can be achieved using techniques of chemi‐ cal quenching, coaxial grey filters and PM tubes defocusing. The two former processes re‐ duce the mean quantity of light emitted and the later reduces the detection probability [71]. Basically, the specific experimental parameter (K) is equal to the ratio of the triple coinciden‐ ces counting rate (NT) to the sum of double coincidences counting rate (ND). Determination of a counting efficiency (εD) for each counting point (ND) leads to the activity of the source (N0). The efficiency functions εT and εD are nonlinear functions for a particular emitter and

**•** The TDCR method of LSC is well established for measuring the activity of pure beta emit‐ ting and electron capture radionuclides. Recently, a new TDCR counting system was de‐ signed by the National Physical Laboratory (NPL) for activity assays of low-energy, pure β-emitting radionuclides and EC nuclides. Three photomultiplier tubes (PMT) were ar‐ ranged in the optical chamber as well as a NaI(Tl) detector was mounted below the opti‐ cal chamber. The detector allows 4πβ-γ coincidence measurements to be performed in

**•** Radionuclides such as P-32, Sr-89, Y-90, Tl-204, and Rh-106 were successfully studied us‐ ing an in-house built new TDCR-Čerenkov counter developed by Kossert. Since Čerenkov counting acts as natural discrimination for αemitters and low-energy β emitters, some po‐ tential radioactive impurities or progenies will not disturb the measurements. Two stand‐ ard sources, e.g. Cl-36 and P-32 were used to determine the free parameter and to

values [68].

276 Latest Research into Quality Control

tion system [62].

counting system [70].

parallel [72].

(e) The triple to double coincidence ratio (TDCR) method

Two innovative TDCR instrumentations were developed:

An ionization chamber system referring to a long living and stable standard source is very adequate for the comparison of γ-ray emitting radio-nuclides. In most cases Ra-226 sealed sources have been used as the reference because the Ra-226 sources were widely used in ra‐ diotherapy [69]. Zimmerman et al. standardized and compared solution of Sn-117m by 4πβ liquid scintillation (LS) spectrometry and 4π γ-ray spectrometry (NaI(Tl) and high-purity germanium detectors). Massic activities were measured for determining the dose calibrator factor settings [17].

#### *3.1.2. Uncertainty of measurement*

Examples for the evaluation of detection efficiency (ε), activity accuracy, and measure‐ ment uncertainty (U) of absolute activity of radiopharmaceuicals and related radioisotopes are shown in Table 2. Components of combined uncertainty were further summarized in this section.

(a) Uncertainty for the efficiency tracing (and extrapolation) method using a non-H-3 stand‐ ard solution

Components of combined uncertainty in the activity determination include counting statis‐ tics, background, dead time, weighing, decay scheme parameter, half-life, and extrapolation of efficiency curve [57]. Source of uncertainty evaluated by Woods et al. in the absolute standardization of low energy β emitter, i.e. Pb-210 are counting, background, half life, β dead time, γ dead time, resolving times, choice of fit, count rate dependence, dead time formula, weighing, separation time, extrapolation range, contaminants, and reproducibility [58].

(b) Uncertainty for the C/N efficiency tracing method

Component of uncertainty in the standardization of Re-186 by the C/N method of LS effi‐ ciency tracing with H-3 include source preparation, scintillator stability, dead time, liquidscintillation measurements, uncertainty due to H-3 reference standard, EC/β- branching ratio, spectral distributions for EC and β branches. [74]

The contributions to the uncertainty of the value of the specific activity are volatility of H2 [GeCl6] during the preparation of solid sources for coincidence measurements, drop mass‐ es, counting statistics, background variation, accidental coincidences and dead time losses, Compton continuum of the 1077 keV peak included in the γ window around the 511 keV peak, decay scheme correct ion factor, correction factor for non-vanishing εEC, impurities and halflife uncertainty, and detection of 511 keV quanta in the β detector due to its γ sensitivity [52].

The components contributing to the uncertainty of 4πβ-γ coincidence method were estimat‐ ed as follows: counting statistics and background variation, instrumental corrections, impur‐ ities, half-life uncertainty, decay scheme correction factor, and mass of droplet. Standard deviation of LSC composed of the following contributions: counting statistics, background variation, scintillator stability, comparison with H-3 tracer, instrumental corrections (dead time), dilution factor, droplet mass, radioactive impurities, half-life uncertainty, main decay data, uncertainty of the ε calculation due to the K-L model, capture probabilities PK, PL, fluo‐ rescence yields, ωK, ωL, spectral distribution of β particles, and average energy of weak Aug‐ er electrons [75].

an injected activity greater than 20% different from the prescribed dosage. Tthe Society of Nuclear Medicine (SNM) guidelines also recommend that the measurement be with 10% of the prescribed dosage. Moreover, the instruments being used are capable of accurate meas‐ urements to within 5% [79]. Therefore, programs for the establishment and dissemination of

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International comparison of standard sources and solutions, such as P-32, Mn-54, Zn-65, Ir-192, Tl-204, and Am-241, which is organized by the International Committee of Weights and Measures (CIPM), the EUROMET system, the former COMECOM, and bilateral com‐

South Africa's national radioactivity measurement standard is maintained by the National Metrology Laboratory (NML) of the Council for Scientific and Industrial Research (CSIR). Standardizations are undertaken by a number of direct methods utilizing liquid scintillation

Comparisons of activity measurements for I-131, Tl-201 and Tc-99m with radionuclide cali‐ brators were organized in Cuba since 2002. During 2002, the Radionuclide Metrology De‐ partment of the Isotope Center (CENTIS-DMR) has organized several comparisons with various radionuclides in order to obtain information on the quality of the activity measure‐

The Australian Radiation Protection and Nuclear Safety Agency (ARPANSA) conducts a series of Radiopharmaceutical Quality Assurance Test Program under a Memorandum of Under‐ standing (MOU) between ARPANSA and the Therapeutic Goods Administration (TGA). For example, in 2005, 46 batches of 24 different types of radiopharmaceuticals, e.g., ready to use radiopharmaceuticals and kits for the preparation of Tc-99m were tested. Two percent in 46

International comparison program of national metrological institutes for the standardization of Fe-55, which is a suitable radionuclide standard for X-ray spectrometers, was held by the Comité Consultative pour les Etalons de Mesures des Rayonnements Ionisants (CCEMRI) of the Bureau International des Poids et Mesures (BIPM) [62]. National Metrology Institute of Japan - Advanced Industrial Science and Technology (NMIJ/AIST, Japan) and National In‐ stitute of Ionizing Radiation Metrology (ENEA-INMRI, Italy) have been involved in recent years, particularly those relevant in the frame of the international cooperation coordinated by the BIPM and the International Committee for Radionuclide Metrology (ICRM). Particu‐ lar research activities are devoted on the field of the nuclear safety, nuclear medicine and environmental radionuclide measurements. [83]. International comparisons held by BIPM also can be traced by laboratories such as National Institute for Physics and Nuclear Engi‐ neering (Romania) [59], Laboratorio de Metrologia Nuclear (Brazil) in collaboration with the Laboratório Nacional de Metrologia das Radiações Ionizantes, from Rio de Janeiro [57], Ra‐ diation Safety Systems Division, Bhabha Atomic Research Centre (India) [61], and Electro‐

The Ce-139 measurements formed part of a regional comparison organized by the Asia Pa‐

ments during production and administration of radiopharmaceuticals in Cuba [81].

batches of radiopharmaceuticals tested was failure to meet full specifications [82].

activity measurement standards in nuclear medicine are held in many countries.

parisons, has been held since 1962 [60].

technical Laboratory (ETL) (Japan) [69].

cific Metrology Programme (APMP) [68].

counting (LSC) [80].

Source of the uncertainty: counting statistics, mass, dead time, background, timing, chemi‐ cal effects (adsorption, sample spread, impurities), input parameters and statistical model, quenching, kB influence, decay scheme parameters, and pulse shape discriminator setting. [76]

(c) Uncertainty for the non-extrapolation tracer method

The quoted total uncertainty (1σ) of 0.85% comprised mainly the components due to count‐ ing statistics (0.28%), afterpulsing (0.40%) and the evaluated decay-scheme data (0.63%). εM: double tube detection efficiency of Mn-54, εM\*: reduced Mn-54 efficiency due to quenching caused by the addition of the Fe-55 aliquot [65].

(d) Uncertainty for the coincidence method by a 4πβ-γ system

Uncertainty components assayed by Koskinas et al. for the standardization of Eu-152 were counting statistics, weighing, dead time, impurities, half life, extrapolation of efficiency curve [77].

(e) Uncertainty for the TDCR method

The main source of uncertainty of TDCR method comes from the model describing the nonlinearity of the scintillator due to the ionization quenching phenomenon [71]. Type A stand‐ ard uncertainty, i.e. counting statistics and type B standard uncertainty, i.e. extrapolation (interception uncertainty), spurious pulses, nonuniformity of sources, tracer activity, E. C. correction, dead-time, background, half-life, weighing were evaluated by Sahagia et al. [59].

(f) Uncertainty for the 4πγ counting method.

Construction of an ionization chamber efficiency curve is not a straightforward process as the curve has to be extracted from experimental calibration points analytically. The efficien‐ cy curve is implicitly contained in individual radionuclide coefficients and these are ob‐ tained experimentally or by Monte Carlo modelling or calculated back from the efficiency curve. Due to this variety, the interpretation and intercomparison of different efficiency curves is often hard and transferring individual radionuclide calibration coefficients be‐ tween ionization chambers of different constructions is not a simple process [78].

#### *3.1.3. International measurement program*

One of the most important components in the quality system of radiopharmaceuticals is to establish the measurement traceability to international standards for ensuring the accurate and consistent of measurement results [5]. Traceability of activity measurements is the criti‐ cal part in the production and use of unsealed radioactive sources in nuclear medicine. The U.S. Nuclear Regulatory Commission (NRC) defines a medical event as a patient receiving an injected activity greater than 20% different from the prescribed dosage. Tthe Society of Nuclear Medicine (SNM) guidelines also recommend that the measurement be with 10% of the prescribed dosage. Moreover, the instruments being used are capable of accurate meas‐ urements to within 5% [79]. Therefore, programs for the establishment and dissemination of activity measurement standards in nuclear medicine are held in many countries.

deviation of LSC composed of the following contributions: counting statistics, background variation, scintillator stability, comparison with H-3 tracer, instrumental corrections (dead time), dilution factor, droplet mass, radioactive impurities, half-life uncertainty, main decay data, uncertainty of the ε calculation due to the K-L model, capture probabilities PK, PL, fluo‐ rescence yields, ωK, ωL, spectral distribution of β particles, and average energy of weak Aug‐

Source of the uncertainty: counting statistics, mass, dead time, background, timing, chemi‐ cal effects (adsorption, sample spread, impurities), input parameters and statistical model, quenching, kB influence, decay scheme parameters, and pulse shape discriminator setting. [76]

The quoted total uncertainty (1σ) of 0.85% comprised mainly the components due to count‐ ing statistics (0.28%), afterpulsing (0.40%) and the evaluated decay-scheme data (0.63%). εM: double tube detection efficiency of Mn-54, εM\*: reduced Mn-54 efficiency due to quenching

Uncertainty components assayed by Koskinas et al. for the standardization of Eu-152 were counting statistics, weighing, dead time, impurities, half life, extrapolation of efficiency

The main source of uncertainty of TDCR method comes from the model describing the nonlinearity of the scintillator due to the ionization quenching phenomenon [71]. Type A stand‐ ard uncertainty, i.e. counting statistics and type B standard uncertainty, i.e. extrapolation (interception uncertainty), spurious pulses, nonuniformity of sources, tracer activity, E. C. correction, dead-time, background, half-life, weighing were evaluated by Sahagia et al. [59].

Construction of an ionization chamber efficiency curve is not a straightforward process as the curve has to be extracted from experimental calibration points analytically. The efficien‐ cy curve is implicitly contained in individual radionuclide coefficients and these are ob‐ tained experimentally or by Monte Carlo modelling or calculated back from the efficiency curve. Due to this variety, the interpretation and intercomparison of different efficiency curves is often hard and transferring individual radionuclide calibration coefficients be‐

One of the most important components in the quality system of radiopharmaceuticals is to establish the measurement traceability to international standards for ensuring the accurate and consistent of measurement results [5]. Traceability of activity measurements is the criti‐ cal part in the production and use of unsealed radioactive sources in nuclear medicine. The U.S. Nuclear Regulatory Commission (NRC) defines a medical event as a patient receiving

tween ionization chambers of different constructions is not a simple process [78].

(c) Uncertainty for the non-extrapolation tracer method

(d) Uncertainty for the coincidence method by a 4πβ-γ system

caused by the addition of the Fe-55 aliquot [65].

(e) Uncertainty for the TDCR method

(f) Uncertainty for the 4πγ counting method.

*3.1.3. International measurement program*

er electrons [75].

278 Latest Research into Quality Control

curve [77].

International comparison of standard sources and solutions, such as P-32, Mn-54, Zn-65, Ir-192, Tl-204, and Am-241, which is organized by the International Committee of Weights and Measures (CIPM), the EUROMET system, the former COMECOM, and bilateral com‐ parisons, has been held since 1962 [60].

South Africa's national radioactivity measurement standard is maintained by the National Metrology Laboratory (NML) of the Council for Scientific and Industrial Research (CSIR). Standardizations are undertaken by a number of direct methods utilizing liquid scintillation counting (LSC) [80].

Comparisons of activity measurements for I-131, Tl-201 and Tc-99m with radionuclide cali‐ brators were organized in Cuba since 2002. During 2002, the Radionuclide Metrology De‐ partment of the Isotope Center (CENTIS-DMR) has organized several comparisons with various radionuclides in order to obtain information on the quality of the activity measure‐ ments during production and administration of radiopharmaceuticals in Cuba [81].

The Australian Radiation Protection and Nuclear Safety Agency (ARPANSA) conducts a series of Radiopharmaceutical Quality Assurance Test Program under a Memorandum of Under‐ standing (MOU) between ARPANSA and the Therapeutic Goods Administration (TGA). For example, in 2005, 46 batches of 24 different types of radiopharmaceuticals, e.g., ready to use radiopharmaceuticals and kits for the preparation of Tc-99m were tested. Two percent in 46 batches of radiopharmaceuticals tested was failure to meet full specifications [82].

International comparison program of national metrological institutes for the standardization of Fe-55, which is a suitable radionuclide standard for X-ray spectrometers, was held by the Comité Consultative pour les Etalons de Mesures des Rayonnements Ionisants (CCEMRI) of the Bureau International des Poids et Mesures (BIPM) [62]. National Metrology Institute of Japan - Advanced Industrial Science and Technology (NMIJ/AIST, Japan) and National In‐ stitute of Ionizing Radiation Metrology (ENEA-INMRI, Italy) have been involved in recent years, particularly those relevant in the frame of the international cooperation coordinated by the BIPM and the International Committee for Radionuclide Metrology (ICRM). Particu‐ lar research activities are devoted on the field of the nuclear safety, nuclear medicine and environmental radionuclide measurements. [83]. International comparisons held by BIPM also can be traced by laboratories such as National Institute for Physics and Nuclear Engi‐ neering (Romania) [59], Laboratorio de Metrologia Nuclear (Brazil) in collaboration with the Laboratório Nacional de Metrologia das Radiações Ionizantes, from Rio de Janeiro [57], Ra‐ diation Safety Systems Division, Bhabha Atomic Research Centre (India) [61], and Electro‐ technical Laboratory (ETL) (Japan) [69].

The Ce-139 measurements formed part of a regional comparison organized by the Asia Pa‐ cific Metrology Programme (APMP) [68].

The National Institute of Standards and Technology (NIST) maintains a program for the establishment and dissemination of activity measurement standards in nuclear medicine, i.e. Ga-67, Y-90, Tc-99m, Mo-99, In-111, I-125, I-131, and Tl-201 for more than ten years. These standards are disseminated through Standard Reference Materials (SRMs), Calibration Serv‐ ices, radionuclide calibrator settings, and the NIST Radioactivity Measurement Assurance Program (NRMAP, formerly the NEI/NIST MAP). For over 3600 comparisons, 96% of the participants' results differed from that of NIST by less than 10%, with 98% being less than 20%. The percentage of participants results within 10% of NIST ranges from 88% to 98% [79].

In PET/CT, truncation artifacts occur due to the difference in size of the field of view be‐ tween the CT (50 cm) and PET (70 cm) tomographs [87,88] and frequently seen in large pa‐ tients or patients scanned with arms down, such as in the case of melanoma and head and neck indications. When a patient extends beyond the CT field of view, the extended part of the anatomy is truncated and consequently is not represented in the reconstructed CT im‐ age. Truncation also causes streaking artifacts at the edge of the CT image, leading to an overestimation of the attenuation coefficients used to correct the PET data. This increase in attenuation coefficients creates a rim of high activity at the truncation edge, resulting in the

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281

The most prevalent artifact in PET/CT imaging is respiratory motion during scanning. The artifact is due to the discrepancy between the chest position on the CT image and the chest position on the PET image. PET images are acquired over time periods (time frames) that can vary from a few seconds to tens of minutes. Therefore, during such time periods various motions may have significant effects on the PET images. Both respiratory and contraction induced heart motions have major effect (source of error) on PET imaging of cardiac and thoracic regions. Some equipment, e.g., dose calibrators for the measurements of quantita‐ tive measurements is calibrated against or traceable to a reference source of whole body to‐ mographs [89]. Because of the long acquisition time of a PET scan, it is acquired while the patient is freely breathing. The final image is hence an average of many breathing cycles. On the other hand, a CT scan is usually acquired during a specific stage of the breathing cycle. This difference in respiratory motion between PET scans and CT scans results in breathing artifacts on PET/CT images. Several literatures have described this problem [90-91]. The arti‐ facts resulted from respiratory motion or patient motion is also commonly found in myocar‐ dial perfusion SPECT. This is because that SPECT requires that the object of interest remains constant for the duration of the acquisition [92-93]. Visually detectable patient motion has

been reported in 36% of clinical studies in one study [94] and 43% in another [95].

Source of clinical problems of the patients were also indicated by Hladik III, including (i) special patient populations, e.g., pregnant or breast-feeding women, pediatric and geriatric patients, patients requiring dialysis, incontinent, catheterized or miscellaneous patients, (ii) insufficient patient care, education, and preparation, e.g. insufficient patient instruction, shielding or protection in exposure and contamination problems, pregnancy testing, with‐ holding xanthine-containing foods and drug-drug interaction prior to imaging, delay in the administration or imaging, metal implants of patient, (iii) improper behavior of patient, e.g., excessive movement, contamination from incontinence, attenuation from jewelry, prosthe‐ ses, or implants, etc., and (iv) unexpected altered biodistributions may be undetectable, ad‐

There are several patient-related artifacts and interpretation pitfalls that can potentially compromise nuclear medicine imaging, as discussed above. In order to minimize or identify these artifacts, technologists play an important role in recognizing and correcting them. For example, technologists should ask patients to remove all metallic objects before imaging and should document the location of non-removable metallic objects to minimize or identify the

misinterpretation of the PET scan.

verse reactions or untoward effects, [96]

(b) Equipment- or technologist-related risks:

Measurements from a variety of types of detectors including, ionization chambers, radionu‐ clide calibrators, solid state detectors, Ge detectors, NaI(Tl) detectors, liquid scintillation counters (LSC), Cherenkov counting, and proportional counter are reported [79].

#### **3.2. Nuclear medicine imaging**

#### *3.2.1. PET, CT, PET/CT, and SPECT imaging*

PET, CT, PET/CT, and SPECT are non-invasive imaging tools and applied for creating two dimensional (2D) cross section images of three dimensional (3D) objects. PET and SPECT can potentially provide functional or biochemical information by measuring distribution and kinetics of radiolabelled molecules, whereas CT visualizes X-ray density in tissues in the body. The PET imaging in oncology has been migrating from the use of dedicated PET scanners to the use of PET/CT tomographs. This is due to the advantages that PET/CT offers over dedicated PET. One of these advantages is that the integration of PET and CT imaging into a single scanning session allows excellent fusion of the acquired data. Although these nuclear medicine imaging tools provide many advantages and applications in diagnosing diseases clinically, they also poses some challenges and induce artifacts and quantitative er‐ rors that can affect the image quality.

#### *3.2.2. Risks of artifact in PET, CT, and SPECT imaging*

Artifacts and pitfalls can arise at any stage in the process of nuclear medicine imaging and can be grouped into issues related to the (i) patient, (ii) the equipment, or the technologist.

#### (a) Patient-related risks:

In PET/CT, the patient-related artifacts commonly found are due to metallic implants, trun‐ cation, and respiratory motion (or patient motion). These artifacts occur because the CT scan is used to replace a PET transmission scan for the purpose of attenuation correction of the PET data.

Metallic implants, such as dental fillings, hip prosthetics, or chemotherapy ports, cause high CT numbers and generate streaking artifacts on CT images due to their high pho‐ ton absorption [85,86]. This increase CT numbers causes correspondingly high PET attenu‐ ation coefficients, resulting in an overestimation of the PET activity and thereby to a falsepositive PET finding.

In PET/CT, truncation artifacts occur due to the difference in size of the field of view be‐ tween the CT (50 cm) and PET (70 cm) tomographs [87,88] and frequently seen in large pa‐ tients or patients scanned with arms down, such as in the case of melanoma and head and neck indications. When a patient extends beyond the CT field of view, the extended part of the anatomy is truncated and consequently is not represented in the reconstructed CT im‐ age. Truncation also causes streaking artifacts at the edge of the CT image, leading to an overestimation of the attenuation coefficients used to correct the PET data. This increase in attenuation coefficients creates a rim of high activity at the truncation edge, resulting in the misinterpretation of the PET scan.

The most prevalent artifact in PET/CT imaging is respiratory motion during scanning. The artifact is due to the discrepancy between the chest position on the CT image and the chest position on the PET image. PET images are acquired over time periods (time frames) that can vary from a few seconds to tens of minutes. Therefore, during such time periods various motions may have significant effects on the PET images. Both respiratory and contraction induced heart motions have major effect (source of error) on PET imaging of cardiac and thoracic regions. Some equipment, e.g., dose calibrators for the measurements of quantita‐ tive measurements is calibrated against or traceable to a reference source of whole body to‐ mographs [89]. Because of the long acquisition time of a PET scan, it is acquired while the patient is freely breathing. The final image is hence an average of many breathing cycles. On the other hand, a CT scan is usually acquired during a specific stage of the breathing cycle. This difference in respiratory motion between PET scans and CT scans results in breathing artifacts on PET/CT images. Several literatures have described this problem [90-91]. The arti‐ facts resulted from respiratory motion or patient motion is also commonly found in myocar‐ dial perfusion SPECT. This is because that SPECT requires that the object of interest remains constant for the duration of the acquisition [92-93]. Visually detectable patient motion has been reported in 36% of clinical studies in one study [94] and 43% in another [95].

Source of clinical problems of the patients were also indicated by Hladik III, including (i) special patient populations, e.g., pregnant or breast-feeding women, pediatric and geriatric patients, patients requiring dialysis, incontinent, catheterized or miscellaneous patients, (ii) insufficient patient care, education, and preparation, e.g. insufficient patient instruction, shielding or protection in exposure and contamination problems, pregnancy testing, with‐ holding xanthine-containing foods and drug-drug interaction prior to imaging, delay in the administration or imaging, metal implants of patient, (iii) improper behavior of patient, e.g., excessive movement, contamination from incontinence, attenuation from jewelry, prosthe‐ ses, or implants, etc., and (iv) unexpected altered biodistributions may be undetectable, ad‐ verse reactions or untoward effects, [96]

#### (b) Equipment- or technologist-related risks:

The National Institute of Standards and Technology (NIST) maintains a program for the establishment and dissemination of activity measurement standards in nuclear medicine, i.e. Ga-67, Y-90, Tc-99m, Mo-99, In-111, I-125, I-131, and Tl-201 for more than ten years. These standards are disseminated through Standard Reference Materials (SRMs), Calibration Serv‐ ices, radionuclide calibrator settings, and the NIST Radioactivity Measurement Assurance Program (NRMAP, formerly the NEI/NIST MAP). For over 3600 comparisons, 96% of the participants' results differed from that of NIST by less than 10%, with 98% being less than 20%. The percentage of participants results within 10% of NIST ranges from 88% to 98% [79].

Measurements from a variety of types of detectors including, ionization chambers, radionu‐ clide calibrators, solid state detectors, Ge detectors, NaI(Tl) detectors, liquid scintillation

PET, CT, PET/CT, and SPECT are non-invasive imaging tools and applied for creating two dimensional (2D) cross section images of three dimensional (3D) objects. PET and SPECT can potentially provide functional or biochemical information by measuring distribution and kinetics of radiolabelled molecules, whereas CT visualizes X-ray density in tissues in the body. The PET imaging in oncology has been migrating from the use of dedicated PET scanners to the use of PET/CT tomographs. This is due to the advantages that PET/CT offers over dedicated PET. One of these advantages is that the integration of PET and CT imaging into a single scanning session allows excellent fusion of the acquired data. Although these nuclear medicine imaging tools provide many advantages and applications in diagnosing diseases clinically, they also poses some challenges and induce artifacts and quantitative er‐

Artifacts and pitfalls can arise at any stage in the process of nuclear medicine imaging and can be grouped into issues related to the (i) patient, (ii) the equipment, or the technologist.

In PET/CT, the patient-related artifacts commonly found are due to metallic implants, trun‐ cation, and respiratory motion (or patient motion). These artifacts occur because the CT scan is used to replace a PET transmission scan for the purpose of attenuation correction of the

Metallic implants, such as dental fillings, hip prosthetics, or chemotherapy ports, cause high CT numbers and generate streaking artifacts on CT images due to their high pho‐ ton absorption [85,86]. This increase CT numbers causes correspondingly high PET attenu‐ ation coefficients, resulting in an overestimation of the PET activity and thereby to a false-

counters (LSC), Cherenkov counting, and proportional counter are reported [79].

**3.2. Nuclear medicine imaging**

280 Latest Research into Quality Control

*3.2.1. PET, CT, PET/CT, and SPECT imaging*

rors that can affect the image quality.

(a) Patient-related risks:

positive PET finding.

PET data.

*3.2.2. Risks of artifact in PET, CT, and SPECT imaging*

There are several patient-related artifacts and interpretation pitfalls that can potentially compromise nuclear medicine imaging, as discussed above. In order to minimize or identify these artifacts, technologists play an important role in recognizing and correcting them. For example, technologists should ask patients to remove all metallic objects before imaging and should document the location of non-removable metallic objects to minimize or identify the artifacts from metallic implants. In PET/CT imaging, it is crucial for technologists to careful‐ ly position patients at the center of the field of view and with arms above head to reduce truncation artifacts. Moreover, in order to minimize the artifacts from respiratory motion and produce accurately quantifiable images, it is also essential that technologists instruct pa‐ tients about breath-hold techniques before the scanning session.

sources of error or uncertainty, were elucidated. Although there still have many hard-tocontrolled quantitative errors and artifacts that can eventually affect the quality of imaging, therapeutic efficacy, or safety, it is important for the facility staffs to be aware and continual improvement of these quality factors. By reducing uncertainty and risk or increasing process knowledge and product understanding resulting from QbD can significantly improve the ef‐

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283

ficiency of manufacturing processes.

API Active pharmaceutical ingredient

BNMS British Nuclear Medicine Society

CFR Code of Federal Regulations (U.S.)

CMC Chemistry, and manufacturing and controls

CSIR Council for Scientific and Industrial Research

CGRPP Current Good Radiopharmacy Practice (EU)

EANM European Association of Nuclear Medicine (EU)

EDQM European Directorate for the Quality of Medicines & HealthCare

IMP Investigational Medicinal Product (for drugs used in clinical trials of EU)

CGMP Current Good Manufacturing Practice

CT Computed Tomography

CQAs Critical quality attributes

EC Ethical Committee (EU)

EIND Exploratory IND (FDA, U.S.)

IND Investigational new drug

LSC Liquid scintillation counting

FDA U.S. Food and Drug Administration

IAEA International Atomic Energy Agency

IRDS Investigational Radiopharmaceutical Drug Service

HPLC High-performance liquid chromatography ICH International Conference on Harmonisation

ARPANSA Australian Radiation Protection and Nuclear Safety Agency

BIPM Bureau International des Poids et Mesures (France)

**5. Abbreviations**

Moreover, sources of clinical problems of error medication also include fail of (i) patient identification, (ii) dosage prescription and administration, (iii) radionuclide administration, (iv) radiopharmaceutical prescription and administration in kinetics or finished product pu‐ rity testing, (v) interventional medications, (vi) injection technique, (vii) radiopharmaceuti‐ cal labelled, (viii) preparation or execution of diagnostic or therapeutic procedure, and (ix) radiation protection [7,96].

QC performed on nuclear medicine cameras provides the confidence to technologists and physicians that a scan supplies an accurate representation of the radioisotope distribution in the patient. The instrumentation for nuclear medicine imaging is more complex than that used for whole-body and planar imaging, and requires careful quality control to ensure op‐ timum performance. According to the standards, the main performance parameters are div‐ ided into two groups. The first group includes basic intrinsic measurements: spatial resolution in axial and transaxial directions, sensitivity, count rate capabilities by measuring the system dead time and the generation of random events at different radioactivity levels, and scatter fraction of γ rays emitted by the annihilation of positron. The second group in‐ cludes measurements of the accuracy of corrections for physical effects, specifically: uni‐ formity correction, scatter correction, attenuation correction, and count rate linearity correction. Other possible tests to be added to the list of acceptance or performance tests such as: noise equivalent count rate, partial volume and spillover, motion artefacts, image quality test, and PET/CT image co-registration [89].

Nuclear medicine imaging increases the accuracy of diagnosis by combining anatomic infor‐ mation with functional imaging. It is highly dependent on a host of technical considerations. Knowledgeable technologists can minimize or reduce artifacts and other potential problems with image acquisition and, in that way, produce better-quality images.

#### **4. Conclusion**

Implement of ICH QbD for the radiopharmaceutical manufacturing and imaging technology can be harmonized to a globalized framework in accordance with the regulations and re‐ quirements of U.S. FDA, IAEA, WHO and EANM. The attributes of the components in the quality unit (QA/QC), including the aspects of organization, staffing and personnel, facili‐ ties, instrumentation and equipment, operation procedure, radiopharmaceuticals, protocol and conduct of a study or a treatment, records and reports, and audit were reviewed and indentified. Critical quality attributes (CQAs) for assuring accurate radioactive dosimetry calculation in the efficiency tracing of absolute activity measurement and in the patient- and technologist-related risks for nuclear medicine imaging (PET, CT, and SPECT), i.e. potential sources of error or uncertainty, were elucidated. Although there still have many hard-tocontrolled quantitative errors and artifacts that can eventually affect the quality of imaging, therapeutic efficacy, or safety, it is important for the facility staffs to be aware and continual improvement of these quality factors. By reducing uncertainty and risk or increasing process knowledge and product understanding resulting from QbD can significantly improve the ef‐ ficiency of manufacturing processes.

#### **5. Abbreviations**

artifacts from metallic implants. In PET/CT imaging, it is crucial for technologists to careful‐ ly position patients at the center of the field of view and with arms above head to reduce truncation artifacts. Moreover, in order to minimize the artifacts from respiratory motion and produce accurately quantifiable images, it is also essential that technologists instruct pa‐

Moreover, sources of clinical problems of error medication also include fail of (i) patient identification, (ii) dosage prescription and administration, (iii) radionuclide administration, (iv) radiopharmaceutical prescription and administration in kinetics or finished product pu‐ rity testing, (v) interventional medications, (vi) injection technique, (vii) radiopharmaceuti‐ cal labelled, (viii) preparation or execution of diagnostic or therapeutic procedure, and (ix)

QC performed on nuclear medicine cameras provides the confidence to technologists and physicians that a scan supplies an accurate representation of the radioisotope distribution in the patient. The instrumentation for nuclear medicine imaging is more complex than that used for whole-body and planar imaging, and requires careful quality control to ensure op‐ timum performance. According to the standards, the main performance parameters are div‐ ided into two groups. The first group includes basic intrinsic measurements: spatial resolution in axial and transaxial directions, sensitivity, count rate capabilities by measuring the system dead time and the generation of random events at different radioactivity levels, and scatter fraction of γ rays emitted by the annihilation of positron. The second group in‐ cludes measurements of the accuracy of corrections for physical effects, specifically: uni‐ formity correction, scatter correction, attenuation correction, and count rate linearity correction. Other possible tests to be added to the list of acceptance or performance tests such as: noise equivalent count rate, partial volume and spillover, motion artefacts, image

Nuclear medicine imaging increases the accuracy of diagnosis by combining anatomic infor‐ mation with functional imaging. It is highly dependent on a host of technical considerations. Knowledgeable technologists can minimize or reduce artifacts and other potential problems

Implement of ICH QbD for the radiopharmaceutical manufacturing and imaging technology can be harmonized to a globalized framework in accordance with the regulations and re‐ quirements of U.S. FDA, IAEA, WHO and EANM. The attributes of the components in the quality unit (QA/QC), including the aspects of organization, staffing and personnel, facili‐ ties, instrumentation and equipment, operation procedure, radiopharmaceuticals, protocol and conduct of a study or a treatment, records and reports, and audit were reviewed and indentified. Critical quality attributes (CQAs) for assuring accurate radioactive dosimetry calculation in the efficiency tracing of absolute activity measurement and in the patient- and technologist-related risks for nuclear medicine imaging (PET, CT, and SPECT), i.e. potential

with image acquisition and, in that way, produce better-quality images.

tients about breath-hold techniques before the scanning session.

quality test, and PET/CT image co-registration [89].

radiation protection [7,96].

282 Latest Research into Quality Control

**4. Conclusion**

API Active pharmaceutical ingredient ARPANSA Australian Radiation Protection and Nuclear Safety Agency BNMS British Nuclear Medicine Society BIPM Bureau International des Poids et Mesures (France) CT Computed Tomography CFR Code of Federal Regulations (U.S.) CMC Chemistry, and manufacturing and controls CQAs Critical quality attributes CSIR Council for Scientific and Industrial Research CGMP Current Good Manufacturing Practice CGRPP Current Good Radiopharmacy Practice (EU) EC Ethical Committee (EU) EANM European Association of Nuclear Medicine (EU) EDQM European Directorate for the Quality of Medicines & HealthCare EIND Exploratory IND (FDA, U.S.) FDA U.S. Food and Drug Administration HPLC High-performance liquid chromatography ICH International Conference on Harmonisation IMP Investigational Medicinal Product (for drugs used in clinical trials of EU) IND Investigational new drug IAEA International Atomic Energy Agency IRDS Investigational Radiopharmaceutical Drug Service LSC Liquid scintillation counting

MA Marketing authorization (EU) MS Mass spectrometry MOU Memorandum of Understanding NRC Nuclear Regulatory Commission NCA National Competent Authority (EU) PET Positron emission tomography QA Quality assurance QC Quality control QP Qualified persons who are professional responsible for the release of a drug in Europe QbD Qulaity by design RPR Responsible person for the small-scale preparation of radiopharmaceuticals RDRC Radioactive Drug Research Committee (FDA, U.S.) SPECT Single photon emission computed tomography SSRP Small-scale "in-house" radiopharmaceutical SOP Standard operating procedure TGA Therapeutic Goods Administration TLC Thin layer chromatography USP United States Pharmacopeia WHO World Health Organization

Public\_Web\_Site/ICH\_Products/Guidelines/Quality/Q8\_R1/Step4/Q8\_R2\_Guide‐

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line.pdf.

#### **Author details**

Kung-Tien Liu1\*, Jian-Hua Zhao2 , Lee-Chung Men2 and Chien-Hsin Chen1

\*Address all correspondence to: ktliu@ecic.com.tw


#### **References**

[1] International Conference on Harmonisation. (2009). *ICH Harmonised Tripartite Guide‐ line, Pharmaceutical Development, Q8 (R2).*, http://www.ich.org/fileadmin/ Public\_Web\_Site/ICH\_Products/Guidelines/Quality/Q8\_R1/Step4/Q8\_R2\_Guide‐ line.pdf.

MA Marketing authorization (EU)

MOU Memorandum of Understanding NRC Nuclear Regulatory Commission

NCA National Competent Authority (EU)

QP Qualified persons who are professional responsible for the release of a drug in Europe

RPR Responsible person for the small-scale preparation of radiopharmaceuticals

, Lee-Chung Men2

[1] International Conference on Harmonisation. (2009). *ICH Harmonised Tripartite Guide‐ line, Pharmaceutical Development, Q8 (R2).*, http://www.ich.org/fileadmin/

and Chien-Hsin Chen1

RDRC Radioactive Drug Research Committee (FDA, U.S.) SPECT Single photon emission computed tomography

SSRP Small-scale "in-house" radiopharmaceutical

\*Address all correspondence to: ktliu@ecic.com.tw

1 Everlight Chemical Industrial Corporation,, Taiwan

2 Chemistry Division, Institute of Nuclear Energy Research,, Taiwan

PET Positron emission tomography

SOP Standard operating procedure

TLC Thin layer chromatography USP United States Pharmacopeia WHO World Health Organization

Kung-Tien Liu1\*, Jian-Hua Zhao2

**Author details**

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*strumentation and methods for use in agriculture, industry and medicine*, 64(10-11), 1454

[69] Hino, Y., Matui, S., Yamada, T., Takeuchi, N., Onoma, K., Iwamoto, S., et al. (2000). Absolute Measurement of 166mHo Radioactivity and Development of Sealed Sour‐ ces for Standardization of γ-Ray Emitting Nuclides. *Applied Radiation and Isotopes*,

[70] Broda, R., Péron, M. N., Cassette, P., Terlikowska, T., & Hainos, D. (1998). Standardi‐ zation of 139Ce by the Liquid Scintillation Counting Using the Triple to Double Co‐

[71] Cassette, P., Broda, R., Hainos, D., & Terlikowska, T. (2000). Analysis of Detection-Efficiency Variation Techniques for the Implementation of the TDCR Method in Liq‐

[72] Johansson, L. C., & Sephton, J. P. (2010). Validation of a New TDCR System at NPL.

[73] Kossert, K. (2010). Activity Standardization by Means of a New Tdcr-Cerenkov

[74] Coursey, B. M., Cessna, J., Garcia-Torano, E., Golas, D. B., Grau, Malonda. A., Gray, D. H., et al. (1991). The Standardization and Decay Scheme of Rhenium-186. *Interna‐ tional Journal of Radiation Applications and Instrumentation Part A Applied Radiation and*

[75] Schönfeld, E., Janssen, H., Schotzig, U., Gunther, E., & Schrader, H. (1994). Standardi‐ zation and Decay Data of 186Re. *Nuclear Instruments and Methods in Physics Research Section A: Accelerators*, *Spectrometers, Detectors and Associated Equipment*, 339(1-2),

[76] Günther, E. (2000). Standardization of 237np by the Ciemat/Nist Lsc Tracer Method.

[77] Koskinas, M. F., Fonseca, K. A., & Dias, M. S. (2002). Disintegration Rate Measure‐

[78] Švec, A. (2009). Interpretation of Ionization Chamber Efficiency Curves. *Metrologia*,

[79] Cessna, J. T., & Golas, D. B. (2012). The Nist Radioactivity Measurement Assurance Program for the Radiopharmaceutical Industry. *Applied radiation and isotopes*.

[80] Simpson, B.R.S. (2002). Radioactivity Standardization in South Africa. *Applied Radia‐*

[81] Oropesa, P., Hernández, A. T., Serra, R., Martinez, E., & Varela, C. (2003). Compari‐ sons of Activity Measurements with Radionuclide Calibrators. *Applied Radiation and*

ment of a 152Eu Solution. *Applied Radiation and Isotopes*, 56, 1-2, 441-445.

incidence Ratio Method. *Applied Radiation and Isotopes*, 1035-1040.

*Applied radiation and isotopes*, 1537 -1539 .

*Applied Radiation and Isotopes.*, 52(3), 471-474.

*tion and Isotopes*, 56(1-2), 301-305.

*Isotopes*, 59(5-6), 383-387.

*Isotopes.*, 42(9), 865-869.

174-179.

43-46.

uid Scintillation Counting. *Applied Radiation and Isotopes*, 52(3), 643-648.

Counting Technique. *Applied radiation and isotopes*, 68(6), 1116-1120.


290 Latest Research into Quality Control

52(3), 545-549.


[95] Wheat, J. M., & Currie, G. M. (2004). Incidence and Characterization of Patient Mo‐ tion in Myocardial Perfusion SPECT: Part 1. *Journal of Nuclear Medicine Technology*, 32(2), 60-65.

**Chapter 14**

**Unified Procedures for Quality Controls in Analogue**

Breast cancer is the most commonly diagnosed cancer in women [1]. Current attempts to control breast cancer concentrate on early detection by means of massive screening cam‐ paign, via periodic mammography and physical examination, because ample evidence in‐ dicates that such screening indeed can be effective in lowering the death rate [2]. Early diagnosis of breast cancer plays a leading role in reducing the mortality and improving

Mammography consists in imaging the female breast using X-rays with low contrast (to keep the delivered dose low), but at the same time high resolution (especially used for

The goal of mammography is to achieve the image quality required for a given detection task, while ensuring that the patient-absorbed dose is kept as low as reasonably achievable [4]. As practised now, it normally requires a dedicated X-ray tube with special anode materials such as molybdenum or rhodium, small focal spots, operating at a tube voltage around 25 to 32 kV, and carefully chosen films and screens in dedicated cassettes. Stationary or moving grids are used as in other branches of plain film radiography. Present-day mammography can be descri‐ bed as a low-dose procedure [5]. In recent years, advances in screen-film technology and filmprocessing techniques have contributed to major improvements in the quality of

> © 2012 Testagrossa et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2012 Testagrossa et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mammographic images. At present, two distinct mammographic techniques exist:

**•** Analogue mammography in which the image is recorded on a film.

**and Digital Mammography**

Raffaele Novario, Maria Giulia Tripepi and

Additional information is available at the end of the chapter

Giuseppe Vermiglio

**1. Introduction**

early detection).

http://dx.doi.org/10.5772/51349

the prognosis of this disease [3].

Barbara Testagrossa, Giuseppe Acri, Federica Causa,

[96] Hladik, W. B., & Norenberg, J. P. (1996). Problems Associated with the Clinical Use of Radiopharmaceuticals: A Proposed Classification System and Troubleshooting Guide. *European Journal of Nuclear Medicine and Molecular Imaging*, 23(8), 997-1002.

## **Unified Procedures for Quality Controls in Analogue and Digital Mammography**

[95] Wheat, J. M., & Currie, G. M. (2004). Incidence and Characterization of Patient Mo‐ tion in Myocardial Perfusion SPECT: Part 1. *Journal of Nuclear Medicine Technology*,

[96] Hladik, W. B., & Norenberg, J. P. (1996). Problems Associated with the Clinical Use of Radiopharmaceuticals: A Proposed Classification System and Troubleshooting Guide. *European Journal of Nuclear Medicine and Molecular Imaging*, 23(8), 997-1002.

32(2), 60-65.

292 Latest Research into Quality Control

Barbara Testagrossa, Giuseppe Acri, Federica Causa, Raffaele Novario, Maria Giulia Tripepi and Giuseppe Vermiglio

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51349

#### **1. Introduction**

Breast cancer is the most commonly diagnosed cancer in women [1]. Current attempts to control breast cancer concentrate on early detection by means of massive screening cam‐ paign, via periodic mammography and physical examination, because ample evidence in‐ dicates that such screening indeed can be effective in lowering the death rate [2]. Early diagnosis of breast cancer plays a leading role in reducing the mortality and improving the prognosis of this disease [3].

Mammography consists in imaging the female breast using X-rays with low contrast (to keep the delivered dose low), but at the same time high resolution (especially used for early detection).

The goal of mammography is to achieve the image quality required for a given detection task, while ensuring that the patient-absorbed dose is kept as low as reasonably achievable [4]. As practised now, it normally requires a dedicated X-ray tube with special anode materials such as molybdenum or rhodium, small focal spots, operating at a tube voltage around 25 to 32 kV, and carefully chosen films and screens in dedicated cassettes. Stationary or moving grids are used as in other branches of plain film radiography. Present-day mammography can be descri‐ bed as a low-dose procedure [5]. In recent years, advances in screen-film technology and filmprocessing techniques have contributed to major improvements in the quality of mammographic images. At present, two distinct mammographic techniques exist:

**•** Analogue mammography in which the image is recorded on a film.

© 2012 Testagrossa et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Testagrossa et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**•** Digital mammography in which the image is digitalised.

The production of analogue or digital mammography images is based on two distinct con‐ cepts of image formation [6].

**a.** Image acquisition, including X-ray generation system, image receptor and (in some sys‐

Unified Procedures for Quality Controls in Analogue and Digital Mammography

http://dx.doi.org/10.5772/51349

295

**c.** Image presentation, including monitor, imaging presentation software, printer and

To produce images with adequate quality, each part of the imaging chain must function within the limits dictated by the standards of screen-film mammography [9], although the

In the EUREF protocol it is assumed that digital mammography should perform at least as

In this context, a unified protocol is proposed here that can be used with either analogue or digital mammography systems, with the view of reducing the volume of verification proce‐ dures to test the operation of such equipment. The advantage of the proposed protocol is that it can be applied as is to both analogue and digital mammography. The results obtained from the application of this protocol to analogue and digital mammography are presented in Section 3, with particular emphasis on image quality. The remaining part of this Section is

In screen-film mammography, the film is used as the medium for both image acquisition and display. However, whilst providing excellent spatial resolution in high contrast struc‐ tures, screen-film mammography has limited detection capability for low-contrast lesions in dense breasts [10]. On phantoms, the highest spatial resolution can be as high as 15–20 lp/mm but with a very low associated contrast. In addition, noise can limit the reliability of detection, especially for the small or subtle structures [11]. Although considerable advances in film-screen mammography have occurred over the past 20 years, some inherent limita‐ tions to further technical improvement exist [12]. One such limitation results from the trade off between dynamic range (latitude) and contrast resolution (gradient) [13]. The relationship between X-ray exposure, image density, and contrast is illustrated by the Hurt‐ er and Driffield (H&D) sigmoid curve (Fig. 1) which uniquely characterises a given type of

Because of the sigmoid shape of the characteristic curve, the range of X-ray exposures over which the film display gradient is significant, i.e., the image latitude, is limited. The parts of the H&D curve where the slope is flat indicate poor contrast (i.e. over- or under-

In screen-film mammography, the automatic exposure control (AEC) has the critical role of ensuring that the appropriate amount of radiation reaches the image receptor to produce a target optical density on the processed film [16]. In AEC systems, an ion chamber or other radiation detector is placed beneath the film cassette and connected electrically to the expo‐ sure time control circuit. When a pre-set amount of radiation has been detected, the expo‐

tems) image receptor corrections;

definition of such limits for digital systems is still in progress.

dedicated to a review of mammographic techniques.

screen-film system under specific conditions [14].

**b.** Image processing software;

viewing box.

screen-film mammography.

**1.1. Screen-film mammography**

exposed images) [12, 16].

The analogue image is a continuous representation of spatial and intensity variations of the X-ray pattern transmitted by the tissue under analysis. Traditionally, the mammo‐ graphic image is analogue, obtained using conventional screen-film image receptors as the standard detector [7]. The advantages of screen-film mammography are: high spatial reso‐ lution and low contrast sensitivity achieved through improvements in X-ray tube design, screen-film combinations, grids, and film processing [8]. Thus, analogue mammography permits high image quality, low patient dose, and most importantly, the ability to detect small, nonpalpable breast cancers.

In digital systems, image acquisition and display are two independent processes [4]. In such systems images are captured as a digital signal, making electronic transfer and storage of images possible. Digital systems offer a large dynamic range of operation, improving visual‐ ization of all areas of the breast and increasing exposure latitude. Also, the digital format allows grayscale adjustment to optimize contrast for any imaging task.

In addition, with the digitalization of the diagnostic image, new medical applications have now emerged, such as Computer-Aided Diagnosis (CAD), stereo mammography, tomosyn‐ thesis, contrast medium imaging and dual energy imaging [7].

For a successful screening function the mammograms should contain sufficient diagnostic information to be able to detect breast cancer, using a radiation dose as low as reasonably achievable (ALARA principle). In this context, it is necessary to establish and actively main‐ tain regular and adequate Quality Assurance (QA) procedures that take into account medi‐ cal, organisational and technical aspects. The QA procedure should include periodic tests to ensure accurate target and critical structure localization. Such tests are referred to as Quality Controls (QC). They are fundamental for the QA procedure because they help ascertain that the equipment performs consistently at a high quality level.

However, whilst the requirement for standardisation is impelling, the Italian legislation (D.L.vo 187/00) is not keeping pace with the advances in mammographic technology. Indeed, at present both analogue and digital formats are used in an un-regulated way, without intro‐ ducing a proper regulation especially for digital mammography. As a consequence, the QA protocols have been adapted ad hoc to the new digital technology, thus resulting in multiple protocols, some of which valid only for specific machines, resulting in high costs of operation.

On the other hand, at the European level, QA procedures for both analogue and digital mam‐ mography systems have been properly addressed and defined, [European guidelines for qual‐ ity assurance in mammography screening – 4th Edition, Section 2]. In both cases, in fact, the QC of the physical and technical aspects must guarantee the best possible diagnostic information obtainable and image quality stability, within the limits imposed by the ALARA principle.

However, for the case of digital systems the imaging chain can be divided into three inde‐ pendent parts, as cited in [9]:


**•** Digital mammography in which the image is digitalised.

cepts of image formation [6].

294 Latest Research into Quality Control

small, nonpalpable breast cancers.

pendent parts, as cited in [9]:

The production of analogue or digital mammography images is based on two distinct con‐

The analogue image is a continuous representation of spatial and intensity variations of the X-ray pattern transmitted by the tissue under analysis. Traditionally, the mammo‐ graphic image is analogue, obtained using conventional screen-film image receptors as the standard detector [7]. The advantages of screen-film mammography are: high spatial reso‐ lution and low contrast sensitivity achieved through improvements in X-ray tube design, screen-film combinations, grids, and film processing [8]. Thus, analogue mammography permits high image quality, low patient dose, and most importantly, the ability to detect

In digital systems, image acquisition and display are two independent processes [4]. In such systems images are captured as a digital signal, making electronic transfer and storage of images possible. Digital systems offer a large dynamic range of operation, improving visual‐ ization of all areas of the breast and increasing exposure latitude. Also, the digital format

In addition, with the digitalization of the diagnostic image, new medical applications have now emerged, such as Computer-Aided Diagnosis (CAD), stereo mammography, tomosyn‐

For a successful screening function the mammograms should contain sufficient diagnostic information to be able to detect breast cancer, using a radiation dose as low as reasonably achievable (ALARA principle). In this context, it is necessary to establish and actively main‐ tain regular and adequate Quality Assurance (QA) procedures that take into account medi‐ cal, organisational and technical aspects. The QA procedure should include periodic tests to ensure accurate target and critical structure localization. Such tests are referred to as Quality Controls (QC). They are fundamental for the QA procedure because they help ascertain that

However, whilst the requirement for standardisation is impelling, the Italian legislation (D.L.vo 187/00) is not keeping pace with the advances in mammographic technology. Indeed, at present both analogue and digital formats are used in an un-regulated way, without intro‐ ducing a proper regulation especially for digital mammography. As a consequence, the QA protocols have been adapted ad hoc to the new digital technology, thus resulting in multiple protocols, some of which valid only for specific machines, resulting in high costs of operation. On the other hand, at the European level, QA procedures for both analogue and digital mam‐ mography systems have been properly addressed and defined, [European guidelines for qual‐ ity assurance in mammography screening – 4th Edition, Section 2]. In both cases, in fact, the QC of the physical and technical aspects must guarantee the best possible diagnostic information obtainable and image quality stability, within the limits imposed by the ALARA principle.

However, for the case of digital systems the imaging chain can be divided into three inde‐

allows grayscale adjustment to optimize contrast for any imaging task.

thesis, contrast medium imaging and dual energy imaging [7].

the equipment performs consistently at a high quality level.

**c.** Image presentation, including monitor, imaging presentation software, printer and viewing box.

To produce images with adequate quality, each part of the imaging chain must function within the limits dictated by the standards of screen-film mammography [9], although the definition of such limits for digital systems is still in progress.

In the EUREF protocol it is assumed that digital mammography should perform at least as screen-film mammography.

In this context, a unified protocol is proposed here that can be used with either analogue or digital mammography systems, with the view of reducing the volume of verification proce‐ dures to test the operation of such equipment. The advantage of the proposed protocol is that it can be applied as is to both analogue and digital mammography. The results obtained from the application of this protocol to analogue and digital mammography are presented in Section 3, with particular emphasis on image quality. The remaining part of this Section is dedicated to a review of mammographic techniques.

#### **1.1. Screen-film mammography**

In screen-film mammography, the film is used as the medium for both image acquisition and display. However, whilst providing excellent spatial resolution in high contrast struc‐ tures, screen-film mammography has limited detection capability for low-contrast lesions in dense breasts [10]. On phantoms, the highest spatial resolution can be as high as 15–20 lp/mm but with a very low associated contrast. In addition, noise can limit the reliability of detection, especially for the small or subtle structures [11]. Although considerable advances in film-screen mammography have occurred over the past 20 years, some inherent limita‐ tions to further technical improvement exist [12]. One such limitation results from the trade off between dynamic range (latitude) and contrast resolution (gradient) [13]. The relationship between X-ray exposure, image density, and contrast is illustrated by the Hurt‐ er and Driffield (H&D) sigmoid curve (Fig. 1) which uniquely characterises a given type of screen-film system under specific conditions [14].

Because of the sigmoid shape of the characteristic curve, the range of X-ray exposures over which the film display gradient is significant, i.e., the image latitude, is limited. The parts of the H&D curve where the slope is flat indicate poor contrast (i.e. over- or underexposed images) [12, 16].

In screen-film mammography, the automatic exposure control (AEC) has the critical role of ensuring that the appropriate amount of radiation reaches the image receptor to produce a target optical density on the processed film [16]. In AEC systems, an ion chamber or other radiation detector is placed beneath the film cassette and connected electrically to the expo‐ sure time control circuit. When a pre-set amount of radiation has been detected, the expo‐ sure is automatically terminated. Other limitations of film-screen mammography include (a) noise caused by the random fluctuation of X-ray quantum absorption by the fluorescent screen and the film emulsion, which can limit the detection of subtle structures, (b) the trade-off between spatial resolution and detection efficiency of the film and screen, and (c) the inefficiency of rejection of scatter radiation by the mammographic grid [12].

(ADC). Once the digital image is stored in the computer memory it can be displayed with

Unified Procedures for Quality Controls in Analogue and Digital Mammography

http://dx.doi.org/10.5772/51349

297

Digital mammography systems, unlike screen-film mammography systems, allow manipu‐ lation of fine differences in image contrast by means of image processing algorithms [10]. The physical properties of the digital image (contrast, resolution and noise) can vary notice‐ ably according to the detection technology used. There are two methods of image capture used in digital mammography that represent different generations of technology: indirect

Indirect conversion digital detectors uses a two step process for X-rays detection, similar to

Direct conversion should not be confused with "direct readout", which is a capability of all

Fully digital mammography (FDM) detectors are the final class of detectors. These detectors are sealed units that are permanently mounted to a mammography system. FDM detectors are electronic devices that directly capture X-ray images. In general, such devices require

Computed Radiography (CR) is at this moment the most common digital radiography mo‐

CR for mammography system employ as the X-ray absorber a storage photostimulable phosphor imaging plate (typically BaFBr:Eu+2, where the atomic energy levels of the europi‐ um activator determine the characteristics of light emission), that replaces the traditional screen-film combination [22]. In this case, the removable detector or the Imaging Plate (IP) is inserted as a cassette in a conventional mammography unit. So, the IP can be used in a

X-ray absorption mechanisms are identical to those of conventional phosphors. The pecu‐ liarity here is that the useful optical signal is not derived from the light that is emitted in

CR digital phosphor plates have shown promise in mammographic imaging because of the

The potential advantages of this technology are the small detector-element size, the fact that the plates can be used also in conventional mammography units, the ease of having multiple plate sizes, and the relatively low cost. In addition the plates are reusable since they can be

However scattering of the light within the phosphor causes the release of traps over a greater area of the image than the size of the incident laser beam. This results in loss of

prompt response to the incident radiation, but rather from the subsequent emission.

dality in radiology departments, in place of conventional screen film systems [21].

contrast independent of the detector properties [12].

that a new mammography system be installed [8].

*1.2.1. Photostimulable phosphors (Computed Radiography systems)*

standard mammography machine without modification [7].

wide exposure latitude and linear response [23].

readily erased optically [8].

spatial resolution [24].

conversion and direct conversion [20].

screen-film [1].

electronic detectors.

**Figure 1.** The Hurted & Driffield (H&D) curve describes optical density (OD) vs. the logarithm (base 10) of exposure [15].

#### **1.2. Digital mammography**

Digital mammography is an emerging technology, first approved in January 2000 [17], in which the image acquisition, display and storage functions can be performed independent‐ ly, allowing for optimisation of each function. It offers several potential advantages includ‐ ing wider dynamic range, improved contrast, increased signal to noise ratio for overcoming the limitations of the film–screen combination (limited latitude, limited display contrast, low detection efficiency and noise), and therefore, increasing the sensitivity and specificity of breast cancer detection [18, 19]. Moreover, digital images offer a variety of new and im‐ proved applications. The digital image will provide image archiving and retrieval advantag‐ es over film, and will facilitate the use of computer-aided diagnosis [11, 20]. Other advanced applications made possible through digital imaging, such as dual energy and 3D tomosyn‐ thesis are expected to further improve diagnostic sensitivity and specificity.

In particular, Full Field Digital Mammography (FFDM) offers the promise of revolutionizing the practice of mammography through its superior dose and contrast performance [20]. In FFDM the screen-film is substituted by a fixed or removable digital detector. The digital image is obtained by sampling the X-ray pattern at discrete increments of spatial position and image signal intensity. Any digital image is a 2-dimensional grid of picture elements (pixels), which is defined by its size and bit depth. The size of an image is given by the length by width (in pixels) product. The bit depth is the number of shades of gray that can be displayed [1].

In a digital imager a detector absorbs the X-rays and produces an electronic signal at each pixel. The signal is then translated into a digital value by an analog-to-digital converter (ADC). Once the digital image is stored in the computer memory it can be displayed with contrast independent of the detector properties [12].

Digital mammography systems, unlike screen-film mammography systems, allow manipu‐ lation of fine differences in image contrast by means of image processing algorithms [10]. The physical properties of the digital image (contrast, resolution and noise) can vary notice‐ ably according to the detection technology used. There are two methods of image capture used in digital mammography that represent different generations of technology: indirect conversion and direct conversion [20].

Indirect conversion digital detectors uses a two step process for X-rays detection, similar to screen-film [1].

Direct conversion should not be confused with "direct readout", which is a capability of all electronic detectors.

Fully digital mammography (FDM) detectors are the final class of detectors. These detectors are sealed units that are permanently mounted to a mammography system. FDM detectors are electronic devices that directly capture X-ray images. In general, such devices require that a new mammography system be installed [8].

#### *1.2.1. Photostimulable phosphors (Computed Radiography systems)*

sure is automatically terminated. Other limitations of film-screen mammography include (a) noise caused by the random fluctuation of X-ray quantum absorption by the fluorescent screen and the film emulsion, which can limit the detection of subtle structures, (b) the trade-off between spatial resolution and detection efficiency of the film and screen, and (c)

**Figure 1.** The Hurted & Driffield (H&D) curve describes optical density (OD) vs. the logarithm (base 10) of exposure [15].

Digital mammography is an emerging technology, first approved in January 2000 [17], in which the image acquisition, display and storage functions can be performed independent‐ ly, allowing for optimisation of each function. It offers several potential advantages includ‐ ing wider dynamic range, improved contrast, increased signal to noise ratio for overcoming the limitations of the film–screen combination (limited latitude, limited display contrast, low detection efficiency and noise), and therefore, increasing the sensitivity and specificity of breast cancer detection [18, 19]. Moreover, digital images offer a variety of new and im‐ proved applications. The digital image will provide image archiving and retrieval advantag‐ es over film, and will facilitate the use of computer-aided diagnosis [11, 20]. Other advanced applications made possible through digital imaging, such as dual energy and 3D tomosyn‐

In particular, Full Field Digital Mammography (FFDM) offers the promise of revolutionizing the practice of mammography through its superior dose and contrast performance [20]. In FFDM the screen-film is substituted by a fixed or removable digital detector. The digital image is obtained by sampling the X-ray pattern at discrete increments of spatial position and image signal intensity. Any digital image is a 2-dimensional grid of picture elements (pixels), which is defined by its size and bit depth. The size of an image is given by the length by width (in pixels)

In a digital imager a detector absorbs the X-rays and produces an electronic signal at each pixel. The signal is then translated into a digital value by an analog-to-digital converter

thesis are expected to further improve diagnostic sensitivity and specificity.

product. The bit depth is the number of shades of gray that can be displayed [1].

**1.2. Digital mammography**

296 Latest Research into Quality Control

the inefficiency of rejection of scatter radiation by the mammographic grid [12].

Computed Radiography (CR) is at this moment the most common digital radiography mo‐ dality in radiology departments, in place of conventional screen film systems [21].

CR for mammography system employ as the X-ray absorber a storage photostimulable phosphor imaging plate (typically BaFBr:Eu+2, where the atomic energy levels of the europi‐ um activator determine the characteristics of light emission), that replaces the traditional screen-film combination [22]. In this case, the removable detector or the Imaging Plate (IP) is inserted as a cassette in a conventional mammography unit. So, the IP can be used in a standard mammography machine without modification [7].

X-ray absorption mechanisms are identical to those of conventional phosphors. The pecu‐ liarity here is that the useful optical signal is not derived from the light that is emitted in prompt response to the incident radiation, but rather from the subsequent emission.

CR digital phosphor plates have shown promise in mammographic imaging because of the wide exposure latitude and linear response [23].

The potential advantages of this technology are the small detector-element size, the fact that the plates can be used also in conventional mammography units, the ease of having multiple plate sizes, and the relatively low cost. In addition the plates are reusable since they can be readily erased optically [8].

However scattering of the light within the phosphor causes the release of traps over a greater area of the image than the size of the incident laser beam. This results in loss of spatial resolution [24].

#### *1.2.2. Optical detector*

The detector consists of a phosphor screen, a charged coupled device (CCD) camera, and a fiberoptic taper to couple the light from the screen to the camera. It now represents the most widely used digital mammography technique for cassette-free imaging [8].

This shortcoming is particularly relevant in the case of mammography because it is well known that both image quality and breast dose depend on the equipment used and the ra‐

Unified Procedures for Quality Controls in Analogue and Digital Mammography

http://dx.doi.org/10.5772/51349

299

For a complete and accurate estimate of image quality and delivered dose, the following

**Screen-film mammography Digital mammography**

Source-to-image distance Alignment of X-ray field/image receptor Film/bucky edge Radiation leakage Output

> Accuracy HVL

> > value

difference per step

reproducibility

Variation in SNR

Variation in dose

AEC Central opt. dens. control settings Exposure control steps: central

Target opt. dens. control settings ------

Long-term reproducibility Long-term

Opt. dens. control step Exposure control steps:

Short-term reproducibility Short-term reproducibility

X-ray source Focal spot size

Tube voltage Reproducibility

diographic technique employed.

**•** Bucky and image receptor

**•** Viewing conditions

X-ray generation

components and system parameters should be monitored [9]:

**•** X-ray generation and exposure control system

**•** Film processing (for screen-film systems)

**•** Monitors and printers (for digital systems)

**•** Image processing (for digital systems)

**•** System properties (including dose)

The imaging performance of these systems depends on a number of factors, including the characteristics of the phosphor screen, the choice of CCD and the method used to optically couple the phosphor to the CCD.

A CCD is an integrated circuit formed by depositing a series of electrodes, called 'gates' on a semiconductor substrate to form an array of metal-oxide-semiconductor (MOS) capacitors [22].

CCDs are particularly well suited to digital radiography because of their high spatial resolu‐ tion capability, wide dynamic range and high degree of linearity with incident signal.

#### *1.2.3. Flat panel*

The active matrix flat panel technology is the most promising digital radiographic tech‐ nique [25, 26].

The active matrix detector is based on large glass substrates on which imaging pixels are deposited.

This flat panel plate consists of a matrix of approximately 5 million photodiodes that form the readout for each image. The charge produced on the diode in response to light emitted from the phosphor surface is collected and digitized [1].

### **2. Quality Control (QC)**

QCs are fundamental to guarantee that the radiological equipment performs consistently, with standard and constant physical and technical operational parameters.

The technological advances of the past ten years have revolutionised imaging techniques for diagnostics. As a consequence, QC procedures need to be updated to suit the new technologies and related protocols. This is particularly true for mammographic equip‐ ment, for which the physical parameters to be monitored to guarantee high-quality imag‐ ing are identified in specific documents.

The European Protocol for "Quality Control of the Physical and Technical Aspects of Mammography Screening" [9] gives guidance on physical, technical and dose measure‐ ments, and the periodicity of the corresponding tests to be performed as part of mam‐ mography screening programmes.

On the other hand, in the case of the Italian regulation, the relevant legislation (D.L.vo 187/00) was approved before the commercialisation of CR and digital mammography. Therefore, guidelines and procedures for CR and digital mammography are missing.

This shortcoming is particularly relevant in the case of mammography because it is well known that both image quality and breast dose depend on the equipment used and the ra‐ diographic technique employed.

For a complete and accurate estimate of image quality and delivered dose, the following components and system parameters should be monitored [9]:


*1.2.2. Optical detector*

298 Latest Research into Quality Control

*1.2.3. Flat panel*

nique [25, 26].

**2. Quality Control (QC)**

ing are identified in specific documents.

mography screening programmes.

deposited.

couple the phosphor to the CCD.

The detector consists of a phosphor screen, a charged coupled device (CCD) camera, and a fiberoptic taper to couple the light from the screen to the camera. It now represents the most

The imaging performance of these systems depends on a number of factors, including the characteristics of the phosphor screen, the choice of CCD and the method used to optically

A CCD is an integrated circuit formed by depositing a series of electrodes, called 'gates' on a semiconductor substrate to form an array of metal-oxide-semiconductor (MOS) capacitors [22]. CCDs are particularly well suited to digital radiography because of their high spatial resolu‐ tion capability, wide dynamic range and high degree of linearity with incident signal.

The active matrix flat panel technology is the most promising digital radiographic tech‐

The active matrix detector is based on large glass substrates on which imaging pixels are

This flat panel plate consists of a matrix of approximately 5 million photodiodes that form the readout for each image. The charge produced on the diode in response to light emitted

QCs are fundamental to guarantee that the radiological equipment performs consistently,

The technological advances of the past ten years have revolutionised imaging techniques for diagnostics. As a consequence, QC procedures need to be updated to suit the new technologies and related protocols. This is particularly true for mammographic equip‐ ment, for which the physical parameters to be monitored to guarantee high-quality imag‐

The European Protocol for "Quality Control of the Physical and Technical Aspects of Mammography Screening" [9] gives guidance on physical, technical and dose measure‐ ments, and the periodicity of the corresponding tests to be performed as part of mam‐

On the other hand, in the case of the Italian regulation, the relevant legislation (D.L.vo 187/00) was approved before the commercialisation of CR and digital mammography.

Therefore, guidelines and procedures for CR and digital mammography are missing.

with standard and constant physical and technical operational parameters.

from the phosphor surface is collected and digitized [1].

widely used digital mammography technique for cassette-free imaging [8].




**Screen-film mammography Digital mammography**

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Exposure time


Spatial resolution MTF and NPS ----- Scanning time ----- Geometric distortion ----- Artifact evaluation ----- Ghost image factor

Some of the above components are suitable only for analogue systems, others only for digital ones, and some are common to both systems although requiring dedicated QC procedures.

With reference to QCs for mammography, the EU legislation is subdivided in two parts: Sec‐ tion 2a for screen-film mammography, Section 2b for digital mammography. In both cases, several measurements should be undertaken by medical physicists. The components that are common to both analogue and digital mammographic systems are listed in Table 1 with corresponding operational parameters specific for the two cases. As expected, the methodol‐ ogy to be used for QC in the two different cases are substantially different particularly with

For example, in the case of traditional, analogue mammography, spatial resolution and threshold contrast visibility can be used to uniquely characterise the image quality. On the other hand, in digital mammography image quality is assessed by monitoring the Modula‐ tion Transfer Function (MTF), Noise Power Spectrum (NPS) and Nyquist frequency. MTF represents the efficiency or fan imaging system in reproducing subject contrast at various spatial frequencies [7, 20, 27]. The Nyquist frequency, instead, indicates the maximum spa‐ tial resolution that can be visualized in an image. NPS provides information on noise at dif‐ ferent spatial frequencies. In digital mammography, in fact, spatial resolution is obtained

The combination of MTF and NPS gives the Detective Quantum Efficiency (DQE), regarded as the best overall indicator of the image quality of digital radiographic systems. DQE is the efficiency with which a detector uses the incident photons to form an image [28]. Systems with higher DQE can produce higher quality images, at the same dose. Further, there are also other parameters that need to be monitored in digital techniques to defined the image

Dosimetry Glandular dose per PMMA thickness

Image quality Threshold contrast visibility

**Table 1.** Operational parameters relevant to analogue and digital mammographs [9].

Inter plate sensitivity variations

respect to image quality monitoring.

from MTF and Nyquist frequency.

quality. These are listed in Table 2.


**Table 1.** Operational parameters relevant to analogue and digital mammographs [9].

**Screen-film mammography Digital mammography**

Object thickness and tube voltage compensation


Back-up timer and security cut-off

Maintain force for 1 minute Compression force indicator Compression plate alignment, symmetric



CNR per PMMA thickness

Object thickness and tube voltage

Correspondence between AEC

Anti scatter grid Grid system factor

Inter cassette sensitivity variation

Screen-film contact -----








Uncorrected dels----- Number of uncorrected

(on image)

images)

defective dels

defective dels

(between images)

Compression Compression force

Screen-film Inter cassette sensitivity variation

(mAs)

(OD range)

Adjustable range ----- Spectra -----

compensation

sensors

Bucky and image receptor

300 Latest Research into Quality Control

Response function

Detector element failure

Missed tissue at chest wall side detector homogeneity

Some of the above components are suitable only for analogue systems, others only for digital ones, and some are common to both systems although requiring dedicated QC procedures.

With reference to QCs for mammography, the EU legislation is subdivided in two parts: Sec‐ tion 2a for screen-film mammography, Section 2b for digital mammography. In both cases, several measurements should be undertaken by medical physicists. The components that are common to both analogue and digital mammographic systems are listed in Table 1 with corresponding operational parameters specific for the two cases. As expected, the methodol‐ ogy to be used for QC in the two different cases are substantially different particularly with respect to image quality monitoring.

For example, in the case of traditional, analogue mammography, spatial resolution and threshold contrast visibility can be used to uniquely characterise the image quality. On the other hand, in digital mammography image quality is assessed by monitoring the Modula‐ tion Transfer Function (MTF), Noise Power Spectrum (NPS) and Nyquist frequency. MTF represents the efficiency or fan imaging system in reproducing subject contrast at various spatial frequencies [7, 20, 27]. The Nyquist frequency, instead, indicates the maximum spa‐ tial resolution that can be visualized in an image. NPS provides information on noise at dif‐ ferent spatial frequencies. In digital mammography, in fact, spatial resolution is obtained from MTF and Nyquist frequency.

The combination of MTF and NPS gives the Detective Quantum Efficiency (DQE), regarded as the best overall indicator of the image quality of digital radiographic systems. DQE is the efficiency with which a detector uses the incident photons to form an image [28]. Systems with higher DQE can produce higher quality images, at the same dose. Further, there are also other parameters that need to be monitored in digital techniques to defined the image quality. These are listed in Table 2.


Acoustic and light signaling

Source-to-image distance

Long-term reproducibility

Short term reproducibility

Object thickness compensation

Tube voltage compensation exposure. Frequency: Daily

Security cut-off Acceptance test, status test and constancy test.

Frequency: Daily

Acceptance test.

Constancy test.

Frequency: Yearly

should be recorded. Frequency: Yearly

Frequency: Yearly

15%.

Acceptance test, status test and constancy test. The acoustic and light signals should function properly.

The security cut-off should function properly.

had to be considerably smaller than the previous one.

Deviations from the reference value of exposures ≤ ± 2%.

The measured counts per second (cps) should be recorded.

Acceptance test, status test and constancy test. Deviations from the mean value of exposures < ± 5%.

Acceptance test, status test and constancy test.

Acceptance test, status test and constancy test.

per second (cps) should be recorded.

Manufacturers specification, typical ≥ 600 mm.

Operating procedure: The test of acoustic and light signalling will be performed with the

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Operating procedure: To verify the correct functioning of the security cut-off produce an exposure with a high mAs value and report the measured dose. Then, produce a second exposure releasing the switch before the set time and report the new measured dose. This value

Operating procedure: if the focal spot is indicated, measure the distance between the focal spot indication mark on the tube housing and the top surface of the bucky. Add the distance between bucky surface and the top of the image receptor to the resulting value. Alternatively, calculate the source-to-image distance by the magnification of an object of known dimension.

Operating procedure: the long term reproducibility of the AEC system is calculated by

determining the deviation of the exposures obtained from the phan-ex and from the reference value (45 mm PMMA test block), with the exposure meter accurately placed on the plate holder.

Operating procedure: the short term reproducibility of the AEC system is calculated by the deviation of the 3 routine exposures (45 mm PMMA test block) of the phan-ex, with the exposure meter accurately placed on the plate holder. The measured counts per second (cps)

Deviations from the reference value of exposures (45 mm PMMA test block) ≤ ± 15%.

Operating procedure: the object thickness compensation of the AEC is calculated by determining the deviation of exposures of the phan-ex detector, accurately placed on PMMA plates of 30, 45 and 60 mm thickness, from the reference value (45 mm PMMA) at 28 kV. The measured counts

Deviations from the reference value of exposures (45 mm PMMA test block, imaged at 28 kV) ≤ ±

**Table 2.** List of parameters for digital image quality control [27].

The problem is to define a unified protocol that can be applied to any (analogue, CR, digital) type of mammographic system.

On the basis of procedures developed previously [29, 30], and to minimise problems arising from the use of different QC procedure to monitor different physical parameters for ana‐ logue and digital mammographs it is proposed here to monitor only parameters related to the beam at the output of the RX tube. The resulting QC procedure is then flexible and ap‐ plicable universally to any type of mammograph.

The only additional pieces of equipment needed to execute the proposed QC is a phantom coupled to a solid-state exposure meter (PHAN-EX).

The phantom is a 4.5 cm thick block of PMMA, simulating a standard breast, including details simulating those of clinical interest (micro-calcification, tumoral mass, fibrous structures). This is coupled to an RX exposure meter composed of a photodiode and a digital counter, thus capable of measuring the exposure and the quality of the mammo‐ graphic image [31]. The proposed protocol was tested on different (analogue and digital) mammographs, to assess its versatility and accuracy, independent of the physical charac‐ teristics of the mammographic system. Results on AEC tests obtained from the implemen‐ tation of the proposed protocol implemented on analogue and digital mammographs, are presented and discussed in Section 3.


**Metric Performance attribute**

302 Latest Research into Quality Control

MTF Resolution properties of the image/detector/system NPS Noise properties of the image/detector/system

DQE SNR transfer properties of the detector eDQE SNR transfer properties of the system

Uniformity Signal uniformity in the absence of an object Exposure Indicator Accuracy of exposure indication by the system Linearity Exposure response behavior of the system

Artifact Non-uniform artifactual features in the images

High-contrast resolution Ability of the system to represent high-contrast patterns Low-contrast resolution Ability of the system to represent low-contrast patterns

Ghosting Appearance of shadows of prior images on subsequent images Throughput Speed by which a system can sequentially capture images Normal exposure Target exposure values for clinical use reflecting system speed

The problem is to define a unified protocol that can be applied to any (analogue, CR, digital)

On the basis of procedures developed previously [29, 30], and to minimise problems arising from the use of different QC procedure to monitor different physical parameters for ana‐ logue and digital mammographs it is proposed here to monitor only parameters related to the beam at the output of the RX tube. The resulting QC procedure is then flexible and ap‐

The only additional pieces of equipment needed to execute the proposed QC is a phantom

The phantom is a 4.5 cm thick block of PMMA, simulating a standard breast, including details simulating those of clinical interest (micro-calcification, tumoral mass, fibrous structures). This is coupled to an RX exposure meter composed of a photodiode and a digital counter, thus capable of measuring the exposure and the quality of the mammo‐ graphic image [31]. The proposed protocol was tested on different (analogue and digital) mammographs, to assess its versatility and accuracy, independent of the physical charac‐ teristics of the mammographic system. Results on AEC tests obtained from the implemen‐ tation of the proposed protocol implemented on analogue and digital mammographs, are

Dark noise Noise in the absence of signal

Distortion Geometrical accuracy of images

**Table 2.** List of parameters for digital image quality control [27].

plicable universally to any type of mammograph.

coupled to a solid-state exposure meter (PHAN-EX).

presented and discussed in Section 3.

type of mammographic system.


should be accurately placed. The resulting measured kV should be recorded. After having assessed that the differences between measured and nominal tube voltage values are within 1 kV, the exposures can be repeated at 1 kV intervals, after positioning the exposure meter, by

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Accuracy for the range of clinically used tube voltages (25 –31 kV): measured mGy vs nominal kV

Operating procedure: Adequately position the exposure meter and report the counts per second

Operating procedure: To determine tube voltage reproducibility, accurately position the kVmeter and make at least three exposures at a fixed tube voltage that is normally used clinically (e.g. 28 kV). When the deviation from the mean value is < ± 0.5 kV and repeat the exposures, after positioning the exposure meter, and record the resulting counts per second (cps).

Operating procedure: Adequately position the exposure meter. Make at least three exposure at a fixed tube voltage that is normally used clinically (e.g. 28 kV) and report the measured counts per

Operating procedure: After accurately positioning the PMMA phantom and the sensor, the time

Entrance dose: ≤10 mGy (40 mm PMMA test block); ≤ 12 mGy (45 mm PMMA test block); ≤ 20

Operating procedure: Accurately position the exposure meter on the PMMA test block of known

Operating procedure: The output rate should be measured using a Mo/Mo target-filter combination at 28 kV, in the absence of scatter material and attenuation, and reporting the counts per second (cps). After calculating the exposure value, calculate the output rate at a

Exposure time needed to image a 45 mm PMMA phantom: < 2 sec.

thickness. Report the counts per second (cps) measured at the entrance.

Output rate must be < / 7.5 mGy/s (at the focus-to-film distance).

distance equal to the focus-to-film distance (FFD).

recording the resulting counts per second (cps).

Acceptance test, status test and constancy test.

curve should be within the error bar.

(cps) measured at intervals of 1 kV.

Reproducibility (at 28 kV): < ± 0.5 kV.

Reproducibility (at 28 kV): < ± 2 %.

for a routine exposure is measured.

Exposure time Acceptance test, status test and constancy test.

Reference dose Acceptance test, status test and constancy test.

Output rate Acceptance test, status test and constancy test.

mGy (50 mm PMMA test block).

Constancy test.

Frequency: Yearly

Constancy test.

second (cps). Frequency: Yearly

Frequency: Yearly

Frequency: Yearly

Frequency: Yearly

Acceptance test, quality control. AGD (45 mm PMMA): < 2 mGy.

Average glandular dose (AGD)

Tube Voltage Reproducibility


Operating procedure: the tube voltage compensation of the AEC is calculated by imaging the 45 mm PMMA test block, setting the tube voltage at 26 kV, 28 kV and 30 kV, with the exposure meter of the phan-ex accurately placed on the test block. The measured counts per second (cps)

All the deviation in the measured exposures between successive steps: 0.1 - 0.2 per step. Operating procedure: The optical density control step can be determined by placing the phan-ex on a 45 mm PMMA plate and taking an exposure at all possible steps, setting the operating

Parallel to the axis tube, the exposure value should decrease by 30-35 % at a height of 12 cm from the chest wall. Perpendicularly to the axis tube, a typical value of exposure decrease is < 7%

Operating procedure: Beam uniformity can be determined by positioning the exposure meter on a 45 mm PMMA plate, first at the centre of the PMMA plate and, successively, at the top, right, bottom and left of the test block. Image the plate and report the measured counts per second

Operating procedure: It can be estimated by imaging two resolution lead bar patterns, up to 20 line pairs per mm (lp/mm) each, placed on a 45 mm-thick PMMA plate. Image the patterns using

Operating procedure: It can be estimated by imaging a suitable phantom containing 5-6 mm circular details. The phantom is accurately placed on a 45 mm PMMA plate. Image the phantom

X-rays must cover the film by no more than 5 mm outside the film parallel to the axis tube,

Accuracy for the range of clinically used tube voltages (25 –31 kV): < ± 1 kV.

Operating procedure: The alignment of the X-ray field and image receptor at the chest wall side can be determined by using two loaded cassettes and two X-ray absorbers. Produce an exposure

Operating procedure: The equipment should be tested over the range of clinically used settings (typically 25 – 31 kV) at intervals of 1 kV. To determine the tube voltage accuracy, the kV-meter

voltage at 28 kV. The measured counts per second (cps) should be recorded.

from the centre of the X-ray field to 10 cm, for each side.

Acceptance test, status test and constancy test.

a Mo/Mo target-filter combination at 28 kV.

Acceptance test, status test and constancy test.

using a Mo/Mo target-filter combination at 28 kV.

Acceptance test, status test and constancy test.

laterally X-rays must totally cover the film.

Frequency: Every three months

Acceptance test, status test.

Spatial resolution should be ≥ 12 line pairs per mm (lp/mm)

Minimum detectable contrast for a 5-6 mm detail < 1.3%.

should be recorded. Frequency: Yearly

304 Latest Research into Quality Control

Frequency: Yearly Uniformity Acceptance test, quality control.

(cps).

Spatial resolution (at high frequency)

Threshold contrast

Alignment of X-ray field/image receptor

Tube Voltage Accuracy

visibility

Frequency: Yearly

Frequency: Yearly

Frequency: Yearly

Difference per step Acceptance test, status test and constancy test.


Maximum automatically applied force: 130 - 200 N.

Acceptance test, status test and constancy test.

paddle should be ≤ 5 mm for symmetrical load.

successfully implemented for both analogue and digital mammographs.

device or a bathroom scale.

Frequency: Yearly

Frequency: Yearly

sure, utilising the same phantom-exposure meter pair.

**3. Results and discussion**

estimate the entrance dose.

tics of the mammograph.

rect operation of the AEC system.

for a 45 mm PMMA test block).

Compression plate alignment

Operating procedure: The compression force can be estimated using a compression force test

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The difference between the measured distances at the left and right side of the compression

Operating procedure: The alignment of the compression device at maximum force can be

visualized and measured when a piece of foam-rubber is compressed.

**Table 3.** Proposed protocol for mammography QC and technical specification of the parameters to be monitored.

The chosen protocol can be used equally for acceptance, status and constancy tests. It was

In particular, it was implemented for constancy tests of all parameters relevant to the expo‐

In addition to the protocol, Table 3, the QC report worksheet is proposed in which the raw results (counts per second, cps) can be reported, Fig. 2-3. The raw data is then elaborated to

The proposed protocol and QC report were tested on different (analogue and digital) mam‐ mographs, to assess their versatility and accuracy, independent of the physical characteris‐

As an example, the AEC test results obtained for a digital mammographic system are report‐

The results obtained from the object thickness compensation are represented in Fig. 4. In particular, in Fig. 4 (a), the value of the dose (mGy) normalised to the tube load value (mAs) for the reference PMMA test block thickness (45 mm), is constant and within the error bar (± 15 %). The dose as a function of the PMMA plate thickness is presented in Fig. 4 (b). This curve shows that, with increasing dose, the normalised dose is constant, indicating the cor‐

The results obtained from the tube voltage compensation are presented in Fig. 5 (a). Differ‐ ently from the previous test, where the tube voltage was kept constant (28 kVp) varying on‐ ly the tube load, in this type of test two parameters are varied: tube voltage and tube load. Therefore, in this test the parameter chosen to assess the tube voltage compensation is the logarithm (base 10) of the dose. Also in this case the results show that the logarithm of the dose is within the limit values (± 15 % calculated for a reference tube voltage of 28 kVp and

ed in Figs. 4-6 to show that the same protocol can also be used on digital instruments.


**Table 3.** Proposed protocol for mammography QC and technical specification of the parameters to be monitored.

#### **3. Results and discussion**

Operating procedure: After determining the tube load (mAs) necessary to image the phan-ex, accurately position the exposure meter on the 45 mm PMMA test block and report the measured counts per second (cps), without backscattering. After calculating the exposure value, calculate the output rate at a distance equal to the focus-to-film distance (FFD) and convert this value into

Operating procedure: The grid system factor can be estimated by accurately positioning the phan-ex and measuring counts per second (at 28 kV), without compression, and with and

Operating procedure: image the bucky at the lowest position of the AEC-selector, without

Operating procedure: Make an exposure of a 1 mm lead sheet and verify if the AEC system

For 28 kV Mo/Mo target-filter combination the HVL must be between 0.30 and 0.40 mm Al

Operating procedure: Position the exposure detector at the reference ROI (since the HVL is position-dependent) on top of the bucky. Place the compression device halfway between focal spot and detector. Select a Mo/Mo target/filter combination, 28 kV tube voltage and an adequate tube loading (mAs-setting), and expose the detector directly. The filters can be placed on the compression device and must intercept the whole radiation field. Use the same tube load

For 28 kV Mo/Mo target-filter combination, focal spots size are reported in the following table.

Operating procedure: Produce a magnified image of the pinhole and measure, on the image, the

the average glandular dose.

Grid system factor must be ≤ 3.

PMMA. Verify the image uniformity.

The back-up timer should function properly.

Acceptance test, status test and constancy test.

(mAs) setting and expose the detector through each filter.

length and the width, in cm. Repeat for all available focal spots.

Focal spot size At acceptance and when resolution has changed, quality control.

without the grid system. Grid imaging Acceptance test, status test and constancy test. No significant non uniformity

Frequency: Yearly Back-up timer Acceptance test, quality control.

equivalent.

Frequency: Yearly

Frequency: Yearly

Compression force Acceptance test, status test and constancy test.

Half Value Layer

(HVL)

terminates the exposure. Frequency: Yearly

Grid system factor At acceptance and when dose or exposure time increases suddenly.

Frequency: Yearly

306 Latest Research into Quality Control

The chosen protocol can be used equally for acceptance, status and constancy tests. It was successfully implemented for both analogue and digital mammographs.

In particular, it was implemented for constancy tests of all parameters relevant to the expo‐ sure, utilising the same phantom-exposure meter pair.

In addition to the protocol, Table 3, the QC report worksheet is proposed in which the raw results (counts per second, cps) can be reported, Fig. 2-3. The raw data is then elaborated to estimate the entrance dose.

The proposed protocol and QC report were tested on different (analogue and digital) mam‐ mographs, to assess their versatility and accuracy, independent of the physical characteris‐ tics of the mammograph.

As an example, the AEC test results obtained for a digital mammographic system are report‐ ed in Figs. 4-6 to show that the same protocol can also be used on digital instruments.

The results obtained from the object thickness compensation are represented in Fig. 4. In particular, in Fig. 4 (a), the value of the dose (mGy) normalised to the tube load value (mAs) for the reference PMMA test block thickness (45 mm), is constant and within the error bar (± 15 %). The dose as a function of the PMMA plate thickness is presented in Fig. 4 (b). This curve shows that, with increasing dose, the normalised dose is constant, indicating the cor‐ rect operation of the AEC system.

The results obtained from the tube voltage compensation are presented in Fig. 5 (a). Differ‐ ently from the previous test, where the tube voltage was kept constant (28 kVp) varying on‐ ly the tube load, in this type of test two parameters are varied: tube voltage and tube load. Therefore, in this test the parameter chosen to assess the tube voltage compensation is the logarithm (base 10) of the dose. Also in this case the results show that the logarithm of the dose is within the limit values (± 15 % calculated for a reference tube voltage of 28 kVp and for a 45 mm PMMA test block).








**Figure 3.** QC report worksheet for raw data recording (part 2).

dose per step are reported in Fig. 6 (b).

logue and digital mammographic system.

The dose radiated by the AEC system as a function of the tube voltage is presented in Fig. 5 (b), as measured with the phan-ex. From the results of Fig 5 (b) it is noticed that as the tube voltage increases, the dose decreases, further confirming that the AEC system is functioning correctly. Results from the test on the "difference per step" are reported in Fig. 6. Also in this case, the logarithm of the dose was calculated at each step. The obtained values are within the limit values (0.2 – 0.4 as the step difference was 2), Fig. 6 (a). The corresponding values of the

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For the short-term reproducibility test, exposure values were measured, from which the average dose value was determined with respect to the tube load supplied by the AEC sys‐ tem (mGy/mAs), Fig. 7, to show the proposed unified protocol is equally applicable to ana‐




**Figure 2.** QC report worksheet for raw data recording (part 1).

#### Unified Procedures for Quality Controls in Analogue and Digital Mammography http://dx.doi.org/10.5772/51349 309


**Figure 3.** QC report worksheet for raw data recording (part 2).

**Figure 2.** QC report worksheet for raw data recording (part 1).

308 Latest Research into Quality Control

The dose radiated by the AEC system as a function of the tube voltage is presented in Fig. 5 (b), as measured with the phan-ex. From the results of Fig 5 (b) it is noticed that as the tube voltage increases, the dose decreases, further confirming that the AEC system is functioning correctly.

Results from the test on the "difference per step" are reported in Fig. 6. Also in this case, the logarithm of the dose was calculated at each step. The obtained values are within the limit values (0.2 – 0.4 as the step difference was 2), Fig. 6 (a). The corresponding values of the dose per step are reported in Fig. 6 (b).

For the short-term reproducibility test, exposure values were measured, from which the average dose value was determined with respect to the tube load supplied by the AEC sys‐ tem (mGy/mAs), Fig. 7, to show the proposed unified protocol is equally applicable to ana‐ logue and digital mammographic system.

eters in analogue and (direct or indirect) digital instruments. The simplification is even more relevant in the latter type of mammographs for which the QC procedures currently used

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The applicability of the proposed phantom can be further extended to the measurement of parameters other than those relevant to the exposure even for the next generation of mam‐ mographs which are still under development. One such instrument is the SYRMEP, equip‐ ped with a Si-based microstrip detector and a synchrotron X-Ray source characterised by

**Figure 5.** Results of tube voltage compensation test: (a) logarithm of the dose, red lines representing the limit values (± 15 %) with respect to 28 kVp reference tube voltage; (b) the dose (mGy) as a function of tube voltage (kVp).

present considerable difficulties in the interpretation of the measurement protocols.

superior performance with respect to typical X-Ray tubes [36].

**Figure 4.** Results of object thickness compensation test: (a) the dose (mGy) normalised to the tube load value (mAs); (b) the dose (mGy) as a function of PMMA plate thickness.

The use of the phan-ex, coupled with the proposed protocol, is useful also to verify parame‐ ters related to the exposure such as tube voltage precision and accuracy, and exposure time.

Most importantly the proposed protocol permits the evaluation of the functional parameters of the instruments by utilising a single phantom, thus significantly reducing the number of additional dedicated equipment and simplifying the task of the Medical Physics Expert.

The results obtained from raw data analysis obtained following the proposed protocol were found to be consistent with those obtained from standard procedures [32-35], thus highlight‐ ing the usefulness and versatility of the proposed unified protocol to test all relevant param‐ eters in analogue and (direct or indirect) digital instruments. The simplification is even more relevant in the latter type of mammographs for which the QC procedures currently used present considerable difficulties in the interpretation of the measurement protocols.

The applicability of the proposed phantom can be further extended to the measurement of parameters other than those relevant to the exposure even for the next generation of mam‐ mographs which are still under development. One such instrument is the SYRMEP, equip‐ ped with a Si-based microstrip detector and a synchrotron X-Ray source characterised by superior performance with respect to typical X-Ray tubes [36].

**Figure 4.** Results of object thickness compensation test: (a) the dose (mGy) normalised to the tube load value (mAs);

The use of the phan-ex, coupled with the proposed protocol, is useful also to verify parame‐ ters related to the exposure such as tube voltage precision and accuracy, and exposure time.

Most importantly the proposed protocol permits the evaluation of the functional parameters of the instruments by utilising a single phantom, thus significantly reducing the number of additional dedicated equipment and simplifying the task of the Medical Physics Expert.

The results obtained from raw data analysis obtained following the proposed protocol were found to be consistent with those obtained from standard procedures [32-35], thus highlight‐ ing the usefulness and versatility of the proposed unified protocol to test all relevant param‐

(b) the dose (mGy) as a function of PMMA plate thickness.

310 Latest Research into Quality Control

**Figure 5.** Results of tube voltage compensation test: (a) logarithm of the dose, red lines representing the limit values (± 15 %) with respect to 28 kVp reference tube voltage; (b) the dose (mGy) as a function of tube voltage (kVp).

**Figure 6.** Results of difference per step test: (a) logarithm of the dose, red lines representing the limit values (0.2 – 0.4 per step); (b) the dose (mGy) per step.

**Figure 7.** Results of short term reproducibility test, dose to tube load ratio (mGy/mAs) for three different exposures :

, Federica Causa1

1 Environmental, Healh, Social and Industrial Department - University of Messina, Italy

2 Department of Biotechnologies and Life Sciences– University of Insubria, Italy

, Raffaele Novario2

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,

(a) digital, (b) analogue mammograph;

, Giuseppe Acri1

\*Address all correspondence to: vermigli@unime.it

and Giuseppe Vermiglio1\*

**Author details**

Barbara Testagrossa1

Maria Giulia Tripepi1

#### Unified Procedures for Quality Controls in Analogue and Digital Mammography http://dx.doi.org/10.5772/51349 313

**Figure 7.** Results of short term reproducibility test, dose to tube load ratio (mGy/mAs) for three different exposures : (a) digital, (b) analogue mammograph;

#### **Author details**

**Figure 6.** Results of difference per step test: (a) logarithm of the dose, red lines representing the limit values (0.2 – 0.4

per step); (b) the dose (mGy) per step.

312 Latest Research into Quality Control

Barbara Testagrossa1 , Giuseppe Acri1 , Federica Causa1 , Raffaele Novario2 , Maria Giulia Tripepi1 and Giuseppe Vermiglio1\*

\*Address all correspondence to: vermigli@unime.it

1 Environmental, Healh, Social and Industrial Department - University of Messina, Italy

2 Department of Biotechnologies and Life Sciences– University of Insubria, Italy

#### **References**

[1] Shah, A. J., Wang, J., Yamada, T., & Fajardo, L. L. (2003). Digital Mammography: A Review of Technical Development and Clinical Applications. *Clinical Breast Cancer*, 4(1), 63-70.

[14] Feig, S. A., & Yaffe, M. J. (1998). Digital mammography. *Radio Graphics*, 18, 893-901. [15] Screen-Film Radiography II. (2012). http://myweb.dal.ca/halem/phyc2250/09\_-

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[16] Pisano, E. D., & Yaffe, M. J. (2005). State of the art: Digital Mammography. *Radiology*,

[17] Berns, E. A., Hendrick, R. E., Solari, M., Barke, L., Reddy, D., Wolfman, J., Segal, L., De Leon, P., Benjamin, S., & Willis, L. (2006). Digital and screen-film mammography: comparison of image acquisition and interpretation times. *American Journal of Roent‐*

[18] Yaffe, in., Haus, A., & Yaffe, M. J., Eds. (1992). *Syllabus of Categorial Course on Techni‐ cal Aspects of Mammography*, Radiological Society of North America, Oak Book, IL. [19] Säbel, M., & Aichinger, H. (1996). Recent development in breast imaging. *Physics in*

[20] Smith, A. P. (2003). Fundamentals of Digital Mammography: Physics, Technology

[21] Rampado, O., Isoardi, P., & Ropolo, R. (2006). Quantitative assessment of computed radiography quality control parameters. *Physics in Medicine and Biology*, 51,

[22] Panagiotakis, G. (2012). Mammographic detectors. *PhD Thesis*, University of Patras, http://www.hep.upatras.gr/class/download/bio\_sim\_eik/mammographic\_detec‐

[23] Kruger, D. G., Abreu, C. C., Hendee, E. G., Kocharian, A., Peppler, W. W., Mistretta, C. A., & Mac Donald, C. A. (1996). Imaging Characteristics of x-ray capillary optics in

[24] Pisano, E. D., Yaffe, M. J., & Kuzmiak, C. M. (2004). *Digital mammography*, Lippencott,

[25] Yaffe, M. J., & Rowlands, J. A. (1998). X-ray detectors for digital radiography. *Physics*

[26] Kasap, S. O., & Rowlands, J. A. (2000). X-ray photoconductors and stabilized a-Se for direct conversion digital flat-panel X-ray image detectors. *Journal of Materials Science:*

[27] Samei, E., & Ravin, C. E. (2008, March 12-13, 2008). Washington DC, USA. *Assuring image quality for classification of digital chest radiographs: conference proceedings*, NIOSH

[28] Ranger, N. T., Samei, E., Dobbins, J. T., I. I. I., & Ravin, C. E. (2007). Assessment of Detective Quantum Efficiency: intercomparison of a recently introduced internation‐

and Practical Consideration. *Radiology Management*, 25(5), 18-24, 26-31.

\_Screen-Film\_Radiography\_II.pdf, accessed 25 June 2012.

234(2), 353-362.

*genology*, 187, 38-41.

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*Medicine and Biology*, 41, 315-368.

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*in Medicine and Biology*, 42, 1-39.

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Scientific Workshop.

digital mammography. *Medical Physics*, 23(2), 187-196.

al standard with prior methods. *Radiology*, 243(3), 785-795.


[14] Feig, S. A., & Yaffe, M. J. (1998). Digital mammography. *Radio Graphics*, 18, 893-901.

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4(1), 63-70.

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*ology*, 232(1), 197-204.

30(3), 442-448.

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[1] Shah, A. J., Wang, J., Yamada, T., & Fajardo, L. L. (2003). Digital Mammography: A Review of Technical Development and Clinical Applications. *Clinical Breast Cancer*,

[2] Feig, S. A. (1988). Decreased breast cancer mortality through mammographic screen‐

[3] Skaane, P., & Skjennald, A. (2004). Screen-film mammography versus full-filled digi‐ tal mammography with soft-copy reading: screening program. *The Oslo II study. Radi‐*

[4] Huda, W., Sajewicz, A. M., & Ogden, K. M. (2003). Experimental investigation of the dose and image quality characteristics of a digital imaging system. *Medical Physics*,

[5] Law, J. (2006). The development of mammography. *Physics in Medicine and Biology*,

[6] Noel, A., & Thibauld, F. (2004). Digital detectors for mammography: the technical

[7] James, J. J. (2004). The current status of digital mammography. *Clinical Radiology*, 59,

[8] Maidment, A. D. A. (2003). Digital mammography. *Seminars in Roentgenology*, 38(3),

[9] Perry, N., Broeders, M., de Wolf, C., Törnberg, S., Holland, R., & von Karsa, L., Ed. (2006). *European guidelines for quality assurance in breast cancer screening and diagnosis* (4th Edition), Luxembourg, Office for Official Publications of the European Commun‐

[10] Pisano, E. D., Cole, E. B., Hemminger, B. M., Yaffe, M. J., Aylward, S. R., Maidment, A. D. A., Johnston, E., Williams, M. B., Niklason, L. T., Conant, E. F., Fayardo, L. L., Kopans, D. B., Brown, M. E., & Pizer, S. M. (2000). Image processing algorithms for

[11] Muller, S. (1999). Full-field digital mammography designed as a complete system.

[12] Feig, S. A., & Yaffe, M. J. (1996). Current status of digital mammography. *Seminars in*

[13] Cherie, M. Kuzmiak. (2012). *Digital Mammography, Imaging of the Breast- Technical As‐ pects and Clinical Implication, Laszlo Tabar (Ed.)*, 978-9-53510-284-7, InTech, Available from, http://www.intechopen.com/books/imaging-of-the-breast-technical-aspects-

and-clinical-implication/digital-mammography-chapteraccessed 5 June 2012.

digital mammography: a pictorial essay. *Radio Graphics*, 20, 1479-1491.

ing: results of clinical trials. *Radiology*, 167, 659-665.

challenges. *European Radiology*, 14, 1990-1998.

*European Journal of Radiology*, 31, 25-34.

*Ultrasound, CT, and MRI*, 17(5), 424-443.


[29] Vermiglio, G., Tripepi, M. G., Mannino, G., Sansotta, C., & Testagrossa, B. (2005, 14-17 June 2005). Prove periodiche di funzionalità in mammografia mediante misure di esposizione. Verona, Italy. *In: Polimetrica S.a.s. (ed.) Proceedings of 4° Congresso Na‐ zionale AIFM*, 672-675.

**Section 5**

**Quality Control in Energy**


## **Quality Control in Energy**

[29] Vermiglio, G., Tripepi, M. G., Mannino, G., Sansotta, C., & Testagrossa, B. (2005, 14-17 June 2005). Prove periodiche di funzionalità in mammografia mediante misure di esposizione. Verona, Italy. *In: Polimetrica S.a.s. (ed.) Proceedings of 4° Congresso Na‐*

[30] Testagrossa, B., Sansotta, C., Acri, G., Tripepi, M. G., & Vermiglio, G. (2007, October 1-3 2007). Vasto Marina (CH), Paper presented at XXXIV Convegno AIRP "Sicurezza e qualità in radioprotezione". *Fantoccio multiuso per controlli di qualità in mammografia*

[32] Italian Regulation 26 Maggio 2000, n. 187. Attuazione della direttiva 97/43/Euratom in materia di protezione sanitaria delle persone contro i pericoli delle radiazioni ion‐ izzanti connesse ad esposizioni mediche. Gazzetta Ufficiale n. 157 del 7 luglio 2000. Suppl. Ordinario n. 105. http://www.camera.it/parlam/leggi/deleghe/00187dl.htm

[33] Linee Guida ANPEQ-ISPESL relative al controllo sugli impianti radiologici e acces‐ sori. (2004). http://www.anpeq.it/download/app\_radiologiche.pdf, accessed 27 June

[34] Corrado, F., Gennaro, G., Golinelli, P., & Rossetti, V. (2004). Protocollo Italiano per il controllo di qualità degli aspetti fisici e tecnici in mammografia. *AIFM Report*, http:// www.fisicamedica.it/aifm/report/2004\_n1\_ReportAIFM.pdf, accessed 27 June 2012.

[35] Norma CEI 62-27 ed.2 (CEI EN 60601-2-7). (1999). Apparecchi elettromedicali. *Parte 2: Norme particolari per la sicurezza di generatori ad alta tensione dei generatori radiologici per*

[36] Castelli, E. (2012). Un sistema di rivelazione per mammografia digitale con luce di sincrotrone. *PhD Thesis*, University of Trieste, http://www.infn.it/thesis/PDF/256-Ber‐

*zionale AIFM*, 672-675.

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(accessed 25 June 2012).

2012.

*diagnostica*.

*tradizionale e digitale: conference proceedings*.

gamaschi-laurea.pdf, accessed 25 June 2012.

[31] *Controllo di qualità in mammografia PHAN-EX*, Operating handbook.

**Chapter 15**

**The Quality Management of The R&D in High Energy**

Particle physics, also recognized as high energy physics, is a basic subject focusing on the research of the elementary elements of materials and their mutual actions. One distinguish‐ ed characteristic of particle physics study is that the experimental equipments involved are always huge and special ones. Therefore, big science projects, including the R&D of large detectors, are usually required in high energy physics experiments. Those projects are com‐ plicated systematic engineering, involving many front and technology fields. It is impossible for a single institute to finish those large projects by its own. Cooperation among different institutes or organizations is necessary for big science projects in particle physics, especially

The Institute of High Energy Physics (IHEP) is the biggest and comprehensive fundamental research center in Chinese Academy of Science. The major research fields of IHEP are parti‐ cle physics, accelerator physics and technologies, radiation technologies and application, Particle physics experiments and Accelerator physics and technology are two of the leading research areas. The main research facilities at IHEP include Beijing Electron Positron Collid‐ er (BEPC) and Beijing Spectrometer (BES), DayaBay Neutrino Experiment, Chinese Spalla‐ tion Neutron Source, etc. IHEP has extensive cooperation with all high energy physics laboratories and participates in many important particle physics experiments in the world.

The Beijing Electron Positron Collider (BEPC) consists of the injector, the storage ring, the transportation line, the Beijing Spectrometer (BES), the Beijing Synchrotron Radiation Facili‐

> © 2012 Zhu and Qian; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Zhu and Qian; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**1.2. The Beijing Electron Positron Collider [2] and the Beijing Spectrometer [3]**

**Physics Detector**

Xuemin Zhu and Sen Qian

http://dx.doi.org/10.5772/51434

**1. Introduction**

international ones.

**1. 1. The Institute of High Energy Physics [1]**

Additional information is available at the end of the chapter

## **The Quality Management of The R&D in High Energy Physics Detector**

Xuemin Zhu and Sen Qian

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51434

### **1. Introduction**

Particle physics, also recognized as high energy physics, is a basic subject focusing on the research of the elementary elements of materials and their mutual actions. One distinguish‐ ed characteristic of particle physics study is that the experimental equipments involved are always huge and special ones. Therefore, big science projects, including the R&D of large detectors, are usually required in high energy physics experiments. Those projects are com‐ plicated systematic engineering, involving many front and technology fields. It is impossible for a single institute to finish those large projects by its own. Cooperation among different institutes or organizations is necessary for big science projects in particle physics, especially international ones.

#### **1. 1. The Institute of High Energy Physics [1]**

The Institute of High Energy Physics (IHEP) is the biggest and comprehensive fundamental research center in Chinese Academy of Science. The major research fields of IHEP are parti‐ cle physics, accelerator physics and technologies, radiation technologies and application, Particle physics experiments and Accelerator physics and technology are two of the leading research areas. The main research facilities at IHEP include Beijing Electron Positron Collid‐ er (BEPC) and Beijing Spectrometer (BES), DayaBay Neutrino Experiment, Chinese Spalla‐ tion Neutron Source, etc. IHEP has extensive cooperation with all high energy physics laboratories and participates in many important particle physics experiments in the world.

#### **1.2. The Beijing Electron Positron Collider [2] and the Beijing Spectrometer [3]**

The Beijing Electron Positron Collider (BEPC) consists of the injector, the storage ring, the transportation line, the Beijing Spectrometer (BES), the Beijing Synchrotron Radiation Facili‐

ty (BSRF) and the computer center. Beijing Spectrometer (BES) is a general purpose magnet‐ ic spectrometer in the South IP of the storage ring. The general layout of the BEPC is shown in Fig.1.

Liquid Scintillator, Muon Veto Detector, Readout Electronic and Data acquisition system (DAQ) etc. While America is in responsible of the construction of water Cherenkov detector

The Quality Management of The R&D in High Energy Physics Detector

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321

Scientists from the Chinese Academy of Sciences (CAS) and the U.S.-based Brookhaven Na‐ tional Laboratory and the Lawrence Berkeley National Laboratory will participate in the un‐ derground experiment. An international funding commission comes into existence in the funding agency to discuss fee issues and instruct the experiment process and fee manage‐ ment through experimental supervision organization. The project management of the Daya Bay Neutrino Experiment adopts the advanced and mature modern management idea used for managing large international joint project and big science experimental research project. An international cooperation group is built and management rules are made. Besides, a co‐ operation group commission is founded, during which executive board and spokesperson is elected for overall supervision of the whole project. The Daya Bay Neutrino Experiment is

Chinese Spallation Neutron Source (CSNS) is designed to build a device with the power of proton beam reaching up to 100 kW effective and the flux of pulsed neutrons coming out top in the world, along with other three spallation neutron sources built in America, Japan and British. CSNS is also a large cooperative project, supported by Chinese Academy of Sci‐ ences and Guangdong government. The normal operation for uses is foreseen in 2018. IHEP is the main construction institution in the project with the Institute of Physics Chinese Acad‐ emy of Sciences as the co-operation unit. The construction team bring together three genera‐ tions of outstanding scientific and technical researchers in China. An international CSNS neutron technology advisory committee is set up for reviewing the key experimental work. The experts of the advisory committee are from well-known laboratories in America, Japan,

**2. Introduction of Quality Management of Scientific Projects in IHEP**

During the process of big science project and research, IHEP has significant advantages in accelerator physics and technology, human resources, international cooperation and aca‐ demic exchange. IHEP owns mature model and advanced experience in the quality manage‐

Before 2011, the project manager is responsible for the big science project management in IHEP. International cooperation group is formed and fees are under the sponsors' supervi‐ sion and review. There is a perfect project management system, though without quality

and so on.

initiated in 2007 and finished in 2012.

**1.4. Chinese Spallation Neutron Source [6]**

Germany, Australia and other countries.

ment of scientific projects.

**2.1. Project Management System**

management system meeting international standards.

**Figure 1.** The airscape of the BEPC.

BEPC started construction in 1984 and the first electron-proton collider was produced in Oct. 1988. BEPCII was installed in 2003 and finished five years later in 2009. IHEP estab‐ lishes comprehensive and long-term cooperation with high energy laboratories and univer‐ sities all over the world, especially in USA, Japan and Europe. With the international cooperation, IHEP have gained huge success in 30 years. For example, IHEP took part in the research of CMS and ATLAS detectors of Large Hadron Collider (LHC), which is the world's largest, highest-energy particle accelerator and the collider at the beginning of 21 centuries, built by CERN [4]. BESIII is also organized by IHEP and participated by 51 insti‐ tutions and universities around the world, 34 from Asian, 12 from Europe and 5 from USA.

#### **1.3. The Daya Bay Neutrino Experiment [5]**

The Daya Bay Neutrino Experiment is a neutrino-oscillation experiment designed to meas‐ ure the mixing angle q13 using anti-neutrinos produced by the reactors of the Daya Bay Nu‐ clear Power Plant (NPP) and the Ling Ao NPP.

The Daya Bay Neutrino Experiment is a major international joint research program, mainly organized by China working closely with researchers from other countries. In terms of both money and people, it is among the largest scientific collaborations between US and China. More than 200 scientists from China, include Hong Kong and Taiwan, the US, Russia, the Czech Republic are involved in the Daya Bay experiment. During the cooperation, China is in change of the laboratory construction, R&D of Anti-neutrino detector (AD), Gd-loaded Liquid Scintillator, Muon Veto Detector, Readout Electronic and Data acquisition system (DAQ) etc. While America is in responsible of the construction of water Cherenkov detector and so on.

Scientists from the Chinese Academy of Sciences (CAS) and the U.S.-based Brookhaven Na‐ tional Laboratory and the Lawrence Berkeley National Laboratory will participate in the un‐ derground experiment. An international funding commission comes into existence in the funding agency to discuss fee issues and instruct the experiment process and fee manage‐ ment through experimental supervision organization. The project management of the Daya Bay Neutrino Experiment adopts the advanced and mature modern management idea used for managing large international joint project and big science experimental research project. An international cooperation group is built and management rules are made. Besides, a co‐ operation group commission is founded, during which executive board and spokesperson is elected for overall supervision of the whole project. The Daya Bay Neutrino Experiment is initiated in 2007 and finished in 2012.

#### **1.4. Chinese Spallation Neutron Source [6]**

ty (BSRF) and the computer center. Beijing Spectrometer (BES) is a general purpose magnet‐ ic spectrometer in the South IP of the storage ring. The general layout of the BEPC is shown

BEPC started construction in 1984 and the first electron-proton collider was produced in Oct. 1988. BEPCII was installed in 2003 and finished five years later in 2009. IHEP estab‐ lishes comprehensive and long-term cooperation with high energy laboratories and univer‐ sities all over the world, especially in USA, Japan and Europe. With the international cooperation, IHEP have gained huge success in 30 years. For example, IHEP took part in the research of CMS and ATLAS detectors of Large Hadron Collider (LHC), which is the world's largest, highest-energy particle accelerator and the collider at the beginning of 21 centuries, built by CERN [4]. BESIII is also organized by IHEP and participated by 51 insti‐ tutions and universities around the world, 34 from Asian, 12 from Europe and 5 from USA.

The Daya Bay Neutrino Experiment is a neutrino-oscillation experiment designed to meas‐ ure the mixing angle q13 using anti-neutrinos produced by the reactors of the Daya Bay Nu‐

The Daya Bay Neutrino Experiment is a major international joint research program, mainly organized by China working closely with researchers from other countries. In terms of both money and people, it is among the largest scientific collaborations between US and China. More than 200 scientists from China, include Hong Kong and Taiwan, the US, Russia, the Czech Republic are involved in the Daya Bay experiment. During the cooperation, China is in change of the laboratory construction, R&D of Anti-neutrino detector (AD), Gd-loaded

in Fig.1.

320 Latest Research into Quality Control

**Figure 1.** The airscape of the BEPC.

**1.3. The Daya Bay Neutrino Experiment [5]**

clear Power Plant (NPP) and the Ling Ao NPP.

Chinese Spallation Neutron Source (CSNS) is designed to build a device with the power of proton beam reaching up to 100 kW effective and the flux of pulsed neutrons coming out top in the world, along with other three spallation neutron sources built in America, Japan and British. CSNS is also a large cooperative project, supported by Chinese Academy of Sci‐ ences and Guangdong government. The normal operation for uses is foreseen in 2018. IHEP is the main construction institution in the project with the Institute of Physics Chinese Acad‐ emy of Sciences as the co-operation unit. The construction team bring together three genera‐ tions of outstanding scientific and technical researchers in China. An international CSNS neutron technology advisory committee is set up for reviewing the key experimental work. The experts of the advisory committee are from well-known laboratories in America, Japan, Germany, Australia and other countries.

#### **2. Introduction of Quality Management of Scientific Projects in IHEP**

During the process of big science project and research, IHEP has significant advantages in accelerator physics and technology, human resources, international cooperation and aca‐ demic exchange. IHEP owns mature model and advanced experience in the quality manage‐ ment of scientific projects.

#### **2.1. Project Management System**

Before 2011, the project manager is responsible for the big science project management in IHEP. International cooperation group is formed and fees are under the sponsors' supervi‐ sion and review. There is a perfect project management system, though without quality management system meeting international standards.

BEPCII completed the construction task successfully by time, with high quality and budget

The Quality Management of The R&D in High Energy Physics Detector

http://dx.doi.org/10.5772/51434

323

**Figure 3.** The Relationship between the quality control system in large scientific experiments and ISO9001 Quality

As described in Fig.3, The quality control in large scientific experiments corresponds with ISO9001 Quality management systems, which is classified according to the production. While, the quality control in large scientific experiments is classified according to the type of different work. The ISO9001 Quality management system is widely adopted by corporations

In 2011, IHEP passed a national quality management certification system: GB/ T19001-2008(idt ISO90012008). After two years' development of quality management system from its very beginning to being passed, it has confirmed that IHEP has the ability to pro‐

The set up of quality management system makes the project management procedure stand‐ ard, and promotes the overall management level in IHEP. The clients' needs are fully met and the quality management of IHEP joined the line of international standard management. The role played by quality management in the scientific research, especially in the big sci‐

The project of BESIII detector began its research and development, according to the scientific project management system and quality management system, like other big science projects.

under control.

management systems.

all over the word and it's more normative.

duce scientific production meeting requirements.

ence project, is invaluable and imponderable.

**3. Quality management in R&D of BESIII detector**

**Figure 2.** The Organization Chart of the BEPCII Project Management.

In the project management system shown in Fig.2, special-purpose management mechanism such as fund, purchasing, quality, safety and archive is established, with clear responsibili‐ ties and authorities. Besides, the internal communication mechanism and interface manage‐ ment mechanism are also set up. CPM Plan is adopted for fund and schedule management. To ensure the quality of the project, during the design and development process, experts are always invited for evaluation. And an international council committee is asked for review in terms of major international cooperation projects.

In fact, the requirements of the project management system have already displayed in the ISO 9001 quality management system. Though without a systematic quality manual and standards and lack of resource, purchasing and archive management. In the project manage‐ ment system, quality management is more focused on the management of various test guidelines and processing of key parts (including outsourced progress)

#### **2.2. Quality Management Systems**

The BEPCII project headquarters has placed great important on the quality management and published "BEPCII project management file" in 2002. In the file, responsibilities and rights of personnel, fund management, file number, document signing and alteration, early stages management, bidding and purchasing are described in detail.

At the beginning of 2005, during the construction of BEPCII project, the headquarters built a quality management system according to GB/T19001-2000(idt ISO9001:2000). Although the system doesn't get a national certification, it is completely in accordance with standards of quality management system requirements and it has played a very good effect. In 2009, BEPCII completed the construction task successfully by time, with high quality and budget under control.

**Figure 3.** The Relationship between the quality control system in large scientific experiments and ISO9001 Quality management systems.

As described in Fig.3, The quality control in large scientific experiments corresponds with ISO9001 Quality management systems, which is classified according to the production. While, the quality control in large scientific experiments is classified according to the type of different work. The ISO9001 Quality management system is widely adopted by corporations all over the word and it's more normative.

In 2011, IHEP passed a national quality management certification system: GB/ T19001-2008(idt ISO90012008). After two years' development of quality management system from its very beginning to being passed, it has confirmed that IHEP has the ability to pro‐ duce scientific production meeting requirements.

The set up of quality management system makes the project management procedure stand‐ ard, and promotes the overall management level in IHEP. The clients' needs are fully met and the quality management of IHEP joined the line of international standard management. The role played by quality management in the scientific research, especially in the big sci‐ ence project, is invaluable and imponderable.

#### **3. Quality management in R&D of BESIII detector**

**Figure 2.** The Organization Chart of the BEPCII Project Management.

322 Latest Research into Quality Control

terms of major international cooperation projects.

**2.2. Quality Management Systems**

In the project management system shown in Fig.2, special-purpose management mechanism such as fund, purchasing, quality, safety and archive is established, with clear responsibili‐ ties and authorities. Besides, the internal communication mechanism and interface manage‐ ment mechanism are also set up. CPM Plan is adopted for fund and schedule management. To ensure the quality of the project, during the design and development process, experts are always invited for evaluation. And an international council committee is asked for review in

In fact, the requirements of the project management system have already displayed in the ISO 9001 quality management system. Though without a systematic quality manual and standards and lack of resource, purchasing and archive management. In the project manage‐ ment system, quality management is more focused on the management of various test

The BEPCII project headquarters has placed great important on the quality management and published "BEPCII project management file" in 2002. In the file, responsibilities and rights of personnel, fund management, file number, document signing and alteration, early

At the beginning of 2005, during the construction of BEPCII project, the headquarters built a quality management system according to GB/T19001-2000(idt ISO9001:2000). Although the system doesn't get a national certification, it is completely in accordance with standards of quality management system requirements and it has played a very good effect. In 2009,

guidelines and processing of key parts (including outsourced progress)

stages management, bidding and purchasing are described in detail.

The project of BESIII detector began its research and development, according to the scientific project management system and quality management system, like other big science projects.

#### **3.1. Mechanism Management**

BESIII detector R&D is part of BEPCII project. So the quality management of the detector research is responsible by the project director. As a whole, BESIII carries out the manage‐ ment system of BEPCII project headquarters strictly and makes some special mechanism to form a mechanism with a clear hierarchy. Quality technician are employed in the project.

**3.2. Fund Management**

quirements of the quality management system

project will receive examination and evaluation.

**3.3. Control of documents and records**

**Figure 5.** The workflow of archive management.

Fund management is important for the whole management of scientific project. Appropriate fund use a basis for carrying out any high energy physics experiment smoothly. As for the R&D of BESIII detector, the experiment design and development planning will affect the ra‐ tionality of the budget and fund use directly. They are also the important contents in the re‐

The Quality Management of The R&D in High Energy Physics Detector

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325

Funds come from Chinese Academy Sciences (CAS) allocation and self-provided funds in the BESIII project. At the end of the year, expenses are counted and reported to CAS and the

Control of documents and records is critical whether for scientific project management or for quality management. For high energy physic experiments, large and complicated equip‐ ments are usually involved. During the project design and scheme phase, rules for docu‐ ments and records reserved need to be made clearly and principles for numbering and

BEPCII project builds up a special mechanism of file control. Because BESIII is part of the whole project, the rules of document management are in accordance with the requirements of BEPCII. *Documents and records* need to be signed according to the regulations, in accord‐

signing the documents and records need to be described specifically.

BESIII detector R&D project has outlined the responsibilities and rights of each person in charge with an appropriate staffing in the organization. The communication methods of the total and sub system and record control requirements are defined.

The director in charge of sub system is responsible for the implementation of the BESIII re‐ search plan, management, arrangement of related resources and coordination with scientific and technical issues. Each division leading person is specifically responsible for the respec‐ tive task implementation plan. Members in the project cooperate with each other closely at reaching difficult goals. The whole project has the characteristic of unified task, defined re‐ sponsibilities, reasonable arrangement and integrated resources.

The high energy physics experiment is a complex project, and the communication in differ‐ ent study cells seems more important. The Task Control Form is widely used in study works, and the forms are preserved and archived as records of the system.


**Figure 4.** The task control form used by different teams.

Researchers in the project communicate with each other in time and have a regular meeting each or twice a week, to make sure the project is under schedule control and discuss some technical problems. Meeting minutes are kept as a reference. Sub-system will report the progress of the project and accept an inspection and evaluation regularly.

#### **3.2. Fund Management**

**3.1. Mechanism Management**

324 Latest Research into Quality Control

BESIII detector R&D is part of BEPCII project. So the quality management of the detector research is responsible by the project director. As a whole, BESIII carries out the manage‐ ment system of BEPCII project headquarters strictly and makes some special mechanism to form a mechanism with a clear hierarchy. Quality technician are employed in the project.

BESIII detector R&D project has outlined the responsibilities and rights of each person in charge with an appropriate staffing in the organization. The communication methods of the

The director in charge of sub system is responsible for the implementation of the BESIII re‐ search plan, management, arrangement of related resources and coordination with scientific and technical issues. Each division leading person is specifically responsible for the respec‐ tive task implementation plan. Members in the project cooperate with each other closely at reaching difficult goals. The whole project has the characteristic of unified task, defined re‐

The high energy physics experiment is a complex project, and the communication in differ‐ ent study cells seems more important. The Task Control Form is widely used in study

Researchers in the project communicate with each other in time and have a regular meeting each or twice a week, to make sure the project is under schedule control and discuss some technical problems. Meeting minutes are kept as a reference. Sub-system will report the

progress of the project and accept an inspection and evaluation regularly.

total and sub system and record control requirements are defined.

sponsibilities, reasonable arrangement and integrated resources.

**Figure 4.** The task control form used by different teams.

works, and the forms are preserved and archived as records of the system.

Fund management is important for the whole management of scientific project. Appropriate fund use a basis for carrying out any high energy physics experiment smoothly. As for the R&D of BESIII detector, the experiment design and development planning will affect the ra‐ tionality of the budget and fund use directly. They are also the important contents in the re‐ quirements of the quality management system

Funds come from Chinese Academy Sciences (CAS) allocation and self-provided funds in the BESIII project. At the end of the year, expenses are counted and reported to CAS and the project will receive examination and evaluation.

#### **3.3. Control of documents and records**

Control of documents and records is critical whether for scientific project management or for quality management. For high energy physic experiments, large and complicated equip‐ ments are usually involved. During the project design and scheme phase, rules for docu‐ ments and records reserved need to be made clearly and principles for numbering and signing the documents and records need to be described specifically.

**Figure 5.** The workflow of archive management.

BEPCII project builds up a special mechanism of file control. Because BESIII is part of the whole project, the rules of document management are in accordance with the requirements of BEPCII. *Documents and records* need to be signed according to the regulations, in accord‐ ance with the whole project and effective as well. *Documents and records* need to be preserved and archived on a regular basis.

further refinement of the time arrangement of the project design report and makes the man‐

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327

The CPM project is highly in accord with the practical progressand BEPCII zero-grade CPM project is modified frequently. The BEPCII project was finished in 2008 and was finally

The R&D of large detectors is involved with bulk purchase. In the BESIII project, purchasing management rules are made according to the relevant laws and regulations on acquisition. Purchasing and approval process are defined clearly. Bidding is strictly adopted in the project to save research money. An appropriate regulation in the purchasing process is a

Abroad purchase has a long life cycle, heads of procurement need to do significant prepara‐ tory work in advance, and the heads should be quite familiar with the procurement proce‐ dures in order to complete the purchase in time. The purchasing department published the

agement of the project construction effective.

guarantee for carrying out the project under the budget..

checked in 2009.

**3.5. Purchasing Management**

flowchart to facilitate the work.

**Figure 7.** Abroad purchase flowchart.

There are several characteristics in archive management work, especially for the high energy experiments. Firstly, this work must be arranged by the project management department at the beginning of the project. Secondly, the document and records which need to be pre‐ served must be clearly described and the responsibility should be defined at the first time. Thirdly, the archive office, the project office and each member working for the project should cooperate to get the work done quickly and perfectly.

All the quality documents of the whole process of each single detector, from design, re‐ search, test, and acceptance are preserved according to the regulations. Technical specifica‐ tions, interface of different tasks, diagrams, test reports are archived as written documents. Regular meeting minutes are kept also as archives. Those *Documents and records* can be used to track and follow the quality of the product in the whole process.

The running cycle of big science project, just like its construction cycle, is as long as to last more than ten years. Therefore, control of documents and records is very essential for the running and maintenance of the big science project, as an important prop and support.

#### **3.4. Schedule Management**

BESIII project has followed Critical Path Method (CPM) to control the schedule of the whole project. The plan in the CPM is in detail and convenient for check. It is easy for revision ac‐ cording to the actual process and make sure it is updated in time within the system.

**Figure 6.** BEPCII zero-grade CPM project (partly,2002).

In order to give a better control of the schedule, CPM is classified. Any sub-system could make its own play and updates in time following the step of the total plan. Therefore inter communication plays an important role in the schedule management. In a word, CPM is a further refinement of the time arrangement of the project design report and makes the man‐ agement of the project construction effective.

The CPM project is highly in accord with the practical progressand BEPCII zero-grade CPM project is modified frequently. The BEPCII project was finished in 2008 and was finally checked in 2009.

#### **3.5. Purchasing Management**

ance with the whole project and effective as well. *Documents and records* need to be preserved

There are several characteristics in archive management work, especially for the high energy experiments. Firstly, this work must be arranged by the project management department at the beginning of the project. Secondly, the document and records which need to be pre‐ served must be clearly described and the responsibility should be defined at the first time. Thirdly, the archive office, the project office and each member working for the project

All the quality documents of the whole process of each single detector, from design, re‐ search, test, and acceptance are preserved according to the regulations. Technical specifica‐ tions, interface of different tasks, diagrams, test reports are archived as written documents. Regular meeting minutes are kept also as archives. Those *Documents and records* can be used

The running cycle of big science project, just like its construction cycle, is as long as to last more than ten years. Therefore, control of documents and records is very essential for the running and maintenance of the big science project, as an important prop and support.

BESIII project has followed Critical Path Method (CPM) to control the schedule of the whole project. The plan in the CPM is in detail and convenient for check. It is easy for revision ac‐

In order to give a better control of the schedule, CPM is classified. Any sub-system could make its own play and updates in time following the step of the total plan. Therefore inter communication plays an important role in the schedule management. In a word, CPM is a

cording to the actual process and make sure it is updated in time within the system.

and archived on a regular basis.

326 Latest Research into Quality Control

**3.4. Schedule Management**

**Figure 6.** BEPCII zero-grade CPM project (partly,2002).

should cooperate to get the work done quickly and perfectly.

to track and follow the quality of the product in the whole process.

The R&D of large detectors is involved with bulk purchase. In the BESIII project, purchasing management rules are made according to the relevant laws and regulations on acquisition. Purchasing and approval process are defined clearly. Bidding is strictly adopted in the project to save research money. An appropriate regulation in the purchasing process is a guarantee for carrying out the project under the budget..

Abroad purchase has a long life cycle, heads of procurement need to do significant prepara‐ tory work in advance, and the heads should be quite familiar with the procurement proce‐ dures in order to complete the purchase in time. The purchasing department published the flowchart to facilitate the work.

**Figure 7.** Abroad purchase flowchart.

#### **4. BESIII-MUC Quality Management in R&D of BESIII-MUC Detector**

**4.2. Quality Management in the R&D of BESIII-MUC Detector**

bugging, running and maintenance.

7 papers have been published in *NIMA*, as follows:

**1.** A new surface treatment for the prototype RPCs[9],

**5.** First results of the RPC commissioning at BESIII [13]

**7.** An underground cosmic-ray detector made of RPC [15] 8 papers have been published in *Chinese Physics C,*as follows:

**3.** A Study of RPC Gas Composition using Daya Bay RPCs [18]

**4.** Quality control and database on RPC for the BES experiment [19]

**6.** The BESIII Muon Identification System [14]

**1.** Cosmic Ray Test Station for BES RPC [16]

**5.** Test of BES RPC in the avalanche mode [20]

**7.** Study of the RPC-Gd as thremal neutron detector [22]

**Design**

**Table 1.** The analysis of the manuscripts published by MUC group.

whole research process.

The whole process of R&D of MUC detector include the design of the basic unit RPC, prop‐ erties investigation, bulk production, SM design; design of MUC detector, installation de‐

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Throughout the research process, the project director managed the project scientifically and effectively, with each research aspect considered carefully, comprehensively and deeply, and made some achievements. From the pre-research in 2003 to the formal data collection in 2009, more than 30 papers have been published by the research group of MUC, covering the

**2.** Cosmic ray test results on resistive plate chamber for the BESIII experiments [10]

**4.** A monitor for the composition of the gas mixture of BESIII muon chambers [12]

**3.** The Design and Mass Production on RPC for the BESIII Experiment [11]

**2.** Research and Development of Large Area Resistive Plate Chamber [17]

**6.** Performance Study of RPC Prototypes for the BES Muon Detector [21]

**Performance Test**

MUC 6 5

SM 6

**Mass Production**

RPC 1 b 2 a c e f 3 d 4 7 g

**Research**

**Work Application**

#### **4.1. Introduction of BESIII Detector and MUC Detector**

The Beijing Spectrometer (BESIII) is designed to measure the properties of the particles pro‐ duced in the collisions of electrons and positrons at BEPCII. The physics goal of the BESIII experiment is to conduct high statistics and highly precise studies on a number of physics topics in this energy region, including light hadron spectroscopy, charmonium spectra, charm meson decay properties, QCD, tau physics, rare decays, search for glueballs and oth‐ er non-pure quark states [3].

The BESIII detector will consist of a 1 T superconducting solenoid magnet, a high precision main drift chamber (MDC), Time-Of-Flight counters (TOF), a CsI crystal Electromagnetic Calorimeter (EMC) and a muon identifier chamber (MUC) that is integrated in the iron mag‐ netic field return yoke [7]. The muon identifier is the outer most subsystem of the BESIII de‐ tector [8], which is constructed by resistive plate chambers (RPCs, shown in Fig.8.a). 962 RPC are used in the whole MUC detector, which consists of 136 RPC superlayer modules (SM, shown in Fig.8.b). And the Fig.8.c shows the status of the MUC detector when it was finished it's barrel part assemblage. The Fig.8.d shown the designed construct of the BESIII MUC detector with the endcap and barrel parts.

**Figure 8.** a). The RPC moduls, (b). The Suprlayer Modul, (c). The overview the barrel part of the MUC detector after it's assemblage, (d) The construct of the BESIII MUC detector.

#### **4.2. Quality Management in the R&D of BESIII-MUC Detector**

The whole process of R&D of MUC detector include the design of the basic unit RPC, prop‐ erties investigation, bulk production, SM design; design of MUC detector, installation de‐ bugging, running and maintenance.

Throughout the research process, the project director managed the project scientifically and effectively, with each research aspect considered carefully, comprehensively and deeply, and made some achievements. From the pre-research in 2003 to the formal data collection in 2009, more than 30 papers have been published by the research group of MUC, covering the whole research process.

7 papers have been published in *NIMA*, as follows:

**4. BESIII-MUC Quality Management in R&D of BESIII-MUC Detector**

The Beijing Spectrometer (BESIII) is designed to measure the properties of the particles pro‐ duced in the collisions of electrons and positrons at BEPCII. The physics goal of the BESIII experiment is to conduct high statistics and highly precise studies on a number of physics topics in this energy region, including light hadron spectroscopy, charmonium spectra, charm meson decay properties, QCD, tau physics, rare decays, search for glueballs and oth‐

The BESIII detector will consist of a 1 T superconducting solenoid magnet, a high precision main drift chamber (MDC), Time-Of-Flight counters (TOF), a CsI crystal Electromagnetic Calorimeter (EMC) and a muon identifier chamber (MUC) that is integrated in the iron mag‐ netic field return yoke [7]. The muon identifier is the outer most subsystem of the BESIII de‐ tector [8], which is constructed by resistive plate chambers (RPCs, shown in Fig.8.a). 962 RPC are used in the whole MUC detector, which consists of 136 RPC superlayer modules (SM, shown in Fig.8.b). And the Fig.8.c shows the status of the MUC detector when it was finished it's barrel part assemblage. The Fig.8.d shown the designed construct of the BESIII

**Figure 8.** a). The RPC moduls, (b). The Suprlayer Modul, (c). The overview the barrel part of the MUC detector after it's

**4.1. Introduction of BESIII Detector and MUC Detector**

MUC detector with the endcap and barrel parts.

assemblage, (d) The construct of the BESIII MUC detector.

er non-pure quark states [3].

328 Latest Research into Quality Control


8 papers have been published in *Chinese Physics C,*as follows:



As shown in Table 1, it is not difficult to come into conclusion that the whole R&D of MUC detector applied scientific project quality management, which promotes the research work. In the phase of initial RPC research, the key point is on the study of the detector's perform‐ ance test. It is the phase for building a standard quality management. After the acceptance of RPC and project review, mass production and SM reassembling come into being. In this phase, scientific quality control and management play a key role.

installation and debugging. In each phase, review and identification is defined. For impor‐ tant phases, such as aging test, assembly test and system test, detailed guidelines and in‐ structor are written. As shown in Table 2, during the outsourcing process, key parts are defined, and acceptance rules are also clearly described. Control point is set up and design files are carried out strictly to ensure the product quality. More detailed could be found in

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331

We could come into conclusion that scientific quality management can promote scientific

The schedule of the project could be arranged and controlled well, especially the adoption of CPM, which could provide a time map for the whole project. Throughout the four years' successful implementation and of BEPCII project, CPM plays an important role in the project acceptance in due. CPM was adjusted in time according to the project status, thus effective management and restriction was formed for all the related sub systems in the project.

The project has been implemented within the budget and cost was controlled. Purchasing procedures and approval process were strictly described, which played a role for the fair use

Documents and records were kept in detail, as reference in the project to find the source of old problems and avoid new problems. Especially for those big scientific projects which will last more than ten years, files about interface management and quality management and various records are significant for the running and maintenance in the following work. They also act as important reference for the future project construction in high energy physics.

Scientific quality management could promote scientific research effectively. At the same time, as the development of scientific research, cooperation among researchers will be in‐ creased. It is good for the communication and exchange in the area of quality management

Experiences in big scientific project are good for the growth of young researchers. With par‐ ticipation in the R&D of big science equipment under quality management system, research‐

In an ongoing scientific project managed launched by IHEP, researchers are from partici‐ pants in BESIII or DayaBay. Although it is non-international, at the beginning the project is managed as required in strict quality management, just like that in big science project. As the development of the project, communication and cooperation among other institutions

ers will learn how to organize and manage scientific programs or projects in future.

and promotes the refining of the quality management system thus.

**5. Significance of Scientific Quality Management in Research**

table 2 for summary.

of the fund.

**5.1. Promote Scientific Projects**

**5.2. Promote Scientific Research**

projects to proceed successfully, in the following ways:

A perfect quality tracking system is established in each session, from the production and test of RPC, assembling and test of modules, to the installation and debugging of MUC detector, to ensure the supervision of the performance of detector is plausible.

Especially for the mass production of RPC and SM, before research and test, a database is built for storage related data and affording date support for quality control and final run‐ ning & maintenance.

#### **4.3. Summary**

All the requirements such as verification, validation, monitoring, measurement, inspection and test activities specific to the detector are described in the design report of the detector in detail. The report plays the same role as in making a particular quality control plan.


**Table 2.** Quality management/control factor distribution of MUC detector.

The design report of the detector divide the R&D process into several phases, including con‐ cept design, project design, sample trail-manufacture, product research and production, test, installation and debugging. In each phase, review and identification is defined. For impor‐ tant phases, such as aging test, assembly test and system test, detailed guidelines and in‐ structor are written. As shown in Table 2, during the outsourcing process, key parts are defined, and acceptance rules are also clearly described. Control point is set up and design files are carried out strictly to ensure the product quality. More detailed could be found in table 2 for summary.

#### **5. Significance of Scientific Quality Management in Research**

#### **5.1. Promote Scientific Projects**

As shown in Table 1, it is not difficult to come into conclusion that the whole R&D of MUC detector applied scientific project quality management, which promotes the research work. In the phase of initial RPC research, the key point is on the study of the detector's perform‐ ance test. It is the phase for building a standard quality management. After the acceptance of RPC and project review, mass production and SM reassembling come into being. In this

A perfect quality tracking system is established in each session, from the production and test of RPC, assembling and test of modules, to the installation and debugging of MUC detector,

Especially for the mass production of RPC and SM, before research and test, a database is built for storage related data and affording date support for quality control and final run‐

All the requirements such as verification, validation, monitoring, measurement, inspection and test activities specific to the detector are described in the design report of the detector in

> **Mass productio n**

article inspection ■ ■ ■ ■

traceability ■ ■ ■ ■ preventive action □ □ ■ ■

The design report of the detector divide the R&D process into several phases, including con‐ cept design, project design, sample trail-manufacture, product research and production, test,

corrective action ■

**Assemblag**

**<sup>e</sup> Debug Running**

detail. The report plays the same role as in making a particular quality control plan.

phase, scientific quality control and management play a key role.

to ensure the supervision of the performance of detector is plausible.

□ ■ **Design Prototype**

characteristic ■ ■ □ critical process ■ ■

improvement ■ ■ ■ effectiveness ■ ■

quality plan □ ■

**Table 2.** Quality management/control factor distribution of MUC detector.

characteristic ■ □

critical

major

quality

ning & maintenance.

330 Latest Research into Quality Control

**4.3. Summary**

We could come into conclusion that scientific quality management can promote scientific projects to proceed successfully, in the following ways:

The schedule of the project could be arranged and controlled well, especially the adoption of CPM, which could provide a time map for the whole project. Throughout the four years' successful implementation and of BEPCII project, CPM plays an important role in the project acceptance in due. CPM was adjusted in time according to the project status, thus effective management and restriction was formed for all the related sub systems in the project.

The project has been implemented within the budget and cost was controlled. Purchasing procedures and approval process were strictly described, which played a role for the fair use of the fund.

Documents and records were kept in detail, as reference in the project to find the source of old problems and avoid new problems. Especially for those big scientific projects which will last more than ten years, files about interface management and quality management and various records are significant for the running and maintenance in the following work. They also act as important reference for the future project construction in high energy physics.

#### **5.2. Promote Scientific Research**

Scientific quality management could promote scientific research effectively. At the same time, as the development of scientific research, cooperation among researchers will be in‐ creased. It is good for the communication and exchange in the area of quality management and promotes the refining of the quality management system thus.

Experiences in big scientific project are good for the growth of young researchers. With par‐ ticipation in the R&D of big science equipment under quality management system, research‐ ers will learn how to organize and manage scientific programs or projects in future.

In an ongoing scientific project managed launched by IHEP, researchers are from partici‐ pants in BESIII or DayaBay. Although it is non-international, at the beginning the project is managed as required in strict quality management, just like that in big science project. As the development of the project, communication and cooperation among other institutions both at home and abroad have increased. To coordinate the partnership among different or‐ ganizations and unites, cooperation group is formed. As shown below, a formality manage‐ ment system and strictness organization is built, which lays a solid foundation for the sustainable development of cooperation group and joint research work in future, whose Or‐ ganization Chart shown in fig.9 for example.

**Author details**

**References**

Xuemin Zhu1\* and Sen Qian1\*

Public/WebHome.

\*Address all correspondence to: zhuxm@ihep.ac.cn

1 The Institute of High Energy Physics, Chinese Academy of Sciences, China

[1] The Institute of High Energy Physics. http://english.ihep.cas.cn/au/bi/.

[4] European Organization for Nuclear Research. http://public.web.cern.ch/public/.

[5] The Bay Reactor Neutrino Experiment. http://dayabay.ihep.ac.cn/twiki/bin/view/

The Quality Management of The R&D in High Energy Physics Detector

http://dx.doi.org/10.5772/51434

333

[6] China Spallation Neutron Source (CSNS). http://csns.ihep.ac.cn/english/index.htm.

[7] Tianchi, Z. (2010). Design and construction of the BESIII detector Nuclear. *Nuclear In‐*

[8] Boxiang, Y. (2009). The construction of the BESIII experiment. *Nuclear Instruments and*

[9] Jiawen, Z. (2005). A new surface treatment for the prototype RPCs. *Nuclear Instru‐*

[10] Jifeng, H. Cosmic ray test results on resistive plate chamber for the BESIII experi‐ ments. *Cosmic ray test results on resistive plate chamber for the BESIII experiments.*

[11] Jiawen, Z. (2007). The Design and Mass Production on RPC for the BESIII Experi‐ ment. *Nuclear Instruments and Methods in Physics Research A*, 580, 1250-1256.

[12] Sen, Q. (2008). A monitor for the composition of the gas mixture of BESIII muon chambers. *Nuclear Instruments and Methods in Physics Research A*, 595, 520-525.

[13] Yuguang, X. (2008). First results of the RPC commissioning at BESIII. *Nuclear Instru‐*

[14] Sen, Q. (2010). The BESIII Muon Identification System. *Nuclear Instruments and Meth‐*

[15] Qingmin, Z. (2007). An underground cosmic-ray detector made of RPC. *Nuclear In‐*

[2] The BEPCII Project. http://english.ihep.cas.cn/rs/fs/bepc/index.html. [3] The Beijing Spectrometer. http://bes3.ihep.ac.cn/orga/institute.htm.

*struments and Methods in Physics Research A*, 614, 345-399.

*ments and Methods in Physics Research A*, 540(2005), 102-112.

*ments and Methods in Physics Research A*, 595, 520-525.

*struments and Methods in Physics Research A*, 583, 278-284.

*ods in Physics Research A*, 614, 196-205.

*Methods in Physics Research A*, 598, 7-11.

**Figure 9.** The organization of the BEPCII

#### **6. Conclusion**

Quality management plays a significant role both in project management and in the scientif‐ ic research. With a scientific and comprehensive quality management system, big science project will be duly executed. The level of scientific projects will be greatly improved by the application and popularization of national and international quality standards.

#### **Acknowledgements**

In the process of writing this manuscript, we have received much understanding and sup‐ port from many departments in our institute. We are particularly grateful to those staffs in the IHEP offices, archives, the purchasing department and the project teams for their strong‐ ly supported. Special thanks go to Prof. Zhao Jingwei for this support and encouragement to finish this work.

#### **Author details**

both at home and abroad have increased. To coordinate the partnership among different or‐ ganizations and unites, cooperation group is formed. As shown below, a formality manage‐ ment system and strictness organization is built, which lays a solid foundation for the sustainable development of cooperation group and joint research work in future, whose Or‐

Quality management plays a significant role both in project management and in the scientif‐ ic research. With a scientific and comprehensive quality management system, big science project will be duly executed. The level of scientific projects will be greatly improved by the

In the process of writing this manuscript, we have received much understanding and sup‐ port from many departments in our institute. We are particularly grateful to those staffs in the IHEP offices, archives, the purchasing department and the project teams for their strong‐ ly supported. Special thanks go to Prof. Zhao Jingwei for this support and encouragement to

application and popularization of national and international quality standards.

ganization Chart shown in fig.9 for example.

332 Latest Research into Quality Control

**Figure 9.** The organization of the BEPCII

**6. Conclusion**

**Acknowledgements**

finish this work.

Xuemin Zhu1\* and Sen Qian1\*

\*Address all correspondence to: zhuxm@ihep.ac.cn

1 The Institute of High Energy Physics, Chinese Academy of Sciences, China

#### **References**


[16] Qian, L. (2006). Cosmic Ray Test Station for BES RPC. *China Physics C (High Energy And Unclear Physics)*, 30(4).

**Section 6**

**Quality Control in Cosmetics**


**Quality Control in Cosmetics**

[16] Qian, L. (2006). Cosmic Ray Test Station for BES RPC. *China Physics C (High Energy*

[17] Jiawen, Z. (2003). Research and Development of Large Area Resistive Plate Chamber.

[18] Malie, H. (2010). Study of RPC gas composition using Daya Bay RPCs. *China Physics*

[19] Jifeng, H. (2008). Quality control and database on RPC for the BES experiment. *Chi‐*

[20] Jifeng, H. (2008). Test of BES RPC in the avalanche mode. *China Physics C (High En‐*

[21] Yuguang, X. (2008). Performance Study of RPC Prototypes for the BES Muon Detec‐

[22] Sen, Q. (2009). Study of the RPC-Gd as thremal neutron detector. *China Physics C*

*China Physics C (High Energy And Unclear Physics)*, 27(7).

*na Physics C (High Energy And Unclear Physics)*, 32(3).

tor. *China Physics C (High Energy And Unclear Physics)*, 31(1).

*C (High Energy And Unclear Physics)*, 34(8).

*ergy And Unclear Physics)*, 32(5).

*(High Energy And Unclear Physics)*, 33(8).

*And Unclear Physics)*, 30(4).

334 Latest Research into Quality Control

**Chapter 16**

**Cosmetics' Quality Control**

Maria Gabriela José de Almeida,

Marcos Antonio Corrêa and Vera Lucia Borges Isaac

The quality of a cosmetic product, in the same way as to other kind of products, is initially defined by the manufacturer that chooses the features that a product should present. On the other hand, the quality control of a product aims to verify if all of these defined features are in accordance with the standard definitions and if it will be maintained during the shelf life

The quality control of cosmetics is important to ensure the efficacy and safety of products and its raw-materials. Due to the rapid growth that cosmetic industries have exhibit all over the world, efficient, low cost and rapid methods to assay cosmetics' quality control are a pri‐ ority. Some current techniques used by the cosmetic industry can be applied to the evalua‐ tion of cosmetics' quality control in an efficient manner, such as: rheology, sensory analysis

Sensory analysis is a powerful tool, since there is no equipment able to measure the human feelings. It applies experimental design and statistical analysis to obtain information about a product in relation to what people feel when use or consume a product, in other words, it is used to indicate consumer acceptance of a particular product. It can be understood as the discipline that interprets, assess and measures characteristics of a product, after stimulating people in relation to their vital senses, as vision, touch, smell and taste (Stone et al., 1992). It is widely used in food industry and recently, it has also been applied in the cosmetic indus‐ try (Almeida et al., 2008; Aust et al., 1987; Backe et al., 1999; Lee et al., 2005; Parente et al.,

> © 2012 Chiari et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Chiari et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Bruna Galdorfini Chiari,

http://dx.doi.org/10.5772/51846

of the product (Shewhart, 1980).

and small angle X-ray scattering (SAXS).

2005; Wortel et al., 2000).

**1. Introduction**

**Chapter 16**

## **Cosmetics' Quality Control**

Bruna Galdorfini Chiari, Maria Gabriela José de Almeida, Marcos Antonio Corrêa and Vera Lucia Borges Isaac

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51846

#### **1. Introduction**

The quality of a cosmetic product, in the same way as to other kind of products, is initially defined by the manufacturer that chooses the features that a product should present. On the other hand, the quality control of a product aims to verify if all of these defined features are in accordance with the standard definitions and if it will be maintained during the shelf life of the product (Shewhart, 1980).

The quality control of cosmetics is important to ensure the efficacy and safety of products and its raw-materials. Due to the rapid growth that cosmetic industries have exhibit all over the world, efficient, low cost and rapid methods to assay cosmetics' quality control are a pri‐ ority. Some current techniques used by the cosmetic industry can be applied to the evalua‐ tion of cosmetics' quality control in an efficient manner, such as: rheology, sensory analysis and small angle X-ray scattering (SAXS).

Sensory analysis is a powerful tool, since there is no equipment able to measure the human feelings. It applies experimental design and statistical analysis to obtain information about a product in relation to what people feel when use or consume a product, in other words, it is used to indicate consumer acceptance of a particular product. It can be understood as the discipline that interprets, assess and measures characteristics of a product, after stimulating people in relation to their vital senses, as vision, touch, smell and taste (Stone et al., 1992). It is widely used in food industry and recently, it has also been applied in the cosmetic indus‐ try (Almeida et al., 2008; Aust et al., 1987; Backe et al., 1999; Lee et al., 2005; Parente et al., 2005; Wortel et al., 2000).

© 2012 Chiari et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Chiari et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The sensory analysis can be applied in the research and development of a new cosmetic (Isaac et al., 2012a), in controlling the manufacturing process to evaluate raw-materials qual‐ ity and, even, to make possible the substitution of a raw-material of a product that is tradi‐ tional in the market without changes in the product's features (Meilgaard et al., 1991; Muñoz et al., 1993).

The sensory analysis was first applied to the food industry, but the high advance in other areas, such as the cosmetic and pharmaceutical industries, and the important da‐ ta obtained with the sensory analysis, demanded this useful technique to describe what

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 339

An important advantage of the use of sensory analysis in the quality control of a cosmetic product is that it yields a complex analysis in relation to all sensorial attributes that a prod‐ uct could present, it means that, the volunteer who participates of the sensorial panel is able to give information about the fragrance, the sensation, the appearance, the consistence, and other features that this person experience when use such product. The description of these characteristics by means of equipment would be an arduous work and would provide not sufficient or not valuable data when compared to the data provided by the human senses. Beyond that, the acquisition of this equipment could be of high cost when compared to the

The association of data obtained from sensory analysis and instrumental analysis (especially physicochemical analysis) provides great information and a more complete profile of the

Nowadays, there are companies specialized in perform sensory analysis of cosmetic prod‐ ucts, and thus, they could be contracted to perform this study for cosmetic industries that

The sensorial performance of cosmetics is essential to the acceptance of consumers (Almeida et al., 2008; Fouéré et al., 2005; Lee et al., 2005; Proksch, 2005), thus, especial attention should

The sensorial features of a formulation are mainly related to the raw-materials and package (Dooley et al., 2009). The raw-materials influence directly in what the consumer feels when applies the cosmetic. The emollients, for example, are raw-materials of marked influence in the tactile sense (Parente et al., 2008; Gorcea and Laura, 2010). Other raw-materials are avail‐ able at the market and are commercialized to be used in formulations as sensorial modifiers. The main representatives of this kind of product are the silicones and Polymethyl Methacry‐

The package influences in the first impression of the consumer about a product, since the first sense used to choose a cosmetic in the market is the vision. After, the smell is used too. The tact is not involved in the first purchase attitude, but it will define if a consumer will

In this context, it is possible to verify that the sensorial features of a cosmetic are of great

Thus, the sensorial analysis could help a company to define the attributes that a product

Another point is that these desired sensorial characteristics should be maintained during the cosmetic shelf life. To obtain that, the raw-materials used should be of good quality, the

should or not present beyond the characteristics and intensity of these attributes.

the consumers fell.

product (Ross, 2009).

be given to this subject.

late (Ozkan et al., 2012).

become a loyal consumer.

importance in the success of it in the market.

sensory analyses' costs (Ross, 2009).

don't have a sector trained to do it.

The application of sensory analysis could be related to the product control, referring to the storage, packaging and maintenance of sensory quality in relation to time and temperature (Muñoz et al., 1993), since these factors can change a sensory attribute that the product present originally (Zague, 2008) and people who participates of the sensorial panel could re‐ alize the changes in the sensorial attributes. Another function of this important tool is to per‐ formance comparative tests between competing products.

Another tool that could be applied to evaluate cosmetics' quality control is the rheology, which studies the flow and deformation of fluids. It has been used in research laboratories and industries as a tool for characterizing ingredients and products, and to predict the per‐ formance of products and consumer acceptance.

Rheology has been widely used because, by means of this tool, the researcher can determine physicochemical properties of a product. Constructing a rheogram, it is possible to check the flow curve, evaluate if there is a yield stress and a hysteresis area, which appears to be relat‐ ed to the release of drugs and actives. It is also possible to construct a creep and recovery curve obtaining information about viscoelasticity of each system.

Specifically, in relation to the quality control of cosmetics, specifically, rheology can be ap‐ plied to help in determining the stability of products by means of the apparent viscosity measured periodically in a determined period exposing the samples to stress conditions (high and low temperatures, solar irradiation), and to monitor the flow characteristics dur‐ ing the shelf life or in the stability assay of a product.

The SAXS technique have being used for the analysis of cosmetics, in order to evaluate the presence of liquid crystalline structures, called liquid-crystals, which are known to increase the stability of formulations becoming, therefore, desirable in cosmetics (Makai et al., 2003).

Combining these three tools, it is possible to test the quality of cosmetics with a rich range of data, and obtain a deep characterization of the system. The results contribute to determining product use, or even, they provide indication of what need to be done to develop a product with predetermined characteristics.

#### **2. Sensory analysis**

Sensory analysis is defined by Piana et al. (2004) as the examination of a product through the evaluation of the attributes perceptible by the five sense organs (organoleptic attributes), such as color, odor, taste, touch, texture and noise, allowing the establishment of the organo‐ leptic profile of diverse products, including cosmetics.

The sensory analysis was first applied to the food industry, but the high advance in other areas, such as the cosmetic and pharmaceutical industries, and the important da‐ ta obtained with the sensory analysis, demanded this useful technique to describe what the consumers fell.

The sensory analysis can be applied in the research and development of a new cosmetic (Isaac et al., 2012a), in controlling the manufacturing process to evaluate raw-materials qual‐ ity and, even, to make possible the substitution of a raw-material of a product that is tradi‐ tional in the market without changes in the product's features (Meilgaard et al., 1991;

The application of sensory analysis could be related to the product control, referring to the storage, packaging and maintenance of sensory quality in relation to time and temperature (Muñoz et al., 1993), since these factors can change a sensory attribute that the product present originally (Zague, 2008) and people who participates of the sensorial panel could re‐ alize the changes in the sensorial attributes. Another function of this important tool is to per‐

Another tool that could be applied to evaluate cosmetics' quality control is the rheology, which studies the flow and deformation of fluids. It has been used in research laboratories and industries as a tool for characterizing ingredients and products, and to predict the per‐

Rheology has been widely used because, by means of this tool, the researcher can determine physicochemical properties of a product. Constructing a rheogram, it is possible to check the flow curve, evaluate if there is a yield stress and a hysteresis area, which appears to be relat‐ ed to the release of drugs and actives. It is also possible to construct a creep and recovery

Specifically, in relation to the quality control of cosmetics, specifically, rheology can be ap‐ plied to help in determining the stability of products by means of the apparent viscosity measured periodically in a determined period exposing the samples to stress conditions (high and low temperatures, solar irradiation), and to monitor the flow characteristics dur‐

The SAXS technique have being used for the analysis of cosmetics, in order to evaluate the presence of liquid crystalline structures, called liquid-crystals, which are known to increase the stability of formulations becoming, therefore, desirable in cosmetics (Makai et al., 2003).

Combining these three tools, it is possible to test the quality of cosmetics with a rich range of data, and obtain a deep characterization of the system. The results contribute to determining product use, or even, they provide indication of what need to be done to develop a product

Sensory analysis is defined by Piana et al. (2004) as the examination of a product through the evaluation of the attributes perceptible by the five sense organs (organoleptic attributes), such as color, odor, taste, touch, texture and noise, allowing the establishment of the organo‐

formance comparative tests between competing products.

curve obtaining information about viscoelasticity of each system.

formance of products and consumer acceptance.

ing the shelf life or in the stability assay of a product.

leptic profile of diverse products, including cosmetics.

with predetermined characteristics.

**2. Sensory analysis**

Muñoz et al., 1993).

338 Latest Research into Quality Control

An important advantage of the use of sensory analysis in the quality control of a cosmetic product is that it yields a complex analysis in relation to all sensorial attributes that a prod‐ uct could present, it means that, the volunteer who participates of the sensorial panel is able to give information about the fragrance, the sensation, the appearance, the consistence, and other features that this person experience when use such product. The description of these characteristics by means of equipment would be an arduous work and would provide not sufficient or not valuable data when compared to the data provided by the human senses. Beyond that, the acquisition of this equipment could be of high cost when compared to the sensory analyses' costs (Ross, 2009).

The association of data obtained from sensory analysis and instrumental analysis (especially physicochemical analysis) provides great information and a more complete profile of the product (Ross, 2009).

Nowadays, there are companies specialized in perform sensory analysis of cosmetic prod‐ ucts, and thus, they could be contracted to perform this study for cosmetic industries that don't have a sector trained to do it.

The sensorial performance of cosmetics is essential to the acceptance of consumers (Almeida et al., 2008; Fouéré et al., 2005; Lee et al., 2005; Proksch, 2005), thus, especial attention should be given to this subject.

The sensorial features of a formulation are mainly related to the raw-materials and package (Dooley et al., 2009). The raw-materials influence directly in what the consumer feels when applies the cosmetic. The emollients, for example, are raw-materials of marked influence in the tactile sense (Parente et al., 2008; Gorcea and Laura, 2010). Other raw-materials are avail‐ able at the market and are commercialized to be used in formulations as sensorial modifiers. The main representatives of this kind of product are the silicones and Polymethyl Methacry‐ late (Ozkan et al., 2012).

The package influences in the first impression of the consumer about a product, since the first sense used to choose a cosmetic in the market is the vision. After, the smell is used too. The tact is not involved in the first purchase attitude, but it will define if a consumer will become a loyal consumer.

In this context, it is possible to verify that the sensorial features of a cosmetic are of great importance in the success of it in the market.

Thus, the sensorial analysis could help a company to define the attributes that a product should or not present beyond the characteristics and intensity of these attributes.

Another point is that these desired sensorial characteristics should be maintained during the cosmetic shelf life. To obtain that, the raw-materials used should be of good quality, the manufacture practices should be appropriate, the preservatives used need to be efficient and the formulation should be stable.

The area where will be performed the analyses should be divided in individual cabins (Fig‐ ure 2) with a window, where the analyst must offer the samples to the volunteer, sink and

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 341

The cabins must be ventilated and odor free, to avoid interferences in the analyses. The tem‐ perature and humidity should be controled around 22 ºC and at 45% of humidity (Isaac et

It is recommended that the walls and furniture of the rooms are colored with neutral and light colors to not disturb the attention of the volunteers and to not interfere in the attributes

The volunteers should not smoke, should be healthy, with ease of memorization and com‐

In the study, the volunteers judges could be an experienced judge or not, depending on the kind of evaluation and the answers that the professional team needs to obtain. In the case of utilization of sensorial analysis in the quality control of a cosmetic, usually the volunteers are regular users of the product in analysis, since they need to be familiarized with the char‐ acteristics of the product and have sensibility to perceive slight modification on it. When the aim of the sensorial analysis is to evaluate the acceptance of a product that should be launched in the market, it is recommended that the volunteers are potencial users of this new product, orienting the formulator to make changes in the formulation and guiding the

company to evaluate if the costs of the product launch are recommended or not.

faucet, to the volunteer use when necessary (Isaac et al., 2012).

**Figure 2.** An example of layout of sensorial analysis cabine (Isaac et al., 2012a).

analyzed by the vision, such as color and appearance of the product.

al., 2012).

munication.

In conclusion, the sensorial analysis is an indispensable technique to help the formula‐ tor to evaluate the quality of its new product, in relation to its sensorial characteristics and to its stability, testing if the product will keep the nice sensorial feelings that trans‐ mit to the consumer during the time of use. This tool is helpful to the research and devel‐ opment area of a company which aims to obtain good quality products of high acceptance by the consumers. The suitable application of sensory evaluation could avoid the outlay of a company with the launching of a product in the market that was reject‐ ed by the volunteers of the preliminary study.

Currently, the sensorial analysis have gained more scientific rigor due to the need to offer to the consumers products that meet their expectations and due to the high competition be‐ tween the major industries of this sector.

To perform the sensorial analysis with rigor and organization, the laboratory destined to it must have the following areas:

A room destined to the analyst who leads the team (Figure 1a)

A conference room (Figure 1b)

A room for the samples preparation (Figure 1c)

An area to the analyses with the volunteers (Figure 1d)

The laboratory should be located in an easy access place.

**Figure 1.** An example of layout of a sensorial analysis laboratory (Isaac et al., 2012a).

The area where will be performed the analyses should be divided in individual cabins (Fig‐ ure 2) with a window, where the analyst must offer the samples to the volunteer, sink and faucet, to the volunteer use when necessary (Isaac et al., 2012).

**Figure 2.** An example of layout of sensorial analysis cabine (Isaac et al., 2012a).

manufacture practices should be appropriate, the preservatives used need to be efficient and

In conclusion, the sensorial analysis is an indispensable technique to help the formula‐ tor to evaluate the quality of its new product, in relation to its sensorial characteristics and to its stability, testing if the product will keep the nice sensorial feelings that trans‐ mit to the consumer during the time of use. This tool is helpful to the research and devel‐ opment area of a company which aims to obtain good quality products of high acceptance by the consumers. The suitable application of sensory evaluation could avoid the outlay of a company with the launching of a product in the market that was reject‐

Currently, the sensorial analysis have gained more scientific rigor due to the need to offer to the consumers products that meet their expectations and due to the high competition be‐

To perform the sensorial analysis with rigor and organization, the laboratory destined to it

the formulation should be stable.

340 Latest Research into Quality Control

ed by the volunteers of the preliminary study.

A room for the samples preparation (Figure 1c)

An area to the analyses with the volunteers (Figure 1d)

The laboratory should be located in an easy access place.

**Figure 1.** An example of layout of a sensorial analysis laboratory (Isaac et al., 2012a).

A room destined to the analyst who leads the team (Figure 1a)

tween the major industries of this sector.

must have the following areas:

A conference room (Figure 1b)

The cabins must be ventilated and odor free, to avoid interferences in the analyses. The tem‐ perature and humidity should be controled around 22 ºC and at 45% of humidity (Isaac et al., 2012).

It is recommended that the walls and furniture of the rooms are colored with neutral and light colors to not disturb the attention of the volunteers and to not interfere in the attributes analyzed by the vision, such as color and appearance of the product.

The volunteers should not smoke, should be healthy, with ease of memorization and com‐ munication.

In the study, the volunteers judges could be an experienced judge or not, depending on the kind of evaluation and the answers that the professional team needs to obtain. In the case of utilization of sensorial analysis in the quality control of a cosmetic, usually the volunteers are regular users of the product in analysis, since they need to be familiarized with the char‐ acteristics of the product and have sensibility to perceive slight modification on it. When the aim of the sensorial analysis is to evaluate the acceptance of a product that should be launched in the market, it is recommended that the volunteers are potencial users of this new product, orienting the formulator to make changes in the formulation and guiding the company to evaluate if the costs of the product launch are recommended or not.

There are four different methods to perform the sensorial analysis that are most used, they are: affective, discriminative, descriptive (Aust et al., 1987) and methods to evaluate the ef‐ fective of the product.

Independent of the method of sensory analysis suitable for each evaluation, the professional team should use printed questionnaires to obtain the answers from each volunteer. The use of printed questionnaires avoids the contact between the professional and the volunteers preventing that the professional is biased in his responses, beyond that, it facilitates the data collection.

In the elaboration of these questionnaires the professional team should use suitable lexi‐ cons for each class of product, for example, the lexicons used to the evaluation of lip products are different from that used for corporal lotions (Dooley et al., 2009). Some re‐ searches had developed suitable lexicons for different classes of cosmetic products (Civ‐ ille and Dus, 1991; Wortel and Wiechers, 2000; Dooley et al., 2009). The manner as the volunteer is questioned is fundamental to obtain the information required from them. An inadequate formulary could invalidate a sensory evaluation. It is interesting also, that a description of all descriptors attributed to the formulation being provided to the volun‐ teer, for example: "Thickness: Viscosity of the cream when picking up from the contain‐ er", "Ease of spreading: Ease of rubbing the sample over the skin", "Absorption: Ease of absorption of the product through the skin", "Residue: Amount of product left on the skin after application" (Parente et al., 2010).

**Figure 3.** Examples of presentation of hedonic scale (Olshan et al., 2000; Barkat et al., 2003).

(Wortel and Wiechers, 2000).

data by the professional team.

The tests to evaluate the effective of the products should be performed in true conditions of use and the volunteer may use only the product that is being assessed. These tests could be conduct by the evaluation of dermatologists, by the evaluation of volunteers, and even, by the measurement of one parameter by un equipment, such as the equipment that measures hydration, sebum and transepidermal water loss, to define if a product is really effective. Based on these clinical evaluations, a company could create an efficacy claim to the product

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 343

The sensory analysis could be also applied when a cosmetic industry needs to replace a rawmaterial of a commercialized product without changes in the performance of it. This re‐ placement could be originated by many factors, such as the reduction of costs, problems with the firm who provides this raw-material problems with same raw material which caus‐ es irritation, comedogenicity or other problems that affect the consumer. In this field, the sensorial analysis helps the formulator, who proposes different raw materials as substitute,

The statistical analysis is indispensable in the sensory studies. The sensory analysis data should be evaluated transforming them in scores which allows the application of statistical analysis to calculate the mean and standard deviation of the results, and the determination if the difference between the scores obtained is statistically significant. Graphics, tables and preference maps could be elaborated with the results obtained to facilitate the analysis of the

The sensory analysis is especially indispensable in the industries of fragrances and per‐ fumes, and because of that, high-resolution instrumental methods for evaluation of flavor

to evaluate if the consumer will notice the adaptation in the cosmetic product.

The affective methods represent the consumer opinion and evaluate how much consumers like or dislike a product. It is a quantitative method that is performed in order to know the consumers preferences (Aust et al., 1987). This technique could be applied in the develop‐ ment of new products and when it is necessary to replace a constituent of a formulation without loss of the product quality. It could be performed in two different ways: offering two different samples to the volunteer asking him about what sample he prefers between them or using a hedonic scale for the volunteer attributes grades of intensity of its accepta‐ tion in relation to the sample.

The hedonic scale either can be presented to the panel of evaluators in different manners, as shown in Figure 3.

The affective methods provide quantitative data and allow more than one attribute in each sample being evaluated at the same time.

The discriminative test is better represented by the Triangular test. It allows differentiating one between three different samples and is very useful in shelf life studies and in the quality control of cosmetics. The ideal is to perform this evaluation with twelve to forty volunteers, who will receive the three samples and should indicate the different one between them (Zenebon et al., 2008).

The descriptive tests provide a broad sensory description about the product that is being evaluated (Almeida et al., 2008), helping to predict the consumer acceptance and what con‐ sumers think about such product (Almeida et al., 2006; Aust et al., 1987).

There are four different methods to perform the sensorial analysis that are most used, they are: affective, discriminative, descriptive (Aust et al., 1987) and methods to evaluate the ef‐

Independent of the method of sensory analysis suitable for each evaluation, the professional team should use printed questionnaires to obtain the answers from each volunteer. The use of printed questionnaires avoids the contact between the professional and the volunteers preventing that the professional is biased in his responses, beyond that, it facilitates the data

In the elaboration of these questionnaires the professional team should use suitable lexi‐ cons for each class of product, for example, the lexicons used to the evaluation of lip products are different from that used for corporal lotions (Dooley et al., 2009). Some re‐ searches had developed suitable lexicons for different classes of cosmetic products (Civ‐ ille and Dus, 1991; Wortel and Wiechers, 2000; Dooley et al., 2009). The manner as the volunteer is questioned is fundamental to obtain the information required from them. An inadequate formulary could invalidate a sensory evaluation. It is interesting also, that a description of all descriptors attributed to the formulation being provided to the volun‐ teer, for example: "Thickness: Viscosity of the cream when picking up from the contain‐ er", "Ease of spreading: Ease of rubbing the sample over the skin", "Absorption: Ease of absorption of the product through the skin", "Residue: Amount of product left on the

The affective methods represent the consumer opinion and evaluate how much consumers like or dislike a product. It is a quantitative method that is performed in order to know the consumers preferences (Aust et al., 1987). This technique could be applied in the develop‐ ment of new products and when it is necessary to replace a constituent of a formulation without loss of the product quality. It could be performed in two different ways: offering two different samples to the volunteer asking him about what sample he prefers between them or using a hedonic scale for the volunteer attributes grades of intensity of its accepta‐

The hedonic scale either can be presented to the panel of evaluators in different manners, as

The affective methods provide quantitative data and allow more than one attribute in each

The discriminative test is better represented by the Triangular test. It allows differentiating one between three different samples and is very useful in shelf life studies and in the quality control of cosmetics. The ideal is to perform this evaluation with twelve to forty volunteers, who will receive the three samples and should indicate the different one between them

The descriptive tests provide a broad sensory description about the product that is being evaluated (Almeida et al., 2008), helping to predict the consumer acceptance and what con‐

sumers think about such product (Almeida et al., 2006; Aust et al., 1987).

fective of the product.

342 Latest Research into Quality Control

skin after application" (Parente et al., 2010).

tion in relation to the sample.

sample being evaluated at the same time.

shown in Figure 3.

(Zenebon et al., 2008).

collection.


**Figure 3.** Examples of presentation of hedonic scale (Olshan et al., 2000; Barkat et al., 2003).

The tests to evaluate the effective of the products should be performed in true conditions of use and the volunteer may use only the product that is being assessed. These tests could be conduct by the evaluation of dermatologists, by the evaluation of volunteers, and even, by the measurement of one parameter by un equipment, such as the equipment that measures hydration, sebum and transepidermal water loss, to define if a product is really effective. Based on these clinical evaluations, a company could create an efficacy claim to the product (Wortel and Wiechers, 2000).

The sensory analysis could be also applied when a cosmetic industry needs to replace a rawmaterial of a commercialized product without changes in the performance of it. This re‐ placement could be originated by many factors, such as the reduction of costs, problems with the firm who provides this raw-material problems with same raw material which caus‐ es irritation, comedogenicity or other problems that affect the consumer. In this field, the sensorial analysis helps the formulator, who proposes different raw materials as substitute, to evaluate if the consumer will notice the adaptation in the cosmetic product.

The statistical analysis is indispensable in the sensory studies. The sensory analysis data should be evaluated transforming them in scores which allows the application of statistical analysis to calculate the mean and standard deviation of the results, and the determination if the difference between the scores obtained is statistically significant. Graphics, tables and preference maps could be elaborated with the results obtained to facilitate the analysis of the data by the professional team.

The sensory analysis is especially indispensable in the industries of fragrances and per‐ fumes, and because of that, high-resolution instrumental methods for evaluation of flavor and aroma have been developed and between them are the breath analysis via mass spec‐ trometry (Dijksterhuis and Piggott, 2001; Ross, 2009). Instrumental measurements are thought to be objective, representing an independent fact or truth, however, the human smell sense is irreplaceable, being considered by Ross (2009) not necessarily valid because instrumental methods cannot account for the complexity of human perception.

Nevertheless, rheological studies have been applied to objectify the sensations when cosmet‐ ic emulsions are applied to the skin (Brummer and Godersky, 1999).

#### **3. Rheology**

Rheology is a tool widely applied in the food, petrochemical and pharmaceutical industries, but to the cosmetic industry it is incipient yet. Until now, the majority of cosmetic industries use viscometers to guarantee that the viscosity of different batches of a product is main‐ tained.

This chapter was elaborated in order to show that many other rheological characteristics could be used to evaluate and to predict the stability of cosmetic products and could be ap‐ plied to compare competing products in the market and to assay if a change in the composi‐ tion will cause alterations that could be perceived by the consumer.

First, it is necessary to define the three parameters of most importance in rheology: shear stress, shear rate and viscosity. Shear stress can be defined as a force applied in an area. Shear rate is the ratio of the velocity of material to its distance from a stationary object (Naé, 1993). The shear rate can be calculated by the ratio between the velocity and the layer or film thickness. In a lipstick application, for example, with a velocity estimated in 5 cm/s and a layer thickness of 0.1 mm, the ratio (shear rate) is 5.102 s-1. Finally, the viscosity can be de‐ fined as the resistance to flow. Thus, a viscous product presents smaller flow than others.

Concluding, rheology is the study of deformation and flow of materials under external forces. Some equations and the units of these parameters are (Naé, 1993):

$$
\sigma = \mathbf{F}/\mathbf{A} \tag{1}
$$

Where:

= viscosity

= shear stress (Pa) *γ*˙ = shear rate (s-1)

Since the unit of shear stress is Pa and the unit of deformation is s-1, the unit of viscosity is

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 345

Using controlled shear rate and measuring shear stress is possible to carry out rotational as‐ says, and determine flow curves and describe the models: Newtonian or non-Newtonian and, among the last one, plastic, pseudoplastic, dilatant, tixotropic and reopetic fluids. New‐ tonian fluids are materials that present constant viscosity, independent of time and tempera‐ ture. These materials present flow curves with proportionality between shear stress and

Shear rate (s-1)

In the case of non-Newtonians materials, this proportionality between shear stress and shear

If in the beginning of the flow curve there is an increasing in the shear stress but the shear rate is equal to zero, and after to it is verified a Newtonian flow, this material is called plas‐ tic. This initial shear stress with shear rate equal to zero is called yield value and it repre‐ sents the shear stress necessary for the material flow. The Figure 5 represents a plastic material. The yield value is related to the energy required to deform the material sufficiently so that they can flow. The value of the yield stress can be determined by measuring the de‐ formation of the material as a function of the applied stress (Abdel-Rahem et al., 2005).

For non-Newtonian materials time-dependents, if the viscosity decreases with the shear rate, the material is called pseudoplastic and if the viscosity increases, the material is called dilatant. On the other hand, if the material is time-independent, it will be called tixotropic if the viscosity decreases with the shear rate or reopetic if the viscosity increases with the shear

Pa.s. These parameters are involved in scientific measurements of rotational assays.

shear rate. The Figure 4 represents the flow curve of a Newtonian material.

45º

Shear stress (Pa)

**Figure 4.** Flow curve of a Newtonian material.

rate does not happen.

Where:

σ = shear stress (Pa = kg.m-1.s-2)

$$\mathbf{F} = \text{force (N or kg.m.s}^2\text{)}$$

$$\mathbf{A} = \text{area (m}^2\text{)}$$

The viscosity can be defined as the ratio between shear stress and shear rate:

$$
\eta = \sigma/\dot{\gamma} \tag{2}
$$

Where:

and aroma have been developed and between them are the breath analysis via mass spec‐ trometry (Dijksterhuis and Piggott, 2001; Ross, 2009). Instrumental measurements are thought to be objective, representing an independent fact or truth, however, the human smell sense is irreplaceable, being considered by Ross (2009) not necessarily valid because

Nevertheless, rheological studies have been applied to objectify the sensations when cosmet‐

Rheology is a tool widely applied in the food, petrochemical and pharmaceutical industries, but to the cosmetic industry it is incipient yet. Until now, the majority of cosmetic industries use viscometers to guarantee that the viscosity of different batches of a product is main‐

This chapter was elaborated in order to show that many other rheological characteristics could be used to evaluate and to predict the stability of cosmetic products and could be ap‐ plied to compare competing products in the market and to assay if a change in the composi‐

First, it is necessary to define the three parameters of most importance in rheology: shear stress, shear rate and viscosity. Shear stress can be defined as a force applied in an area. Shear rate is the ratio of the velocity of material to its distance from a stationary object (Naé, 1993). The shear rate can be calculated by the ratio between the velocity and the layer or film thickness. In a lipstick application, for example, with a velocity estimated in 5 cm/s and a layer thickness of 0.1 mm, the ratio (shear rate) is 5.102 s-1. Finally, the viscosity can be de‐ fined as the resistance to flow. Thus, a viscous product presents smaller flow than others.

Concluding, rheology is the study of deformation and flow of materials under external

σ =F / A (1)

η= σ / γ˙ (2)

instrumental methods cannot account for the complexity of human perception.

ic emulsions are applied to the skin (Brummer and Godersky, 1999).

tion will cause alterations that could be perceived by the consumer.

forces. Some equations and the units of these parameters are (Naé, 1993):

The viscosity can be defined as the ratio between shear stress and shear rate:

**3. Rheology**

344 Latest Research into Quality Control

tained.

Where:

A = area (m2

σ = shear stress (Pa = kg.m-1.s-2)

F = force (N or kg.m.s-2)

)

= viscosity

= shear stress (Pa)

*γ*˙ = shear rate (s-1)

Since the unit of shear stress is Pa and the unit of deformation is s-1, the unit of viscosity is Pa.s. These parameters are involved in scientific measurements of rotational assays.

Using controlled shear rate and measuring shear stress is possible to carry out rotational as‐ says, and determine flow curves and describe the models: Newtonian or non-Newtonian and, among the last one, plastic, pseudoplastic, dilatant, tixotropic and reopetic fluids. New‐ tonian fluids are materials that present constant viscosity, independent of time and tempera‐ ture. These materials present flow curves with proportionality between shear stress and shear rate. The Figure 4 represents the flow curve of a Newtonian material.

In the case of non-Newtonians materials, this proportionality between shear stress and shear rate does not happen.

If in the beginning of the flow curve there is an increasing in the shear stress but the shear rate is equal to zero, and after to it is verified a Newtonian flow, this material is called plas‐ tic. This initial shear stress with shear rate equal to zero is called yield value and it repre‐ sents the shear stress necessary for the material flow. The Figure 5 represents a plastic material. The yield value is related to the energy required to deform the material sufficiently so that they can flow. The value of the yield stress can be determined by measuring the de‐ formation of the material as a function of the applied stress (Abdel-Rahem et al., 2005).

For non-Newtonian materials time-dependents, if the viscosity decreases with the shear rate, the material is called pseudoplastic and if the viscosity increases, the material is called dilatant. On the other hand, if the material is time-independent, it will be called tixotropic if the viscosity decreases with the shear rate or reopetic if the viscosity increases with the shear rate (Naé, 1993). When the ascending and the descending curves of the flow curve do not overlap it shows thixotropy which is a desirable feature for cosmetics and semisolid drug carriers for topical application (Lippacher et al., 2004). The Figures 6, 7, 8 and 9 represent the flow curves of non-Newtonian materials (Naé, 1993).

Shear stress (Pa)

Shear stress (Pa)

Shear stress (Pa)

viscosity.

Shear stress (Pa)

12 11 plotted as a plotted as viscosity as a function

13 8 sep- se – (and in the next line: paration)

13 10 to instability to an instability because of low viscosity of

13 9 due the emulsifier due to the emulsifier

stress applied on the emulsioned system

14 5 has a viscous has viscous

14 11 These author related These authors verified

15 12 in the viscoelastic in the linear viscoelastic

15 23 Madsen *et al.,* 1998) Madsen *et al.,* 1998).

15 30 stress and the identify stress and to identify

14 1 This low viscosity can occur due to a high shear

(Samavati, *et al*., 2011).

12 12 plotting them versus plotting it versus

12 23 bane, 2008 bane and Bekkour, 2008

**Figure 7.** Flow curve of a dilatant material.

**Figure 8.** Flow curve of a thixotropic material.

**Figure 9.** Flow curve of a reopetic material.

11 Figure 9. Flow curve of a reopetic material.

13 Figure 11

Shear rate (s-1)

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 347

Shear rate (s-1)

Shear rate (s-1)

the condition to the measurement of apparent

Figure 11. Flow curve with hysteresis area.

Shear rate (s


dispersed phase (Corrêa & Isaac,

shear stress (Samavati, et al., 2011).

This low viscosity can occur because of high

3

**Figure 5.** Flow curve of a plastic material.

**Figure 6.** Flow curve of a pseudoplastic material.

For screening purposes and in the initial phases of the formulation development, the rheo‐ logical tests proved to be very useful for the study of stability.

In a stability assay to determine the shelf life of a recently developed product, the formula‐ tion should be exposed to stress conditions, such as storage at -5 ºC, 45 ºC, and cycles of -5 ºC during 24 hours followed by exposure to 45 ºC during more 24 hours. This procedure is done in order to induce the appearance of instability signals in the formulations, where can be cited the darkening of the formulation, the precipitation of a constituent, the phase sepa‐ ration in the case of emulsions, and other signals. These stressing conditions are kept for a period around 2 or 3 months. 12 Figure 10. Flow curve example. Figure 10. Example of a flow curve indicating

**Figure 7.** Flow curve of a dilatant material.

rate (Naé, 1993). When the ascending and the descending curves of the flow curve do not overlap it shows thixotropy which is a desirable feature for cosmetics and semisolid drug carriers for topical application (Lippacher et al., 2004). The Figures 6, 7, 8 and 9 represent the

> Shear rate (s-1 )

Shear rate (s-1 )

For screening purposes and in the initial phases of the formulation development, the rheo‐

In a stability assay to determine the shelf life of a recently developed product, the formula‐ tion should be exposed to stress conditions, such as storage at -5 ºC, 45 ºC, and cycles of -5 ºC during 24 hours followed by exposure to 45 ºC during more 24 hours. This procedure is done in order to induce the appearance of instability signals in the formulations, where can be cited the darkening of the formulation, the precipitation of a constituent, the phase sepa‐ ration in the case of emulsions, and other signals. These stressing conditions are kept for a

flow curves of non-Newtonian materials (Naé, 1993).

Shear stress (Pa)

Shear stress (Pa)

logical tests proved to be very useful for the study of stability.

**Figure 5.** Flow curve of a plastic material.

346 Latest Research into Quality Control

**Figure 6.** Flow curve of a pseudoplastic material.

period around 2 or 3 months.

45º

**Figure 8.** Flow curve of a thixotropic material.

11 Figure 9. Flow curve of a reopetic material.

13 Figure 11

the condition to the measurement of apparent

Figure 11. Flow curve with hysteresis area.

Shear rate (s


dispersed phase (Corrêa & Isaac,

shear stress (Samavati, et al., 2011).

This low viscosity can occur because of high

3

**Figure 9.** Flow curve of a reopetic material.

viscosity.

Shear stress (Pa)

12 Figure 10. Flow curve example. Figure 10. Example of a flow curve indicating

12 11 plotted as a plotted as viscosity as a function

13 8 sep- se – (and in the next line: paration)

13 10 to instability to an instability because of low viscosity of

13 9 due the emulsifier due to the emulsifier

stress applied on the emulsioned system

14 5 has a viscous has viscous

14 11 These author related These authors verified

15 12 in the viscoelastic in the linear viscoelastic

15 23 Madsen *et al.,* 1998) Madsen *et al.,* 1998).

15 30 stress and the identify stress and to identify

14 1 This low viscosity can occur due to a high shear

(Samavati, *et al*., 2011).

12 12 plotting them versus plotting it versus

12 23 bane, 2008 bane and Bekkour, 2008

It is usually measured the viscosity of the stressed formulations periodically during the sta‐ bility assay. It could be done by means of a viscometer or by using a rheometer. 13 Figure 11

11 Figure 9. Flow curve of a reopetic material.

Shear stress (Pa)

viscosity.

Shear stress (Pa)

**Figure 11.** Flow curve with hysteresis area.

12 Figure 10. Flow curve example. Figure 10. Example of a flow curve indicating

12 11 plotted as a plotted as viscosity as a function

12 12 plotting them versus plotting it versus

12 23 bane, 2008 bane and Bekkour, 2008

Shear rate (s-1)

the condition to the measurement of apparent

Figure 11. Flow curve with hysteresis area.

Using the flow curve is possible to compare two samples in relation to its hysteresis area and viscosity. A simple way to verify what formulation have a higher viscosity is by simple observation of the rheogram, since the curve that forms a bigger inclination in relation to the *x* axis of the graphic is the one with higher viscosity. On Figure 12 is showed an example of it, where sample 2 is more viscous than sample 1. It happens because the tangent of the an‐

Shear rate (s-1)

dispersed phase (Corrêa & Isaac,

gle formed is correspondent to the viscosity of the formulation in each shear rate.

shear stress (Samavati, et al., 2011).

0 20 40 60 80 100

**Figure 12.** Comparison between flow curves of different samples (a thixotropic and a plastic fluid).

Shear rate (s-1)

Beyond the different sensorial features caused by the differences in viscosity is known that the viscosity of emulsioned systems is one of the factors that retards or avoids the phase sep‐ aration processes. The coalescence of dispersed phase can be due to the emulsifier agent and can be related to an instability because of low viscosity of dispersed phase (Corrêa & Isaac,

2012). This low viscosity can occur because of high shear stress (Samavati et al., 2011).

Shear stress (Pa)

This low viscosity can occur because of high

 sample 1 sample 2

With a viscometer, it is possible to carry out rotational assays or measurements by steadystate flow. On the other hand, the rheometer allows the development of oscillatory assays or dynamic measurements (Biradar, 2009).

When using an oscillatory rheometer it is necessary to carry out a flow curve assay and de‐ termine the apparent viscosity of the formulation in a defined shear rate. It is recommended to use the higher shear rate in the ascendant curve of the flow curve, since in this point the sample is in a suitable condition, it means that the formulation is not starting to flow and is not excessively sheared (Figure 10).

**Figure 10.** Example of a flow curve indicating the condition to the measurement of apparent viscosity. 14 1 This low viscosity can occur due to a high shear

In some papers, the flow curves have been plotted as viscosity as a function of shear stress instead of the traditional approach of plotting it versus shear rate because has been previ‐ ously found that such curves are more discriminating and give better results for evaluation (Roberts, 2001; Samavati, 2011). stress applied on the emulsioned system (Samavati, *et al*., 2011). 14 5 has a viscous has viscous

After obtaining, periodically, the minimum apparent viscosity of the samples exposed to stress conditions during a period, they should be compared with the initial value, and also compared the viscosity values of the control with the samples exposed to stress conditions, which allows the verification of the increase, decrease or maintenance of this attribute of the formulations. 14 11 These author related These authors verified 15 12 in the viscoelastic in the linear viscoelastic

Further exploiting the same assay, it is possible to calculate the hysteresis area of the formu‐ lation in each flow curve performed during the stability assay. The hysteresis loop areas can be obtained through a three-step experiment: upward curve, plateau, downward (Bencha‐ bane and Bekkour, 2008) and represents a way to measure, indirectly, the spreadability of the formulation, so it is possible to define if the formulation losses or gains easiness on spreadability during the shelf life. How much bigger is the hysteresis area, higher is the spreadability. 15 23 Madsen *et al.,* 1998) Madsen *et al.,* 1998). 15 30 stress and the identify stress and to identify

3

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 349

3

Shear rate (s-1)

the condition to the measurement of apparent

Figure 11. Flow curve with hysteresis area. **Figure 11.** Flow curve with hysteresis area.

Shear stress (Pa)

viscosity.

12 Figure 10. Flow curve example. Figure 10. Example of a flow curve indicating

12 11 plotted as a plotted as viscosity as a function

12 12 plotting them versus plotting it versus

14 1 This low viscosity can occur due to a high shear

(Samavati, *et al*., 2011).

stress applied on the emulsioned system

12 23 bane, 2008 bane and Bekkour, 2008

It is usually measured the viscosity of the stressed formulations periodically during the sta‐

With a viscometer, it is possible to carry out rotational assays or measurements by steadystate flow. On the other hand, the rheometer allows the development of oscillatory assays or

When using an oscillatory rheometer it is necessary to carry out a flow curve assay and de‐ termine the apparent viscosity of the formulation in a defined shear rate. It is recommended to use the higher shear rate in the ascendant curve of the flow curve, since in this point the sample is in a suitable condition, it means that the formulation is not starting to flow and is

maximum shear rate

13 Figure 11

11 Figure 9. Flow curve of a reopetic material.

bility assay. It could be done by means of a viscometer or by using a rheometer.

descendant curve

**Figure 10.** Example of a flow curve indicating the condition to the measurement of apparent viscosity.

ascendant curve

Shear rate (s-1 )

In some papers, the flow curves have been plotted as viscosity as a function of shear stress instead of the traditional approach of plotting it versus shear rate because has been previ‐ ously found that such curves are more discriminating and give better results for evaluation

After obtaining, periodically, the minimum apparent viscosity of the samples exposed to stress conditions during a period, they should be compared with the initial value, and also compared the viscosity values of the control with the samples exposed to stress conditions, which allows the verification of the increase, decrease or maintenance of this attribute of the

Further exploiting the same assay, it is possible to calculate the hysteresis area of the formu‐ lation in each flow curve performed during the stability assay. The hysteresis loop areas can be obtained through a three-step experiment: upward curve, plateau, downward (Bencha‐ bane and Bekkour, 2008) and represents a way to measure, indirectly, the spreadability of the formulation, so it is possible to define if the formulation losses or gains easiness on spreadability during the shelf life. How much bigger is the hysteresis area, higher is the

dynamic measurements (Biradar, 2009).

348 Latest Research into Quality Control

not excessively sheared (Figure 10).

(Roberts, 2001; Samavati, 2011).

formulations.

spreadability.

Shear stress (Pa)

13 8 sep- se – (and in the next line: paration) 13 9 due the emulsifier due to the emulsifier 13 10 to instability to an instability because of low viscosity of Using the flow curve is possible to compare two samples in relation to its hysteresis area and viscosity. A simple way to verify what formulation have a higher viscosity is by simple observation of the rheogram, since the curve that forms a bigger inclination in relation to the *x* axis of the graphic is the one with higher viscosity. On Figure 12 is showed an example of it, where sample 2 is more viscous than sample 1. It happens because the tangent of the an‐ gle formed is correspondent to the viscosity of the formulation in each shear rate.

dispersed phase (Corrêa & Isaac,

15 23 Madsen *et al.,* 1998) Madsen *et al.,* 1998). **Figure 12.** Comparison between flow curves of different samples (a thixotropic and a plastic fluid).

15 30 stress and the identify stress and to identify Beyond the different sensorial features caused by the differences in viscosity is known that the viscosity of emulsioned systems is one of the factors that retards or avoids the phase sep‐ aration processes. The coalescence of dispersed phase can be due to the emulsifier agent and can be related to an instability because of low viscosity of dispersed phase (Corrêa & Isaac, 2012). This low viscosity can occur because of high shear stress (Samavati et al., 2011).

In general, for emulsioned systems, the continuos phase is shear thinning, which means that its viscosity decreases with the increasing on shear rate and viscoelastic, which means that it has viscous and elastic components (Tadros, 2004).

viscous modulus is a measure of the energy lost per cycle and represents the liquid-like

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 351

In the stress sweep analyses, the structure of the sample is progressively destroyed by ap‐ plying oscillations with an increasing stress amplitude at a fixed frequency (Callens et al., 2003). The linear viscoelasticity region occurs over that region of strain where the complex modulus is independent of the strain (Hemar, 2000). The linear viscoelastic region is deter‐ mined by the maximum stress which can be applied without affecting G' and G''. Further‐ more, the relative magnitude of the moduli is a qualitative indication for the structure in the sample. Two different situations can occur: G' > G'' for a network consisting of secondary

Frequency sweep tests are performed in the linear viscoelastic region of each sample, keeping the structure of the system intact during the measurement. By performing such small stress amplitude oscillations at a whole range of frequencies, the type of network structure can be revealed. The main difference between a network of secondary bonds and one of physical entanglements is located in the low frequency range: in an entan‐ gled network the polymers can disentangle if the available time is long enough (low fre‐ quency). In a network with secondary bonds the bonds are fixed irrespective of the time scale. This results for an entangled solution in a limiting slope of 2 for G' and 1 for G'' at low frequency in a log-log plot of moduli versus frequency, while at intermediate fre‐ quency a plateau develops. For a network of secondary bonds an almost constant value of G' and G'' is observed over the whole frequency range, with the value of G' exceeding

The stress sweep is important to evaluate the linear viscoelastic region of a sample that is a range of shear stress in which the formulation does not suffer profound alterations on it structure, being not disrupted. When a shear stress of the linear viscoelastic region is ap‐ plied in an oscillatory assay, only the intermolecular and interparticle forces are being evalu‐ ated (Martin, 1993). To determine the linear viscoelastic region, the oscillating stress sweeps are carried out for the most extreme values. These measurements are used to determine where the reological properties are independent of the applied stress and the identify the

Knowing the values of shear stress that do not cause the disrupt in the formulation by means of the stress sweep, the analyst could perform a frequency sweep of the formulation. The frequency sweep is carried out in a constant shear stress found in the linear viscoelastic region. With this assay it is possible to evaluate the elastic or storage modulus (G') and the viscous or loss modulus (G''). The cosmetic excipients most used, emulsions and gels, are often viscoelastic samples. The viscoelastic samples when evaluated by means of the fre‐ quency sweep present G' and G'' values. When the G' value is higher than G'' it is an indica‐

Emulsions which exhibits G' values higher than G'' (Figure 14) are described as more stable than formulations with G'' values higher than G' (Figure 15), since they tends to recovery its initial structure faster and more efficiently than the others, and are less susceptible to the

tive that the formulation is more elastic than viscous. It is a characteristic of gels.

bonds and G'≤ G'' for a physically entangled polymer solution (Callens et al., 2003).

component. If a sample is viscous the viscous modulus will be high.

that of G''(Callens et al., 2003; Madsen et al., 1998).

critical rheological properties (Tuarez, 2011).

An example of the verification of differences in viscosity and thixotropy between two sam‐ ples is shown on Figure 13.

**Figure 13.** Comparison between flow curves of different samples.

Sample 1 is less viscous but more thixotropic than formulation 2. This simple verification gives to the analyst wide information, depending on what he needs.

Lescanne et al. (2004) studied organogels and aging properties of them. Organogels can be obtained by precipitation processes. These authors verified that, when aggregates are formed by the cooling rate, can be observed a elastic behavior, however, these aggregates can be aligned in the direction of the flow without lost the structure and when the flow is stopped, the aggregates are quickly rearranged and it inducing an thixotropic behavior. When the hot solution is introduced between the flat and the conical plates of the rheometer cell it is cooled to 5 ºC with a cooling rate of 20ºC/min, during the first hour of the gel life, it was measured the elastic properties of a gel as a function of time just after the cooling. Five minutes after its formation, the gel was submitted to a periodic stress (0.5 Pa) at a constant frequency (*f* = 1Hz). The authors showed that the shear moduli are constants and the aging phenomenon did not modify the elastic properties at least in a period of 1 h. However, when more than a week of aging is waited the samples lost most of its elastic properties.

The flow curve is a rotational assay, but using a rheometer it is possible to perform os‐ cillatory assays too. Among the oscillatory assays are stress sweep and the frequency sweep assays.

The elastic (storage) modulus G' and the viscous (loss) modulus G'' are determined as a function of frequency or stress. The elastic modulus is a measure of energy stored and re‐ covered per cycle of deformation and represents the solid-like component of a viscoelastic material. If a sample is elastic or highly structured then the elastic modulus will be high. The viscous modulus is a measure of the energy lost per cycle and represents the liquid-like component. If a sample is viscous the viscous modulus will be high.

In general, for emulsioned systems, the continuos phase is shear thinning, which means that its viscosity decreases with the increasing on shear rate and viscoelastic, which means that it

An example of the verification of differences in viscosity and thixotropy between two sam‐

 sample 1 sample 2

0 20 40 60 80 100

Shear rate (s-1 )

Sample 1 is less viscous but more thixotropic than formulation 2. This simple verification

Lescanne et al. (2004) studied organogels and aging properties of them. Organogels can be obtained by precipitation processes. These authors verified that, when aggregates are formed by the cooling rate, can be observed a elastic behavior, however, these aggregates can be aligned in the direction of the flow without lost the structure and when the flow is stopped, the aggregates are quickly rearranged and it inducing an thixotropic behavior. When the hot solution is introduced between the flat and the conical plates of the rheometer cell it is cooled to 5 ºC with a cooling rate of 20ºC/min, during the first hour of the gel life, it was measured the elastic properties of a gel as a function of time just after the cooling. Five minutes after its formation, the gel was submitted to a periodic stress (0.5 Pa) at a constant frequency (*f* = 1Hz). The authors showed that the shear moduli are constants and the aging phenomenon did not modify the elastic properties at least in a period of 1 h. However, when

more than a week of aging is waited the samples lost most of its elastic properties.

The flow curve is a rotational assay, but using a rheometer it is possible to perform os‐ cillatory assays too. Among the oscillatory assays are stress sweep and the frequency

The elastic (storage) modulus G' and the viscous (loss) modulus G'' are determined as a function of frequency or stress. The elastic modulus is a measure of energy stored and re‐ covered per cycle of deformation and represents the solid-like component of a viscoelastic material. If a sample is elastic or highly structured then the elastic modulus will be high. The

has viscous and elastic components (Tadros, 2004).

gives to the analyst wide information, depending on what he needs.

Shear stress (Pa)

**Figure 13.** Comparison between flow curves of different samples.

ples is shown on Figure 13.

350 Latest Research into Quality Control

sweep assays.

In the stress sweep analyses, the structure of the sample is progressively destroyed by ap‐ plying oscillations with an increasing stress amplitude at a fixed frequency (Callens et al., 2003). The linear viscoelasticity region occurs over that region of strain where the complex modulus is independent of the strain (Hemar, 2000). The linear viscoelastic region is deter‐ mined by the maximum stress which can be applied without affecting G' and G''. Further‐ more, the relative magnitude of the moduli is a qualitative indication for the structure in the sample. Two different situations can occur: G' > G'' for a network consisting of secondary bonds and G'≤ G'' for a physically entangled polymer solution (Callens et al., 2003).

Frequency sweep tests are performed in the linear viscoelastic region of each sample, keeping the structure of the system intact during the measurement. By performing such small stress amplitude oscillations at a whole range of frequencies, the type of network structure can be revealed. The main difference between a network of secondary bonds and one of physical entanglements is located in the low frequency range: in an entan‐ gled network the polymers can disentangle if the available time is long enough (low fre‐ quency). In a network with secondary bonds the bonds are fixed irrespective of the time scale. This results for an entangled solution in a limiting slope of 2 for G' and 1 for G'' at low frequency in a log-log plot of moduli versus frequency, while at intermediate fre‐ quency a plateau develops. For a network of secondary bonds an almost constant value of G' and G'' is observed over the whole frequency range, with the value of G' exceeding that of G''(Callens et al., 2003; Madsen et al., 1998).

The stress sweep is important to evaluate the linear viscoelastic region of a sample that is a range of shear stress in which the formulation does not suffer profound alterations on it structure, being not disrupted. When a shear stress of the linear viscoelastic region is ap‐ plied in an oscillatory assay, only the intermolecular and interparticle forces are being evalu‐ ated (Martin, 1993). To determine the linear viscoelastic region, the oscillating stress sweeps are carried out for the most extreme values. These measurements are used to determine where the reological properties are independent of the applied stress and the identify the critical rheological properties (Tuarez, 2011).

Knowing the values of shear stress that do not cause the disrupt in the formulation by means of the stress sweep, the analyst could perform a frequency sweep of the formulation. The frequency sweep is carried out in a constant shear stress found in the linear viscoelastic region. With this assay it is possible to evaluate the elastic or storage modulus (G') and the viscous or loss modulus (G''). The cosmetic excipients most used, emulsions and gels, are often viscoelastic samples. The viscoelastic samples when evaluated by means of the fre‐ quency sweep present G' and G'' values. When the G' value is higher than G'' it is an indica‐ tive that the formulation is more elastic than viscous. It is a characteristic of gels.

Emulsions which exhibits G' values higher than G'' (Figure 14) are described as more stable than formulations with G'' values higher than G' (Figure 15), since they tends to recovery its initial structure faster and more efficiently than the others, and are less susceptible to the gravitational forces which retards or avoids the coalescence process and the phase separa‐ tion of emulsions (Alam and Aramaki, 2009). So, the G' values higher than G'' in emulsions is a desirable feature, being an indicative of stability of the cosmetic system.

Analyzing the result obtained in the first 300 seconds is verified that sample 1 exhibited low‐ er compliance values than sample 2, which represents a higher difficult on being deformed than sample 1. The difficult on being deformed is always linked to higher viscosity values. In the second part of the assay, where the shear stress imposed to the sample is removed, represented in the graphic by the time 301 to 600 seconds, is verified the viscoelastic proper‐ ties of the samples. Formulations that are able to recovery its initial structure or part of it exhibit a gradually decrease in the compliance values. On Figure 17 there is an example of a formulation that is not a viscoelastic sample, it means that it do not exhibits storage modu‐

0 100 200 300 400 500 600

Time (s)

0 100 200 300 400 500 600 700

Time (s)

In addition, the rheology can be used to evaluate the stability over the time by dynamic and oscillatory rheological measurements (Pénzes et al., 2004; Vasiljevic et al., 2006) and the re‐ lease of active principles. According to Martinez et al. (2007), the transdermal absorption of

 sample 1 sample 2

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 353

lus, and is not able to recovery its structure when the shear stress is ceased.

0,000 0,001 0,002 0,003 0,004 0,005 0,006 0,007 0,008 0,009 0,010 0,011 0,012

0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18

**Figure 17.** A creep and recovery example of a non-viscoelastic sample.

J (1/Pa)

**Figure 16.** A creep and recovery example of viscoelastic samples.

J (1/Pa)

**Figure 14.** A frequency sweep example (G'>G'').

**Figure 15.** A frequency sweep example (G''>G').

Another assay that could be conducted using an oscillatory rheometer is the creep and re‐ covery assay. It is done by submitting the samples to a constant shear stress during a period, and after, removing this shear stress and monitoring the formulation in relation to the defor‐ mation (measured by the compliance - J) during the same period. The compliance parameter is the resulting strain divided by the applied stress (Koop, 2009; Toro-Vazquez et al., 2010). If the compliance parameter is the relationship between strain and the applied stress, the strain is dimensionless and stress is measured in Pa, then, the compliance can be measured in 1/Pa.

In the example showed on the Figure 16 the samples were submitted to a shear stress during 300 seconds, and after removing this shear stress it was monitored during more 300 seconds.

Analyzing the result obtained in the first 300 seconds is verified that sample 1 exhibited low‐ er compliance values than sample 2, which represents a higher difficult on being deformed than sample 1. The difficult on being deformed is always linked to higher viscosity values.

In the second part of the assay, where the shear stress imposed to the sample is removed, represented in the graphic by the time 301 to 600 seconds, is verified the viscoelastic proper‐ ties of the samples. Formulations that are able to recovery its initial structure or part of it exhibit a gradually decrease in the compliance values. On Figure 17 there is an example of a formulation that is not a viscoelastic sample, it means that it do not exhibits storage modu‐ lus, and is not able to recovery its structure when the shear stress is ceased.

**Figure 16.** A creep and recovery example of viscoelastic samples.

gravitational forces which retards or avoids the coalescence process and the phase separa‐ tion of emulsions (Alam and Aramaki, 2009). So, the G' values higher than G'' in emulsions

0 20 40 60 80 100

0 20 40 60 80 100

Another assay that could be conducted using an oscillatory rheometer is the creep and re‐ covery assay. It is done by submitting the samples to a constant shear stress during a period, and after, removing this shear stress and monitoring the formulation in relation to the defor‐ mation (measured by the compliance - J) during the same period. The compliance parameter is the resulting strain divided by the applied stress (Koop, 2009; Toro-Vazquez et al., 2010). If the compliance parameter is the relationship between strain and the applied stress, the strain is dimensionless and stress is measured in Pa, then, the compliance can be measured

In the example showed on the Figure 16 the samples were submitted to a shear stress during 300 seconds, and after removing this shear stress it was monitored during more 300 seconds.

Frequency (Hz)

Frequency (Hz)

G' G''

> G' G''

is a desirable feature, being an indicative of stability of the cosmetic system.

1

1

10

100

G' (Pa), G'' (Pa)

10

100

G' (Pa), G'' (Pa)

**Figure 14.** A frequency sweep example (G'>G'').

352 Latest Research into Quality Control

**Figure 15.** A frequency sweep example (G''>G').

in 1/Pa.

1000

**Figure 17.** A creep and recovery example of a non-viscoelastic sample.

In addition, the rheology can be used to evaluate the stability over the time by dynamic and oscillatory rheological measurements (Pénzes et al., 2004; Vasiljevic et al., 2006) and the re‐ lease of active principles. According to Martinez et al. (2007), the transdermal absorption of topically administered drugs depends on the rate of release and the permeability of them into the skin and also of the viscosity of the formulation (Martinez et al., 2007).

rheological analysis, since were verified that the thicker the interlamellar water layers, the higher the viscosity of the cream (Eccleston et al., 2000). Thus, liquid crystals could be re‐ sponsible by the emulsion stabilization and by the increasing in the viscosity (Klein, 2002), being the presence of this structures desirable in cosmetic emulsions which could be an indi‐

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 355

**Figure 19.** Schemes of the microscopic visualization of lamellar gel networks surrounding emulsion droplets proposed

This kind of structure is more commonly found in cosmetics due to the high diversity of components used in it in order to obtain a moisturizer, emollient, humectant, good sensory and, above all, stable cosmetic. In other pharmaceutical forms, usually are used a less diver‐ sified composition, which gives a system easier to understand, described as emulsion or liq‐ uid crystal, or even, a gel, a suspension, etc. The quantity of these lamellar structures, found in cosmetic emulsions, probably is dependent of three main factors: the raw-materials, the amount of it used and the process of preparation, where should be cited, the temperature

cative of quality of them.

by Klein (2002).

and the speed of agitation.

**Figure 18.** Scheme of a cosmetic emulsion containing liquid crystals.

Thus, it is possible to say that different categories of products should present peculiar rheo‐ logical properties inherent to its application (Gregolin et al., 2010).

In this way, the rheology can influence the diffusion coefficient, altering the release and per‐ meation of cosmetics active substances (Welin-Berger et al., 2001; A-sadutjarit et al., 2005; Vasiljevic et al., 2006). Some authors have related the influence of rheological characteristics on the release profiles and consequently in the permeation of active substances in the skin; thus, the addition of thickening agents or attainment of a weak-gel because of physical en‐ tanglement of polymer chains must be considered in the choice of cosmetics bases (Spiclin, et al., 2003). Thus, rheology can help in the assay of release and permeation in the skin. Some studies have been published about it.

So, in a short way, the rheology is a valuable tool that helps in the quality control of cosmet‐ ics, being used in the stability tests, in the comparison between competing samples, in the comparison between an original product and a product with an alteration in a constituent, and in the development of new products, aiming to develop cosmetic with rheological char‐ acteristics which indicate stability.

#### **4. Small Angle X-ray Scattering (SAXS)**

The use of this technique in determining the quality control of a cosmetic is closely related to the stability of the product, which could be improved with the presence of liquid crystals.

Liquid crystals are described as a state of matter between solids and liquids, it means that, they are fluid like liquids but are organized like solids, being called mesophases (Marsh, 1973; Kelker and Hatz, 1980; Müller-Goymann, 2004). These organization contributes to the highly stability of systems.

The formation of liquid crystals in emulsions could be induced by some components present in this system, such as surfactants (Müller-Goymann, 2004). So, what happens is that it is possible to find a peculiar system that is not a simple emulsion and not a genuine liquid crystal, but an emulsioned system that contains liquid crystals, commonly lamellar struc‐ tures, that are formed around of the inner phase of the emulsion (Oka et al., 2008), making difficult the coalescence, flocculation and the separation of the oily and water phases, what makes the system formed more stable than a simple emulsion (Figures 18 and 19). Floccula‐ tion is defined as the formation of aggregates of droplets of an emulsion under the influence of interparticle colloidal forces which are net attractive (Dickinson, 1992) and the formation of lamellar structures avoid or prevent the occurrence of this phenomenon. The formation of lamellar structures is essential to obtain emulsified oil/water systems finely dispersed, with balanced hydrophilic-lipophilic properties, resulting in minimal interfacial tension between aqueous and oily phases, thus contributing to the stability of the system (Engels et al., 1995). Previous studies have also shown that it is possible to make correlation between SAXS and rheological analysis, since were verified that the thicker the interlamellar water layers, the higher the viscosity of the cream (Eccleston et al., 2000). Thus, liquid crystals could be re‐ sponsible by the emulsion stabilization and by the increasing in the viscosity (Klein, 2002), being the presence of this structures desirable in cosmetic emulsions which could be an indi‐ cative of quality of them.

**Figure 18.** Scheme of a cosmetic emulsion containing liquid crystals.

topically administered drugs depends on the rate of release and the permeability of them

Thus, it is possible to say that different categories of products should present peculiar rheo‐

In this way, the rheology can influence the diffusion coefficient, altering the release and per‐ meation of cosmetics active substances (Welin-Berger et al., 2001; A-sadutjarit et al., 2005; Vasiljevic et al., 2006). Some authors have related the influence of rheological characteristics on the release profiles and consequently in the permeation of active substances in the skin; thus, the addition of thickening agents or attainment of a weak-gel because of physical en‐ tanglement of polymer chains must be considered in the choice of cosmetics bases (Spiclin, et al., 2003). Thus, rheology can help in the assay of release and permeation in the skin.

So, in a short way, the rheology is a valuable tool that helps in the quality control of cosmet‐ ics, being used in the stability tests, in the comparison between competing samples, in the comparison between an original product and a product with an alteration in a constituent, and in the development of new products, aiming to develop cosmetic with rheological char‐

The use of this technique in determining the quality control of a cosmetic is closely related to the stability of the product, which could be improved with the presence of liquid crystals.

Liquid crystals are described as a state of matter between solids and liquids, it means that, they are fluid like liquids but are organized like solids, being called mesophases (Marsh, 1973; Kelker and Hatz, 1980; Müller-Goymann, 2004). These organization contributes to the

The formation of liquid crystals in emulsions could be induced by some components present in this system, such as surfactants (Müller-Goymann, 2004). So, what happens is that it is possible to find a peculiar system that is not a simple emulsion and not a genuine liquid crystal, but an emulsioned system that contains liquid crystals, commonly lamellar struc‐ tures, that are formed around of the inner phase of the emulsion (Oka et al., 2008), making difficult the coalescence, flocculation and the separation of the oily and water phases, what makes the system formed more stable than a simple emulsion (Figures 18 and 19). Floccula‐ tion is defined as the formation of aggregates of droplets of an emulsion under the influence of interparticle colloidal forces which are net attractive (Dickinson, 1992) and the formation of lamellar structures avoid or prevent the occurrence of this phenomenon. The formation of lamellar structures is essential to obtain emulsified oil/water systems finely dispersed, with balanced hydrophilic-lipophilic properties, resulting in minimal interfacial tension between aqueous and oily phases, thus contributing to the stability of the system (Engels et al., 1995). Previous studies have also shown that it is possible to make correlation between SAXS and

into the skin and also of the viscosity of the formulation (Martinez et al., 2007).

logical properties inherent to its application (Gregolin et al., 2010).

Some studies have been published about it.

**4. Small Angle X-ray Scattering (SAXS)**

acteristics which indicate stability.

354 Latest Research into Quality Control

highly stability of systems.

**Figure 19.** Schemes of the microscopic visualization of lamellar gel networks surrounding emulsion droplets proposed by Klein (2002).

This kind of structure is more commonly found in cosmetics due to the high diversity of components used in it in order to obtain a moisturizer, emollient, humectant, good sensory and, above all, stable cosmetic. In other pharmaceutical forms, usually are used a less diver‐ sified composition, which gives a system easier to understand, described as emulsion or liq‐ uid crystal, or even, a gel, a suspension, etc. The quantity of these lamellar structures, found in cosmetic emulsions, probably is dependent of three main factors: the raw-materials, the amount of it used and the process of preparation, where should be cited, the temperature and the speed of agitation.

In cosmetics, other kinds of systems could be used, such as genuine liquid crystals aiming to explore its characteristics of controlled delivery systems.

that, the scattering of the rays in small angle should be analyzed (Glatter and Kratky, 1982; Urban, 2004; Koch, 2010). Liquid crystals can be analyzed by SAXS since they are able to dis‐ perse the X-rays focused on it. In the SAXS line is used an X-rays detector and an multichan‐ nel analyzer to capture the intense of the SAXS measures (*I(q)*) in function of the modulus of

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 357

Analyzing the data obtained (Figure 19), the *d* value obtained represents the distance be‐ tween the particles able to scatter the X-rays. It is calculated by the equation: *d = 2*π */ q max*, where *q max*, is the maximum intensity of scattering (Craievich, 2002). The relation between the *d* values obtained indicates the type of arrangement found in the system (Glatter and

0,0 0,5 1,0 1,5 2,0 2,5

In the case of the hypothetical curve showed in Figure 19, *d*1 / *d*<sup>2</sup> would result in 2, which

Beyond the advantages already mentioned, in a research conducted by Moaddel and Friberg (1995), the authors showed that the presence of lamellar liquid crystals in an emulsion avoids the water evaporation rate in this system, thus contributing in another way to the sta‐

According to the advantages obtained with the presence of liquid crystals, these mesophases can be of great importance to the Cosmetic Industry in the development of very stable cos‐ metics and, the SAXS technique, an efficient tool to confirm the presence of these desirable

Camerel et al. (2003) pointed the importance in correlate the microstructure of a colloidal suspension with its rheological behavior to define its better use in industry and in life, be‐

yond that, according to these authors there are few reports correlating these analyses.

0.00.51.01.5 2.02.5*q* max. 1

*q* (nm)-1

*q* max. 2

the scattering vector (*q*) (Glatter and Kratky, 1982; Molina et al., 2006; Koch, 2010).

Kratky, 1982; Craievich, 2002; Alexandridis et al., 1998).

10

describe lamellar structures (Alexandridis et al., 1998).

structures that helps in the maintenance of cosmetics' quality control.

bility and maintenance of the cosmetic quality.

100

Intensity (*q*)

**Figure 21.** Hypothetical SAXS curve.

1000

There are different kinds of liquid crystals and different classifications, but this chapter has not the function of describe them, since it have been done by many authors (Bechtold, 2005; Formariz et al., 2005; Atkins and Jones, 2006), the aim was to demonstrate the importance of these structures in the maintenance of the cosmetics' quality. Nevertheless, according to the literature data (Klein, 2002) and to our experience in this subject, it is possible to say that the lamellar arrangement is the most commonly found in cosmetic emulsions.

An initial analysis of the presence of liquid crystals in a cosmetic emulsion could be done using a polarized light microscope, but it should be confirmed and better analyzed by means of Small Angle X-Ray Scattering. When a microscope slide containing a sample of the system is studied and it presents structures that reflect the incident light, it is an evidence of the presence of liquid crystals (Figure 20). So, they should be submitted to SAXS analysis to confirm this expectation (Savic et al., 2011).

**Figure 20.** Photomicrographs of liquid-crystal present in emulsions evaluated by polarized light microscope.

The SAXS method requires a synchrotron light source that is formed by means of a particle accelerator, and using a monochromatic beam, that is used to irradiate the sample. After that, the scattering of the rays in small angle should be analyzed (Glatter and Kratky, 1982; Urban, 2004; Koch, 2010). Liquid crystals can be analyzed by SAXS since they are able to dis‐ perse the X-rays focused on it. In the SAXS line is used an X-rays detector and an multichan‐ nel analyzer to capture the intense of the SAXS measures (*I(q)*) in function of the modulus of the scattering vector (*q*) (Glatter and Kratky, 1982; Molina et al., 2006; Koch, 2010).

Analyzing the data obtained (Figure 19), the *d* value obtained represents the distance be‐ tween the particles able to scatter the X-rays. It is calculated by the equation: *d = 2*π */ q max*, where *q max*, is the maximum intensity of scattering (Craievich, 2002). The relation between the *d* values obtained indicates the type of arrangement found in the system (Glatter and Kratky, 1982; Craievich, 2002; Alexandridis et al., 1998).

**Figure 21.** Hypothetical SAXS curve.

In cosmetics, other kinds of systems could be used, such as genuine liquid crystals aiming to

There are different kinds of liquid crystals and different classifications, but this chapter has not the function of describe them, since it have been done by many authors (Bechtold, 2005; Formariz et al., 2005; Atkins and Jones, 2006), the aim was to demonstrate the importance of these structures in the maintenance of the cosmetics' quality. Nevertheless, according to the literature data (Klein, 2002) and to our experience in this subject, it is possible to say that the

An initial analysis of the presence of liquid crystals in a cosmetic emulsion could be done using a polarized light microscope, but it should be confirmed and better analyzed by means of Small Angle X-Ray Scattering. When a microscope slide containing a sample of the system is studied and it presents structures that reflect the incident light, it is an evidence of the presence of liquid crystals (Figure 20). So, they should be submitted to SAXS analysis to

**Figure 20.** Photomicrographs of liquid-crystal present in emulsions evaluated by polarized light microscope.

The SAXS method requires a synchrotron light source that is formed by means of a particle accelerator, and using a monochromatic beam, that is used to irradiate the sample. After

lamellar arrangement is the most commonly found in cosmetic emulsions.

explore its characteristics of controlled delivery systems.

356 Latest Research into Quality Control

confirm this expectation (Savic et al., 2011).

In the case of the hypothetical curve showed in Figure 19, *d*1 / *d*<sup>2</sup> would result in 2, which describe lamellar structures (Alexandridis et al., 1998).

Beyond the advantages already mentioned, in a research conducted by Moaddel and Friberg (1995), the authors showed that the presence of lamellar liquid crystals in an emulsion avoids the water evaporation rate in this system, thus contributing in another way to the sta‐ bility and maintenance of the cosmetic quality.

According to the advantages obtained with the presence of liquid crystals, these mesophases can be of great importance to the Cosmetic Industry in the development of very stable cos‐ metics and, the SAXS technique, an efficient tool to confirm the presence of these desirable structures that helps in the maintenance of cosmetics' quality control.

Camerel et al. (2003) pointed the importance in correlate the microstructure of a colloidal suspension with its rheological behavior to define its better use in industry and in life, be‐ yond that, according to these authors there are few reports correlating these analyses.

Our research group has invested in researches to assess the stability of cosmetics (Isaac et al., 2008); evaluating of the influence of the addition of thickening agents in creams using rheo‐ logical measurements (Isaac et al., 2012a); evaluating the thickeners' influence on the rheo‐ logical properties of a cosmetic (Isaac et al., 2012b,c); proposing alternative methods to assay the efficacy and safety of them (Chiari et al., 2012a; Chiari et al., 2012b) and using of the sen‐ sory analysis in the cosmetics development (Isaac et al., 2012a) which, in different points of view of what was demonstrated in this chapter, also influence in the product quality.

[5] Almeida, I.F., Gaio, A.R., & Bahia, M.F. (2008). Hedonic and descriptive skinfeel anal‐ ysis of two oleogels: comparison with other topical formulations. J. Sens. Stud., 23(1),

Cosmetics' Quality Control http://dx.doi.org/10.5772/51846 359

[6] A-Sadutjarit, R., Sirivat, A., & Vayumhasuwan, P. (2005). Viscoelastic properties of carbopol 940 gels and their relationships to piroxicam diffusion coefficients in gel

[7] Atkins, P., & Jones, L. (2006). Princípios de química: questionando a vida moderna e

[8] Aust, L.B., Oddo, P., Wild, J.E., Mills, O.H., & Deupree, J.S. (1987). The descriptive analysis of skin care products by a trained panel of judges. J. Soc. Cosmet. Chem., 38,

[9] Backe, I., Meges, S., Lauze, C., Macleod, P., & Dupuy, P. (1999). Sensory analysis of four medical spa spring waters containing various mineral concentrations. Int. J. Der‐

[10] Barkat, S., Thomas-Danguin, T., Bensafi, M., Rouby, C., & Sicard, G. (2003). Odor and color of cosmetic products: correlations between subject judgement and autonomous nervous system response. International Journal of Cosmetic Science, 25, 273-283. [11] Bechtold, I. H. (2005). Liquid crystals: A complex system of simple application. Rev.

[12] Benchabane, A., & Bekkour, K. (2008). Rheological properties of carboxymethyl cellu‐

[13] Biradar, S.V., Dhumal, R.S., & Paradkar, A. (2009). Rheological investigation of selfemulsification process. Journal of Pharmacy and Pharmaceutical Science, 12(1), 17-31.

[14] Brummer, R., & Godersky, S. (1999). Rheological studies to objectify sensations oc‐ curring when cosmetic emulsions are applied to the skin. Colloids and Surfaces A:

[15] Callens, C., Ceulemans, J., Ludwig, A., Foreman, P., & Remon, J.P. (2003). Rheologi‐ cal study on mucoadhesivity of some nasal powder formulations. European Journal

[16] Camerel, F., Gabriel, J.C.P., Batail, P., Panine, P., & Davidson, P. (2003). Combined SAXS – Rheological studies of liquid-cristalline colloidal dispersions of mineral parti‐

[17] Chiari, B.G., Magnani, C., Salgado, H.R.N., Côrrea, M.A., & Isaac, V.L.B. (2012a). Es‐ tudo da segurança de cosméticos: presente e futuro. Revista Brasileira de Ciências

[18] Chiari, B.G., Martini, P.C., Moraes, J.D.D., Andréo, R., Corrêa, M.A., Cicarelli, R.M.B., & Isaac, V.L.B. (2012b). Use of HepG2 cells to assay the safety of cosmetic active sub‐

stances. International Journal of Research in Cosmetic Science, 2(2), 8-14.

lose (CMC) solutions. Colloid Polymer Science, 286, 1173-1180.

Physicochemical and Engineering Aspects, 152, 89-94.

of Pharmaceutics and Biopharmaceutics, 55, 323-328.

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#### **5. Conclusion**

This chapter aimed to show the facility that some simple or advanced techniques already used, sometimes to other finalities, could offer to the quality control of cosmetic products. The sensory analysis, rheology and SAXS technique have earned attention due to the impor‐ tant contribution that they can offer to the cosmetic area.

### **Author details**

Bruna Galdorfini Chiari, Maria Gabriela José de Almeida, Marcos Antonio Corrêa and Vera Lucia Borges Isaac\*

\*Address all correspondence to: veraisaac@fcfar.unesp.br

Faculdade de Ciências Farmacêuticas, UNESP - Univ Estadual Paulista, Departamento de Fármacos e Medicamentos, Laboratório de Cosmetologia – LaCos, Araraquara, Laboratório de Cosmetologia, São Paulo, Brazil

#### **References**


[5] Almeida, I.F., Gaio, A.R., & Bahia, M.F. (2008). Hedonic and descriptive skinfeel anal‐ ysis of two oleogels: comparison with other topical formulations. J. Sens. Stud., 23(1), 92-113.

Our research group has invested in researches to assess the stability of cosmetics (Isaac et al., 2008); evaluating of the influence of the addition of thickening agents in creams using rheo‐ logical measurements (Isaac et al., 2012a); evaluating the thickeners' influence on the rheo‐ logical properties of a cosmetic (Isaac et al., 2012b,c); proposing alternative methods to assay the efficacy and safety of them (Chiari et al., 2012a; Chiari et al., 2012b) and using of the sen‐ sory analysis in the cosmetics development (Isaac et al., 2012a) which, in different points of

This chapter aimed to show the facility that some simple or advanced techniques already used, sometimes to other finalities, could offer to the quality control of cosmetic products. The sensory analysis, rheology and SAXS technique have earned attention due to the impor‐

Bruna Galdorfini Chiari, Maria Gabriela José de Almeida, Marcos Antonio Corrêa and

Faculdade de Ciências Farmacêuticas, UNESP - Univ Estadual Paulista, Departamento de Fármacos e Medicamentos, Laboratório de Cosmetologia – LaCos, Araraquara, Laboratório

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\*Address all correspondence to: veraisaac@fcfar.unesp.br

**5. Conclusion**

358 Latest Research into Quality Control

**Author details**

**References**

Vera Lucia Borges Isaac\*

de Cosmetologia, São Paulo, Brazil

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[70] Stone, H.S., & Sidel, J.L. (1992). Sensory evaluation practices. San Diego, CA: Aca‐

[71] Tadros, T. (2004). Application of rheology for assessment and prediction of the longterm physical stability of emulsions. Advances in Colloid and Interface Science, 108 –

[72] Toro-Vazquez, J.F., Morales-Rueda, J., Ajay Mallia, V., & Weiss, R.G. (2010). Relation‐ ship between molecular structure and thermo-mechanical properties of candelilla wax and amides derived from (R)-12-hydroxystearic acid as gelators of safflower oil.

[73] Tuarez, E.P., Sadtler, V., Marchal, P, Choplin, L., & Salager, J.L. (2011). Making use of formulation-composition map to prepare highly concentrated emulsions with partic‐

ular rheological properties. Ind. Eng. Chem. Res., 50, 2380-87.

emollients: recent perspectives. J. Dtsch. Dermatol. Ges., 10(5), 768-774.

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[52] Müller-Goymann, C.C. (2004). Physicochemical characterization of colloidal drug de‐ livery systems such as reverse micelles, vesicles, liquid crystals and nanoparticles for topical administration. European Journal of Pharmaceutics and Biopharmaceutics 58,

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[58] Parente, M.E., Ares, G., & Manzoni, A.V. (2010). Application of two consumer profil‐ ing techniques to cosmetic emulsions. Journal of Sensory Studies, 25, 685-705. [59] Parente, M.E., Gámbaro, A., & Ares, G. (2008). Sensory characterization of emollients.

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**Section 7**

**Sops: What Are They Good For?**


**Section 7**

**Sops: What Are They Good For?**

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[77] Wortel, V.A.L., & Wiechers, J.W. (2000). Skin sensory performance of individual per‐ sonal care ingredients and marketed personal care products. Food Qual. Pref.,

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cal emulsions containing encapsulated menthol. J. Sens. Stud., 23(1), 26-34.

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versidade Estadual Paulista Júlio de Mesquita Filho, Araraquara.

of Pharmaceutics, 309, 171-177.

11(1-2), 121-127.

364 Latest Research into Quality Control

**Chapter 17**

**Standard Operating Procedures**

Additional information is available at the end of the chapter

Standardization is defined as an activity that gives rise to solutions for repetitive application to problems in various disciplines. Generally, the activity constitudes the process of estab‐ lishing (determining, formulating, and issuing) and implementing standards. Thus, stand‐ ards are the perfect result of a standardization activity and inside the context of quality systems consist of quality documents or documents related to the quality system. High lev‐ els of quality are important to accomplish Company business objectives. Quality, a source of competitive benefit, should stay a symbol of Company products and services. High quality is not an additional value; it is an important elemantary necessity. Each employee in all or‐ ganizational units is responsible for guaranteeing that their work processes are effective and continually getting better. Top management should provide the training and an appropriate motivating environment to support teamwork both inside and across organizational units for employees to advance processes. Ultimately, everyone in an institution is responsible for the quality of its products and services. An institution in the role of a sponsor of clinical tri‐ als can best achieve its business objectives by establishing and managing robust quality sys‐ tems with their integral quality documents including standard operating procedures (SOPs) (Manghani, K. 2011). The Quality Management system must evolve by trial and error, with enlarging experience, by group discussions and with changing understanding. In the begin‐ ning, attention will be focused on basic operational SOPs, afterwards moving to record keeping (as more and more SOPs are issued) and filling gaps as practice admits missing links in the chain of Quality Assurance. Essentially problems will turn up. One way to react to them is to talk with people in other laboratories who have faced similar problems. It

> © 2012 Akyar; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Akyar; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**(What Are They Good For ?)**

Isin Akyar

http://dx.doi.org/10.5772/50439

**1. Introduction**

#### **Chapter 17**

## **Standard Operating Procedures (What Are They Good For ?)**

Isin Akyar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50439

#### **1. Introduction**

Standardization is defined as an activity that gives rise to solutions for repetitive application to problems in various disciplines. Generally, the activity constitudes the process of estab‐ lishing (determining, formulating, and issuing) and implementing standards. Thus, stand‐ ards are the perfect result of a standardization activity and inside the context of quality systems consist of quality documents or documents related to the quality system. High lev‐ els of quality are important to accomplish Company business objectives. Quality, a source of competitive benefit, should stay a symbol of Company products and services. High quality is not an additional value; it is an important elemantary necessity. Each employee in all or‐ ganizational units is responsible for guaranteeing that their work processes are effective and continually getting better. Top management should provide the training and an appropriate motivating environment to support teamwork both inside and across organizational units for employees to advance processes. Ultimately, everyone in an institution is responsible for the quality of its products and services. An institution in the role of a sponsor of clinical tri‐ als can best achieve its business objectives by establishing and managing robust quality sys‐ tems with their integral quality documents including standard operating procedures (SOPs) (Manghani, K. 2011). The Quality Management system must evolve by trial and error, with enlarging experience, by group discussions and with changing understanding. In the begin‐ ning, attention will be focused on basic operational SOPs, afterwards moving to record keeping (as more and more SOPs are issued) and filling gaps as practice admits missing links in the chain of Quality Assurance. Essentially problems will turn up. One way to react to them is to talk with people in other laboratories who have faced similar problems. It

© 2012 Akyar; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Akyar; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

should not be forgotten that Quality Management is a tool rather than a goal. The goal is quality performance of the laboratory. The philosopher Kant saw autonomy as self-govern‐ ment originning from morality, with morality proceeding from knowledge and self-disci‐ pline. Conger & Kanungo noted that an appropriate level of authority, discretion, formalization, and rule structure is a requirement for worker empowerment, which we see as consistent with the concept of self-government. Merriam-Webster defined autonomy as 'the quality or state of being self-governing; especially: the right of self-government; self-di‐ recting freedom and especially moral independence'. Necessitated SOP use will be absolute‐ ly related to the sense of self-determination experienced by workers. Worker participation in SOP advancenment and clarification controls the affiliation between required SOP use and the sense of self-determination experienced by workers.

**2. Overview**

without loss of effectiveness (Saxena).

tection Agency 2001).

The quality documents constitudes of Company policies, quality management plan, SOPs, working instructions, conventions, guidelines, forms, templates, logs, tags and labels. They are organized by consensus and approved by a nominated body and they provide for com‐ mon and repeated use, rules, guidelines or characteristics for activities or their results with a view to promote transparency, consistency, reproducibility, interchangeability and to facili‐ tate communication. The hierarchy and types of quality documents relevant to quality sys‐ tems will depend upon Company business objectives and business model. SOPs are Level 2 quality documents and, along with other related quality documents, guarantee the efficacy and effectiveness of quality systems (Manghani, K. 2011). Standard operating procedures (SOPs) are a vital component in any quality management system (Hattamer-Apostel, R. 2001). Every good quality system is based on its Standard Operating Procedures (SOPs) (Saxena). The advancement and use of SOPs are a necessary part of a successful quality sys‐ tem as it supplies individuals with the information to carry out a job adequately, and aids precision in the quality and integrity of a product or end-result (United States Environmen‐ tal Protection Agency, 2007). They assign all processes involved in an organization (Frank, D. 2010). A quality system is defined as the organizational structure, responsibilities, proc‐ esses, procedures and resources for implementing quality management (Manghani, K. 2011).

Standard Operating Procedures (What Are They Good For ?)

http://dx.doi.org/10.5772/50439

369

*Standard Operating Procedures* are sets of instructions having the force of a directive, covering those features of operations which lend themselves to a definite or standardized procedure

The purpose of a SOP is to reach out the operations correctly and always in the same man‐ ner. A SOP should be available at the place where the work is done". SOPs assist the prog‐ ress of constant application of processes and procedures so even when there are changes in personnel, organizations avoid inconsistencies and safety risks (Frank, D. 2010). Standard operating procedures or SOPs are written step-by-step procedures that quality control (QC), quality assurance (QA), and production units use in order to assure the accuracy and preci‐ sion of the quantitative experimental results and materials that they generate and provide in support of other units. SOP's are needed to guarantee the continuity of processes to obtain quality performance and quality products/preparations (Natural Resources Management and Environment Dept.). SOP's are alive documents that detail written instructions describ‐ ing specific steps to follow in all activities under defined conditions (Jain, SK. 2008). They are used to accomplish standardization when performing specific functions and is used to set out the way practice and procedures necessitated to be performed. SOPs are written in‐ structions and records of procedures agreed and adopted as standard practice (Cardiff Uni‐ versity, 2009). SOP's are necessary to guarantee the progression of processes to accomplish quality performance and quality products/preparations (Jain, SK. 2008). A Standard Operat‐ ing Procedure (SOP) document is a routine or repetitive activity followed by an organiza‐ tion. SOPs describe both technical and administrative operational elements of an organization that would be managed under a Quality Assurance Project Plan and under an organization's Quality Management Plan (Almeida S.L.), (United States Environmental Pro‐

Standard Operating Procedures (SOP) is a process document that describes in detail the way that an operator should perform a given operation. SOPs involve the purpose of the opera‐ tion, the equipment and materials required, how to perform the set-up and operations re‐ quired for the process, how to perform the maintenance and shutdown operations carried out by the worker, a description of safety issues, trouble-shooting, a list of spare parts and where to find them, illustrations, and checklists. The SOP is one of many process documents which is needed for consistent operation of a given process, with other documents involving process flow charts, material specifications, and so forth.

The purpose of SOPs today is to guarantee that all workers are performing tasks in the same way, which is a needed for condition to get expected output from the process. When all workers perform their tasks constantly, it becomes possible to run controlled experiments to test the impact of changing various process parameters. When a process change is shown to improve process performance, SOPs are updated and workers are trained to the new proce‐ dures. All over the process, it is adorable to involve workers in SOP development and to praise worker ideas for the SOP improvement. For constant organizational advance, organ‐ ized processes need to be constantly improved, hence necessitating ideas from those work‐ ers using those procedures. Ideas are not creative simply because they deviate from organized knowledge; ideas are creative when they are novel and suitable to the task at hand. Workers may have many ideas; nevertheless, what they choose to do with their ideas will depend on various organizational and individual-difference factors. The most impor‐ tant factor, however, for the advancement of creative behaviours is worker intrinsic motiva‐ tion– a sine qua non of worker creative contribution. By the help of confirmatory factor analysis, the Spreitzer construct validated the four dimensions of intrinsic motivation (i.e. psychological authorization): (a) Competence (example item includes 'I am confident about my ability to do my job'); (b) Meaning (example item includes 'The work I do is very impor‐ tant to me'; (c) Impact (example item includes 'I have a great deal of control over what hap‐ pens in my department'; (d) Self-determination (example item includes 'I can decide on my own how to go about doing my work'). Furthermore, Spreitzer argued and empirically es‐ tablished that an antecedent condition to innovation (i.e. creativity) and effectiveness is in‐ trinsic motivation (De Trevil et al. 2005).

#### **2. Overview**

should not be forgotten that Quality Management is a tool rather than a goal. The goal is quality performance of the laboratory. The philosopher Kant saw autonomy as self-govern‐ ment originning from morality, with morality proceeding from knowledge and self-disci‐ pline. Conger & Kanungo noted that an appropriate level of authority, discretion, formalization, and rule structure is a requirement for worker empowerment, which we see as consistent with the concept of self-government. Merriam-Webster defined autonomy as 'the quality or state of being self-governing; especially: the right of self-government; self-di‐ recting freedom and especially moral independence'. Necessitated SOP use will be absolute‐ ly related to the sense of self-determination experienced by workers. Worker participation in SOP advancenment and clarification controls the affiliation between required SOP use and

Standard Operating Procedures (SOP) is a process document that describes in detail the way that an operator should perform a given operation. SOPs involve the purpose of the opera‐ tion, the equipment and materials required, how to perform the set-up and operations re‐ quired for the process, how to perform the maintenance and shutdown operations carried out by the worker, a description of safety issues, trouble-shooting, a list of spare parts and where to find them, illustrations, and checklists. The SOP is one of many process documents which is needed for consistent operation of a given process, with other documents involving

The purpose of SOPs today is to guarantee that all workers are performing tasks in the same way, which is a needed for condition to get expected output from the process. When all workers perform their tasks constantly, it becomes possible to run controlled experiments to test the impact of changing various process parameters. When a process change is shown to improve process performance, SOPs are updated and workers are trained to the new proce‐ dures. All over the process, it is adorable to involve workers in SOP development and to praise worker ideas for the SOP improvement. For constant organizational advance, organ‐ ized processes need to be constantly improved, hence necessitating ideas from those work‐ ers using those procedures. Ideas are not creative simply because they deviate from organized knowledge; ideas are creative when they are novel and suitable to the task at hand. Workers may have many ideas; nevertheless, what they choose to do with their ideas will depend on various organizational and individual-difference factors. The most impor‐ tant factor, however, for the advancement of creative behaviours is worker intrinsic motiva‐ tion– a sine qua non of worker creative contribution. By the help of confirmatory factor analysis, the Spreitzer construct validated the four dimensions of intrinsic motivation (i.e. psychological authorization): (a) Competence (example item includes 'I am confident about my ability to do my job'); (b) Meaning (example item includes 'The work I do is very impor‐ tant to me'; (c) Impact (example item includes 'I have a great deal of control over what hap‐ pens in my department'; (d) Self-determination (example item includes 'I can decide on my own how to go about doing my work'). Furthermore, Spreitzer argued and empirically es‐ tablished that an antecedent condition to innovation (i.e. creativity) and effectiveness is in‐

the sense of self-determination experienced by workers.

368 Latest Research into Quality Control

process flow charts, material specifications, and so forth.

trinsic motivation (De Trevil et al. 2005).

The quality documents constitudes of Company policies, quality management plan, SOPs, working instructions, conventions, guidelines, forms, templates, logs, tags and labels. They are organized by consensus and approved by a nominated body and they provide for com‐ mon and repeated use, rules, guidelines or characteristics for activities or their results with a view to promote transparency, consistency, reproducibility, interchangeability and to facili‐ tate communication. The hierarchy and types of quality documents relevant to quality sys‐ tems will depend upon Company business objectives and business model. SOPs are Level 2 quality documents and, along with other related quality documents, guarantee the efficacy and effectiveness of quality systems (Manghani, K. 2011). Standard operating procedures (SOPs) are a vital component in any quality management system (Hattamer-Apostel, R. 2001). Every good quality system is based on its Standard Operating Procedures (SOPs) (Saxena). The advancement and use of SOPs are a necessary part of a successful quality sys‐ tem as it supplies individuals with the information to carry out a job adequately, and aids precision in the quality and integrity of a product or end-result (United States Environmen‐ tal Protection Agency, 2007). They assign all processes involved in an organization (Frank, D. 2010). A quality system is defined as the organizational structure, responsibilities, proc‐ esses, procedures and resources for implementing quality management (Manghani, K. 2011).

*Standard Operating Procedures* are sets of instructions having the force of a directive, covering those features of operations which lend themselves to a definite or standardized procedure without loss of effectiveness (Saxena).

The purpose of a SOP is to reach out the operations correctly and always in the same man‐ ner. A SOP should be available at the place where the work is done". SOPs assist the prog‐ ress of constant application of processes and procedures so even when there are changes in personnel, organizations avoid inconsistencies and safety risks (Frank, D. 2010). Standard operating procedures or SOPs are written step-by-step procedures that quality control (QC), quality assurance (QA), and production units use in order to assure the accuracy and preci‐ sion of the quantitative experimental results and materials that they generate and provide in support of other units. SOP's are needed to guarantee the continuity of processes to obtain quality performance and quality products/preparations (Natural Resources Management and Environment Dept.). SOP's are alive documents that detail written instructions describ‐ ing specific steps to follow in all activities under defined conditions (Jain, SK. 2008). They are used to accomplish standardization when performing specific functions and is used to set out the way practice and procedures necessitated to be performed. SOPs are written in‐ structions and records of procedures agreed and adopted as standard practice (Cardiff Uni‐ versity, 2009). SOP's are necessary to guarantee the progression of processes to accomplish quality performance and quality products/preparations (Jain, SK. 2008). A Standard Operat‐ ing Procedure (SOP) document is a routine or repetitive activity followed by an organiza‐ tion. SOPs describe both technical and administrative operational elements of an organization that would be managed under a Quality Assurance Project Plan and under an organization's Quality Management Plan (Almeida S.L.), (United States Environmental Pro‐ tection Agency 2001).

SOPs are determined to be specific to the organization whose activities are defined and as‐ sist that organization to maintain their quality control and quality assurance processes (United States Environmental Protection Agency 2001).

achiavable the staff or the people for whom they were written are not either following them. It therefore becomes very essential for the personnel to be trained on these SOPs so that they are absolutely aware of why and how SOPs can play important role in fulfilling the specific organizatory requirements from WHO, FDA, EMEA or other national health authorities. Health authorities world wide like the FDA or EMEA expect pharmaceutical, cosmetic and food producers to describe their manufacturing processes in written SOPs (GMP7.com). An organization's SOP manual is an important training document and provides workers with increased confidence, motivation and a sense of achievement (Frank, D. 2010). A SOP is a compulsory instruction. If deviations from this instruction are allowed, the conditions for these should be documented including who can give permission for this and what exactly the complete procedure will be. The original should rest at a secure place while working copies should be authenticated with stamps and/or signatures of authorized persons. The advancement and use of *SOPs* are a basic part of a successful quality system. It supplies in‐ formation to perform a job regularly, and constantly in order to access pre-determined spec‐

Standard Operating Procedures (What Are They Good For ?)

http://dx.doi.org/10.5772/50439

371

*SOP* clarifies the followings; what is the objective of SOP (Purpose), what are applicability and use of SOP (Scope)?, who will perform tasks (Responsibility), who will ensure imple‐

Reviewing and approving the SOP prior to its use. Immediate supervisor and the QA

Staff

Author

Author

Manager

Staff and the QAM

mentation of procedure (Accountability), how tasks will be performed (Procedure).

**Responsibility Responsible**

Identifying the need for development or revision of a standard operating procedure (SOP) and to convey that need to their immediate supervisor and/or the QA Manager (QAM).

An individual SOP to include sufficient detail that the process or procedure can be followed by another person when needed.

Requesting peers to review the SOP to determine whether it

Ensuring that the procedure or process follows the details noted in the individual SOP and to detail in writing when the SOP or a

Ensuring that all routine operations and activities in their area

Overseeing the appropriate preparation, numbering, retention, indexing, revision, and use of SOPs. QAM

(United States Environmental Protection Agency, 2007).

are documented by SOPs. Manager

component of that SOP has not been followed.

ification and quality end-result.

contains sufficient detail.

**Table 1.** Responsibility distribution in SOP.

All organizations, businesses, etc. should have SOPs (Jain, SK. 2008). SOPs support employ‐ ees with the information necessitated to perform their jobs regularly and help guarantee consistency in the quality of performance (Frank, D. 2010). SOPs are used by the govern‐ mental agencies, private industry, and academic laboratories by scientists and engineers from all of the science, technology, engineering, and mathematical disciplines. SOPs can also be intensely valuable in academic laboratories and can be employed anytime there is proc‐ ess that likely more than one person will use in a research group (Natural Resources Man‐ agement and Environment Dept.). SOPs are mainly associated with specific documentation necessities. It should not be forgotten that "If you don't document, it didn't happen! (Jain, SK. 2008). The International Conference on Harmonization Good Clinical Practice (ICH GCP) guideline ascertaines SOPs as "detailed, written instructions to achieve uniformity of the performance of a specific function". SOPs must be well written in order to supply an ef‐ ficacious control of good clinical practice (GCP) and prevent errors from occurring, thereby lessening waste and rework. Poorly written SOPs are a source of misinformation. To be user friendly, they should be absolute, unambiguous and must be written in plain language. SOPs are controlled documents and are best written by persons involved in the activity, process or function that is required to be specified or covered in the SOP. SOPs must be re‐ viewed prior to their approval for release, for adequacy, completeness and compliance with Company standards and all applicable legal, ethical and regulatory requirements. They must be checked out and updated as necessitated over their life cycle and any changes made to the SOPs must be re-approved. They must bear a revision status on them and their distri‐ bution must continually be documented and controlled. When obsolete SOPs are needed to be hold for any purpose, they should be suitably identified to prevent unintended use. Only relevant SOPs in their current version must be available at points of use and must remain legible. SOPs are mandatory for the implementation of GCP and other GxPs, namely, cGMP (Good Manufacturing Practice) and GLP (Good Laboratory Practice), within the scope of quality systems; therefore, it is well said that without SOPs there are no GxPs: no SOPs, no quality systems, and no GxPs (Manghani, K. 2011). SOPs are necessary for a clinical research organization whether it concerns a pharmaceutical company, a sponsor, a contract research organization, an investigator site, an Ethics Committee or any other party involved in clini‐ cal research to achieve maximum safety and efficiency of the performed clinical research Operatings. It is therefore a must that all people and sites involved in clinical studies (both at the sponsor and at the investigative sites) have suitable SOPs in place so as to conduct clinical research and to ensure compliance with the current regulations.

The presence of these quality documents is important when regulatory inspections (FDA, EMEA) take place since the most frequent reported deficiencies during inspections are the lack of written SOPs and/or the failure to adhere to them. The risk of GMP non-compliance is high at organizations with a poor suitability of specific SOPs and also if at all they are achiavable the staff or the people for whom they were written are not either following them. It therefore becomes very essential for the personnel to be trained on these SOPs so that they are absolutely aware of why and how SOPs can play important role in fulfilling the specific organizatory requirements from WHO, FDA, EMEA or other national health authorities. Health authorities world wide like the FDA or EMEA expect pharmaceutical, cosmetic and food producers to describe their manufacturing processes in written SOPs (GMP7.com). An organization's SOP manual is an important training document and provides workers with increased confidence, motivation and a sense of achievement (Frank, D. 2010). A SOP is a compulsory instruction. If deviations from this instruction are allowed, the conditions for these should be documented including who can give permission for this and what exactly the complete procedure will be. The original should rest at a secure place while working copies should be authenticated with stamps and/or signatures of authorized persons. The advancement and use of *SOPs* are a basic part of a successful quality system. It supplies in‐ formation to perform a job regularly, and constantly in order to access pre-determined spec‐ ification and quality end-result.

*SOP* clarifies the followings; what is the objective of SOP (Purpose), what are applicability and use of SOP (Scope)?, who will perform tasks (Responsibility), who will ensure imple‐ mentation of procedure (Accountability), how tasks will be performed (Procedure).


**Table 1.** Responsibility distribution in SOP.

SOPs are determined to be specific to the organization whose activities are defined and as‐ sist that organization to maintain their quality control and quality assurance processes

All organizations, businesses, etc. should have SOPs (Jain, SK. 2008). SOPs support employ‐ ees with the information necessitated to perform their jobs regularly and help guarantee consistency in the quality of performance (Frank, D. 2010). SOPs are used by the govern‐ mental agencies, private industry, and academic laboratories by scientists and engineers from all of the science, technology, engineering, and mathematical disciplines. SOPs can also be intensely valuable in academic laboratories and can be employed anytime there is proc‐ ess that likely more than one person will use in a research group (Natural Resources Man‐ agement and Environment Dept.). SOPs are mainly associated with specific documentation necessities. It should not be forgotten that "If you don't document, it didn't happen! (Jain, SK. 2008). The International Conference on Harmonization Good Clinical Practice (ICH GCP) guideline ascertaines SOPs as "detailed, written instructions to achieve uniformity of the performance of a specific function". SOPs must be well written in order to supply an ef‐ ficacious control of good clinical practice (GCP) and prevent errors from occurring, thereby lessening waste and rework. Poorly written SOPs are a source of misinformation. To be user friendly, they should be absolute, unambiguous and must be written in plain language. SOPs are controlled documents and are best written by persons involved in the activity, process or function that is required to be specified or covered in the SOP. SOPs must be re‐ viewed prior to their approval for release, for adequacy, completeness and compliance with Company standards and all applicable legal, ethical and regulatory requirements. They must be checked out and updated as necessitated over their life cycle and any changes made to the SOPs must be re-approved. They must bear a revision status on them and their distri‐ bution must continually be documented and controlled. When obsolete SOPs are needed to be hold for any purpose, they should be suitably identified to prevent unintended use. Only relevant SOPs in their current version must be available at points of use and must remain legible. SOPs are mandatory for the implementation of GCP and other GxPs, namely, cGMP (Good Manufacturing Practice) and GLP (Good Laboratory Practice), within the scope of quality systems; therefore, it is well said that without SOPs there are no GxPs: no SOPs, no quality systems, and no GxPs (Manghani, K. 2011). SOPs are necessary for a clinical research organization whether it concerns a pharmaceutical company, a sponsor, a contract research organization, an investigator site, an Ethics Committee or any other party involved in clini‐ cal research to achieve maximum safety and efficiency of the performed clinical research Operatings. It is therefore a must that all people and sites involved in clinical studies (both at the sponsor and at the investigative sites) have suitable SOPs in place so as to conduct

(United States Environmental Protection Agency 2001).

370 Latest Research into Quality Control

clinical research and to ensure compliance with the current regulations.

The presence of these quality documents is important when regulatory inspections (FDA, EMEA) take place since the most frequent reported deficiencies during inspections are the lack of written SOPs and/or the failure to adhere to them. The risk of GMP non-compliance is high at organizations with a poor suitability of specific SOPs and also if at all they are Procedures are not an end in themselves - they do not ensure good performance or results. More important are well-designed systems and processes, qualified employees, and a moti‐ vating company culture. Procedures provide process people – environment but do not cre‐ ate processes, qualified people, or a good working environment (Jain, SK. 2008). The responsibility distribution in a SOP is shown in Table 1.

SOPs can signify agreement with organizational and governmental needs and can be used as a part of a personnel training program, since they should supply detailed work instruc‐ tions. It minimizes opportunities for miscommunication and can address safety concerns.

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When historical data are being estimated for current use, SOPs can also be very important for reconstructing project activities when no other references are accessible. Besides, SOPs are commonly used as checklists by inspectors when auditing procedures. Eventually, the benefits of a valid SOP are decreased work effort, along with developed comparability, cred‐

The advancement and use of SOPs is a basic part of a successful quality system. It supplies individuals with the information to perform a job regularly and aids constancy in the quali‐ ty and integrity of a product or end-result through constant implementation of a process or

SOPs can also be used as a part of a personnel training program, hence they should support detailed work instructions. When historical data are being assessed for current use, SOPs can be beneficial for reconstructing project activities. Additionally, SOPs are commonly used as checklists by inspectors when auditing procedures. Finally, the benefits of a valid SOP are minimized work effort, together with improved data comparability, credibility, and legal defensibility. SOPs are necessary even when published methods are being administered be‐ cause cited published methods may not include appropriate information for conducting the

For example, if the SOP is written for a standard analytical method, the SOP should desig‐ nate the procedures to be followed in greater detail than appear in the published method, detailing how, if at all, the SOP differs from the standard method and any options, changes or alterations that the organization follows (United States Environmental Protection Agency, 2007). The significance regularly set up and managed quality control and quality assurance systems with their integral well-written SOPs and other quality documents for the achieve‐ ment of Company business objectives cannot be ignored. They serve as a passport to success by assisting the Company to accomplish high-quality processes, procedures, systems, and people, with eventual high-quality products and services and enhancement of the following: Customer satisfaction, and therefore, customer loyalty and repeat business and referral; timely registration of drugs by eliminating waste and the requirement for rework; opera‐ tional results such as revenue, profitability, market share and export opportunities; align‐ ment of processes with achievement of better results; understanding and motivation of employees toward the Company quality policy and business objectives, as well as participa‐ tion in continuous quality improvement initiatives; and confidence of interested parties in the effectiveness and efficiency of the Company as demonstrated by the financial and social gains from Company performance and reputation (Manghani, K. 2011). Benefits of SOPs are

ibility, and legal defensibility.

procedure inside the arrangement.

procedure in-house.

shown in Table 2.

#### **3. Purpose**

The purpose of SOP is to assign the procedures for the preparation, approval, distribution, amendment and storage of Standard Operating Procedures (Cardiff University, 2009). The purpose or objective of the procedure should express and expand well written title (Jain SK., 2008). SOPs serve as frame for organizational action – support direction and structure. They tell what, how, when, why, and who. (Iowa State University, 2010). In order to be active, SOPs need to define not only what needs to be, but who is qualified to carry it out, and un‐ der what conditions the procedure can be performed reliably (Levine D.I., 2010). They should aid constant conformance support data quality. They should be determined to be specific to the organization and assist that organization to obtain their quality control and quality assurance processes and ensure compliance (Almeida S.L.) SOPs specify the com‐ monly recurring work processes that are to be conducted or followed inside an arrange‐ ment. They approve the way activities are to be performed to alleviate constant conformance to technical and quality system necessities and to provide data quality. They may define, for example, basic programmatic actions and technical actions such as analytical processes, and procedures for maintaining, calibrating, and using equipment. If not written appropriately, SOPs are of limited value. Additionally, the best written SOPs will fail if they are not followed. Therefore, the use of SOPs needs to be checked out and re-enforced by management, alternatively the direct supervisor. Current copies of the SOPs also need to be readily accessible for reference in the work areas of those individuals absolutely carrying out the activity, either in hard copy or electronic format, otherwise SOPs serve little purpose (United States Environmental Protection Agency, 2007).

#### **4. Benefits**

The improvement and use of SOPs promotes quality through consistent implementation of a process or procedure within the organization reduced work effort, along with advanced da‐ ta comparability, credibility, and legal defensibility (Almeida S.L.) The details in an SOP *stand‐ ardize* the process and support step-by-step how-to instructions that enable *anyone* within your operation to perform the task in a consistent manner (Iowa State University, 2010). They ab‐ breviate difference and advance quality through constant impact of a process or procedure inside the organization, although there are temporary or permanent personnel changes.

SOPs can signify agreement with organizational and governmental needs and can be used as a part of a personnel training program, since they should supply detailed work instruc‐ tions. It minimizes opportunities for miscommunication and can address safety concerns.

Procedures are not an end in themselves - they do not ensure good performance or results. More important are well-designed systems and processes, qualified employees, and a moti‐ vating company culture. Procedures provide process people – environment but do not cre‐ ate processes, qualified people, or a good working environment (Jain, SK. 2008). The

The purpose of SOP is to assign the procedures for the preparation, approval, distribution, amendment and storage of Standard Operating Procedures (Cardiff University, 2009). The purpose or objective of the procedure should express and expand well written title (Jain SK., 2008). SOPs serve as frame for organizational action – support direction and structure. They tell what, how, when, why, and who. (Iowa State University, 2010). In order to be active, SOPs need to define not only what needs to be, but who is qualified to carry it out, and un‐ der what conditions the procedure can be performed reliably (Levine D.I., 2010). They should aid constant conformance support data quality. They should be determined to be specific to the organization and assist that organization to obtain their quality control and quality assurance processes and ensure compliance (Almeida S.L.) SOPs specify the com‐ monly recurring work processes that are to be conducted or followed inside an arrange‐ ment. They approve the way activities are to be performed to alleviate constant conformance to technical and quality system necessities and to provide data quality. They may define, for example, basic programmatic actions and technical actions such as analytical processes, and procedures for maintaining, calibrating, and using equipment. If not written appropriately, SOPs are of limited value. Additionally, the best written SOPs will fail if they are not followed. Therefore, the use of SOPs needs to be checked out and re-enforced by management, alternatively the direct supervisor. Current copies of the SOPs also need to be readily accessible for reference in the work areas of those individuals absolutely carrying out the activity, either in hard copy or electronic format, otherwise SOPs serve little purpose

The improvement and use of SOPs promotes quality through consistent implementation of a process or procedure within the organization reduced work effort, along with advanced da‐ ta comparability, credibility, and legal defensibility (Almeida S.L.) The details in an SOP *stand‐ ardize* the process and support step-by-step how-to instructions that enable *anyone* within your operation to perform the task in a consistent manner (Iowa State University, 2010). They ab‐ breviate difference and advance quality through constant impact of a process or procedure inside the organization, although there are temporary or permanent personnel changes.

responsibility distribution in a SOP is shown in Table 1.

(United States Environmental Protection Agency, 2007).

**3. Purpose**

372 Latest Research into Quality Control

**4. Benefits**

When historical data are being estimated for current use, SOPs can also be very important for reconstructing project activities when no other references are accessible. Besides, SOPs are commonly used as checklists by inspectors when auditing procedures. Eventually, the benefits of a valid SOP are decreased work effort, along with developed comparability, cred‐ ibility, and legal defensibility.

The advancement and use of SOPs is a basic part of a successful quality system. It supplies individuals with the information to perform a job regularly and aids constancy in the quali‐ ty and integrity of a product or end-result through constant implementation of a process or procedure inside the arrangement.

SOPs can also be used as a part of a personnel training program, hence they should support detailed work instructions. When historical data are being assessed for current use, SOPs can be beneficial for reconstructing project activities. Additionally, SOPs are commonly used as checklists by inspectors when auditing procedures. Finally, the benefits of a valid SOP are minimized work effort, together with improved data comparability, credibility, and legal defensibility. SOPs are necessary even when published methods are being administered be‐ cause cited published methods may not include appropriate information for conducting the procedure in-house.

For example, if the SOP is written for a standard analytical method, the SOP should desig‐ nate the procedures to be followed in greater detail than appear in the published method, detailing how, if at all, the SOP differs from the standard method and any options, changes or alterations that the organization follows (United States Environmental Protection Agency, 2007). The significance regularly set up and managed quality control and quality assurance systems with their integral well-written SOPs and other quality documents for the achieve‐ ment of Company business objectives cannot be ignored. They serve as a passport to success by assisting the Company to accomplish high-quality processes, procedures, systems, and people, with eventual high-quality products and services and enhancement of the following: Customer satisfaction, and therefore, customer loyalty and repeat business and referral; timely registration of drugs by eliminating waste and the requirement for rework; opera‐ tional results such as revenue, profitability, market share and export opportunities; align‐ ment of processes with achievement of better results; understanding and motivation of employees toward the Company quality policy and business objectives, as well as participa‐ tion in continuous quality improvement initiatives; and confidence of interested parties in the effectiveness and efficiency of the Company as demonstrated by the financial and social gains from Company performance and reputation (Manghani, K. 2011). Benefits of SOPs are shown in Table 2.


**5. Writing style**

Protection Agency, 2007).

during the design stage.

a risk assessment of the task.

protective equipment during the task.

SOPs should be written in a step-by-step, easy-to-read format by subject-matter experts who know the processes and the structure of the organization (Frank, D. 2010). They should be written by individuals aware of the activity and the organization's internal structure. These individuals are basically subject-matter experts who actually perform the work or use the process. A team accession can also be followed, particularly for multi-tasked processes where the experiences of a number of individuals are critical (United States Environmental

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Well-written SOPs should first shortly define the purpose of the work or process, involving any regulatory information or standards that are suitable to the SOP process, and the scope to show what is covered. Any specialized or different terms either in a separate definition

The information presented should be clear and easy to understand. The active voice and present verb tense should be used. SOP shall be simple and short. Information should be transported clearly and absolutely to remove any doubt as to what is needed. Flow charts should be used to illustrate the process being defined (Jain SK., 2008), (United States Envi‐ ronmental Protection Agency, 2007). (United States Environmental Protection Agency, 2001), (Almeida S.L.). It may be helpful to include additional experts to help gather informa‐

The most commonly used method of task analysis is *Hierarchical Task Analysis (HTA)*. Oper‐ ating instructions should be close to the user and kept up to date. The following issues

**1.** There should be no easier, more dangerous opportunities than following the procedure.

**2.** There should be an appropriate QA system in place to guarantee that the procedures can be kept up to date and that any errors are rapidly detected and corrected.

**3.** The procedures should not be needlessly prescriptive. The best way of guaranteeing that procedures do not become overly prescriptive is through involving the operator

**4.** Procedures should contain information on the necessities for the wearing of personal

**5.** Any risks to the operator should be documented at the start of the procedure, based on

**8.** There should be no uncertainty between which procedures apply to which situations.

section or in the suitable discussion section should be explained.

tion and to review, test and approve draft SOPs (Frank D., 2010).

should be considered in evaluating operating procedure documentation:

**6.** An appropriate method of coding each procedure should be used.

**7.** Each time a procedure is produced it should be dated.

**9.** Procedures do not always have to be paper based.

**Table 2.** Benefits Of SOPs.

#### **5. Writing style**

**Benefit Explanation**

later

Placing value only on production while disregarding safety, health and environment is costly finally. It is better to train employees in all aspects of doing a job than to face accidents, fines and litigation

Consumers, from individuals to companies, want products of consistent quality and specifications. SOPs specify job steps that

By following SOPs, you help to guarantee against process shutdowns caused by equipment failure or other facility damage

Following health and environmental steps in SOPs guarantees against spills and emissions that threaten plant neighbors and

Well-written SOPs help to gurantee that government regulations are satisfied. They also show a company's good-faith intention to operate perfectly. Failure to write and use good SOPs only signals government regulators that your company is not serious about

Thorough SOPs can be used as the basis for supplying standardized training for employees who are new to a particular job and for

The process of actively caring about fellow workers involves one worker coaching another in all aspects of proper job performance. When the proper procedures are outlined in a good SOP, any co-

mentioned in the previous item only it usually involves record keeping. SOPs should serve as a strong basis when detailed audit

As people move from job to job inside and between companies, unwritten knowledge and skills disappear from the workplace. Regularly maintained written SOPs can chronicle the best knowledge that can serve new workers when older ones move on.

Although accidents are unfortunate, view them as opportunities to learn how to improve conditions. A good SOP gives you a basis

from which to being investigating accidents

worker can coach another to help improve work skills.

help standardize products and consequently quality.

create community outrage

those who need re-training.

To serve as a checklist for auditors. Auditing job performance is a process similar to observation

checklists are developed.

compliance.

To provide people with all the safety, health, environmental and functional information necessitated to perform a

To guarantee that production operations are performed constantly to obtain quality control of processes and

To guarantee that processes continue uninterrupted and are completed on a

To guarantee that no failures occur in manufacturing and other processes that would harm anyone in the surrounding

To guarantee that acknowledged procedures are followed in compliance with company and government

To serve as a training document for teaching users about the process for

To serve as a checklist for co-workers who observe job performance to

To serve as an historical record of the how, why and when of steps in an existing process so there is a factual basis for revising those steps when a process or equipment are changed.

To serve as an explanation of steps in a process so they can be reviewed in

accident investigations.

(Jain, SK. 2008)

**Table 2.** Benefits Of SOPs.

which the SOP was written.

reinforce proper performance.

job properly.

374 Latest Research into Quality Control

products.

community.

regulations.

prescribed schedule.

SOPs should be written in a step-by-step, easy-to-read format by subject-matter experts who know the processes and the structure of the organization (Frank, D. 2010). They should be written by individuals aware of the activity and the organization's internal structure. These individuals are basically subject-matter experts who actually perform the work or use the process. A team accession can also be followed, particularly for multi-tasked processes where the experiences of a number of individuals are critical (United States Environmental Protection Agency, 2007).

Well-written SOPs should first shortly define the purpose of the work or process, involving any regulatory information or standards that are suitable to the SOP process, and the scope to show what is covered. Any specialized or different terms either in a separate definition section or in the suitable discussion section should be explained.

The information presented should be clear and easy to understand. The active voice and present verb tense should be used. SOP shall be simple and short. Information should be transported clearly and absolutely to remove any doubt as to what is needed. Flow charts should be used to illustrate the process being defined (Jain SK., 2008), (United States Envi‐ ronmental Protection Agency, 2007). (United States Environmental Protection Agency, 2001), (Almeida S.L.). It may be helpful to include additional experts to help gather informa‐ tion and to review, test and approve draft SOPs (Frank D., 2010).

The most commonly used method of task analysis is *Hierarchical Task Analysis (HTA)*. Oper‐ ating instructions should be close to the user and kept up to date. The following issues should be considered in evaluating operating procedure documentation:


#### **6. Preparation of SOP**

When actualizing a SOP one can choose number of different ways to organize and format them. There are some factors which determine what type of SOP to use or create: How many decisions will user need to make during process? How many steps and sub steps are there in procedure? Routine procedures that are short and necessitate few decisions can be writ‐ ten using simple steps format. Long procedures consisting of more than 10 steps, with few decisions should be written along with graphical format or hierarchical steps. Procedures that necessitate many decisions should be written along with flow chart. Requirement for document identification and control, accountability and traceability responsibility must be involved with every SOP; this can be obtained by supporting constant format.

**Figure 1.** A SOP should be written by a team in that field.

will ultimately increase the confidence of the user.

The most substantial step for administering the SOP in working area, train or retrain the user. Every one should follow the procedure accurately with each and every step in detail, it is very significant to train the user otherwise individual may interpret meaning in different ways.

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The trainer should share the reason WHY, SOP must performed correctly while training the user. People can follow better when they understand significance of procedure. Trainer should explain and demonstrate how each step in the SOP will be performed and should assure them this will increase Quality of product by providing safety and accuracy which

The people in the writing team can write or edit parts of an SOP independently and then one person can combine the individual contributions. After combination the SOP should be circulated the draft SOP for review among the initiator before editing a final draft for review by supervisors and subsequent supervised testing by employees. Ideally a writing team should meet at least once in the beginning of a project to establish writing objectives, targets and responsibilities, but then can work semi-independently with one person serving as co‐

**7. Implementing SOP**

The need for an SOP or the revision of an existing one should be identified by informing the appropriate supervisor. Written instructions on standardized procedures supply guidance to guarantee that activities are conducted in a constant way, hence leading to reliable prod‐ uct and service quality. SOPs should be prepared in full compliance with guidelines and or‐ ganizations and must mirror current organizational practices (Hattamer-Apostel, R. 2001). Ideally, SOP's should be written by teams that involve some or all of the following people: Those who will perform the job, those who will perform maintenance on equipment in‐ volved in an SOP, engineers or others who design equipment and processes, technical initia‐ tor, safety personnel, environmental personnel, equipment manufacturers (Jain, SK. 2008).

**Figure 1.** A SOP should be written by a team in that field.

#### **7. Implementing SOP**

**10.** At the start of the procedure an overview of the task should be provided.

operator can check that it is safe to proceed.

signed to be the most prominent information.

step to which they refer.

376 Latest Research into Quality Control

(Health and Safety Executive).

**6. Preparation of SOP**

erator.

**11.** Prerequisites should presented clearly at the start of the procedure to guarantee that the

**12.** The most important information on the page should be defined and this should be de‐

**14.** Any warnings, cautions or notes should be placed immediately prior to the instruction

**15.** Language should be kept as simple as possible, i.e. use nomenclature familiar to the op‐

**17.** Symbols, colours, and shapes used for graphics should conform to industry standards

When actualizing a SOP one can choose number of different ways to organize and format them. There are some factors which determine what type of SOP to use or create: How many decisions will user need to make during process? How many steps and sub steps are there in procedure? Routine procedures that are short and necessitate few decisions can be writ‐ ten using simple steps format. Long procedures consisting of more than 10 steps, with few decisions should be written along with graphical format or hierarchical steps. Procedures that necessitate many decisions should be written along with flow chart. Requirement for document identification and control, accountability and traceability responsibility must be

The need for an SOP or the revision of an existing one should be identified by informing the appropriate supervisor. Written instructions on standardized procedures supply guidance to guarantee that activities are conducted in a constant way, hence leading to reliable prod‐ uct and service quality. SOPs should be prepared in full compliance with guidelines and or‐ ganizations and must mirror current organizational practices (Hattamer-Apostel, R. 2001). Ideally, SOP's should be written by teams that involve some or all of the following people: Those who will perform the job, those who will perform maintenance on equipment in‐ volved in an SOP, engineers or others who design equipment and processes, technical initia‐ tor, safety personnel, environmental personnel, equipment manufacturers (Jain, SK. 2008).

involved with every SOP; this can be obtained by supporting constant format.

**13.** Separate headings should be used to discriminate apparently between sub tasks.

**16.** The nomenclature should be consistent with that on controls or panels.

The most substantial step for administering the SOP in working area, train or retrain the user. Every one should follow the procedure accurately with each and every step in detail, it is very significant to train the user otherwise individual may interpret meaning in different ways.

The trainer should share the reason WHY, SOP must performed correctly while training the user. People can follow better when they understand significance of procedure. Trainer should explain and demonstrate how each step in the SOP will be performed and should assure them this will increase Quality of product by providing safety and accuracy which will ultimately increase the confidence of the user.

The people in the writing team can write or edit parts of an SOP independently and then one person can combine the individual contributions. After combination the SOP should be circulated the draft SOP for review among the initiator before editing a final draft for review by supervisors and subsequent supervised testing by employees. Ideally a writing team should meet at least once in the beginning of a project to establish writing objectives, targets and responsibilities, but then can work semi-independently with one person serving as co‐ ordinator. SOPs should be checked out by several people qualified to assess the SOP in terms of its completeness and clarity of subject matter.

**9. Revising SOPS**

If the SOP does not definitely describe the procedure, then the SOP must be revised. Any change in the procedure must be included into the SOP. Nevertheless, prior to any change to

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If there are some errors in the finalized SOPs, such as typographical errors, printing errors, e.g., wrong page numbers or misaligned sentences) or any errors that do not affect the scope of the procedure, they may be correctly immediately and reprinted. These types of errors do not require full SOP revision, thus a revision number will not be generated and manage‐ ment approval is not needed. If the error occurs on the signature page then the signature page will be resigned. These types of corrections will be traceable since the historical file will reflect all corrections including typographical errors. Specifically, the historical SOP file will contain both the SOP with the correct page(s) as well as the page(s) containing the error. The page with the error will not be removed from the historical file. Additions can be made to an SOP via a clarification or an addendum. Explanations and addenda must be attached to the appropriate SOP until such time that the SOP can be revised. Usually, the revision will be organized during the biannual review process. When the SOP is revised, the revision num‐ ber is updated. Revisions, explanations, and addenda are prepared by appropriate person‐ nel, but must be approved by management. An SOP can be eliminated when it is no longer applicable. Management must approve the elimination of an SOP. Two or more SOPs can be consolidated; in this case one SOP supersedes the other, but management approval is re‐ quired for consolidation of procedures. The signed revised SOP must be sent to the histori‐

the SOP, management must be advised of, and approve, the change.

cal file for archiving (United States Environmental Protection Agency, 2007).

SOPs necessitate to remain current to be useful. The review process should not be overly cumbersome to encourage timely review. Therefore, whenever procedures are changed, SOPs should be updated and re-approved. If desired, only the pertinent section of an SOP can be modified and indicate the change date/revision number for that section in the Table

SOPs should also be reviewed systematically on a periodic basis, e.g. every 1-2 years, to en‐ sure that the policies and procedures remain current and suitable, or to decide whether the SOPs are even needed. The review date should be added to each SOP that has been re‐ viewed. If an SOP defines a process that is no longer followed, it should be removed from the current file and archived (Almeida S.L.) The frequency of review should be indicated by

**10. Frequency of revisions & Reviews**

of Contents and the document control notation.

management in the organization's Quality (Jain, SK., 2008).

SOPs should at least mention:


It should be organized and recorded who is responsible for the proper distribution of the documents, the filing and administration (e.g. of the original and further copies). Finally, it should be indicated how frequently a valid SOP should be periodically evaluated (usually 2 years) and by whom. Only officially issued copies may be used, only then the use of the proper instruction is guaranteed (United States Environmental Protection Agency, 2007).

#### **8. SOP Review and approval**

SOPs should be reviewed (that is, validated) by one or more individuals with appropriate training and experience with the process (Almeida S.L.).

It is especially helpful if draft SOPs are completely tested by individuals other than the orig‐ inal writer before the SOPs are finalized. The completed SOPs then must be checked out and approved by peer reviewers, the QA Manager, and appropriate management prior to the use of the SOP. A set format in styling, information necessitated, and a numbering system is re‐ quired, as well as biannual or annual review to ensure that the procedure is up-to-date. An archival system is needed to ensure that an historical record can be maintained and only cur‐ rent SOPs are available for staff use (United States Environmental Protection Agency, 2007).

The finalized SOPs should be approved as described in the organization's Quality Manage‐ ment Plan or its own SOP for preparation of SOPs. Generally the immediate supervisor, such as a section or branch chief, and the organization's quality assurance officer review and approve each SOP. Signature approval indicates that an SOP has been both reviewed and approved by management. When practical, use of electronic signatures, as well as electronic maintenance and submission, is an acceptable substitution for paper.

SOP general form defines an integrated system of management activities involving plan‐ ning, implementation, documentation, assessment, and improvement to ensure that a proc‐ ess, or item, is of the type and quality needed for the project (United States Environmental Protection Agency, 2007), (Jain, SK. 2008).

#### **9. Revising SOPS**

ordinator. SOPs should be checked out by several people qualified to assess the SOP in

It should be organized and recorded who is responsible for the proper distribution of the documents, the filing and administration (e.g. of the original and further copies). Finally, it should be indicated how frequently a valid SOP should be periodically evaluated (usually 2 years) and by whom. Only officially issued copies may be used, only then the use of the proper instruction is guaranteed (United States Environmental Protection Agency, 2007).

SOPs should be reviewed (that is, validated) by one or more individuals with appropriate

It is especially helpful if draft SOPs are completely tested by individuals other than the orig‐ inal writer before the SOPs are finalized. The completed SOPs then must be checked out and approved by peer reviewers, the QA Manager, and appropriate management prior to the use of the SOP. A set format in styling, information necessitated, and a numbering system is re‐ quired, as well as biannual or annual review to ensure that the procedure is up-to-date. An archival system is needed to ensure that an historical record can be maintained and only cur‐ rent SOPs are available for staff use (United States Environmental Protection Agency, 2007).

The finalized SOPs should be approved as described in the organization's Quality Manage‐ ment Plan or its own SOP for preparation of SOPs. Generally the immediate supervisor, such as a section or branch chief, and the organization's quality assurance officer review and approve each SOP. Signature approval indicates that an SOP has been both reviewed and approved by management. When practical, use of electronic signatures, as well as electronic

SOP general form defines an integrated system of management activities involving plan‐ ning, implementation, documentation, assessment, and improvement to ensure that a proc‐ ess, or item, is of the type and quality needed for the project (United States Environmental

maintenance and submission, is an acceptable substitution for paper.

Protection Agency, 2007), (Jain, SK. 2008).

*b.* to whom proposals for a SOP should be submitted, and who estimates the draft;

*d.* who decides on the date of implementation, and who should be informed;

*e.* how revisions can be made or how a SOP can be withdrawn.

training and experience with the process (Almeida S.L.).

terms of its completeness and clarity of subject matter.

*a.* who can or should make which type of SOP;

SOPs should at least mention:

378 Latest Research into Quality Control

*c.* the procedure of approval;

**8. SOP Review and approval**

If the SOP does not definitely describe the procedure, then the SOP must be revised. Any change in the procedure must be included into the SOP. Nevertheless, prior to any change to the SOP, management must be advised of, and approve, the change.

If there are some errors in the finalized SOPs, such as typographical errors, printing errors, e.g., wrong page numbers or misaligned sentences) or any errors that do not affect the scope of the procedure, they may be correctly immediately and reprinted. These types of errors do not require full SOP revision, thus a revision number will not be generated and manage‐ ment approval is not needed. If the error occurs on the signature page then the signature page will be resigned. These types of corrections will be traceable since the historical file will reflect all corrections including typographical errors. Specifically, the historical SOP file will contain both the SOP with the correct page(s) as well as the page(s) containing the error. The page with the error will not be removed from the historical file. Additions can be made to an SOP via a clarification or an addendum. Explanations and addenda must be attached to the appropriate SOP until such time that the SOP can be revised. Usually, the revision will be organized during the biannual review process. When the SOP is revised, the revision num‐ ber is updated. Revisions, explanations, and addenda are prepared by appropriate person‐ nel, but must be approved by management. An SOP can be eliminated when it is no longer applicable. Management must approve the elimination of an SOP. Two or more SOPs can be consolidated; in this case one SOP supersedes the other, but management approval is re‐ quired for consolidation of procedures. The signed revised SOP must be sent to the histori‐ cal file for archiving (United States Environmental Protection Agency, 2007).

#### **10. Frequency of revisions & Reviews**

SOPs necessitate to remain current to be useful. The review process should not be overly cumbersome to encourage timely review. Therefore, whenever procedures are changed, SOPs should be updated and re-approved. If desired, only the pertinent section of an SOP can be modified and indicate the change date/revision number for that section in the Table of Contents and the document control notation.

SOPs should also be reviewed systematically on a periodic basis, e.g. every 1-2 years, to en‐ sure that the policies and procedures remain current and suitable, or to decide whether the SOPs are even needed. The review date should be added to each SOP that has been re‐ viewed. If an SOP defines a process that is no longer followed, it should be removed from the current file and archived (Almeida S.L.) The frequency of review should be indicated by management in the organization's Quality (Jain, SK., 2008).

### **11. Checklists**

SOPs should describe how the checklist is to be prepared or on what it is to be based (Almei‐ da S.L.)

The Quality Management Plan should indicate the individual (s) responsible for assuring that only the current version is used. That plan should also designate where, and how, out‐ dated versions are to be maintained or archived in a manner to prevent their continued use,

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Electronic storage and retrieval mechanisms are generally easier to access than a hard-copy document format. For the user, electronic access can be limited to a read-only format, there‐ by protecting against unauthorized changes made to the document (United States Environ‐

This term describes an integrated system of management activities involving planning, im‐ plementation, documentation, assessment, and improvement to ensure that a process, or

*How should a SOP be organized?* A SOP should be organized to ensure ease and efficiency in use and to be specific to the organization which develops it. There is no one 'correct' format; and internal formatting will vary with each organization and with the type of SOP being written. *How much detail needs to be included in a SOP?* A SOP should be written with sufficient detail so that someone with a basic understanding of the field, can successfully reproduce the activ‐ ity or procedure when unsupervised (United States Environmental Protection Agency, 2007). The QA systems in place will be covered in general by 'standard operating procedures'

SOPs should be organized to guarantee ease and efficiency in use and to be specific to the organization which develops it. There is no one "correct" format; and internal formatting will vary with each organization and with the type of SOP being written. Where possible break the information into a series of logical steps to avoid a long list. The level of detail pro‐ vided in the SOP may differ based on, e.g., whether the process is critical, the frequency of that procedure being followed, the number of people who will use the SOP, and where training is not routinely available. A generalized format is discussed next (Levine, D.I. 2010). Organization shall have SOP on Preparation, approval, revision and control of standard Op‐ erating Procedure for more excellent control and management of SOPs. Before finalizing and distributing SOPs, organizations should get the documentation reviewed and validated by people with training and experience on the processes. If the SOP does not definitely de‐ fine the procedure, then the SOP must be revised. Any change in the procedure must be in‐ cluded into the SOP. After all, prior to any change to the SOP, management must be advised of, and approve, the change. Finalized SOPs, containing typographical errors, printing er‐ rors, e.g., wrong page numbers or misaligned sentences) or any errors that do not act on the

An organization's SOPs should be written in a format that is tailored to the organization type and its unique requirements. In general, administrative/programmatic SOPs will con‐ sist of five elements: Title page, Table of Contents, Purpose, Procedures, Quality Assurance/ Quality Control, and Reference (Frank, D. 2010). General SOP format is shown in Table 3.

item, is of the type and quality needed for the project (Levine, D.I. 2010).

(SOP) and will be made up of the following essential components:

scope of the procedure may be corrected immediately and reprinted.

as well as to be available for historical data review.

mental Protection Agency, 2007).

**14. SOP General Format**

Many activities use checklists to guarantee that steps are followed in order. Checklists are also used to document completed actions. Any checklists or forms involved as part of an ac‐ tivity should be referenced at the points in the procedure where they are to be used and then attached to the SOP (United States Environmental Protection Agency, 2007).

In some cases, detailed checklists are prepared specifically for a given activity. In those cas‐ es, the SOP should describe, at least generally, how the checklist is to be prepared, or on what it is to be based. Copies of specific checklists should be then maintained in the file with the activity results and/or with the SOP.

Many activities use checklists to guarantee that steps are followed in order. Checklists are also used to document completed actions. Any checklists or forms involved as part of an ac‐ tivity should be referenced at the points in the procedure where they are to be used and then attached to the SOP. In some cases, detailed checklists are prepared specifically for a given activity. In those cases, the SOP should describe, at least generally, how the checklist is to be prepared, or on what it is to be based. Copies of specific checklists should be then main‐ tained in the file with the activity results and/or with the SOP. Remember that the checklist is not the SOP, but a part of the SOP (Jain SK., 2008).

#### **12. Document Control**

Each organization should develop a numbering system to systematically identify and label their SOPs, and the document control should be described in its Quality Management Plan. Usually, each page of an SOP should have control documentation notation. A short title and identification (ID) number can serve as a reference designation. The revision number and date are very useful in identifying the SOP in use when reviewing historical data and is criti‐ cal when the requirement for observable records is included and when the activity is being reviewed (United States Environmental Protection Agency, 2007).

#### **13. SOP Document Tracking and Archival**

The organization should sustain a master list of all SOPs. This file or database should show the SOP number, version number, date of issuance, title, author, status, organizational di‐ vision, branch, section, and any historical information regarding past versions. The QA Man‐ ager (or designee) is usually the individual responsible for sustaining a file listing all current quality-related SOPs used inside the organization. If an electronic database is used, auto‐ matic "Review SOP" notices can be sent. Note that this list may be used also when au‐ dits are being considered or when questions are raised as to practices being followed within the organization.

The Quality Management Plan should indicate the individual (s) responsible for assuring that only the current version is used. That plan should also designate where, and how, out‐ dated versions are to be maintained or archived in a manner to prevent their continued use, as well as to be available for historical data review.

Electronic storage and retrieval mechanisms are generally easier to access than a hard-copy document format. For the user, electronic access can be limited to a read-only format, there‐ by protecting against unauthorized changes made to the document (United States Environ‐ mental Protection Agency, 2007).

### **14. SOP General Format**

**11. Checklists**

380 Latest Research into Quality Control

the activity results and/or with the SOP.

**12. Document Control**

the organization.

is not the SOP, but a part of the SOP (Jain SK., 2008).

reviewed (United States Environmental Protection Agency, 2007).

**13. SOP Document Tracking and Archival**

da S.L.)

SOPs should describe how the checklist is to be prepared or on what it is to be based (Almei‐

Many activities use checklists to guarantee that steps are followed in order. Checklists are also used to document completed actions. Any checklists or forms involved as part of an ac‐ tivity should be referenced at the points in the procedure where they are to be used and then

In some cases, detailed checklists are prepared specifically for a given activity. In those cas‐ es, the SOP should describe, at least generally, how the checklist is to be prepared, or on what it is to be based. Copies of specific checklists should be then maintained in the file with

Many activities use checklists to guarantee that steps are followed in order. Checklists are also used to document completed actions. Any checklists or forms involved as part of an ac‐ tivity should be referenced at the points in the procedure where they are to be used and then attached to the SOP. In some cases, detailed checklists are prepared specifically for a given activity. In those cases, the SOP should describe, at least generally, how the checklist is to be prepared, or on what it is to be based. Copies of specific checklists should be then main‐ tained in the file with the activity results and/or with the SOP. Remember that the checklist

Each organization should develop a numbering system to systematically identify and label their SOPs, and the document control should be described in its Quality Management Plan. Usually, each page of an SOP should have control documentation notation. A short title and identification (ID) number can serve as a reference designation. The revision number and date are very useful in identifying the SOP in use when reviewing historical data and is criti‐ cal when the requirement for observable records is included and when the activity is being

The organization should sustain a master list of all SOPs. This file or database should show the SOP number, version number, date of issuance, title, author, status, organizational di‐ vision, branch, section, and any historical information regarding past versions. The QA Man‐ ager (or designee) is usually the individual responsible for sustaining a file listing all current quality-related SOPs used inside the organization. If an electronic database is used, auto‐ matic "Review SOP" notices can be sent. Note that this list may be used also when au‐ dits are being considered or when questions are raised as to practices being followed within

attached to the SOP (United States Environmental Protection Agency, 2007).

This term describes an integrated system of management activities involving planning, im‐ plementation, documentation, assessment, and improvement to ensure that a process, or item, is of the type and quality needed for the project (Levine, D.I. 2010).

*How should a SOP be organized?* A SOP should be organized to ensure ease and efficiency in use and to be specific to the organization which develops it. There is no one 'correct' format; and internal formatting will vary with each organization and with the type of SOP being written.

*How much detail needs to be included in a SOP?* A SOP should be written with sufficient detail so that someone with a basic understanding of the field, can successfully reproduce the activ‐ ity or procedure when unsupervised (United States Environmental Protection Agency, 2007).

The QA systems in place will be covered in general by 'standard operating procedures' (SOP) and will be made up of the following essential components:

SOPs should be organized to guarantee ease and efficiency in use and to be specific to the organization which develops it. There is no one "correct" format; and internal formatting will vary with each organization and with the type of SOP being written. Where possible break the information into a series of logical steps to avoid a long list. The level of detail pro‐ vided in the SOP may differ based on, e.g., whether the process is critical, the frequency of that procedure being followed, the number of people who will use the SOP, and where training is not routinely available. A generalized format is discussed next (Levine, D.I. 2010).

Organization shall have SOP on Preparation, approval, revision and control of standard Op‐ erating Procedure for more excellent control and management of SOPs. Before finalizing and distributing SOPs, organizations should get the documentation reviewed and validated by people with training and experience on the processes. If the SOP does not definitely de‐ fine the procedure, then the SOP must be revised. Any change in the procedure must be in‐ cluded into the SOP. After all, prior to any change to the SOP, management must be advised of, and approve, the change. Finalized SOPs, containing typographical errors, printing er‐ rors, e.g., wrong page numbers or misaligned sentences) or any errors that do not act on the scope of the procedure may be corrected immediately and reprinted.

An organization's SOPs should be written in a format that is tailored to the organization type and its unique requirements. In general, administrative/programmatic SOPs will con‐ sist of five elements: Title page, Table of Contents, Purpose, Procedures, Quality Assurance/ Quality Control, and Reference (Frank, D. 2010). General SOP format is shown in Table 3.


**Procedures (continued)**

here (Levine, D.I. 2010).

(United States Environmental Protection Agency, 2007).

the temperatures at which the steps are performed.

only the current versions available in the biorepository file.

represents year.

If calculations are involved in analyzing the data, then an example of the calculation should be provided. Figures and tables showing laboratory apparatus, representative data, etc. can be included

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Once the necessity for a particular SOP is organized, it should be drafted immediately. SOPs are drafted by laboratory or supervisory staff qualified to perform the procedure. Next the SOP is reviewed by other staff, where possible, and then approved by the QA Manager (QAM) and management, such as immediate supervisor. Circulation to staff members for review/comment is advisable prior to acquiring management approval. The SOPs should be written to define study methods or procedures in sufficient detail so as to guarantee the quality and integrity of the data or procedure to be followed. When writing SOPs, the detail used may include both procedural requirements (exact instructions) and guidance information (general information) on the procedure. Procedural requirements must be followed accurately, while guidance information is used to help perform the procedure; it is not a mandatory requirement and, therefore, it does not have to be followed exactly. Procedural requirements can be distinguished from guidance elements, based on the context they are used. Office standard format for margins, font, and font size should be followed. Official SOPs will have a colored header and footer on each page, dated signatures on the front title page, and be printed on ivory colored paper with a watermark. An outline format should be used and include alpha and/or numeric characters are to be included to indicate levels of information

A SOP should be written as soon as the need for a standard written procedure for an activity is required (Cardiff University, 2009). How much someone knows about an entire process or job affects the way he or she does that job. Incorporate safety, health and environment into the traditional how-to-operate or how-to-do steps. Based on best practice/standards, the procedure should be written in specific detail to ensure that the procedure can be repeated in a reproducible fashion to include the order of steps that should be followed, the times allowed for each step (as needed) and

It should be kept in mind that many people do not read all the steps before starting on step one. ASOP should be written as long as necessary for a specific job. People tend to ignore long SOPs because they cannot remember more than 6 to 12 steps. If the SOP goes beyond 10 steps, the following solutions should be considered; The long SOPs should be broken into several logical subjob SOPs, an accompanying shortened SOP should be written that lists only the steps but not detailed explanations of those steps, and the long-form SOP should be made as a training document

All SOPs before implementation or after revision will be approved by the management committee before implementation. Previous versions of all documentation will be stored electronically, with

All SOPs will be checked out on an annual basis by the management committee. Protective equipment that should be worn by staff when performing the procedure described. A list of the equipment needed to perform the procedure. All materials and supplies should be recorded. The date the procedure was first introduced as well as the date of the most recent version. The date format should be based on the ddmmyyyy system where d represents day, m represents month and y

or manual to supplement the shorter sub-job SOPs mentioned earlier (Jain, SK. 2008).

**Procedures (continued)**

**Element Explanation**

382 Latest Research into Quality Control

the SOP.

training tool.

**Table of Contents**

**Title page** The SOP should be arranged to guarantee ease and efficiency in use and to be specific to the

organization (Almeida S.L.) Each SOP produced will be issued with a unique SOP number for reference purposes. This will be located in the table on the front page and in the footer of the document. This number will state where the SOP originated, the year it was produced, the SOP number and also state the version number. The SOP reference and effective date should be included in the footer on each page of the SOP. The first page or cover page of each SOP should contain the following information: a title that clearly identifies the activity or procedure, an SOP identification (ID) number, date of issue and/or revision, the name of the applicable agency, division, and/or branch to which this SOP applies, and the signatures and signature dates of those individuals who prepared and approved the SOP. Electronic signatures are satisfactory for SOPs obtained on a computerized database. (Jain SK., 2008), (United States Environmental Protection Agency, 2001), (Almeida S.L.), (Frank D., 2010). The Author shall be the individual primarily responsible for writing

**Chapter pages:** Chapter pages can help divide content by area or task type. Chapter pages serve as

**Title** – a clear, brief title describing the aim of the SOP and the conditions under which it can be accurately used (Levine D.I., 2010).The title should use directive language to declare what is being done to what (United States Environmental Protection Agency, 2007). Each SOP should be given a

A table of contents may be necessitated for quick reference, particularly if the SOP is long, for locating information and to designate changes or revisions made only to certain sections of an SOP. Denotes changes or revisions made only to certain sections of a SOP (Almeida S.L.), (United States

regulatory information or standards that are appropriate to the process (Levine, D.I. 2010). It is

guidance; an aid to meeting operating aims; mandatory and prescribe behaviour; and used as a

The key to any program striving for quality is the set of Standard Operating Procedures (SOPs) that describe how work is to be done. The procedure section will identify how the aims will be achieved. This will clearly indicate a step by step description of how the procedure to be followed. Steps should include products and equipment required, possible obstacles, personnel qualifications and safety considerations. For lengthy process descriptions, a flow chart might be necessary to define processes

mini title pages introducing each section and indicate dates for the most recent revisions.

unique name which captures the significance of the practice described (Levine D.I. ,2010).

**Definitions** There should be a part defining any words, phrases, or acronyms having special meaning or

**Purpose** Each chapter should first briefly describe the purpose of the work or process, including any

**Procedures** In general there are four major types of procedure: Procedures that supply general operating

recommended to include criteria for the control of the described system during operation.

Table of contents is not required if SOP is three pages or less.

application (United States Environmental Protection Agency 2001).

Environmental Protection Agency, 2007).

that often involve interferences or variances.

If calculations are involved in analyzing the data, then an example of the calculation should be provided. Figures and tables showing laboratory apparatus, representative data, etc. can be included here (Levine, D.I. 2010).

Once the necessity for a particular SOP is organized, it should be drafted immediately. SOPs are drafted by laboratory or supervisory staff qualified to perform the procedure. Next the SOP is reviewed by other staff, where possible, and then approved by the QA Manager (QAM) and management, such as immediate supervisor. Circulation to staff members for review/comment is advisable prior to acquiring management approval. The SOPs should be written to define study methods or procedures in sufficient detail so as to guarantee the quality and integrity of the data or procedure to be followed. When writing SOPs, the detail used may include both procedural requirements (exact instructions) and guidance information (general information) on the procedure. Procedural requirements must be followed accurately, while guidance information is used to help perform the procedure; it is not a mandatory requirement and, therefore, it does not have to be followed exactly. Procedural requirements can be distinguished from guidance elements, based on the context they are used. Office standard format for margins, font, and font size should be followed. Official SOPs will have a colored header and footer on each page, dated signatures on the front title page, and be printed on ivory colored paper with a watermark. An outline format should be used and include alpha and/or numeric characters are to be included to indicate levels of information (United States Environmental Protection Agency, 2007).

A SOP should be written as soon as the need for a standard written procedure for an activity is required (Cardiff University, 2009). How much someone knows about an entire process or job affects the way he or she does that job. Incorporate safety, health and environment into the traditional how-to-operate or how-to-do steps. Based on best practice/standards, the procedure should be written in specific detail to ensure that the procedure can be repeated in a reproducible fashion to include the order of steps that should be followed, the times allowed for each step (as needed) and the temperatures at which the steps are performed.

It should be kept in mind that many people do not read all the steps before starting on step one. ASOP should be written as long as necessary for a specific job. People tend to ignore long SOPs because they cannot remember more than 6 to 12 steps. If the SOP goes beyond 10 steps, the following solutions should be considered; The long SOPs should be broken into several logical subjob SOPs, an accompanying shortened SOP should be written that lists only the steps but not detailed explanations of those steps, and the long-form SOP should be made as a training document or manual to supplement the shorter sub-job SOPs mentioned earlier (Jain, SK. 2008).

All SOPs before implementation or after revision will be approved by the management committee before implementation. Previous versions of all documentation will be stored electronically, with only the current versions available in the biorepository file.

All SOPs will be checked out on an annual basis by the management committee. Protective equipment that should be worn by staff when performing the procedure described. A list of the equipment needed to perform the procedure. All materials and supplies should be recorded. The date the procedure was first introduced as well as the date of the most recent version. The date format should be based on the ddmmyyyy system where d represents day, m represents month and y represents year.

added.

**Procedures** Personnel Qualifications/Responsibilities (identifying any special qualifications users should have

**(continued)**

such as certification or training experience and/or any individual or positions having responsibility for the activity being described)(United States Environmental Protection Agency 2001) Any related SOPs (of operations used in the present SOP); possible safety instructions should be **Quality Assurance/ Quality Control**

the method should be defined.

Protection Agency 2001).

(Frank, D. 2010).

The preparation of appropriate QC procedures (self-checks, such as calibrations, recounting, reidentification) and QC material (such as blanks - rinsate, trip, field, or method; replicates; splits; spikes; and performance evaluation samples) that are needed to display successful performance of

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All SOPs should be checked out annually to certify all SOPs are in line with current processes, guidelines and regulations. They should be checked out in regards with the review date assigned and recorded on the front page of each SOP. The designated individuals will approve all SOP amendments. All significant amendments will be required 2 months in advance of the annual review. Any control steps and provisions for review or oversight should be defined prior to acceptance of the product or deliverable. This can involve test plans such as verification and validation plans for software or running a "spell-check" program on the finished document (United States Environmental

Finally, next all appropriate QA and quality control (QC) activities for that procedure should be defined, and list any cited or significant references (United States Environmental Protection Agency, 2007). Before finalizing and distributing SOPs, organizations must get the documentation reviewed and validated by people with training and experience on the processes. Additionally, it is a good idea to have the SOPs tested by staff who will be asked to comply with them. By following these steps, the author can identify missing information or needed revisions. Once SOPs are approved, they should be made readily available to facility management, building occupants and cleaning employees. The SOP will require final approval and authorization. The signature on an SOP will authoise the associated forms which should show an identical issue date to the SOP. When an SOP is issued and become effective, adequate time is required for training purposes. Finally, SOPs must remain current, so they should be updated and re-approved at least annually or whenever procedures change. Though the SOP development process takes time and effort, it can provide significant improvement to a cleaning organization's operational results and workers' understanding and job performance

All SOPs are reviewed by the applicable supervisor at least every two years in order to maintain their relevancy. Names of those individuals who have reviewed and approved the SOP for use in the laboratory. Signatures and dates should be supplied whenever possible as well. For those SOPs which do not necessitate a revision, documentation attesting to that fact must be submitted to the QAM who in turn initials and dates the table located at the bottom of the title page of the original SOP (United States Environmental Protection Agency, 2007). All SOPs require version control to ensure that individuals are using the correct verision of SOP. It is good to practice to assign a document a version number, in the format N. n where N represents a finalized document and n represents draft versions. Each new, approved and finalized document a major verison number…. should be assigned. When taking a document for revision or as draft, assign a new minor version. During the review cycle assign each new revision of the draft the next minor version, upon approval/finalization of the document assign the next major version (United States Environmental Protection Agency

2001), (Natural Resources Management and Environment Dept.).

**References** References relating to the development of the SOP are required to be listed. These may include other

SOPs, regulatory guidelines and published papers etc. Documents listed in the SOP must be recorded in the appendices and listed accordingly (United States Environmental Protection Agency 2001).

Generally there are four types of procedure: (Health and Safety Executive).

General operating guidance procedures

Procedures that an aid providing procedures to meeting operating aims

Mandatory and behaviour prescribing procedures.

Training tool procedures

**Scope and Applicability**–under what specific conditions can this protocol be used reliably; are there any known interferents or other limitations on the protocol's effective use?

**Introduction**–Appropriate background information on the system, methods, and instruments are used. The background section should plan the procedure and the specific aim of the SOP.

**Materials and Supplies**–There should be a list of any reagents involving names of suppliers used in this procedure. If the suppliers are obscure sources, a list of addresses and contact information should be supplied as well. **Cautions** – If there are some specific health and safety precautions they should be considered. For example, should gloves be worn? If so, what kind? How should spills, if they occur, be cleaned up? Are there any special procedures that should be followed in order to safely dispose of waste? (Levine, D.I. 2010). Some SOPs should be written for people who perform under different interpersonal circumstances, people who work alone, two or more people who work together as a team, for people who will control other people doing a job, for people who not familiar with rules generally understood by your employees (Jain, SK. 2008).

Well-written SOPs should first briefly describe the purpose of the work or process, including any regulatory information or standards that are appropriate to the SOP process, and the scope to indicate what is covered. Diagrams and flow charts should be used to help to break up long sections of text and to briefly summarize a series of steps for the reader(Almeida S.L.)

The age, education, knowledge, skill, experience and training, and work culture of the individuals should be considered who will be performing the SOP steps.

Criteria, checklists, or other standards should be applied during the procedure such as citing the document as guidance for reviewing SOPs Records Management (specifically,e.g.,as forms to be used and locations of files.

Once writing of an SOP have been completed, there should be several workers test it and give you feedback.

**Health and** Primarily for technical SOPs

**Safety**

**Warnings**

**Cautions** Primarily for technical SOPs

**Interferences** Primarily for technical SOPs

**Quality Assurance/ Quality Control** The preparation of appropriate QC procedures (self-checks, such as calibrations, recounting, reidentification) and QC material (such as blanks - rinsate, trip, field, or method; replicates; splits; spikes; and performance evaluation samples) that are needed to display successful performance of the method should be defined.

**Procedures (continued)**

384 Latest Research into Quality Control

added.

Personnel Qualifications/Responsibilities (identifying any special qualifications users should have such as certification or training experience and/or any individual or positions having responsibility for

Any related SOPs (of operations used in the present SOP); possible safety instructions should be

**Scope and Applicability**–under what specific conditions can this protocol be used reliably; are there

**Introduction**–Appropriate background information on the system, methods, and instruments are

**Materials and Supplies**–There should be a list of any reagents involving names of suppliers used in this procedure. If the suppliers are obscure sources, a list of addresses and contact information should be supplied as well. **Cautions** – If there are some specific health and safety precautions they should be considered. For example, should gloves be worn? If so, what kind? How should spills, if they occur, be cleaned up? Are there any special procedures that should be followed in order to safely dispose of waste? (Levine, D.I. 2010). Some SOPs should be written for people who perform under different interpersonal circumstances, people who work alone, two or more people who work together as a team, for people who will control other people doing a job, for people who not

Well-written SOPs should first briefly describe the purpose of the work or process, including any regulatory information or standards that are appropriate to the SOP process, and the scope to indicate what is covered. Diagrams and flow charts should be used to help to break up long sections

The age, education, knowledge, skill, experience and training, and work culture of the individuals

Criteria, checklists, or other standards should be applied during the procedure such as citing the document as guidance for reviewing SOPs Records Management (specifically,e.g.,as forms to be used

Once writing of an SOP have been completed, there should be several workers test it and give you

used. The background section should plan the procedure and the specific aim of the SOP.

the activity being described)(United States Environmental Protection Agency 2001)

Generally there are four types of procedure: (Health and Safety Executive).

Procedures that an aid providing procedures to meeting operating aims

any known interferents or other limitations on the protocol's effective use?

familiar with rules generally understood by your employees (Jain, SK. 2008).

of text and to briefly summarize a series of steps for the reader(Almeida S.L.)

should be considered who will be performing the SOP steps.

General operating guidance procedures

Training tool procedures

and locations of files.

Primarily for technical SOPs

feedback.

**Cautions** Primarily for technical SOPs

**Interferences** Primarily for technical SOPs

**Health and Safety Warnings**

Mandatory and behaviour prescribing procedures.

All SOPs should be checked out annually to certify all SOPs are in line with current processes, guidelines and regulations. They should be checked out in regards with the review date assigned and recorded on the front page of each SOP. The designated individuals will approve all SOP amendments. All significant amendments will be required 2 months in advance of the annual review. Any control steps and provisions for review or oversight should be defined prior to acceptance of the product or deliverable. This can involve test plans such as verification and validation plans for software or running a "spell-check" program on the finished document (United States Environmental Protection Agency 2001).

Finally, next all appropriate QA and quality control (QC) activities for that procedure should be defined, and list any cited or significant references (United States Environmental Protection Agency, 2007). Before finalizing and distributing SOPs, organizations must get the documentation reviewed and validated by people with training and experience on the processes. Additionally, it is a good idea to have the SOPs tested by staff who will be asked to comply with them. By following these steps, the author can identify missing information or needed revisions. Once SOPs are approved, they should be made readily available to facility management, building occupants and cleaning employees. The SOP will require final approval and authorization. The signature on an SOP will authoise the associated forms which should show an identical issue date to the SOP. When an SOP is issued and become effective, adequate time is required for training purposes. Finally, SOPs must remain current, so they should be updated and re-approved at least annually or whenever procedures change. Though the SOP development process takes time and effort, it can provide significant improvement to a cleaning organization's operational results and workers' understanding and job performance (Frank, D. 2010).

All SOPs are reviewed by the applicable supervisor at least every two years in order to maintain their relevancy. Names of those individuals who have reviewed and approved the SOP for use in the laboratory. Signatures and dates should be supplied whenever possible as well. For those SOPs which do not necessitate a revision, documentation attesting to that fact must be submitted to the QAM who in turn initials and dates the table located at the bottom of the title page of the original SOP (United States Environmental Protection Agency, 2007). All SOPs require version control to ensure that individuals are using the correct verision of SOP. It is good to practice to assign a document a version number, in the format N. n where N represents a finalized document and n represents draft versions. Each new, approved and finalized document a major verison number…. should be assigned. When taking a document for revision or as draft, assign a new minor version. During the review cycle assign each new revision of the draft the next minor version, upon approval/finalization of the document assign the next major version (United States Environmental Protection Agency 2001), (Natural Resources Management and Environment Dept.).

**References** References relating to the development of the SOP are required to be listed. These may include other SOPs, regulatory guidelines and published papers etc. Documents listed in the SOP must be recorded in the appendices and listed accordingly (United States Environmental Protection Agency 2001).

**Contact list** It should involve contact details for relevant individuals such as author of document.

**Appendices** This section should list appendices of other SOPs referenced in the document, or related to the procedure.

**15. Types of SOP**

pieces of paper) (Almeida S.L.)

**•** SOPs for safety precautions

**•** SOPs for analytical methods.

**•** SOPs for Quality Assurance.

**•** SOPs for the preparation of reagents.

administrative procedure (Almeida S.L.).

**16. Guidelines for Technical SOP Text**

written for a wide variety of activities.

**•** SOPs for receiving and registration of samples.

**•** SOPs for archiving and how to deal with complaints.

Some of the most important SOP types:

Several categories and types of SOPs can be distinguished. The name "SOP" may not always be appropriate, e.g., the description of situations or other matters may better designated *pro‐ tocols, instructions* or simply *registration forms.* Also *worksheets* belonging to an analytical pro‐ cedure have to be standardized (to avoid jotting down readings and calculations on odd

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387

**•** Fundamental SOPs. These give instructions how to make SOPs of the other categories. **•** Methodic SOPs. These describe a complete testing system or method of investigation.

Generally the SOPs may be written for any repetitive technical activity, as well as for any

SOPs may be written for any repetitive technical activity, as well as for any authoritative or functional programmatic procedure, that is being followed inside an organization. General guidance for preparing both technical and administrative SOPs follows and examples of each are located in the Appendix (United States Environmental Protection Agency, 2007).

Technical SOP and Administrative SOP are typical structures of SOPs. Technical and admin‐ istrative SOPs need to involve the specific steps aimed at initiating, coordinating, and record‐ ing and/or reporting the results of the activity, and should be tailored only to that activity. A technical SOP is a standard operating procedure which involves environmental data gen‐ eration, manipulation, or accumulation, e.g., an analytical process. Technical SOPs can be

Examples are SOPs instructing the user how to perform a specific analytical method to be fol‐ lowed in the laboratory or field (such as field testing using an immunoassay kit), or how to collect a sample in order to preserve the sample integrity and representativeness (such as col‐ lection of samples for future analysis of volatile organic compounds or trace metals), or how to conduct a bioassessment of a freshwater site. Technical SOPs are also needed to cover ac‐

**•** Standard procedures for operating instruments, apparatus and other equipment.

**Distribution** Once approved the original paper SOP folder, it will also contain supporting documentation relating to each approved SOP for referencing purposes. An electronic copy of the SOP will be held. Approved SOPs will be distributed in hard copy to PDs and will be published. The paper version of the abandoned SOP will be filled into the archived SOP folder. All SOPs will be checked out and approved annually before it is superseded, unless a specific reason for a 6 month review can be justified. All SOPs must be kept for the duration of the project.

> When the Sop fulfils all the necessary requirements it is printed. The author hands over the manuscript (or the floppy disk with text) to the SOP administrator who is responsible for the printing. The number of copies is decided by him/her and the author. Copying SOPs is forbidden. Extra copies can be obtained from the SOP administrator. The author (or his successor) signs all copies in the presence of the administrator before distribution. As the new copies are distributed the old ones (if there was one) are taken in. For each SOP a list of holders is made. The holder signs for receipt of a copy. The list is kept with the spare copies. Users are responsible for proper keeping of the SOPs. If necessary, copies can be protected by a cover or foil, and/or be kept in a loose-leaf binding.

> Appropriate SOPs will be placed in green binders to be found in a designated spot in each work area, e.g., laboratory, equipment rooms, the library, etc., and shall be available to staff and managers. These binders will not be located in the supervisor's office. Removal of an individual SOP requires completion of the sign-out located on the insider of the binder. The binder must not be removed from its designated spot by anyone other than the QAM or laboratory director. It is the responsibility of the QAM to update each binder as individual SOPs are revised. The staff is required to read any revised SOP within 7 working days of issuance if the SOP is applicable to their work. Reading of the updated SOP requires signature on the SOP review sheet (United States Environmental Protection Agency, 2007).

**Archiving** Proper archiving is essential for good administration of SOPs. All operating instructions should be kept up-to-date and be accesible to personnel. Good Laboratory Practice requires that all documentation pertaining to a test or investigation should be kept for a certain period. SOPs belong to this documentation. An historical file is created for each SOP that is approved by management and will be maintained in the company's archives by the QAM. The historical file will consist of the original signed SOP and all subsequent modifications thereof. Official SOPs will have both colored header and footer lines, and be printed on watermarked ivory colored paper. All copies of the original will be black and white, initialed, numbered, and placed in the appropriate binder located in each office. If a procedure is incorporated into another SOP (superseded), a copy of the superseded version is placed in the historical file of both SOPs (United States Environmental Protection Agency, 2007).

**Table 3.** General SOP Format.

### **15. Types of SOP**

**Contact list** It should involve contact details for relevant individuals such as author of document.

SOPs must be kept for the duration of the project.

procedure.

386 Latest Research into Quality Control

Agency, 2007).

2007).

**Table 3.** General SOP Format.

**Appendices** This section should list appendices of other SOPs referenced in the document, or related to the

**Distribution** Once approved the original paper SOP folder, it will also contain supporting documentation relating

to each approved SOP for referencing purposes. An electronic copy of the SOP will be held. Approved SOPs will be distributed in hard copy to PDs and will be published. The paper version of the abandoned SOP will be filled into the archived SOP folder. All SOPs will be checked out and approved annually before it is superseded, unless a specific reason for a 6 month review can be justified. All

When the Sop fulfils all the necessary requirements it is printed. The author hands over the manuscript (or the floppy disk with text) to the SOP administrator who is responsible for the printing. The number of copies is decided by him/her and the author. Copying SOPs is forbidden. Extra copies can be obtained from the SOP administrator. The author (or his successor) signs all copies in the presence of the administrator before distribution. As the new copies are distributed the old ones (if there was one) are taken in. For each SOP a list of holders is made. The holder signs for receipt of a copy. The list is kept with the spare copies. Users are responsible for proper keeping of the SOPs. If

necessary, copies can be protected by a cover or foil, and/or be kept in a loose-leaf binding.

**Archiving** Proper archiving is essential for good administration of SOPs. All operating instructions should be

Appropriate SOPs will be placed in green binders to be found in a designated spot in each work area, e.g., laboratory, equipment rooms, the library, etc., and shall be available to staff and managers. These binders will not be located in the supervisor's office. Removal of an individual SOP requires completion of the sign-out located on the insider of the binder. The binder must not be removed from its designated spot by anyone other than the QAM or laboratory director. It is the responsibility of the QAM to update each binder as individual SOPs are revised. The staff is required to read any revised SOP within 7 working days of issuance if the SOP is applicable to their work. Reading of the updated SOP requires signature on the SOP review sheet (United States Environmental Protection

kept up-to-date and be accesible to personnel. Good Laboratory Practice requires that all documentation pertaining to a test or investigation should be kept for a certain period. SOPs belong to this documentation. An historical file is created for each SOP that is approved by management and will be maintained in the company's archives by the QAM. The historical file will consist of the original signed SOP and all subsequent modifications thereof. Official SOPs will have both colored header and footer lines, and be printed on watermarked ivory colored paper. All copies of the original will be black and white, initialed, numbered, and placed in the appropriate binder located in each office. If a procedure is incorporated into another SOP (superseded), a copy of the superseded version is placed in the historical file of both SOPs (United States Environmental Protection Agency, Several categories and types of SOPs can be distinguished. The name "SOP" may not always be appropriate, e.g., the description of situations or other matters may better designated *pro‐ tocols, instructions* or simply *registration forms.* Also *worksheets* belonging to an analytical pro‐ cedure have to be standardized (to avoid jotting down readings and calculations on odd pieces of paper) (Almeida S.L.)

Some of the most important SOP types:


Generally the SOPs may be written for any repetitive technical activity, as well as for any administrative procedure (Almeida S.L.).

SOPs may be written for any repetitive technical activity, as well as for any authoritative or functional programmatic procedure, that is being followed inside an organization. General guidance for preparing both technical and administrative SOPs follows and examples of each are located in the Appendix (United States Environmental Protection Agency, 2007).

#### **16. Guidelines for Technical SOP Text**

Technical SOP and Administrative SOP are typical structures of SOPs. Technical and admin‐ istrative SOPs need to involve the specific steps aimed at initiating, coordinating, and record‐ ing and/or reporting the results of the activity, and should be tailored only to that activity.

A technical SOP is a standard operating procedure which involves environmental data gen‐ eration, manipulation, or accumulation, e.g., an analytical process. Technical SOPs can be written for a wide variety of activities.

Examples are SOPs instructing the user how to perform a specific analytical method to be fol‐ lowed in the laboratory or field (such as field testing using an immunoassay kit), or how to collect a sample in order to preserve the sample integrity and representativeness (such as col‐ lection of samples for future analysis of volatile organic compounds or trace metals), or how to conduct a bioassessment of a freshwater site. Technical SOPs are also needed to cover ac‐ tivities such as data processing and evaluation (including verification and validation), mod‐ eling, risk assessment, and auditing of equipment operation. Citing published methods in SOPs is not always acceptable, because cited published methods may not contain pertinent information for conducting the procedure-in-house. Technical SOPs need to include the spe‐ cific steps aimed at initiating, coordinating, and recording and/or reporting the results of the activity, and should be tailored only to that activity. Technical SOPs should fit within the framework presented here, but this format can be modified, reduced, or expanded as required.

be positive. Finally, our model high- lights the importance of worker participation. Produc‐ tion pressures, high capacity utilization, and lack of management – especially supervisor – support are likely to reduce opportunities for worker participation, and hence lower intrin‐

Standard Operating Procedures (What Are They Good For ?)

http://dx.doi.org/10.5772/50439

389

*Purpose:* Provide an example of a standard operating protocol or SOP that can be appreciat‐

*Scope and Applicability:* The following protocol can be used wherever quality coffee beans,

*Introduction:* Coffee is the beverage of choice of many college students. Properly prepared the beverage provides an invigorating and revitalizing effect. One of the most frequently used methods of preparation is the drip method. In this method, water, heated to near boil‐ ing temperatures, is slowly added to finely ground coffee beans held in a filter unit. The cof‐ fee beverage is collected below the filter unit in a glass carafe. Today this procedure is frequently accomplished using a semi-automated process in an electronic coffee maker. The procedure below outlines a reliable method for preparing drip coffee using any commercial‐

*References:* For information on coffee beans, the standard methods of preparation of coffee,

*Materials and Supplies:* Freshly ground Starbucks® coffee (any flavor you prefer; medium grind works best with most commercial coffee makers), commercial 4-c drip coffee maker including filter (gold mesh preferred but high quality paper filter may be used), good quali‐ ty drinking water (Polar Springs®, Brita®-filtered, or similar quality source recommended),

*Cautions:* Hot coffee can scald and burn. Water is an electrical conductor. If spills occur dur‐ ing the brewing process, wait until the brewing process is complete, turn of the electricity, and disconnect the unit from the electricity before attempting to clean up any spills. Acci‐ dental spills may be cleaned up with a kitchen sponge and dish washing detergent such as ….®. Used coffee grounds can be disposed of in the regular trash. Be sure to carefully read the directions that accompanied your coffee maker unit before attempting to use it. In partic‐ ular, it is important to find out if your unit has (1) a pause feature that will allow you to remove the carafe while the coffee is brewing; and (2) an auto-off feature that turns off the

ly available drip coffee maker, high quality ground coffee beans, and filtered water.

coffee cup, and additives (as desired: sugar or sugar alterative, cream or milk).

heater unit located beneath the carafe at a set time after the coffee has been brewed.

cedure who is less than ten years old be actively supervised by an adult.

*Personnel Qualifications:* No special knowledge or training is required to make coffee. How‐ ever, due to the potential risk of burns, it is recommended that anyone performing this pro‐

sic motivation and creativity (De Trevil et al. 2005).

*Submitted by:* Ay Dot Student; *Approved by:* Professor Ex

good drinking water, and a drip coffee maker are available.

*TITLE:* Preparation of the Perfect Cup of Coffee by the Drip Method *Date of Preparation:* 11/29/05; *Date of Revision:* N/A; *Revision No.:* N/A

ed by undergraduate research students from all academic disciplines.

*Example SOP*

and recipes see:

#### **17. Guidelines for Administrative or Fundamental Programmatic SOP**

An administrative SOP is a standard operating procedure which does not include environ‐ mental data manipulation activities, e.g., how to conduct an inspection. As with the techni‐ cal SOPs, these SOPs can be written for a wide variety of activities, e.g., reviewing documentation such as contracts, QA Project Plans and Quality Management Plans; inspect‐ ing (auditing) the work of others; determining organizational training needs; developing in‐ formation on records maintenance; validating data packages; or describing office correspondence procedures.

Administrative SOPs need to include a number of specific steps aimed at initiating the activ‐ ity, coordinating the activity, and recording and/or reporting the results of the activity, tail‐ ored to that activity. For example, audit or assessment SOPs should specify the authority for the assessment, how auditees are to be selected, what will be done with the results, and who is responsible for corrective action. Administrative SOPs should fit within the framework presented here, but this format can be modified, reduced, or expanded (United States Envi‐ ronmental Protection Agency, 2007).

#### **18. Conclusion**

Eventually, SOPs serve as a fundamental means of communication for all levels of the organ‐ ization. Not only do they include employees departmentally, but they also allow manage‐ ment and employees to gain a cross-functional view of the organization. This attitude encourages employees to think about how process change may affect other functional areas. A good system forces employee to think through processes and examine how procedure might influence product, personnel, production, and equipment. *It should not be forgotten that the "Best written SOPs will fail if they are not followed"* (Hattamer-Apostel, R. 2001), (Jain, SK. 2008).

What happens to workers' intrinsic task motivation and creativity when they are required to follow SOPs in completing their tasks? Job design and work motivation theory literatures have suggested a negative relationship; the OM literature has suggested a positive relation. We suggest that the discussion has been hindered by differences in conceptualizing required SOP use, by not explicitly incorporating the multidimensional nature of intrinsic motivation into the analysis, by an ambiguous definition of autonomy, and by ignoring important con‐ textual moderators. When these three elements are included in the discussion, we showed that the relationship between required SOP use and intrinsic motivation could theoretically be positive. Finally, our model high- lights the importance of worker participation. Produc‐ tion pressures, high capacity utilization, and lack of management – especially supervisor – support are likely to reduce opportunities for worker participation, and hence lower intrin‐ sic motivation and creativity (De Trevil et al. 2005).

#### *Example SOP*

tivities such as data processing and evaluation (including verification and validation), mod‐ eling, risk assessment, and auditing of equipment operation. Citing published methods in SOPs is not always acceptable, because cited published methods may not contain pertinent information for conducting the procedure-in-house. Technical SOPs need to include the spe‐ cific steps aimed at initiating, coordinating, and recording and/or reporting the results of the activity, and should be tailored only to that activity. Technical SOPs should fit within the framework presented here, but this format can be modified, reduced, or expanded as required.

**17. Guidelines for Administrative or Fundamental Programmatic SOP**

correspondence procedures.

388 Latest Research into Quality Control

ronmental Protection Agency, 2007).

**18. Conclusion**

An administrative SOP is a standard operating procedure which does not include environ‐ mental data manipulation activities, e.g., how to conduct an inspection. As with the techni‐ cal SOPs, these SOPs can be written for a wide variety of activities, e.g., reviewing documentation such as contracts, QA Project Plans and Quality Management Plans; inspect‐ ing (auditing) the work of others; determining organizational training needs; developing in‐ formation on records maintenance; validating data packages; or describing office

Administrative SOPs need to include a number of specific steps aimed at initiating the activ‐ ity, coordinating the activity, and recording and/or reporting the results of the activity, tail‐ ored to that activity. For example, audit or assessment SOPs should specify the authority for the assessment, how auditees are to be selected, what will be done with the results, and who is responsible for corrective action. Administrative SOPs should fit within the framework presented here, but this format can be modified, reduced, or expanded (United States Envi‐

Eventually, SOPs serve as a fundamental means of communication for all levels of the organ‐ ization. Not only do they include employees departmentally, but they also allow manage‐ ment and employees to gain a cross-functional view of the organization. This attitude encourages employees to think about how process change may affect other functional areas. A good system forces employee to think through processes and examine how procedure might influence product, personnel, production, and equipment. *It should not be forgotten that the "Best written SOPs will fail if they are not followed"* (Hattamer-Apostel, R. 2001), (Jain, SK. 2008).

What happens to workers' intrinsic task motivation and creativity when they are required to follow SOPs in completing their tasks? Job design and work motivation theory literatures have suggested a negative relationship; the OM literature has suggested a positive relation. We suggest that the discussion has been hindered by differences in conceptualizing required SOP use, by not explicitly incorporating the multidimensional nature of intrinsic motivation into the analysis, by an ambiguous definition of autonomy, and by ignoring important con‐ textual moderators. When these three elements are included in the discussion, we showed that the relationship between required SOP use and intrinsic motivation could theoretically *TITLE:* Preparation of the Perfect Cup of Coffee by the Drip Method

*Date of Preparation:* 11/29/05; *Date of Revision:* N/A; *Revision No.:* N/A

*Submitted by:* Ay Dot Student; *Approved by:* Professor Ex

*Purpose:* Provide an example of a standard operating protocol or SOP that can be appreciat‐ ed by undergraduate research students from all academic disciplines.

*Scope and Applicability:* The following protocol can be used wherever quality coffee beans, good drinking water, and a drip coffee maker are available.

*Introduction:* Coffee is the beverage of choice of many college students. Properly prepared the beverage provides an invigorating and revitalizing effect. One of the most frequently used methods of preparation is the drip method. In this method, water, heated to near boil‐ ing temperatures, is slowly added to finely ground coffee beans held in a filter unit. The cof‐ fee beverage is collected below the filter unit in a glass carafe. Today this procedure is frequently accomplished using a semi-automated process in an electronic coffee maker. The procedure below outlines a reliable method for preparing drip coffee using any commercial‐ ly available drip coffee maker, high quality ground coffee beans, and filtered water.

*References:* For information on coffee beans, the standard methods of preparation of coffee, and recipes see:

*Materials and Supplies:* Freshly ground Starbucks® coffee (any flavor you prefer; medium grind works best with most commercial coffee makers), commercial 4-c drip coffee maker including filter (gold mesh preferred but high quality paper filter may be used), good quali‐ ty drinking water (Polar Springs®, Brita®-filtered, or similar quality source recommended), coffee cup, and additives (as desired: sugar or sugar alterative, cream or milk).

*Cautions:* Hot coffee can scald and burn. Water is an electrical conductor. If spills occur dur‐ ing the brewing process, wait until the brewing process is complete, turn of the electricity, and disconnect the unit from the electricity before attempting to clean up any spills. Acci‐ dental spills may be cleaned up with a kitchen sponge and dish washing detergent such as ….®. Used coffee grounds can be disposed of in the regular trash. Be sure to carefully read the directions that accompanied your coffee maker unit before attempting to use it. In partic‐ ular, it is important to find out if your unit has (1) a pause feature that will allow you to remove the carafe while the coffee is brewing; and (2) an auto-off feature that turns off the heater unit located beneath the carafe at a set time after the coffee has been brewed.

*Personnel Qualifications:* No special knowledge or training is required to make coffee. How‐ ever, due to the potential risk of burns, it is recommended that anyone performing this pro‐ cedure who is less than ten years old be actively supervised by an adult.

#### *Protocol*

1. Make sure that the coffee maker is off. Locate water reservoir unit on coffee maker and carefully add 4-cups of clean drinking water to the reservoir. Note that the outside or inside of most quality coffee makers' water reservoir units are marked for the user's convenience.

[4] Frank, Dave. (2010). How to write SOPs that help increase consistency and improve performance quality in Standard Operating Procedures: A Writing Guide. Available at: http://www.cmmonline.com/management-training/article/standard-operating-

Standard Operating Procedures (What Are They Good For ?)

http://dx.doi.org/10.5772/50439

391

[5] GMP7.com (GMP online consultancy). What is a standard operating procedure? Available at: http://www.gmp7.com/whatisastandardoperatingprocedure\_cm

[6] Hattamer-Apostel, Rita. (2001). Standard operating procedures-a novel perspective. *The Quality Assurance Journal*, 5(4), 207-219, DOI:10.1002/qaj.155, available at:, http:// onlinelibrary.wiley.com/doi/10.1002/qaj.155/abstract?systemMessage=Wiley+Online

[7] Health and Safety Executive. Operating procedures. Available at: http://

[8] Iowa State University (2010). Hotel, restaurant and management extension. Available

[9] Jain, Sanjay Kumar. (2008). Standard operating procedures (SOP) - Back Bone of Pharmaceutical Industries. Pharma info.net Available at: http://www.pharmain‐ fo.net/reviews/standard-operating-procedures-sop-back-bone-pharmaceutical-indus‐

[10] Levine, David I., & Toffel, Michael W. (2010). Quality management and job quality: How the ISO 9001 standard for quality management systems affects employees and

[11] Manghani, Kishu. (2011). Quality assurance: Importance of systems and standard op‐ erating procedures. *Perspect Clin Res.*, 2(1), 34-37, Doi: 10.4103/2229-3485.76288.

[12] Natural Resources Management and Environment Dept. Guidelines for quality man‐ agement in soil and plant laboratories. FAO corporate document repository. Availa‐

[13] Saxena, Akanksha. SOP Writing for Clinical Trials: Staff Training Aspects. Interna‐ tional Biopharmaceutical Association Publication. Available at: http://www.ibpasso‐

[14] United States Environmental Protection Agency (2001). Guidance for Preparing Standard Operating Procedures (SOPs)." 10 Aug. 2010. United States Environmental Protection Agency. EPA QA/G-6. Available at: http://www.cluin.org/download/tool‐

[15] United States Environmental Protection Agency (2007). Guidance for Preparing Standard Operating Procedures (SOPs) EPA QA/G-6. Available at: http://

at: http://www.extension.iastate.edu/HRIM/HACCP/restaurants.htm.

procedures-a-writing-guide.

+Library+will+be+disrupted+3+Mar+from+.

www.hse.gov.uk/comah/sragtech/techmeasoperatio.htm.

employers. *Journal Management Science*, 56(6), 978-96.

ble at: http://www.fao.org/docrep/W7295E/w7295e04.htm.

ciation.org/IBPA\_articles/sop\_writing.htm.

kit/thirdednew/guidanceprepsops.pdf.

www.epa.gov/quality/qs-docs/g6-final.pdf.

380.html.

tries.

2. Locate the coffee filter assembly on the coffee unit. If you are preparing the standard 4-c carafe of coffee, carefully measure one coffee measure of ground coffee into your units cof‐ fee filter assembly. Note that one standard coffee measure is equivalent to 1/8-c of coffee. Close the coffee filter assembly.

3. Plug in the coffee maker and turn the unit on. Wait until the carafe located beneath the coffee filter unit is filled with coffee. Note that some units may have a "pause" feature that will allow you to temporarily remove the carafe and pour a cup of coffee while the unit is working. If you are unfamiliar with your unit, be sure to wait until the unit is done filtering before attempting to remove the carafe.

4. If coffee spills beneath the base of the carafe unit, be sure to turn off the unit and discon‐ nect the electricity before attempting to clean up the spill.

5. Pour yourself a cup of coffee. Most coffee units will keep the carafe warm for a set period of time before turning off automatically. Some however, do not turn off automatically. Be sure to read your coffee maker's instructions beforehand. If in doubt, be sure to turn off the electricity to your unit after the brewing process is complete. (Levine D.I et al, 2010)

#### **Author details**

Isin Akyar\*

Address all correspondence to: isinakyar@gmail.com

Acibadem University Faculty of Medicine Department of Microbiology, Turkey

#### **References**


[4] Frank, Dave. (2010). How to write SOPs that help increase consistency and improve performance quality in Standard Operating Procedures: A Writing Guide. Available at: http://www.cmmonline.com/management-training/article/standard-operatingprocedures-a-writing-guide.

*Protocol*

390 Latest Research into Quality Control

Close the coffee filter assembly.

**Author details**

Isin Akyar\*

**References**

before attempting to remove the carafe.

nect the electricity before attempting to clean up the spill.

Address all correspondence to: isinakyar@gmail.com

1. Make sure that the coffee maker is off. Locate water reservoir unit on coffee maker and carefully add 4-cups of clean drinking water to the reservoir. Note that the outside or inside of most quality coffee makers' water reservoir units are marked for the user's convenience. 2. Locate the coffee filter assembly on the coffee unit. If you are preparing the standard 4-c carafe of coffee, carefully measure one coffee measure of ground coffee into your units cof‐ fee filter assembly. Note that one standard coffee measure is equivalent to 1/8-c of coffee.

3. Plug in the coffee maker and turn the unit on. Wait until the carafe located beneath the coffee filter unit is filled with coffee. Note that some units may have a "pause" feature that will allow you to temporarily remove the carafe and pour a cup of coffee while the unit is working. If you are unfamiliar with your unit, be sure to wait until the unit is done filtering

4. If coffee spills beneath the base of the carafe unit, be sure to turn off the unit and discon‐

5. Pour yourself a cup of coffee. Most coffee units will keep the carafe warm for a set period of time before turning off automatically. Some however, do not turn off automatically. Be sure to read your coffee maker's instructions beforehand. If in doubt, be sure to turn off the

electricity to your unit after the brewing process is complete. (Levine D.I et al, 2010)

Acibadem University Faculty of Medicine Department of Microbiology, Turkey

=psy-ab&q=Guidance+for+Preparing+Standard+Operating+Proced.

ioural Outcomes. *Total Quality Management*, 16(2), 231-41.

[1] Almeida-Lynne, Sherri. Guidance for Preparing Standard Operating Procedures ppt. Available at: http://www.google.com.tr/#hl=tr&safe=active&output=search&sclient

[2] Cardiff University (2009). Standard operating procedure for the use or storage of hu‐ man tissue for the purposes of research or education. Available at: http:// www.cf.ac.uk/govrn/cocom/resources/standard%20Operating%20procedures.pdf.

[3] De Trevil, Suzanne., Antonakis, John., & Edelson, Norman. (2005). Can Standard Op‐ erating Procedures be Motivating? Reconciling Process Variability Issues and Behav‐


**Section 8**

**Quality Control in Clinical Laboratory Medicine**

**Quality Control in Clinical Laboratory Medicine**

**Chapter 18**

**Postmortem DNA: QC Considerations for Sequence and**

Long QT syndrome is a rare disorder of cardiac ion channels, characterised by a prolonged QT interval and T-wave abnormalities on electrocardiogram (ECG) and the occurrence of the ventricular tachycardia t*orsade de pointes*. Sodium, potassium or calcium channels present in heart muscle may be affected, altering the regulation of electrical current in the cells [1-3]. Individuals with this condition will be predisposed to cardiac events such as arrhythmias and polymorphic ventricular tachycardia, which may lead, if untreated, to sudden cardiac death [2,3]. Thirteen genes are associated with the condition, and hundreds of mutations have been identified [3-5]. Currently, more than 95% of the pathogenic mutations listed in disease databases (Gene Connection For the Heart, http://www.fsm.it/cardmoc/; online Hu‐ man Gene Mutation Database, www.hgmd.cf.ac.uk/) are sequence variants (including point mutations and small insertions or deletions), but the importance of whole or multi-exon de‐ letions and duplications has more recently been recognised [6] and it is now recommended to use both sequence and dosage techniques in order to provide comprehensive analysis [3]. In New Zealand, the majority of specimens referred for Long QT syndrome diagnostic test‐ ing are retrieved after death. Postmortem specimens are often difficult to handle as they are usually either tissue samples or severely haemolysed blood. The extracted DNA is frequent‐ ly of low quality, due to the presence of unwanted material such as short fragments pro‐ duced by degradation and chemical modifications from oxidation and hydrolysis processes [7]. As a result, only short sequences can be reliably amplified [7]. Moreover, capillary-based

> © 2012 Lai et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Lai et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Dosage Analysis of Genes Implicated in Long QT**

**Syndrome**

Stella Lai, Renate Marquis-Nicholson,

Jonathan R. Skinner and Donald R. Love

http://dx.doi.org/10.5772/51685

**1. Introduction**

Additional information is available at the end of the chapter

Chuan-Ching Lan, Jennifer M. Love, Elaine Doherty,

## **Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome**

Stella Lai, Renate Marquis-Nicholson, Chuan-Ching Lan, Jennifer M. Love, Elaine Doherty, Jonathan R. Skinner and Donald R. Love

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51685

#### **1. Introduction**

Long QT syndrome is a rare disorder of cardiac ion channels, characterised by a prolonged QT interval and T-wave abnormalities on electrocardiogram (ECG) and the occurrence of the ventricular tachycardia t*orsade de pointes*. Sodium, potassium or calcium channels present in heart muscle may be affected, altering the regulation of electrical current in the cells [1-3]. Individuals with this condition will be predisposed to cardiac events such as arrhythmias and polymorphic ventricular tachycardia, which may lead, if untreated, to sudden cardiac death [2,3]. Thirteen genes are associated with the condition, and hundreds of mutations have been identified [3-5]. Currently, more than 95% of the pathogenic mutations listed in disease databases (Gene Connection For the Heart, http://www.fsm.it/cardmoc/; online Hu‐ man Gene Mutation Database, www.hgmd.cf.ac.uk/) are sequence variants (including point mutations and small insertions or deletions), but the importance of whole or multi-exon de‐ letions and duplications has more recently been recognised [6] and it is now recommended to use both sequence and dosage techniques in order to provide comprehensive analysis [3].

In New Zealand, the majority of specimens referred for Long QT syndrome diagnostic test‐ ing are retrieved after death. Postmortem specimens are often difficult to handle as they are usually either tissue samples or severely haemolysed blood. The extracted DNA is frequent‐ ly of low quality, due to the presence of unwanted material such as short fragments pro‐ duced by degradation and chemical modifications from oxidation and hydrolysis processes [7]. As a result, only short sequences can be reliably amplified [7]. Moreover, capillary-based

© 2012 Lai et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Lai et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

sequencing, the gold standard first-line diagnostic test for Long QT syndrome, is very sensi‐ tive to the presence of contaminants, such as proteins, RNA and residual salt. The presence of such contaminants leads to poor quality electropherograms for analysis, which tend to be compromised by the presence of dye blobs, C-shoulders and a variable degree of baseline noise (Figure 1). Although the QC requirements for array comparative genomic hybridisa‐ tion (aCGH), which can be used to detect whole exon deletion and duplication mutations, are less stringent than those for sequencing, contamination or degradation of sample DNA can lead to suboptimal efficiency of labelling and hybridisation. Such difficulties arising in the practical procedure mean that the analysis of postmortem DNA can be time-consuming and challenging, and obtaining high quality data within a reasonable timeframe can be ex‐ tremely difficult.

presented below concerns both sequence analysis and dosage analysis. The former uses con‐ ventional sequencing technology, while the latter involves the simultaneous high resolution screening of deletion and duplication mutations in multiple cardiac genes as opposed to the more conventional multiplex ligation dependent probe amplification (MLPA) technique,

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

http://dx.doi.org/10.5772/51685

397

We have used two approaches to design primers flanking each of the coding exons of the genes *KCNQ1* (LQT1), *KCNH2* (LQT2), *SCN5A* (LQT3), *KCNE1* (LQT5), *KCNE2* (LQT6), and *KCNJ2* (LQT7), including at least 50 base pairs of the flanking intronic regions. The first used the primer design program called PrimerQuest (Integrated DNA Technologies Inc; http:// www.idtdna.com/Scitools/Applications/Primerquest/Advanced.aspx). This program allows the region in a sequence file to be specified, against which primers are designed to flank the targeted region. The designed primers for each exon are then checked *in silico* for annealing characteristics using the Macintosh-based program, Amplify. Finally, all primers were checked for single nucleotide polymorphisms (SNPs) using the software tool available from the National Genetic Reference Laboratory, Manchester (http://ngrl.man.ac.uk/SNPCheck/ SNPCheck.html). This bioinformatics program uses the current National Center for Biotech‐ nology Information (NCBI) build of the human genome and the current release of the Single Nucleotide Polymorphism database (dbSNP) to identify the position in the sequence where

Following the above approach, we developed an alternate design protocol [12,13]. Each mRNA sequence of interest was identified through the public UCSC genome browser page http://genome.ucsc.edu. This website provides a direct link to ExonPrimer for the design of primers specific to the mRNA sequence. ExonPrimer uses exon position information provid‐ ed by the UCSC genome browser in combination with the primer design tool Primer3 to cre‐ ate primer pairs according to set parameters, while avoiding pairing to homologous regions within the genome. Exon and amplicon size are provided and multiple alternative primer sets are given. Following primer design, all primers were checked for single nucleotide poly‐ morphisms (SNPs), as described above. Following a negative SNP check the primer sequen‐ ces were evaluated using the UCSC genome browser to confirm the identification of single amplicons. Each primer was then tailed with an M13 sequence and manufactured by Inte‐ grated DNA Technologies Inc or Life Technologies. In this way, primers are designed to al‐ low amplification of all exons of interest and the corresponding splice sites using a single set of PCR conditions such that PCR (and subsequent sequencing) can be performed under

which many diagnostic laboratories still use [6].

the primers bind and to detect any known SNPs at these sites

identical conditions within a 96-well PCR plate.

**2. Materials and methods**

**2.1. Sequencing**

*2.1.1. Primer design*

**Figure 1.** Electropherograms showing poor quality sequence data.

Long QT syndrome affects 1 in 2000 individuals [8] and contributes to 15-25% of sudden un‐ explained death in 1-40 year olds [9,10] and 10% of sudden unexplained deaths in infancy (SIDs) [11]. As a consequence, it is an important differential diagnosis to be considered in all cases of autopsy negative young sudden death. Molecular genetic testing is essential to make a postmortem diagnosis, given that screening for the electrocardiogram characteristics is no longer possible after death. Historically the turnaround time for diagnosis has been up to six months, due to the large number of genes to be analysed and the difficulties inherent in dealing with postmortem specimens (as detailed above). However, the demand by coro‐ ners for diagnostic laboratories to undertake more rapid analysis has been steadily increas‐ ing. A protocol tailored specifically to the treatment of postmortem specimens is necessary to meet this demand. Within our laboratory, we have successfully developed a robust proc‐ ess for sequence and dosage analysis of postmortem samples and have achieved an excellent turnaround time of 6-12 weeks. Here, we address the critical QC parameters that should be considered in order to obtain high quality data for rapid, accurate analysis. The discussion presented below concerns both sequence analysis and dosage analysis. The former uses con‐ ventional sequencing technology, while the latter involves the simultaneous high resolution screening of deletion and duplication mutations in multiple cardiac genes as opposed to the more conventional multiplex ligation dependent probe amplification (MLPA) technique, which many diagnostic laboratories still use [6].

#### **2. Materials and methods**

#### **2.1. Sequencing**

sequencing, the gold standard first-line diagnostic test for Long QT syndrome, is very sensi‐ tive to the presence of contaminants, such as proteins, RNA and residual salt. The presence of such contaminants leads to poor quality electropherograms for analysis, which tend to be compromised by the presence of dye blobs, C-shoulders and a variable degree of baseline noise (Figure 1). Although the QC requirements for array comparative genomic hybridisa‐ tion (aCGH), which can be used to detect whole exon deletion and duplication mutations, are less stringent than those for sequencing, contamination or degradation of sample DNA can lead to suboptimal efficiency of labelling and hybridisation. Such difficulties arising in the practical procedure mean that the analysis of postmortem DNA can be time-consuming and challenging, and obtaining high quality data within a reasonable timeframe can be ex‐

Long QT syndrome affects 1 in 2000 individuals [8] and contributes to 15-25% of sudden un‐ explained death in 1-40 year olds [9,10] and 10% of sudden unexplained deaths in infancy (SIDs) [11]. As a consequence, it is an important differential diagnosis to be considered in all cases of autopsy negative young sudden death. Molecular genetic testing is essential to make a postmortem diagnosis, given that screening for the electrocardiogram characteristics is no longer possible after death. Historically the turnaround time for diagnosis has been up to six months, due to the large number of genes to be analysed and the difficulties inherent in dealing with postmortem specimens (as detailed above). However, the demand by coro‐ ners for diagnostic laboratories to undertake more rapid analysis has been steadily increas‐ ing. A protocol tailored specifically to the treatment of postmortem specimens is necessary to meet this demand. Within our laboratory, we have successfully developed a robust proc‐ ess for sequence and dosage analysis of postmortem samples and have achieved an excellent turnaround time of 6-12 weeks. Here, we address the critical QC parameters that should be considered in order to obtain high quality data for rapid, accurate analysis. The discussion

tremely difficult.

396 Latest Research into Quality Control

**Figure 1.** Electropherograms showing poor quality sequence data.

#### *2.1.1. Primer design*

We have used two approaches to design primers flanking each of the coding exons of the genes *KCNQ1* (LQT1), *KCNH2* (LQT2), *SCN5A* (LQT3), *KCNE1* (LQT5), *KCNE2* (LQT6), and *KCNJ2* (LQT7), including at least 50 base pairs of the flanking intronic regions. The first used the primer design program called PrimerQuest (Integrated DNA Technologies Inc; http:// www.idtdna.com/Scitools/Applications/Primerquest/Advanced.aspx). This program allows the region in a sequence file to be specified, against which primers are designed to flank the targeted region. The designed primers for each exon are then checked *in silico* for annealing characteristics using the Macintosh-based program, Amplify. Finally, all primers were checked for single nucleotide polymorphisms (SNPs) using the software tool available from the National Genetic Reference Laboratory, Manchester (http://ngrl.man.ac.uk/SNPCheck/ SNPCheck.html). This bioinformatics program uses the current National Center for Biotech‐ nology Information (NCBI) build of the human genome and the current release of the Single Nucleotide Polymorphism database (dbSNP) to identify the position in the sequence where the primers bind and to detect any known SNPs at these sites

Following the above approach, we developed an alternate design protocol [12,13]. Each mRNA sequence of interest was identified through the public UCSC genome browser page http://genome.ucsc.edu. This website provides a direct link to ExonPrimer for the design of primers specific to the mRNA sequence. ExonPrimer uses exon position information provid‐ ed by the UCSC genome browser in combination with the primer design tool Primer3 to cre‐ ate primer pairs according to set parameters, while avoiding pairing to homologous regions within the genome. Exon and amplicon size are provided and multiple alternative primer sets are given. Following primer design, all primers were checked for single nucleotide poly‐ morphisms (SNPs), as described above. Following a negative SNP check the primer sequen‐ ces were evaluated using the UCSC genome browser to confirm the identification of single amplicons. Each primer was then tailed with an M13 sequence and manufactured by Inte‐ grated DNA Technologies Inc or Life Technologies. In this way, primers are designed to al‐ low amplification of all exons of interest and the corresponding splice sites using a single set of PCR conditions such that PCR (and subsequent sequencing) can be performed under identical conditions within a 96-well PCR plate.

#### *2.1.2. DNA extraction*

Genomic DNA (gDNA) was extracted from peripheral blood leucocytes (EDTA blood sam‐ ples) using the Gentra Puregene DNA Extraction kit (Qiagen), according to the manufactur‐ er's instructions.

The analysis of sequence traces is performed using Variant Reporter v1.0 (Applied Biosys‐ tems). Variant Reporter uses advanced algorithms and quality metrics to automate the de‐

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

http://dx.doi.org/10.5772/51685

399

tection of variants and to streamline the analysis process.

**Figure 2.** Flowchart of the sequencing method used in our laboratory.

A standard phenol/chloroform protease protocol was used to extract gDNA from postmor‐ tem tissue specimens. A small section (2mm x 2mm x 4mm) is usually cut from frozen tissue and diced as finely as possible using a scalpel blade. The tissue is placed into a 1.5ml micro‐ centrifuge tube with 450μl of 1x TES Buffer (1M NaCl, 0.5M Tris-HCl, 10mM EDTA), 60μl of 20μg/μl Proteinase K (Roche) and 10μl of 10% SDS, and incubated overnight with vigorous shaking. Following digestion, an equal volume of phenol is added and the sample is vor‐ texed vigorously. Once homogenous, the sample is centrifuged to separate the layers and the top aqueous layer is removed and transferred to a fresh 1.5ml centrifuge tube. An equal volume of chloroform is then added to the aqueous layer. This is vortexed, centrifuged, and the aqueous (top) layer again transferred to a fresh tube. A 2x volume of 100% ethanol is added to the aqueous layer to precipitate the DNA, followed by centrifugation at 13,000rpm and the supernatant is removed. The pellet is then washed with 70% ethanol and re-centri‐ fuged for 2 minutes at 13,000rpm. The supernatant is again removed, and the pellet air dried prior to re-suspension in TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.0-8.0).

The quality and quantity of extracted gDNA is measured using a NanoDrop ND-1000 Spec‐ trophotometer.

#### *2.1.3. PCR*

PCR amplification is performed in a final 25μl reaction volume with the following reagents: Faststart buffer (Roche Applied Science), 2mM MgCl2 (Roche Applied Science), M13-tailed forward and reverse primers at 0.8μM each (synthesised by Integrated DNA Technologies Inc), 0.4mM dNTPs (GE Healthcare Ltd), 1 unit Faststart Taq DNA polymerase (Roche Ap‐ plied Science) and 5μl GC-rich solution (Roche Applied Science). 50ng of gDNA is included in each reaction. PCR amplification is carried out with the following conditions: denatura‐ tion at 95°C for 5 minutes, followed by 35 cycles of 94°C for 45 seconds, 60°C for 30 seconds and 72°C for 30 seconds, with a final extension of 72°C for 10 minutes.

#### *2.1.4. Sequencing (Figure 2)*

5μL of each PCR is cleaned with ExoSAP-IT (Affymetrix, USB) prior to bidirectional DNA sequencing using M13 forward and reverse primers and Big-Dye Terminator v3.0 (Applied Biosystems Ltd). 20μl of each sequenced product is manually purified using the CleanSEQ Sequencing Purification System (Agencourt Bioscience). Four different drying times prior to elution (20 minutes, 24 hours, four days and seven days) were assessed to establish an opti‐ mal drying time for generating high quality sequencing data. 15μL of purified product was then subjected to capillary electrophoresis using the Applied Biosystems model 3130xl Ge‐ netic Analyzer.

The analysis of sequence traces is performed using Variant Reporter v1.0 (Applied Biosys‐ tems). Variant Reporter uses advanced algorithms and quality metrics to automate the de‐ tection of variants and to streamline the analysis process.

*2.1.2. DNA extraction*

398 Latest Research into Quality Control

er's instructions.

trophotometer.

*2.1.4. Sequencing (Figure 2)*

netic Analyzer.

*2.1.3. PCR*

Genomic DNA (gDNA) was extracted from peripheral blood leucocytes (EDTA blood sam‐ ples) using the Gentra Puregene DNA Extraction kit (Qiagen), according to the manufactur‐

A standard phenol/chloroform protease protocol was used to extract gDNA from postmor‐ tem tissue specimens. A small section (2mm x 2mm x 4mm) is usually cut from frozen tissue and diced as finely as possible using a scalpel blade. The tissue is placed into a 1.5ml micro‐ centrifuge tube with 450μl of 1x TES Buffer (1M NaCl, 0.5M Tris-HCl, 10mM EDTA), 60μl of 20μg/μl Proteinase K (Roche) and 10μl of 10% SDS, and incubated overnight with vigorous shaking. Following digestion, an equal volume of phenol is added and the sample is vor‐ texed vigorously. Once homogenous, the sample is centrifuged to separate the layers and the top aqueous layer is removed and transferred to a fresh 1.5ml centrifuge tube. An equal volume of chloroform is then added to the aqueous layer. This is vortexed, centrifuged, and the aqueous (top) layer again transferred to a fresh tube. A 2x volume of 100% ethanol is added to the aqueous layer to precipitate the DNA, followed by centrifugation at 13,000rpm and the supernatant is removed. The pellet is then washed with 70% ethanol and re-centri‐ fuged for 2 minutes at 13,000rpm. The supernatant is again removed, and the pellet air dried

prior to re-suspension in TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.0-8.0).

and 72°C for 30 seconds, with a final extension of 72°C for 10 minutes.

The quality and quantity of extracted gDNA is measured using a NanoDrop ND-1000 Spec‐

PCR amplification is performed in a final 25μl reaction volume with the following reagents: Faststart buffer (Roche Applied Science), 2mM MgCl2 (Roche Applied Science), M13-tailed forward and reverse primers at 0.8μM each (synthesised by Integrated DNA Technologies Inc), 0.4mM dNTPs (GE Healthcare Ltd), 1 unit Faststart Taq DNA polymerase (Roche Ap‐ plied Science) and 5μl GC-rich solution (Roche Applied Science). 50ng of gDNA is included in each reaction. PCR amplification is carried out with the following conditions: denatura‐ tion at 95°C for 5 minutes, followed by 35 cycles of 94°C for 45 seconds, 60°C for 30 seconds

5μL of each PCR is cleaned with ExoSAP-IT (Affymetrix, USB) prior to bidirectional DNA sequencing using M13 forward and reverse primers and Big-Dye Terminator v3.0 (Applied Biosystems Ltd). 20μl of each sequenced product is manually purified using the CleanSEQ Sequencing Purification System (Agencourt Bioscience). Four different drying times prior to elution (20 minutes, 24 hours, four days and seven days) were assessed to establish an opti‐ mal drying time for generating high quality sequencing data. 15μL of purified product was then subjected to capillary electrophoresis using the Applied Biosystems model 3130xl Ge‐


**Figure 2.** Flowchart of the sequencing method used in our laboratory.

#### **2.2. Array comparative genomic hybridization (aCGH)**

A Roche NimbleGen 12x135K Custom CGH Array was used for dosage analysis. This be‐ spoke CGH array has been designed to screen for dosage changes within the genes responsi‐ ble for LQT1-12 (*KCNQ1, KCNH2, SCN5A, ANK2, KCNE1, KCNE2, KCNJ2, CACNA1C, CAV3, SCN4B, AKAP9,* and *SNTA1*), the LQT-associated genes *GPD1L*, *KCNE3, SCN1B, SCN3B, CACNB2*, and the CPVT1 (*RYR2*) and CPVT2 (*CASQ2*) genes.

should be considered. In our laboratory, we perform this cleanup using another phenol ex‐

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

http://dx.doi.org/10.5772/51685

401

**Figure 3.** Agarose gel image of PCR amplification from an impure DNA with an acceptable A260/280 ratio (1.88) but an acceptable A260/230 ratio (2.5) . NOTE: very few exons amplified effectively and strong primer dimers are visible.

**Figure 4.** Agarose gel image of PCR products amplified from the same DNA sample after purification. NOTE: success‐

ful amplification and significantly reduced primer dimers.

traction and ethanol precipitation to further purify the gDNA sample [17].

Two hundred and fifty nanograms of gDNA are processed according to the manufacturer's instructions (NimbleGen Array User's Guide: CGH and CNV Arrays v8.0; http:// www.nimblegen.com). In brief, extracted gDNA from samples and Promega controls are de‐ natured in the presence of a Cy3- (test) or Cy5- (control) labelled random primers and incu‐ bated with the Klenow fragment of DNA polymerase, together with dNTPs (5mM of each dNTP), at 37ºC for 2 hours. The reaction is terminated by the addition of 21.5μL of 0.5M ED‐ TA, prior to isopropanol precipitation and ethanol washing. Following quantification, the test and sex-matched control samples are combined in equimolar amounts and applied to one of the twelve arrays on a microarray slide. Hybridisation is carried out in a Roche Nim‐ bleGen Hybridisation Chamber for a period of 48 hours. Slides are washed and scanned us‐ ing a NimbleGen MS 200 Microarray Scanner. Array image files (.tif) produced by the MS 200 Data Collection Software are imported into DEVA v1.2.1 (Roche NimbleGen Inc) for analysis. Each genomic region exhibiting a copy number change within one of the LQT genes of interest were examined using the UCSC genome browser (http://genome.ucsc.edu/) to determine the location and significance of the change.

#### **3. Results**

In order to overcome the historical difficulties faced when performing Long QT syndrome testing using postmortem specimens and meet the demand by coroners for rapid results, we addressed the following parameters:

#### **3.1. DNA purity**

Since poor quality gDNA leads to suboptimal PCR amplification affecting downstream ap‐ plications, the purity of gDNA is an important criterion for success in generating high quali‐ ty sequencing data [14,15]. A NanoDrop ND-1000 spectrophotometer was used to measure the quality and quantity of the extracted gDNA. The ratio of absorbance at 260nm and 280nm (A260/280 ratio) is used to assess the purity of gDNA, which should be in the range of 1.8 to 2.0 to be accepted as pure gDNA. The ratio of absorbance at 260nm and 230nm (A260/230 ratio) is used as a secondary measure of nucleic acid purity, and for pure gDNA should be in the range of 2.0-2.2 [16]. Postmortem gDNA isolated using a standard phenol/ chloroform protease protocol may contain residual phenol, chloroform or ethanol. These contaminants inhibit the activity of DNA polymerase in downstream applications (Figures 3-5) [14], so the purity of the gDNA must be strictly monitored. If a suboptimal A260/280 or A260/230 ratio indicates that the gDNA is of low quality, a secondary cleanup process should be considered. In our laboratory, we perform this cleanup using another phenol ex‐ traction and ethanol precipitation to further purify the gDNA sample [17].

**2.2. Array comparative genomic hybridization (aCGH)**

400 Latest Research into Quality Control

to determine the location and significance of the change.

addressed the following parameters:

**3. Results**

**3.1. DNA purity**

*SCN3B, CACNB2*, and the CPVT1 (*RYR2*) and CPVT2 (*CASQ2*) genes.

A Roche NimbleGen 12x135K Custom CGH Array was used for dosage analysis. This be‐ spoke CGH array has been designed to screen for dosage changes within the genes responsi‐ ble for LQT1-12 (*KCNQ1, KCNH2, SCN5A, ANK2, KCNE1, KCNE2, KCNJ2, CACNA1C, CAV3, SCN4B, AKAP9,* and *SNTA1*), the LQT-associated genes *GPD1L*, *KCNE3, SCN1B,*

Two hundred and fifty nanograms of gDNA are processed according to the manufacturer's instructions (NimbleGen Array User's Guide: CGH and CNV Arrays v8.0; http:// www.nimblegen.com). In brief, extracted gDNA from samples and Promega controls are de‐ natured in the presence of a Cy3- (test) or Cy5- (control) labelled random primers and incu‐ bated with the Klenow fragment of DNA polymerase, together with dNTPs (5mM of each dNTP), at 37ºC for 2 hours. The reaction is terminated by the addition of 21.5μL of 0.5M ED‐ TA, prior to isopropanol precipitation and ethanol washing. Following quantification, the test and sex-matched control samples are combined in equimolar amounts and applied to one of the twelve arrays on a microarray slide. Hybridisation is carried out in a Roche Nim‐ bleGen Hybridisation Chamber for a period of 48 hours. Slides are washed and scanned us‐ ing a NimbleGen MS 200 Microarray Scanner. Array image files (.tif) produced by the MS 200 Data Collection Software are imported into DEVA v1.2.1 (Roche NimbleGen Inc) for analysis. Each genomic region exhibiting a copy number change within one of the LQT genes of interest were examined using the UCSC genome browser (http://genome.ucsc.edu/)

In order to overcome the historical difficulties faced when performing Long QT syndrome testing using postmortem specimens and meet the demand by coroners for rapid results, we

Since poor quality gDNA leads to suboptimal PCR amplification affecting downstream ap‐ plications, the purity of gDNA is an important criterion for success in generating high quali‐ ty sequencing data [14,15]. A NanoDrop ND-1000 spectrophotometer was used to measure the quality and quantity of the extracted gDNA. The ratio of absorbance at 260nm and 280nm (A260/280 ratio) is used to assess the purity of gDNA, which should be in the range of 1.8 to 2.0 to be accepted as pure gDNA. The ratio of absorbance at 260nm and 230nm (A260/230 ratio) is used as a secondary measure of nucleic acid purity, and for pure gDNA should be in the range of 2.0-2.2 [16]. Postmortem gDNA isolated using a standard phenol/ chloroform protease protocol may contain residual phenol, chloroform or ethanol. These contaminants inhibit the activity of DNA polymerase in downstream applications (Figures 3-5) [14], so the purity of the gDNA must be strictly monitored. If a suboptimal A260/280 or A260/230 ratio indicates that the gDNA is of low quality, a secondary cleanup process

**Figure 3.** Agarose gel image of PCR amplification from an impure DNA with an acceptable A260/280 ratio (1.88) but an acceptable A260/230 ratio (2.5) . NOTE: very few exons amplified effectively and strong primer dimers are visible.

**Figure 4.** Agarose gel image of PCR products amplified from the same DNA sample after purification. NOTE: success‐ ful amplification and significantly reduced primer dimers.

**3.3. CleanSEQ treatment of sequencing reactions**

rious baseline noise and incorrect basecalling.

**3.4. Drying of DNA-bound beads prior to elution**

quality trace.

seven days.

using CleanSEQ reagent diluted 1:2 in ddH20 (figures 8,9).

The sequenced product is purified using an Agencourt CleanSEQ system, which uses SPRI (Solid Phase Reversible Immobilization) magnetic bead-based technology. According to the manufacturer's recommendation, the sequencing product should be cleaned using undilut‐ ed CleanSEQ reagent, but in our experience, postmortem DNA samples should be cleaned

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

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403

**Figure 8.** Electropherogram obtained from sequencing product cleaned with 10µl of undiluted CleanSEQ reagent: se‐

**Figure 9.** Electropherogram obtained from sequencing product cleaned using diluted CleanSEQ reagent: good

The length of drying time following ethanol washing and prior to elution of the sequencing product from the CleanSEQ beads is one of the critical parameters for obtaining high quality sequence data. We assessed 4 different drying periods: 20 minutes, 24 hours, four days and

A drying time of 20 minutes, as recommended by the manufacturer, is frequently associated with the presence of large 'dye blobs', most probably as a result of residual ethanol in the eluted product (Figure 10); this problem resolves if drying time is extended to 24 hours (Fig‐ ure 11). However, variable baseline noise on the electropherogram will be evident if the dry‐ ing time exceeds four days (Figure 12); further increased background noise and loss of

resolution occur when the drying time exceeds seven days (Figure 13).

**Figure 5.** Electropherogram generated from an impure DNA sample with an acceptable A260/280 ratio (1.95) but an un‐ acceptable A260/230 ratio (0.95). NOTE: significant baseline noise and data unable to be analysed.

#### **3.2. Amount of DNA template used in a sequencing reaction**

The extent of dilution of the cleaned PCR product prior to sequencing determines the amount of DNA template used in the sequencing reaction, which can affect the data quality for analysis [15]. The presence of excessive DNA template in a sequencing reaction will lead to rapidly progressive signal loss on the electropherogram (Figure 6), while using insuffi‐ cient DNA template in a sequencing reaction will result in weak signal strength and a loss of peak shape (Figure 7), causing difficulties in basecalling and accurate analysis [15]. In order to obtain high quality data with good signal strength, the amplified product should be dilut‐ ed in ddH20 to as close as possible to 2.5ng per 100bp.

**Figure 6.** Electropherogram begins with strong high peaks, which fade rapidly.

**Figure 7.** Electropherogram with increased background noise and loss of sharp peak shape.

#### **3.3. CleanSEQ treatment of sequencing reactions**

**Figure 5.** Electropherogram generated from an impure DNA sample with an acceptable A260/280 ratio (1.95) but an un‐

The extent of dilution of the cleaned PCR product prior to sequencing determines the amount of DNA template used in the sequencing reaction, which can affect the data quality for analysis [15]. The presence of excessive DNA template in a sequencing reaction will lead to rapidly progressive signal loss on the electropherogram (Figure 6), while using insuffi‐ cient DNA template in a sequencing reaction will result in weak signal strength and a loss of peak shape (Figure 7), causing difficulties in basecalling and accurate analysis [15]. In order to obtain high quality data with good signal strength, the amplified product should be dilut‐

acceptable A260/230 ratio (0.95). NOTE: significant baseline noise and data unable to be analysed.

**3.2. Amount of DNA template used in a sequencing reaction**

402 Latest Research into Quality Control

ed in ddH20 to as close as possible to 2.5ng per 100bp.

**Figure 6.** Electropherogram begins with strong high peaks, which fade rapidly.

**Figure 7.** Electropherogram with increased background noise and loss of sharp peak shape.

The sequenced product is purified using an Agencourt CleanSEQ system, which uses SPRI (Solid Phase Reversible Immobilization) magnetic bead-based technology. According to the manufacturer's recommendation, the sequencing product should be cleaned using undilut‐ ed CleanSEQ reagent, but in our experience, postmortem DNA samples should be cleaned using CleanSEQ reagent diluted 1:2 in ddH20 (figures 8,9).

**Figure 8.** Electropherogram obtained from sequencing product cleaned with 10µl of undiluted CleanSEQ reagent: se‐ rious baseline noise and incorrect basecalling.

**Figure 9.** Electropherogram obtained from sequencing product cleaned using diluted CleanSEQ reagent: good quality trace.

#### **3.4. Drying of DNA-bound beads prior to elution**

The length of drying time following ethanol washing and prior to elution of the sequencing product from the CleanSEQ beads is one of the critical parameters for obtaining high quality sequence data. We assessed 4 different drying periods: 20 minutes, 24 hours, four days and seven days.

A drying time of 20 minutes, as recommended by the manufacturer, is frequently associated with the presence of large 'dye blobs', most probably as a result of residual ethanol in the eluted product (Figure 10); this problem resolves if drying time is extended to 24 hours (Fig‐ ure 11). However, variable baseline noise on the electropherogram will be evident if the dry‐ ing time exceeds four days (Figure 12); further increased background noise and loss of resolution occur when the drying time exceeds seven days (Figure 13).

**4. Discussion**

**4.1. DNA quality**

*4.1.1. Sequence analysis*

*4.1.2. Dosage analysis (aCGH)*

should be considered before processing any further.

creased volume of template gDNA should be undertaken.

**Figure 14.** Agarose gel (2%) check of gDNA quality prior to aCGH.

The gDNA used in a PCR should be as pure as possible in order to optimise the quality of the template that will be used in downstream applications. The purity of DNA is assessed in our laboratory using a NanoDrop ND-1000 spectrophotometer. It is important to consider both A260/280 and A260/230 as poor DNA quality will affect downstream applications [16,18]. An unacceptable A260/280 or A260/230 ratio indicates the presence of contaminants in the DNA; an abnormal A260/230 indicates the presence of residual phenol or other chemi‐ cal from the extraction process, while an abnormal A260/280 most frequently indicates the presence of protein [16,18]. If any of the ratios appear to be abnormal, DNA purification

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405

The quality requirements for gDNA used in an aCGH assay are not as stringent as those for a sequencing assay. An awareness of the presence of significant degradation is important; however, partial compensation for this can be made by increasing the volume of gDNA used in the amplification and labelling step. In our laboratory, we have found that doubling the volume of gDNA when degradation is visible on the 'check gel' (present in lanes 5-8 of Figure 14) is an effective counter-measure. The adequacy of amplification is measured using a NanoDrop ND-1000 spectrophotometer in order to allow the hybridisation of equimolar amounts of test and control DNA to the array slide. A post-amplification concentration of greater than 2500ng/μl is ideal for further processing; a sample with a post-amplification concentration of less than 1500ng/μl is unlikely to produce good quality data for analysis. Therefore, if there is sufficient volume of gDNA available, a repeat amplification with an in‐

**Figure 10.** Electropherogram of purified products dried for 20 minutes prior to elution; presence of large dye blob.

**Figure 11.** Electropherogram of purified products dried for 24 hours prior to elution; free of dye blobs, minimal base‐ line noise.

**Figure 12.** Electropherogram of purified products dried for 4 days prior to elution; presence of shoulders and messy baseline.

**Figure 13.** Electropherogram of purified products dried for 7 days prior to elution; extremely messy baseline and early loss of resolution.

#### **4. Discussion**

#### **4.1. DNA quality**

**Figure 10.** Electropherogram of purified products dried for 20 minutes prior to elution; presence of large dye blob.

**Figure 11.** Electropherogram of purified products dried for 24 hours prior to elution; free of dye blobs, minimal base‐

**Figure 12.** Electropherogram of purified products dried for 4 days prior to elution; presence of shoulders and

**Figure 13.** Electropherogram of purified products dried for 7 days prior to elution; extremely messy baseline and early

line noise.

404 Latest Research into Quality Control

messy baseline.

loss of resolution.

#### *4.1.1. Sequence analysis*

The gDNA used in a PCR should be as pure as possible in order to optimise the quality of the template that will be used in downstream applications. The purity of DNA is assessed in our laboratory using a NanoDrop ND-1000 spectrophotometer. It is important to consider both A260/280 and A260/230 as poor DNA quality will affect downstream applications [16,18]. An unacceptable A260/280 or A260/230 ratio indicates the presence of contaminants in the DNA; an abnormal A260/230 indicates the presence of residual phenol or other chemi‐ cal from the extraction process, while an abnormal A260/280 most frequently indicates the presence of protein [16,18]. If any of the ratios appear to be abnormal, DNA purification should be considered before processing any further.

#### *4.1.2. Dosage analysis (aCGH)*

The quality requirements for gDNA used in an aCGH assay are not as stringent as those for a sequencing assay. An awareness of the presence of significant degradation is important; however, partial compensation for this can be made by increasing the volume of gDNA used in the amplification and labelling step. In our laboratory, we have found that doubling the volume of gDNA when degradation is visible on the 'check gel' (present in lanes 5-8 of Figure 14) is an effective counter-measure. The adequacy of amplification is measured using a NanoDrop ND-1000 spectrophotometer in order to allow the hybridisation of equimolar amounts of test and control DNA to the array slide. A post-amplification concentration of greater than 2500ng/μl is ideal for further processing; a sample with a post-amplification concentration of less than 1500ng/μl is unlikely to produce good quality data for analysis. Therefore, if there is sufficient volume of gDNA available, a repeat amplification with an in‐ creased volume of template gDNA should be undertaken.

**Figure 14.** Agarose gel (2%) check of gDNA quality prior to aCGH.

#### **4.2. Amount of DNA template used in a sequencing reaction**

The DNA template should be diluted to 2.5ng per 100bp prior to sequencing. Incorrect quantification and dilution will alter the amount of input DNA template in a sequencing reaction, which can lead to problematic data for analysis. In our laboratory, we use Var‐ iant ReporterTM (Applied Biosystems Ltd) for automated analysis of sequence data. The signal strength of the sequence data is automatically assessed. Data with good signal strength is above the value of 200 in the Variant ReporterTM software. Sequence data with weak signal strength, a value below 100, indicates insufficient DNA template has been used in the sequencing reaction. Noisy background and a loss of sharp peak shape are al‐ so commonly evident. A repeat treatment with EXOSAP-IT and less extensive dilution of product is necessary to avoid inaccurate analysis using sequence data with unacceptable quality. In contrast, when excess DNA template is used in a sequencing reaction, a rapid‐ ly progressive signal loss is seen on the electropherogram. In this instance, trace quality can be improved by diluting the eluted product with water and reloading the sample on the capillary sequencing platform (we use an Applied Biosystems model 3130xl Genetic Analyzer).

rate final concentration of ethanol in an attempt to achieve consistency of evaporation/ drying between sequencing runs. Ethanol is highly volatile and it is therefore difficult to achieve consistency - even the short period of time in which the vessel containing the ethanol is open to allow access can result in a decrease in concentration. Here, we showed that the issue of dye blobs can be resolved by simply extending the drying time to 24 hours, allowing adequate time for all ethanol to evaporate completely, despite any

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

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407

On the other hand, we found that the quality of sequence data is adversely affected if the drying time is extended to more than 4 days. This suggests that the stability of purified sequencing products is another factor that affects data quality. High quality sequence da‐ ta was obtained with a drying time of 24 hours; however, the sequences were subopti‐ mal when elution and electrophoresis were carried out at day 4 or day 7 of drying. This indicates that purified sequencing product is most stable for the first 24 hours and that consideration of the sample stability should be taken into account when aiming for high

All sequence traces are analysed in our laboratory using Variant Reporter Software v1.0 (Applied Biosystems). The current CMGS best practice guidelines for Sanger sequence analysis in diagnostic laboratories [19] recommend a PHRED score of at least 20 for bidir‐ ectional data (corresponding to 99% confidence that the base is called correctly), and a PHRED score of at least 30 for unidirectional data (99.9% confidence that each base is called correctly). The procedure we describe above produces bidirectional sequence data that meet our laboratory's even more stringent analysis criteria: a Variant Reporter trace

In order to provide simultaneous dosage analysis of all the Long QT syndrome genes, we developed a custom designed Roche NimbleGen 12x135K CGH array. As part of the vali‐ dation of this array we analysed twenty patients with known copy number abnormalities [20]. Analysis of the data from these individuals with known copy number changes al‐ lowed precise threshold criteria to be developed. It was determined that a log2ratio ≤-0.4 over 6 contiguous probes is indicative of a deletion, and a log2ratio of ≥0.4 over 15 con‐

minor differences in the concentration of ethanol used.

score of 35, corresponding to a false base call rate of 0.031%.

quality data.

*4.5.1. Sequencing*

*4.5.2. Dosage analysis*

tiguous probes indicative of a duplication.

**4.5. Analysis parameters**

#### **4.3. CleanSEQ treatment of sequencing reactions**

Agencourt CleanSEQ is routinely used to purify the sequenced products. According to the manufacturer's recommendation, 20μl of sequencing product should be cleaned with 10μl of CleanSEQ reagent. However, we found that this leads to an excess of CleanSEQ beads in relation to the amount of sequence product, therefore unwanted short fragments (e.g unincorporated dNTPs and excess primers) were able to bind to beads during purifi‐ cation, and the quality of sequence data generated was consequently reduced. We found that diluting the CleanSEQ beads 1:2 with ddH20 leads to much higher quality data.

#### **4.4. Drying of DNA-bound beads prior to elution**

This drying step is a critical QC consideration that significantly affects sequence quality. When the purified product is dried for 24 hours prior to elution, the subsequent electro‐ pherograms are free of dye blobs, C-shoulders and baseline noise, and consistently pass all QC metrics in Variant Reporter.

A drying time of 20 minutes, as suggested by manufacturer, was found to be problemat‐ ic, causing the presence of dye blobs. Dye blobs are a common artefact due to either the presence of excess unincorporated dye or residual ethanol following product purification [15]. Previously, operators have focused on purifying sequenced products using an accu‐ rate final concentration of ethanol in an attempt to achieve consistency of evaporation/ drying between sequencing runs. Ethanol is highly volatile and it is therefore difficult to achieve consistency - even the short period of time in which the vessel containing the ethanol is open to allow access can result in a decrease in concentration. Here, we showed that the issue of dye blobs can be resolved by simply extending the drying time to 24 hours, allowing adequate time for all ethanol to evaporate completely, despite any minor differences in the concentration of ethanol used.

On the other hand, we found that the quality of sequence data is adversely affected if the drying time is extended to more than 4 days. This suggests that the stability of purified sequencing products is another factor that affects data quality. High quality sequence da‐ ta was obtained with a drying time of 24 hours; however, the sequences were subopti‐ mal when elution and electrophoresis were carried out at day 4 or day 7 of drying. This indicates that purified sequencing product is most stable for the first 24 hours and that consideration of the sample stability should be taken into account when aiming for high quality data.

#### **4.5. Analysis parameters**

#### *4.5.1. Sequencing*

**4.2. Amount of DNA template used in a sequencing reaction**

**4.3. CleanSEQ treatment of sequencing reactions**

**4.4. Drying of DNA-bound beads prior to elution**

all QC metrics in Variant Reporter.

Analyzer).

406 Latest Research into Quality Control

The DNA template should be diluted to 2.5ng per 100bp prior to sequencing. Incorrect quantification and dilution will alter the amount of input DNA template in a sequencing reaction, which can lead to problematic data for analysis. In our laboratory, we use Var‐ iant ReporterTM (Applied Biosystems Ltd) for automated analysis of sequence data. The signal strength of the sequence data is automatically assessed. Data with good signal strength is above the value of 200 in the Variant ReporterTM software. Sequence data with weak signal strength, a value below 100, indicates insufficient DNA template has been used in the sequencing reaction. Noisy background and a loss of sharp peak shape are al‐ so commonly evident. A repeat treatment with EXOSAP-IT and less extensive dilution of product is necessary to avoid inaccurate analysis using sequence data with unacceptable quality. In contrast, when excess DNA template is used in a sequencing reaction, a rapid‐ ly progressive signal loss is seen on the electropherogram. In this instance, trace quality can be improved by diluting the eluted product with water and reloading the sample on the capillary sequencing platform (we use an Applied Biosystems model 3130xl Genetic

Agencourt CleanSEQ is routinely used to purify the sequenced products. According to the manufacturer's recommendation, 20μl of sequencing product should be cleaned with 10μl of CleanSEQ reagent. However, we found that this leads to an excess of CleanSEQ beads in relation to the amount of sequence product, therefore unwanted short fragments (e.g unincorporated dNTPs and excess primers) were able to bind to beads during purifi‐ cation, and the quality of sequence data generated was consequently reduced. We found that diluting the CleanSEQ beads 1:2 with ddH20 leads to much higher quality data.

This drying step is a critical QC consideration that significantly affects sequence quality. When the purified product is dried for 24 hours prior to elution, the subsequent electro‐ pherograms are free of dye blobs, C-shoulders and baseline noise, and consistently pass

A drying time of 20 minutes, as suggested by manufacturer, was found to be problemat‐ ic, causing the presence of dye blobs. Dye blobs are a common artefact due to either the presence of excess unincorporated dye or residual ethanol following product purification [15]. Previously, operators have focused on purifying sequenced products using an accu‐ All sequence traces are analysed in our laboratory using Variant Reporter Software v1.0 (Applied Biosystems). The current CMGS best practice guidelines for Sanger sequence analysis in diagnostic laboratories [19] recommend a PHRED score of at least 20 for bidir‐ ectional data (corresponding to 99% confidence that the base is called correctly), and a PHRED score of at least 30 for unidirectional data (99.9% confidence that each base is called correctly). The procedure we describe above produces bidirectional sequence data that meet our laboratory's even more stringent analysis criteria: a Variant Reporter trace score of 35, corresponding to a false base call rate of 0.031%.

#### *4.5.2. Dosage analysis*

In order to provide simultaneous dosage analysis of all the Long QT syndrome genes, we developed a custom designed Roche NimbleGen 12x135K CGH array. As part of the vali‐ dation of this array we analysed twenty patients with known copy number abnormalities [20]. Analysis of the data from these individuals with known copy number changes al‐ lowed precise threshold criteria to be developed. It was determined that a log2ratio ≤-0.4 over 6 contiguous probes is indicative of a deletion, and a log2ratio of ≥0.4 over 15 con‐ tiguous probes indicative of a duplication.

#### **4.6. Other parameters**

It is important to monitor other QC parameters when it comes to sequence analysis of Long QT specimens: the freshness of the Polymer (POP-7) and the usage of the capillaries in the Applied Biosystems model 3130xl Genetic Analyzer.

Polymer (POP-7) is used to separate DNA fragments on genetic analyzers and the poly‐ mer remains stable for up to 7 days [15]. The capillary in the genetic analyzer should be replaced after 1000 injections. Within our laboratory, we perform a regular weekly mainte‐ nance of the analyzer, and the capillary is replaced when 600 injections are reached. From past experience, both the freshness of polymer and the usage of the capillary play a vital role in the data quality. Electropherograms with reduced resolution and peak shape are produced when either of these two parameters is suboptimal. In order to achieve high quality data, both freshness of polymer and usage of the capillary should be closely monitored.

#### **5. Conclusions**

We have successfully established a robust method for processing postmortem specimens for Long QT diagnostic testing in a timely manner. The electropherograms in Figure 15 are indi‐ cative of the high quality data routinely produced, despite the limitations inherent in the types of specimens that are referred. The blood sample used to extract gDNA for sequencing in this example was heavily haemolysed on arrival at the laboratory.

**Figure 15.** High quality sequence data generated from a haemolysed blood specimen; flat baseline along with distinct and evenly spaced peaks allow accurate basecalling.

**Figure 16.** Flowchart of potential QC problems and their solutions.

We acknowledge the assistance of Dr Andrew Dodd in the initial design of primers against the coding regions of the LQT genes, and Mr Daniel Lai for his assistance in optimizing as‐

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

http://dx.doi.org/10.5772/51685

409

**Acknowledgements**

pects of our amplicon clean-up.

The QC parameters described above should be monitored closely in order to consistently achieve optimal results (Figure 16). Although the basic procedure is essentially the same as that routinely used for sequence and aCGH analysis, the poor quality of postmortem speci‐ mens as a source of template DNA mean that particular attention needs to be paid to each step, in particular the critical initial assessment of DNA quality, the addition of the appro‐ priate volume of template DNA to the sequencing reaction, and the drying time of the beads used in the purification procedure prior to elution.

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome http://dx.doi.org/10.5772/51685 409

**Figure 16.** Flowchart of potential QC problems and their solutions.

#### **Acknowledgements**

**4.6. Other parameters**

408 Latest Research into Quality Control

**5. Conclusions**

Applied Biosystems model 3130xl Genetic Analyzer.

It is important to monitor other QC parameters when it comes to sequence analysis of Long QT specimens: the freshness of the Polymer (POP-7) and the usage of the capillaries in the

Polymer (POP-7) is used to separate DNA fragments on genetic analyzers and the poly‐ mer remains stable for up to 7 days [15]. The capillary in the genetic analyzer should be replaced after 1000 injections. Within our laboratory, we perform a regular weekly mainte‐ nance of the analyzer, and the capillary is replaced when 600 injections are reached. From past experience, both the freshness of polymer and the usage of the capillary play a vital role in the data quality. Electropherograms with reduced resolution and peak shape are produced when either of these two parameters is suboptimal. In order to achieve high quality data, both freshness of polymer and usage of the capillary should be closely monitored.

We have successfully established a robust method for processing postmortem specimens for Long QT diagnostic testing in a timely manner. The electropherograms in Figure 15 are indi‐ cative of the high quality data routinely produced, despite the limitations inherent in the types of specimens that are referred. The blood sample used to extract gDNA for sequencing

**Figure 15.** High quality sequence data generated from a haemolysed blood specimen; flat baseline along with distinct

The QC parameters described above should be monitored closely in order to consistently achieve optimal results (Figure 16). Although the basic procedure is essentially the same as that routinely used for sequence and aCGH analysis, the poor quality of postmortem speci‐ mens as a source of template DNA mean that particular attention needs to be paid to each step, in particular the critical initial assessment of DNA quality, the addition of the appro‐ priate volume of template DNA to the sequencing reaction, and the drying time of the beads

in this example was heavily haemolysed on arrival at the laboratory.

and evenly spaced peaks allow accurate basecalling.

used in the purification procedure prior to elution.

We acknowledge the assistance of Dr Andrew Dodd in the initial design of primers against the coding regions of the LQT genes, and Mr Daniel Lai for his assistance in optimizing as‐ pects of our amplicon clean-up.

#### **Author details**

Stella Lai1 , Renate Marquis-Nicholson1,2\*, Chuan-Ching Lan1 , Jennifer M. Love1 , Elaine Doherty1 , Jonathan R. Skinner3 and Donald R. Love1,2\*

\*Address all correspondence to: donaldl@adhb.govt.nz


3 Inherited Disease Group New Zealand, Paediatric Cardiac Services, Starship Childrens' Hospital, New Zealand

[9] Skinner, J. R., Crawford, J., Smith, W., Aitken, A., Heaven, D., & Evans, CA. (2011). Prospective, population-based long QT molecular autopsy study of postmortem neg‐

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

http://dx.doi.org/10.5772/51685

411

[10] Gladding, P. A., Evans, C. A., Crawford, J., Chung, S. K., Vaughan, A., Webster, D., et al. (2010). Posthumous diagnosis of long QT syndrome from neonatal screening

[11] Skinner, J. R. (2010). Sudden Unexplained Death in Infancy and Long QT syndrome.

[12] Doherty, E., Marquis-Nicholson, R., Love, J. M., Brookes, C., Prosser, D., & Love, D. R. (2011). Primer Design to Sequence Analysis- a Pipeline for a Molecular Genetic Di‐ agnostic Laboratory, Applications and Experiences of Quality Control, Ognyan Iva‐ nov (Ed.). 978-9-53307-236-4, InTech, Available from, http://www.intechopen.com/ books/applications-and-experiences-of-quality-control/primer-design-to-sequence-

[13] Lai, D., & Love, D. R. (2012). Automation of a primer design and evaluation pipeline for subsequent sequencing of the coding regions of all human Refseq genes. *Bioinfor‐*

[14] Verhagen, O. J., Wijkhuijs, A. J., van der Sluijs-Gelling, A. J., Szczepanski, T., van der Linden-Schrever, B. E., Pongers-Willemse van, M. J., Wering, E. R., van Dongen, J. J., & van der Schoot, C. E. (1999). Suitable DNA isolation method for the detection of

[15] Applied Biosystems Chemistry Guide, second edition. (2009). DNA Sequencing by Capillary Electrophoresis. Available from:, http:// www3.appliedbiosystems.com/cms/groups/mcb\_support/documents/generaldocu‐

[16] T009 - Technical Support Bulletin and 260/230Ratios. (2008). Available from:, http:// www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucle‐

[17] Powell, R., & Gannon, F. (2002). Purification of DNA by phenol extraction and etha‐ nol precipitation. *Oxford Practical Approach Series, Oxford University Press.*, Available

[18] T042 - Technical Bulletin. (2011). Assessment of Nucleic Acid Purity. Available from:, http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-

[19] Ellard, S., Charlton, R., Yau, M., Gokhale, D., Taylor, G., Wallace, A., & Ramsden, S. C. (2009). Practice guidelines for Sanger Sequencing Analysis and Interpretation. Available from:, http://www.cmgs.org/BPGs/pdfs%20current%20bpgs/Sequen‐

from:, http://fds.oup.com/www.oup.co.uk/pdf/pas/9v1-7 -3.pdf.

minimal residual disease by PCR techniques. *Leukemia*, 13(8), 1298-1299.

ative sudden death in 1 to 40 year olds. *Heart Rhythm*, 8(3), 412-419.

analysis-a-pipeline-for-a-molecular-genetic-diagnostic-laboratory.

cards. *Heart Rhythm*, 7(4), 481-486.

*Current Pediatric Reviews*, 6, 48-55.

*mation*, 8(8), 363-366.

ments/cms\_041003.pdf.

ic-Acid-Purity-Ratios.pdf.

Acid-Purity-Ratios.pdf.

cingv2.pdf.

#### **References**


[9] Skinner, J. R., Crawford, J., Smith, W., Aitken, A., Heaven, D., & Evans, CA. (2011). Prospective, population-based long QT molecular autopsy study of postmortem neg‐ ative sudden death in 1 to 40 year olds. *Heart Rhythm*, 8(3), 412-419.

**Author details**

410 Latest Research into Quality Control

Elaine Doherty1

Hospital, New Zealand

872-880.

1761-1767.

*Crit Care Pain.*, 8, 67-70.

**References**

, Renate Marquis-Nicholson1,2\*, Chuan-Ching Lan1

2 School of Biological Sciences, The University of Auckland, New Zealand

channelopathies: a HuGE review. *Genet Med.*, 8(3), 143-55.

www.ncbi.nlm.nih.gov/books/NBK1129/.

mortem tissues. *Mol Pathol.*, 56, 184-186.

and Donald R. Love1,2\*

3 Inherited Disease Group New Zealand, Paediatric Cardiac Services, Starship Childrens'

[1] Hunter, J. D., Sharma, P., & Rathi, S. (2008). Long QT syndrome. *Contin Educ Anaesth*

[2] Modell, S. M., & Lehmann, M. H. (2006). The long QT syndrome family of cardiac ion

[3] Alders, M., & Mannens, M. M. A. M. (2003). Romano-Ward Syndrome. Feb 20 [Up‐ dated 2012 May 31]., *In: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle;*, 1993Available from:, http://

[4] Yang, Y., Yang, Y., Liang, B., Liu, J., Li, J., Grunnet, M., Olesen, S. P., Rasmussen, H. B., Ellinor, P. T., Gao, L., Lin, X., Li, L., Wang, L., Junjie, J. X., Liu, Y. Y., Liu, Y. Y., Zhang, S. S., Lian, D. D., Peng, L. Y., Jespersen, T., & Chen, Y. H. (2010). Identifica‐ tion of a Kir3.4 mutation in congenital long QT syndrome. *Am. J. Hum. Genet.*, 86,

[5] Dan, M., & Roden, M. D. (2008). Long-QT Syndrome. *N Engl J Med.*, 358, 169-176.

KCNH2 in patients with long QT syndrome. *Heart Rhythm*, 5(9), 1275-1281.

[6] Eddy, C. A., MacCormick, J. M., Chung, S. K., Crawford, J. R., Love, D. R., Rees, M. I., et al. (2008). Identification of large gene deletions and duplications in KCNQ1 and

[7] Bonin, S., Petrera, F., Niccolini, B., & Stanta, G. (2003). PCR analysis in archival post

[8] Schwartz, P. J., Stramba-Badiale, M., Crotti, L., Pedrazzini, M., Besana, A., Bosi, G., et al. (2009). Prevalence of the congenital long-QT syndrome. *Circulation*, 120(18),

, Jonathan R. Skinner3

\*Address all correspondence to: donaldl@adhb.govt.nz

1 Diagnostic Genetics, Auckland City Hospital, New Zealand

, Jennifer M. Love1

,

Stella Lai1


[20] Marquis-Nicholson, R., Doherty, E., Thrush, A., Love, J. M., Lan-C, C., George, A. M., & Love, D. R. (2012). Array-based identification of copy number changes: simultane‐ ous gene-focused and low resolution whole genome analysis. *Sultan Qaboos Universi‐ ty Medical Journal*, submitted.

**Chapter 19**

**Quality Assurance in Antimicrobial Susceptibility**

Most of the clinically important bacteria causing infections in humans are capable of exhibit‐ ing resistance to antimicrobial agents commonly used for the treatment. Therefore, upon iso‐ lation of the organism in the clinical microbiology laboratory, characterization frequently also employs tests to detect its antimicrobial susceptibility. Thus, the report produced by clinical microbiology laboratory for the physician, also includes organism's susceptibility profile to different antimicrobials along with its identification [1]. Antimicrobial susceptibility testing (AST) is performed on bacteria that are isolated from clinical specimens to determine if the bacterial etiology of concern can be killed or inhibited by antimicrobial drugs that are poten‐ tial choices for therapy, at the concentrations of the drugs that are attainable at the site of infec‐ tion using the dosing regimen indicated in the drug product's labeling. The results of AST are generally reported with interpretive categories. The category "susceptible" indicates that the bacteria are inhibited by the usually achievable concentrations of antimicrobial agent when the dosage recommended to treat the site of infection is used. The "intermediate" category de‐ fines the bacteria for which the response rates to usually attainable blood and tissue levels of antimicrobial agent are lower compared to susceptible isolates. The intermediate category plays the role of a buffer zone between the susceptible and resistant categories, but also indi‐ cates a number of other possibilities; the antimicrobials which are concentrated at the site of infection may be regarded as options for treatment (e.g., nitrofurantoin for the urinary tract infections). The "resistant" category, however, defines the bacteria which are not inhibited by the usually achievable concentrations of the agent with normal dosage regimens and that the clinical efficacy of the agent against the isolate may not be sufficient [2]. Clinicians consider these interpretations to determine which antimicrobial agent might be effective in treating the particular patient. The primary role of routine microbiology laboratories is to provide accu‐

> © 2012 Karatuna; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Karatuna; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

**Testing**

Onur Karatuna

**1. Introduction**

http://dx.doi.org/10.5772/51998

## **Quality Assurance in Antimicrobial Susceptibility Testing**

#### Onur Karatuna

[20] Marquis-Nicholson, R., Doherty, E., Thrush, A., Love, J. M., Lan-C, C., George, A. M.,

*ty Medical Journal*, submitted.

412 Latest Research into Quality Control

& Love, D. R. (2012). Array-based identification of copy number changes: simultane‐ ous gene-focused and low resolution whole genome analysis. *Sultan Qaboos Universi‐*

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51998

#### **1. Introduction**

Most of the clinically important bacteria causing infections in humans are capable of exhibit‐ ing resistance to antimicrobial agents commonly used for the treatment. Therefore, upon iso‐ lation of the organism in the clinical microbiology laboratory, characterization frequently also employs tests to detect its antimicrobial susceptibility. Thus, the report produced by clinical microbiology laboratory for the physician, also includes organism's susceptibility profile to different antimicrobials along with its identification [1]. Antimicrobial susceptibility testing (AST) is performed on bacteria that are isolated from clinical specimens to determine if the bacterial etiology of concern can be killed or inhibited by antimicrobial drugs that are poten‐ tial choices for therapy, at the concentrations of the drugs that are attainable at the site of infec‐ tion using the dosing regimen indicated in the drug product's labeling. The results of AST are generally reported with interpretive categories. The category "susceptible" indicates that the bacteria are inhibited by the usually achievable concentrations of antimicrobial agent when the dosage recommended to treat the site of infection is used. The "intermediate" category de‐ fines the bacteria for which the response rates to usually attainable blood and tissue levels of antimicrobial agent are lower compared to susceptible isolates. The intermediate category plays the role of a buffer zone between the susceptible and resistant categories, but also indi‐ cates a number of other possibilities; the antimicrobials which are concentrated at the site of infection may be regarded as options for treatment (e.g., nitrofurantoin for the urinary tract infections). The "resistant" category, however, defines the bacteria which are not inhibited by the usually achievable concentrations of the agent with normal dosage regimens and that the clinical efficacy of the agent against the isolate may not be sufficient [2]. Clinicians consider these interpretations to determine which antimicrobial agent might be effective in treating the particular patient. The primary role of routine microbiology laboratories is to provide accu‐

© 2012 Karatuna; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Karatuna; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

rate and timely antimicrobial susceptibility test results for guiding the treatment of infectious diseases. In order to achieve that, the microbiologist should inform the clinician about wheth‐ er an infectious agent is present in the patient's specimen and which antimicrobial agent should provide the optimum therapy. Although the importance of antimicrobial susceptibili‐ ty testing is well established, the procedure itself is very sensitive to changes in the environ‐ ment and test conditions. Therefore, it is crucial that each variable in the procedure should be standardized and carefully controlled. With more reliable susceptibility results, infectious dis‐ ease specialists and public health leaders can be able to recognize emerging resistance and novel resistance patterns. Additionally, the results of AST can be applied to define the agent of choice for empirical therapy, establish institutional or nationwide policies for prescribing of antibiotics, conduct epidemiological studies or resistance surveillance, and to evaluate the ef‐ ficacy of newly developed agents. Owing to numerous variables that may affect the results, rigorous quality control is of utmost importance for susceptibility testing. Properly per‐ formed quality control would aid in providing accurate, reproducible and timely results. In this chapter the components of a quality assurance program for antimicrobial susceptibility testing will be highlighted.

Nationwide attempts were made to standardize AST methodologies; Clinical and Laborato‐ ry Standards Institute (CLSI, formerly NCCLS) (USA) [11], Werkgroep Richtlijnen Gevoe‐ ligheidsbepalingen (Netherlands) [12], Comité de l'Antibiogramme de la Société Française de Microbiologie (France) [13], the Swedish Reference Group for Antibiotics (Sweden) [14], Deutsches Institut für Normung (Germany) [15], the British Society for Antimicrobial Che‐ motherapy (UK) [16], they all published guidelines to improve the methodology and inter‐ pretation of AST. Recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST), a non-profit organization under the auspices of European Society of Clinical Mi‐ crobiology and Infectious Diseases (ESCMID), developed and published AST guidelines. Breakpoint and QC tables for disk diffusion and minimum inhibitory concentration (MIC)

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In clinical laboratories, widely adopted AST methods are disk diffusion and broth dilution methods. In disk diffusion method, disks impregnated with antimicrobial agents are used. The disks are placed onto agar plates which are preinoculated with the suspension of the microorganism being tested. The basic principle of the disk diffusion method is the diffusion of the antimicrobial agent into the medium which occurs when the disks come into contact with the moist surface of the plate. The concentration of the agent reduces logarithmically as the distance from the disk is increased. After the incubation period the plates are observed for the circular inhibiton zone created around the disk which is due to the inhibitory effect of the antimicrobial agent on the microorganism. Within the zone the concentration of the agent is sufficient to inhibit growth, whereas at the point where the concentration of the agent is no longer enough to inhibit growth, the organism is able to grow and forms a lawn of bacteria around the disk. To interprete the test results, the radius of the inhibition zone is measured and compared against the predefined values provided by the guidelines [18]. The most widely used guidelines are the CLSI and EUCAST guidelines [2, 17]. CLSI divides the results into three categories for most of the organism-agent combinations; susceptible, inter‐ mediate and resistant, whereas EUCAST uses only two categories, susceptible and resistant.

In the dilution methods, however, the susceptibility of the microorganisms to antimicrobial agents is determined whether in tubes (macrobroth dilution method) or in microtube wells molded into a plastic plate (microbroth dilution method). Both broth dilution methods use the same principle; first serial two-fold dilutions of the antimicrobial agent to be tested are made in the tubes/wells containing broth, and then same amount of bacterial suspension is distributed on each tube/well. At the end of the incubation period, the tubes/wells are exam‐ ined for turbidity which is the indicator of bacterial growth in broth. The tubes/wells remain clear where the concentration of the agent is high enough to inhibit the bacterial growth, whereas at lower concentrations of the agent, the bacteria may grow which causes the tube/ well become turbid. The lowest concentration of antimicrobial agent that prevents the *in vi‐ tro* growth of bacteria is defined as the minimal inhibitory concentration (MIC) [18]. As in the disk diffusion method, the MIC values are compared against the predefined values pro‐

vided by the guidelines and their intrepretive category is determined and reported.

testing can freely be accessed on organization's website [17].

#### **2. Overview of the antimicrobial susceptibility testing methodologies**

Fleming was first to report the inhibitory effect of penicillin on agar by observing a zone of growth inhibition of staphylococcal colonies grown next to a *Penicillium* contaminant on an agar plate. Fleming also made two significant contributions to the field of AST in the 1920s. In 1924, he introduced the use of the ditch plate technique for evaluating antimicrobial qual‐ ities of antiseptic solutions [3]. Fleming's second contribution to modern AST was the devel‐ opment of broth dilution technique using turbidity as an end-point determination [4]. Filter paper disks incorporating penicillin were utilized by Vincent & Vincent for assaying this newly discovered compound in 1940s [5]. Agar dilution AST method was also described in the 1940s [6]. At an early stage, it was realized that there were many variables affecting AST methods [7]. In 1961, World Health Organization (WHO) published a report on standardiza‐ tion of AST methodology [8]. The broad application of AST was introduced to clinical labo‐ ratories by the efforts of Bauer, Kirby and co-workers, with the method known as Kirby-Bauer disk diffusion method which is still the most widely used AST technique in the world [9]. Bergeron & Ouellette highlighted the shortcomings of the phenotypic approach to AST and concluded that different bacterial species have different susceptibilities to the same anti‐ biotic, and that there is no international aggreement on breakpoints for interpretation of an‐ timicrobial susceptibility tests [10]. The need for developing standardized AST methods became a necessity soon after antibiotics became commercially available. During World War II, following penicillin, other antibiotics were discovered and used. Altough these new anti‐ biotics were regarded as "wonder drugs" at the time of their introduction, emergence of re‐ sistant strains followed. With the emergence of bacterial resistance to antimicrobials and the changing properties of different bacteria to different classes of antimicrobials, the need for the performance of AST on pathogens became a practical necessity.

Nationwide attempts were made to standardize AST methodologies; Clinical and Laborato‐ ry Standards Institute (CLSI, formerly NCCLS) (USA) [11], Werkgroep Richtlijnen Gevoe‐ ligheidsbepalingen (Netherlands) [12], Comité de l'Antibiogramme de la Société Française de Microbiologie (France) [13], the Swedish Reference Group for Antibiotics (Sweden) [14], Deutsches Institut für Normung (Germany) [15], the British Society for Antimicrobial Che‐ motherapy (UK) [16], they all published guidelines to improve the methodology and inter‐ pretation of AST. Recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST), a non-profit organization under the auspices of European Society of Clinical Mi‐ crobiology and Infectious Diseases (ESCMID), developed and published AST guidelines. Breakpoint and QC tables for disk diffusion and minimum inhibitory concentration (MIC) testing can freely be accessed on organization's website [17].

rate and timely antimicrobial susceptibility test results for guiding the treatment of infectious diseases. In order to achieve that, the microbiologist should inform the clinician about wheth‐ er an infectious agent is present in the patient's specimen and which antimicrobial agent should provide the optimum therapy. Although the importance of antimicrobial susceptibili‐ ty testing is well established, the procedure itself is very sensitive to changes in the environ‐ ment and test conditions. Therefore, it is crucial that each variable in the procedure should be standardized and carefully controlled. With more reliable susceptibility results, infectious dis‐ ease specialists and public health leaders can be able to recognize emerging resistance and novel resistance patterns. Additionally, the results of AST can be applied to define the agent of choice for empirical therapy, establish institutional or nationwide policies for prescribing of antibiotics, conduct epidemiological studies or resistance surveillance, and to evaluate the ef‐ ficacy of newly developed agents. Owing to numerous variables that may affect the results, rigorous quality control is of utmost importance for susceptibility testing. Properly per‐ formed quality control would aid in providing accurate, reproducible and timely results. In this chapter the components of a quality assurance program for antimicrobial susceptibility

**2. Overview of the antimicrobial susceptibility testing methodologies**

the performance of AST on pathogens became a practical necessity.

Fleming was first to report the inhibitory effect of penicillin on agar by observing a zone of growth inhibition of staphylococcal colonies grown next to a *Penicillium* contaminant on an agar plate. Fleming also made two significant contributions to the field of AST in the 1920s. In 1924, he introduced the use of the ditch plate technique for evaluating antimicrobial qual‐ ities of antiseptic solutions [3]. Fleming's second contribution to modern AST was the devel‐ opment of broth dilution technique using turbidity as an end-point determination [4]. Filter paper disks incorporating penicillin were utilized by Vincent & Vincent for assaying this newly discovered compound in 1940s [5]. Agar dilution AST method was also described in the 1940s [6]. At an early stage, it was realized that there were many variables affecting AST methods [7]. In 1961, World Health Organization (WHO) published a report on standardiza‐ tion of AST methodology [8]. The broad application of AST was introduced to clinical labo‐ ratories by the efforts of Bauer, Kirby and co-workers, with the method known as Kirby-Bauer disk diffusion method which is still the most widely used AST technique in the world [9]. Bergeron & Ouellette highlighted the shortcomings of the phenotypic approach to AST and concluded that different bacterial species have different susceptibilities to the same anti‐ biotic, and that there is no international aggreement on breakpoints for interpretation of an‐ timicrobial susceptibility tests [10]. The need for developing standardized AST methods became a necessity soon after antibiotics became commercially available. During World War II, following penicillin, other antibiotics were discovered and used. Altough these new anti‐ biotics were regarded as "wonder drugs" at the time of their introduction, emergence of re‐ sistant strains followed. With the emergence of bacterial resistance to antimicrobials and the changing properties of different bacteria to different classes of antimicrobials, the need for

testing will be highlighted.

414 Latest Research into Quality Control

In clinical laboratories, widely adopted AST methods are disk diffusion and broth dilution methods. In disk diffusion method, disks impregnated with antimicrobial agents are used. The disks are placed onto agar plates which are preinoculated with the suspension of the microorganism being tested. The basic principle of the disk diffusion method is the diffusion of the antimicrobial agent into the medium which occurs when the disks come into contact with the moist surface of the plate. The concentration of the agent reduces logarithmically as the distance from the disk is increased. After the incubation period the plates are observed for the circular inhibiton zone created around the disk which is due to the inhibitory effect of the antimicrobial agent on the microorganism. Within the zone the concentration of the agent is sufficient to inhibit growth, whereas at the point where the concentration of the agent is no longer enough to inhibit growth, the organism is able to grow and forms a lawn of bacteria around the disk. To interprete the test results, the radius of the inhibition zone is measured and compared against the predefined values provided by the guidelines [18]. The most widely used guidelines are the CLSI and EUCAST guidelines [2, 17]. CLSI divides the results into three categories for most of the organism-agent combinations; susceptible, inter‐ mediate and resistant, whereas EUCAST uses only two categories, susceptible and resistant.

In the dilution methods, however, the susceptibility of the microorganisms to antimicrobial agents is determined whether in tubes (macrobroth dilution method) or in microtube wells molded into a plastic plate (microbroth dilution method). Both broth dilution methods use the same principle; first serial two-fold dilutions of the antimicrobial agent to be tested are made in the tubes/wells containing broth, and then same amount of bacterial suspension is distributed on each tube/well. At the end of the incubation period, the tubes/wells are exam‐ ined for turbidity which is the indicator of bacterial growth in broth. The tubes/wells remain clear where the concentration of the agent is high enough to inhibit the bacterial growth, whereas at lower concentrations of the agent, the bacteria may grow which causes the tube/ well become turbid. The lowest concentration of antimicrobial agent that prevents the *in vi‐ tro* growth of bacteria is defined as the minimal inhibitory concentration (MIC) [18]. As in the disk diffusion method, the MIC values are compared against the predefined values pro‐ vided by the guidelines and their intrepretive category is determined and reported.

#### **3. Quality assurance program for antimicrobial susceptibility testing**

**•** Proficiency surveys (5%)

the testing of QC strains.

The goals of the QC program as set by the CLSI [24, 25] includes to monitor the following:

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The continuous monitorization of the performance is best achieved, but not limited to, by

Only organisms likely to be the cause of an infection should be tested for antimicrobial sus‐ ceptibility which necessitates the differentiation should be done between the normal flora that resides at the site of the infection and the actual organism causing the infection. Some important factors are to be considered to decide which bacterium or bacteria from a clinical specimen must be included in the AST; such as the body site from which the organism was isolated, the presence of other bacteria and the quality of the specimen from which the or‐ ganism was grown, the host's status, the ability of the bacterial species to cause infection at

Each laboratory is unique in its capability, resources, level of experience or institutional needs. Therefore, the decision of which antimicrobials to test depends on each laboratory's specifications and cannot be generalized. The decision involves the opinions of infectious diseases specialist and the pharmacist and should also be in concordance with the hospital formulary. Generally, a laboratory defines 10 to 15 antimicrobial agents for routine testing against various organisms or organism groups, which is called antimicrobial panel or bat‐ tery. In CLSI's M100 documents Table 1A (Suggested Groupings of Antimicrobial Agents With FDA Clinical Indications That Should Be Considered for Routine Testing and Report‐ ing on Nonfastidious Organisms by Clinical Microbiology Laboratories in the United States) is a valuable source of information to refer to when such tables are to be created at the local level [2]. Because the identity of the bacterial isolate is often not known at the time the AST is performed, some drugs, which are inappropriate to report for that particular isolate, may

The goal of the clinical microbiology laboratory is to create a report which will direct the clinician to use the least toxic, most cost-effective and most clinically effective agent that is available. This is accomplished by using the selective-reporting protocol provided by the CLSI. CLSI categorizes antimicrobial agents generally into four groups, Group A, B, C and U. Group A includes the primary agents whose results to be reported first. The results of

**•** the precision (repeatability) and accuracy of AST procedures

**•** the performance of persons who carry out the tests and read the results

**3.1. Developing relevant antimicrobial susceptibility testing strategies**

the body site from which the specimen was obtained, etc. [1, 26].

be tested. These results, however, should be supressed in the final report.

**3.2. Selecting antimicrobials to test and to report**

**•** the performance of reagents used in the tests

**•** Other (5%)

Clinical microbiology laboratories are an integral part of the total healthcare delivery sys‐ tem. Quality assurance (QA) is the overall process by which a laboratory can verify that a laboratory does its job well. While QA and quality control (QC) share the similar purposes, their meanings and functions are different [19]. QA can be defined as the overall program by which the quality of the test results can be guaranteed [20]. It evaluates and ensures that procedures provide relevant and timely data in the delivery of healthcare services. QA is primarily concerned with broader measures and monitors the performance of laboratory in total and covers all three phases of testing; pre-analytical, analytical and post-analytical. QC, in the other hand, is responsible for monitoring of the analytical phase of testing only and ensures that the daily tests are working properly [21]. QC and QA, only together provide measures for controlling how correct the tests are being performed because QC by itself of‐ ten does not detect problems in time to prevent harmful results. For example, if >5% of *En‐ terobacter*, *Serratia*, or *Citrobacter* isolates are susceptible to ampicillin, it likely indicates a problem with insufficient inoculum [22]. Although daily or weekly QC test results are in ac‐ ceptable limits, such an error can be overlooked until enough data have been accumulated and evaluated which can sometimes take weeks.

Standard processes are required to establish quality measures to be monitored. Standardiza‐ tion of AST has been achieved by CLSI, and in part by EUCAST. The processes defined in CLSI guidelines help clinical laboratories to perform QC tests, measure their results and pro‐ vide corrective action recommendations covering a broad spectrum of error types. Each lab‐ oratory should establish its own quality requirements for testing processes. Only with established quality goals, laboratories can determine whether acceptable quality is being achieved, identify processes that are not performing satisfactorily and are in need of im‐ provement, or to plan new processes to reach a specified level of quality [21]. And to ensure that all the established quality goals are achieved, a comprehensive QA program should be functional in a clinical laboratory.

The major components of a comprehensive QA program for AST, with the relative amount of effort required to be spent on each component given in parantheses, can be listed as fol‐ lows [23]:


**3. Quality assurance program for antimicrobial susceptibility testing**

and evaluated which can sometimes take weeks.

functional in a clinical laboratory.

416 Latest Research into Quality Control

**•** Clinically relevant testing strategies (15%)

**•** Organism antibiogram verification (15%)

**•** Testing of reference QC strains (15%)

**•** Supervisor review of results (15%)

**•** Technical competency (15%)

**•** Procedure manual (10%)

**•** Cumulative antibiogram (5%)

lows [23]:

Clinical microbiology laboratories are an integral part of the total healthcare delivery sys‐ tem. Quality assurance (QA) is the overall process by which a laboratory can verify that a laboratory does its job well. While QA and quality control (QC) share the similar purposes, their meanings and functions are different [19]. QA can be defined as the overall program by which the quality of the test results can be guaranteed [20]. It evaluates and ensures that procedures provide relevant and timely data in the delivery of healthcare services. QA is primarily concerned with broader measures and monitors the performance of laboratory in total and covers all three phases of testing; pre-analytical, analytical and post-analytical. QC, in the other hand, is responsible for monitoring of the analytical phase of testing only and ensures that the daily tests are working properly [21]. QC and QA, only together provide measures for controlling how correct the tests are being performed because QC by itself of‐ ten does not detect problems in time to prevent harmful results. For example, if >5% of *En‐ terobacter*, *Serratia*, or *Citrobacter* isolates are susceptible to ampicillin, it likely indicates a problem with insufficient inoculum [22]. Although daily or weekly QC test results are in ac‐ ceptable limits, such an error can be overlooked until enough data have been accumulated

Standard processes are required to establish quality measures to be monitored. Standardiza‐ tion of AST has been achieved by CLSI, and in part by EUCAST. The processes defined in CLSI guidelines help clinical laboratories to perform QC tests, measure their results and pro‐ vide corrective action recommendations covering a broad spectrum of error types. Each lab‐ oratory should establish its own quality requirements for testing processes. Only with established quality goals, laboratories can determine whether acceptable quality is being achieved, identify processes that are not performing satisfactorily and are in need of im‐ provement, or to plan new processes to reach a specified level of quality [21]. And to ensure that all the established quality goals are achieved, a comprehensive QA program should be

The major components of a comprehensive QA program for AST, with the relative amount of effort required to be spent on each component given in parantheses, can be listed as fol‐ The goals of the QC program as set by the CLSI [24, 25] includes to monitor the following:


The continuous monitorization of the performance is best achieved, but not limited to, by the testing of QC strains.

#### **3.1. Developing relevant antimicrobial susceptibility testing strategies**

Only organisms likely to be the cause of an infection should be tested for antimicrobial sus‐ ceptibility which necessitates the differentiation should be done between the normal flora that resides at the site of the infection and the actual organism causing the infection. Some important factors are to be considered to decide which bacterium or bacteria from a clinical specimen must be included in the AST; such as the body site from which the organism was isolated, the presence of other bacteria and the quality of the specimen from which the or‐ ganism was grown, the host's status, the ability of the bacterial species to cause infection at the body site from which the specimen was obtained, etc. [1, 26].

#### **3.2. Selecting antimicrobials to test and to report**

Each laboratory is unique in its capability, resources, level of experience or institutional needs. Therefore, the decision of which antimicrobials to test depends on each laboratory's specifications and cannot be generalized. The decision involves the opinions of infectious diseases specialist and the pharmacist and should also be in concordance with the hospital formulary. Generally, a laboratory defines 10 to 15 antimicrobial agents for routine testing against various organisms or organism groups, which is called antimicrobial panel or bat‐ tery. In CLSI's M100 documents Table 1A (Suggested Groupings of Antimicrobial Agents With FDA Clinical Indications That Should Be Considered for Routine Testing and Report‐ ing on Nonfastidious Organisms by Clinical Microbiology Laboratories in the United States) is a valuable source of information to refer to when such tables are to be created at the local level [2]. Because the identity of the bacterial isolate is often not known at the time the AST is performed, some drugs, which are inappropriate to report for that particular isolate, may be tested. These results, however, should be supressed in the final report.

The goal of the clinical microbiology laboratory is to create a report which will direct the clinician to use the least toxic, most cost-effective and most clinically effective agent that is available. This is accomplished by using the selective-reporting protocol provided by the CLSI. CLSI categorizes antimicrobial agents generally into four groups, Group A, B, C and U. Group A includes the primary agents whose results to be reported first. The results of Group B drugs should be selectively reported because these are generally broader spectrum agents. However, if the isolate is resistant to the primary agents, the patient cannot tolerate drugs in Group A, the infection has not responded to the therapy with the primary agents, a secondary agent would be a better clinical choice for the particular infection or that the pa‐ tient has organisms isolated from another site also, and a secondary agent might be more appropriate for treating both organisms, then the results of Group B drugs can be reported [26]. Group C includes alternative or supplemental agents for special cases; such as resistant strains, for patients allergic to primary drugs, for treatment of unusual isolates or for epide‐ miological purposes. And finally, Group U, includes the agents that are used only or pri‐ marily in the treatment of urinary tract infections (e.g., nitrofurantoin, norfloxacin).

*Incubation conditions (atmosphere, temperature, duration):* Different organisms require different incubation conditions. Moreover, some antimicrobial agents require different incubation length or temperature than the other disks used for the same organism (e.g., oxacillin with *Staphylococcus* spp.). The user should refer to CLSI M100 tables which give detailed testing

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*Antimicrobials concentrations to be tested:* The contents of antimicrobial disks in disk diffusion test and concentrations of antibiotic solutions to be tested in dilution tests are also included

Routine QC testing with a range of QC strains is the backbone of the internal QC testing. QC strains are well characterized organisms with defined susceptibility or resistance mecha‐ nisms to the antimicrobial agent(s) tested. Testing of QC strains helps to concurrently moni‐ tor the performance of the test and ensures that the test is being performed properly. The results obtained with the QC strains should be in predefined, acceptable ranges; for disk dif‐ fusion test, between the predefined inhibition zone diameters, and for MIC tests in prede‐ fined MIC ranges. If deviations from the acceptable limits are observed, it indicates unacceptable performance and the source(s) of the error should be investigated. CLSI rec‐ ommends to use various QC strains for different aspects of AST. The list of QC strains can be found in the M100 tables which are updated on a yearly basis. Because of the introduc‐ tion of new drugs, the changes effecting the existing drugs, or the emergence of new resist‐ ance mechanisms which should be investigated by the laboratory, the users are always referred to the latest update available. The QC strains recommended by CLSI are divided in two as being regular "QC strains" and "supplemental QC strains". Each laboratory perform‐ ing AST with CLSI's reference methods should include QC strains in regular QC tests, how‐ ever, the supplemental strains are only required if they are used to assess a new test, for training new personnel, investigation of special susceptibility or resistance characteristics,

CLSI's European counterpart, EUCAST, also publishes guidelines for the use of QC strains for AST, however, compared with the comprehensive battery of QC strains suggested by the CLSI, EUCAST is limited to six QC strains at the moment [27]. The guidelines of EUCAST are continously evolving and on areas where EUCAST's experience is not able to cover yet, EUCAST does not refrain from making referrals to relevant CLSI documents. However, one big difference between the QC strains recommended by CLSI and EUCAST is that, EU‐ CAST's recommendation for *Haemophilus influenzae* NCTC 8468 in contrast to CLSI's *H. in‐ fluenzae* ATCC® 49247. The strain EUCAST chose as a QC strain is susceptible to β-lactam antibiotics whose inhibition zones are easier to read than the ATCC® strain which is a βlactamase negative, ampicillin resistant (BLNAR) strain. The suggested QC strains by CLSI

conditions for each organism or organism group [2].

**3.4. Quality control testing with reference quality control strains**

etc., and are not required to be included in the routine QC of AST [2].

with their specifications are listed in Table 1 [2].

in CLSI documents [2].

Selective-reporting, also called cascade-reporting, improves the clinical relevance of the re‐ ports produced and minimizes the selection of multiresistant strains by avoiding the use of broad spectrum agents when narrow spectrum option is susceptible.

#### **3.3. Standardization of the antimicrobial susceptibility testing methodology**

The procedural steps of each method must be followed strictly in order to obtain reproduci‐ ble results. Standardization of AST methodology helps to optimize bacterial growth condi‐ tions so that the inhibition of growth can be attributed to the antimicrobial agent and the effects of nutrient limitations, temperature differences or other environmental conditions can be eliminated. And it also optimizes conditions for maintaining antimicrobial integrity and activity so that the failure to inhibit bacterial growth can be attributed to the organism's resistance mechanisms [1].

The standardized components of AST include:

*Bacterial inoculum size:* Preparation of the inoculum is one of the most critical steps in any susceptibility test method. Inoculum suspensions are prepared using either a log-phase or direct-colony suspension. When direct-colony suspension method is used, 4 to 5, fresh (16 to 24-hour old) colonies, rather than a single colony, should be selected to minimize the pos‐ sibility of testing a susceptible colony only and missing the resistant mutants dispersed in other colonies. McFarland turbidity standards are used to standardize the number of bacte‐ ria in the inoculum. McFarland standards can be prepared by adding specific volumes of 1% sulfuric acid and 1.175% barium chloride to obtain a barium sulfate solution with a specific optical density. The most commonly used is the McFarland 0.5 standard, which provides turbidity comparable with that of a bacterial suspension containing approximately 1.5 108 CFU/mL (CFU: colony-forming unit). Once standardized, the inoculum suspensions should be used within 15 minutes of preparation. False-susceptible results may occur if too few bac‐ teria are tested, and false-resistant results may be the outcome of testing too many bacteria [26].

*Growth medium:* The most frequently used growth media are Mueller-Hinton broth and Mu‐ eller-Hinton agar. The standardized variables regarding these media should include; its for‐ mulation, pH, cation concentration and thymidine content, thickness of agar (disk diffusion test), and supplements such as blood and serum.

*Incubation conditions (atmosphere, temperature, duration):* Different organisms require different incubation conditions. Moreover, some antimicrobial agents require different incubation length or temperature than the other disks used for the same organism (e.g., oxacillin with *Staphylococcus* spp.). The user should refer to CLSI M100 tables which give detailed testing conditions for each organism or organism group [2].

*Antimicrobials concentrations to be tested:* The contents of antimicrobial disks in disk diffusion test and concentrations of antibiotic solutions to be tested in dilution tests are also included in CLSI documents [2].

#### **3.4. Quality control testing with reference quality control strains**

Group B drugs should be selectively reported because these are generally broader spectrum agents. However, if the isolate is resistant to the primary agents, the patient cannot tolerate drugs in Group A, the infection has not responded to the therapy with the primary agents, a secondary agent would be a better clinical choice for the particular infection or that the pa‐ tient has organisms isolated from another site also, and a secondary agent might be more appropriate for treating both organisms, then the results of Group B drugs can be reported [26]. Group C includes alternative or supplemental agents for special cases; such as resistant strains, for patients allergic to primary drugs, for treatment of unusual isolates or for epide‐ miological purposes. And finally, Group U, includes the agents that are used only or pri‐

marily in the treatment of urinary tract infections (e.g., nitrofurantoin, norfloxacin).

**3.3. Standardization of the antimicrobial susceptibility testing methodology**

broad spectrum agents when narrow spectrum option is susceptible.

resistance mechanisms [1].

418 Latest Research into Quality Control

[26].

The standardized components of AST include:

test), and supplements such as blood and serum.

Selective-reporting, also called cascade-reporting, improves the clinical relevance of the re‐ ports produced and minimizes the selection of multiresistant strains by avoiding the use of

The procedural steps of each method must be followed strictly in order to obtain reproduci‐ ble results. Standardization of AST methodology helps to optimize bacterial growth condi‐ tions so that the inhibition of growth can be attributed to the antimicrobial agent and the effects of nutrient limitations, temperature differences or other environmental conditions can be eliminated. And it also optimizes conditions for maintaining antimicrobial integrity and activity so that the failure to inhibit bacterial growth can be attributed to the organism's

*Bacterial inoculum size:* Preparation of the inoculum is one of the most critical steps in any susceptibility test method. Inoculum suspensions are prepared using either a log-phase or direct-colony suspension. When direct-colony suspension method is used, 4 to 5, fresh (16 to 24-hour old) colonies, rather than a single colony, should be selected to minimize the pos‐ sibility of testing a susceptible colony only and missing the resistant mutants dispersed in other colonies. McFarland turbidity standards are used to standardize the number of bacte‐ ria in the inoculum. McFarland standards can be prepared by adding specific volumes of 1% sulfuric acid and 1.175% barium chloride to obtain a barium sulfate solution with a specific optical density. The most commonly used is the McFarland 0.5 standard, which provides turbidity comparable with that of a bacterial suspension containing approximately 1.5 108 CFU/mL (CFU: colony-forming unit). Once standardized, the inoculum suspensions should be used within 15 minutes of preparation. False-susceptible results may occur if too few bac‐ teria are tested, and false-resistant results may be the outcome of testing too many bacteria

*Growth medium:* The most frequently used growth media are Mueller-Hinton broth and Mu‐ eller-Hinton agar. The standardized variables regarding these media should include; its for‐ mulation, pH, cation concentration and thymidine content, thickness of agar (disk diffusion Routine QC testing with a range of QC strains is the backbone of the internal QC testing. QC strains are well characterized organisms with defined susceptibility or resistance mecha‐ nisms to the antimicrobial agent(s) tested. Testing of QC strains helps to concurrently moni‐ tor the performance of the test and ensures that the test is being performed properly. The results obtained with the QC strains should be in predefined, acceptable ranges; for disk dif‐ fusion test, between the predefined inhibition zone diameters, and for MIC tests in prede‐ fined MIC ranges. If deviations from the acceptable limits are observed, it indicates unacceptable performance and the source(s) of the error should be investigated. CLSI rec‐ ommends to use various QC strains for different aspects of AST. The list of QC strains can be found in the M100 tables which are updated on a yearly basis. Because of the introduc‐ tion of new drugs, the changes effecting the existing drugs, or the emergence of new resist‐ ance mechanisms which should be investigated by the laboratory, the users are always referred to the latest update available. The QC strains recommended by CLSI are divided in two as being regular "QC strains" and "supplemental QC strains". Each laboratory perform‐ ing AST with CLSI's reference methods should include QC strains in regular QC tests, how‐ ever, the supplemental strains are only required if they are used to assess a new test, for training new personnel, investigation of special susceptibility or resistance characteristics, etc., and are not required to be included in the routine QC of AST [2].

CLSI's European counterpart, EUCAST, also publishes guidelines for the use of QC strains for AST, however, compared with the comprehensive battery of QC strains suggested by the CLSI, EUCAST is limited to six QC strains at the moment [27]. The guidelines of EUCAST are continously evolving and on areas where EUCAST's experience is not able to cover yet, EUCAST does not refrain from making referrals to relevant CLSI documents. However, one big difference between the QC strains recommended by CLSI and EUCAST is that, EU‐ CAST's recommendation for *Haemophilus influenzae* NCTC 8468 in contrast to CLSI's *H. in‐ fluenzae* ATCC® 49247. The strain EUCAST chose as a QC strain is susceptible to β-lactam antibiotics whose inhibition zones are easier to read than the ATCC® strain which is a βlactamase negative, ampicillin resistant (BLNAR) strain. The suggested QC strains by CLSI with their specifications are listed in Table 1 [2].


**QC Strain Test(s), for which strain is primarily used**

group (susceptible)

susceptible)

growth)

*Staphylococcus aureus* ATCC® 43300 Screening test for oxacillin resistance in *Staphylococcus aureus* group (resistant)

positive)

growth)

(growth)

susceptible)

*Staphylococcus aureus* ATCC® BAA-1708 Screening test for high-level mupirocin resistance in *Staphylococcus*

*aureus* group (susceptible)

*Staphylococcus aureus* ATCC® BAA-976 Screening test for inducible clindamycin resistance in

*Staphylococcus aureus* ATCC® BAA-977 Screening test for inducible clindamycin resistance in

*Enterococcus faecalis* ATCC® 29212 MIC of *Enterococcus spp*.

Screening test for β-lactamase production in *Staphylococcus aureus*

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*Staphylococcus aureus* group, coagulase negative *Staphylococci* and *Streptococcus* spp. β-hemolytic group with broth microdilution (no

Screening test for high-level mupirocin resistance in *Staphylococcus*

*Staphylococcus aureus* group, coagulase negative *Staphylococci* and *Streptococcus* spp. β-hemolytic group with broth microdilution (no

*Staphylococcus aureus* group, coagulase negative *Staphylococci* and *Streptococcus* spp. β-hemolytic group with broth microdilution

*aureus* group (*mupA* positive; disk diffusion and MIC resistant)

Screening test for vancomycin MIC ≥8 µg/mL in *Staphylococcus*

*Enterococcus spp*. (disk diffusion, broth microdilution, agar dilution:

Screening test for high-level aminoglycoside resistance in

Screening test for *mecA*-mediated oxacillin resistance using cefoxitin in *Staphylococcus aureus* group (disk diffusion and MIC) and coagulase negative *Staphylococci* (disk diffusion) (*mecA*

Screening test for oxacillin resistance in *Staphylococcus aureus*

Screening test for *mecA*-mediated oxacillin resistance using cefoxitin in *Staphylococcus aureus* group (*mecA* negative; MIC

Screening test for inducible clindamycin resistance in

*aureus* group (*mupA* negative; MIC susceptible)

group and coagulase negative *Staphylococci* (positive)


**QC Strain Test(s), for which strain is primarily used**

420 Latest Research into Quality Control

*Escherichia coli* ATCC® 25922 Disk diffusion and MIC of *Enterobacteriaceae*, *Pseudomonas*

*Escherichia coli* ATCC® 35218 Disk diffusion and MIC for β-lactam/β-lactamase inhibitor

*Klebsiella pneumoniae* ATCC® 700603 Screening and confirmatory tests for ESBLs (positive)

*Klebsiella pneumoniae* ATCC® BAA-1705 Confirmatory test for suspected carbapenemase production in

*Klebsiella pneumoniae* ATCC® BAA-1706 Confirmatory test for suspected carbapenemase production in

*Pseudomonas aeruginosa* ATCC® 27853 Disk diffusion and MIC of *Pseudomonas aeruginosa*, *Acinetobacter*

*Staphylococcus aureus* ATCC® 25923 Disk diffusion of *Staphylococcus spp.* and *Enterococcus spp.*

*Staphylococcus aureus* ATCC® 29213 MIC of *Staphylococcus spp.*

*aeruginosa*, *Acinetobacter spp.*, *Burkholderia cepacia*,

Screening and confirmatory tests for ESBLs (negative)

nalidixic acid, minocycline, and sulfisoxazole)

Disk diffusion and MIC of *Neisseria meningitidis* (for ciprofloxacin,

combination drugs of *Enterobacteriaceae*, *Pseudomonas aeruginosa*, *Acinetobacter spp.*, *Burkholderia cepacia*, *Stenotrophomonas*

MIC for β-lactam/β-lactamase inhibitor combination drugs of other

Testing of amoxicillin-clavulanic acid for *Haemophilus spp*.

*spp.*, *Burkholderia cepacia*, *Stenotrophomonas maltophilia*

group and coagulase negative *Staphylococci* (negative) Screening test for *mecA*-mediated oxacillin resistance using cefoxitin in *Staphylococcus aureus* group and coagulase negative *Staphylococci* (*mecA* negative; disk diffusion susceptible) Screening test for inducible clindamycin resistance in

Screening test for β-lactamase production of *Staphylococcus aureus*

*Staphylococcus aureus* group and coagulase negative *Staphylococci*

Screening test for high-level mupirocin resistance in *Staphylococcus*

*aureus* group (*mupA* negative; disk diffusion susceptible)

*Stenotrophomonas maltophilia*

*maltophilia, Staphylococcus spp.*

*Enterobacteriaceae* (MHT positive)

*Enterobacteriaceae* (MHT negative)

MIC of other non-*Enterobacteriaceae*

with disk diffusion (D-zone test) (negative)

non-*Enterobacteriaceae*

MIC of other non-*Enterobacteriaceae*


ry's procedures. For example, if a laboratory does not perform Modified Hodge Test (MHT) to confirm suspected carbapenemase production in *Enterobacteriaceae*, the *Klebsiella pneumo‐ niae* ATCC® BAA-1705 (MHT-positive) and *Klebsiella pneumoniae* ATCC® BAA-1706 (MHTnegative) strains are not necessary for that particular laboratory. QC organisms susceptible to the tested antimicrobials are generally used but resistant QC strains are also necessary

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The QC strains can be obtained from various suppliers and in many formats. What impor‐ tant is, no matter in what format the strain has been received, the initial reconstitution should be performed according to supplier's recommendations. For long term storage, stock cultures can be stored in a suitable stabilizer (e.g., trypticase soy broth with 10 to 15% glyc‐ erol, 50% fetal calf serum in broth, defibrinated sheep blood or skim milk) at -20°C or below (preferably at -60°C or below). To obtain working control cultures, subcultures from the per‐ manent stock culture are made onto agar plates. Isolated colonies (4 to 5) are selected and subcultured to an agar slant (trypticase soy agar slants for non-fastidious organisms and chocolate agar slants for fastidious organisms) and incubated overnight. These working cul‐ tures on agar slants are stored at 2 – 8°C, for no more than three successive weeks. New working control cultures should be prepared at least monthly from permanent stock cul‐ tures. Prior to QC testing, growth from an agar slant is subcultured to agar plates and incu‐ bated overnight. To use for QC testing, 4 to 5 isolated colonies from the plate are selected. A new working culture should be prepared each day the QC test is being performed [2, 23].

Working control cultures can be used to monitor precision (repeatability) and accuracy of the AST as long as no significant change in the mean zone diameter or MIC value, not attrib‐ utable to faulty methodology, is observed. Laboratories usually do not have problems with the maintenance of susceptible QC strains owing to the stability of these strains, however, QC strains with particular resistance mechanisms are harder to maintain since they may be less genetically stable. Repeated subcultures can cause the loss of resistance mechanisms and unsatisfactory performances can be experienced. Documented problems have arisen with the QC strains which carry their specific resistance mechanism on a plasmid (e.g., *E. coli* ATCC® 35218 and *K. pneumoniae* ATCC® 700603) [2]. Suboptimal storage conditions and repeated cultures may cause the spontaneous loss of the plasmid encoding the β-lacta‐

Appropriate QC organisms should be tested daily for all antimicrobial agents routinely in‐ cluded in the antimicrobial battery until a laboratory achieves "satisfactory performance". CLSI makes the definition of "satisfactory performance" as obtaining unacceptable results in no more than 1 out of 20 or 3 out of 30 results obtained in consecutive test days for each antimicrobial agent/organism combination. Once this satisfactory performance is obtained, a laboratory can convert from daily QC testing to weekly QC testing. As long as all QC test results are within the acceptable limits, the laboratory can continue weekly testing, however on occasions when a modification in the test is made, consecutive QC testing is required

when testing for special resistance mechanisms.

mase and off-the-limit results may be encountered.

**3.6. Frequency of QC testing**

(Table 2., adapted from reference 2).

**Table 1.** Quality Control Strains Suggested for Antimicrobial Susceptibility Testing by CLSI

#### **3.5. Selection, obtaining and maintenance of reference QC strains**

When selecting QC strains for routine internal QC testing; the strains that most closely re‐ semble the patient's isolate should be tested [23]. This will provide that the drugs planned to be tested for the patient can be concomitantly tested with the QC strain. Additionally, same materials and testing conditions used for the clinical isolates can be evaluated. Before ob‐ taining the QC strains, laboratories should decide which strains do fit best to the laborato‐ ry's procedures. For example, if a laboratory does not perform Modified Hodge Test (MHT) to confirm suspected carbapenemase production in *Enterobacteriaceae*, the *Klebsiella pneumo‐ niae* ATCC® BAA-1705 (MHT-positive) and *Klebsiella pneumoniae* ATCC® BAA-1706 (MHTnegative) strains are not necessary for that particular laboratory. QC organisms susceptible to the tested antimicrobials are generally used but resistant QC strains are also necessary when testing for special resistance mechanisms.

The QC strains can be obtained from various suppliers and in many formats. What impor‐ tant is, no matter in what format the strain has been received, the initial reconstitution should be performed according to supplier's recommendations. For long term storage, stock cultures can be stored in a suitable stabilizer (e.g., trypticase soy broth with 10 to 15% glyc‐ erol, 50% fetal calf serum in broth, defibrinated sheep blood or skim milk) at -20°C or below (preferably at -60°C or below). To obtain working control cultures, subcultures from the per‐ manent stock culture are made onto agar plates. Isolated colonies (4 to 5) are selected and subcultured to an agar slant (trypticase soy agar slants for non-fastidious organisms and chocolate agar slants for fastidious organisms) and incubated overnight. These working cul‐ tures on agar slants are stored at 2 – 8°C, for no more than three successive weeks. New working control cultures should be prepared at least monthly from permanent stock cul‐ tures. Prior to QC testing, growth from an agar slant is subcultured to agar plates and incu‐ bated overnight. To use for QC testing, 4 to 5 isolated colonies from the plate are selected. A new working culture should be prepared each day the QC test is being performed [2, 23].

Working control cultures can be used to monitor precision (repeatability) and accuracy of the AST as long as no significant change in the mean zone diameter or MIC value, not attrib‐ utable to faulty methodology, is observed. Laboratories usually do not have problems with the maintenance of susceptible QC strains owing to the stability of these strains, however, QC strains with particular resistance mechanisms are harder to maintain since they may be less genetically stable. Repeated subcultures can cause the loss of resistance mechanisms and unsatisfactory performances can be experienced. Documented problems have arisen with the QC strains which carry their specific resistance mechanism on a plasmid (e.g., *E. coli* ATCC® 35218 and *K. pneumoniae* ATCC® 700603) [2]. Suboptimal storage conditions and repeated cultures may cause the spontaneous loss of the plasmid encoding the β-lacta‐ mase and off-the-limit results may be encountered.

#### **3.6. Frequency of QC testing**

**QC Strain Test(s), for which strain is primarily used**

422 Latest Research into Quality Control

*Enterococcus faecalis* ATCC® 51299 Screening test for vancomycin MIC ≥8 µg/mL for *Staphylococcus*

*aureus* group (resistant)

dilution: resistant) *Haemophilus influenzae* ATCC® 49247 Disk diffusion and MIC of *Haemophilus spp*. (BLNAR; β-lactamase

*Haemophilus influenzae* ATCC® 10211 Checking growth capabilities of medium used for disk diffusion and

*Haemophilus influenzae* ATCC® 49766 Disk diffusion and MIC of *Haemophilus spp*. with selected

*Neisseria gonorrhoeae* ATCC® 49226 Disk diffusion and MIC of *Neisseria gonorrhoeae* (CMRNG;

*Bacteroides fragilis* ATCC® 25285 MIC of anaerobes *Bacteroides thetaiotaomicron* ATCC® 29741 MIC of anaerobes *Clostridium difficile* ATCC® 700057 MIC of anaerobes *Eubacterium lentum* ATCC® 43055 MIC of anaerobes

**Table 1.** Quality Control Strains Suggested for Antimicrobial Susceptibility Testing by CLSI

**3.5. Selection, obtaining and maintenance of reference QC strains**

*Streptococcus pneumoniae* ATCC® 49619 Disk diffusion and MIC of *Streptococcus pneumoniae* (penicillin

microdilution (negative)

When selecting QC strains for routine internal QC testing; the strains that most closely re‐ semble the patient's isolate should be tested [23]. This will provide that the drugs planned to be tested for the patient can be concomitantly tested with the QC strain. Additionally, same materials and testing conditions used for the clinical isolates can be evaluated. Before ob‐ taining the QC strains, laboratories should decide which strains do fit best to the laborato‐

negative, ampicillin resistant)

MIC tests for *Haemophilus* spp.

cephalosporins (β-lactamase positive)

spp. viridans group and *Neisseria meningitidis*

Screening test for vancomycin resistance in *Enterococcus spp*. (agar dilution: susceptible) checking that medium is acceptable for testing sulfonamides, trimethoprim, and trimethoprim/sulfamethoxazole

Screening test for high-level aminoglycoside resistance in *Enterococcus spp*. (broth microdilution, agar dilution: resistant) Screening test for vancomycin resistance in *Enterococcus spp*. (agar

chromosomally mediated (penicillin) resistant *N. gonorrhoeae*)

intermediate), S*treptococcus* spp. β-hemolytic group *Streptococcus*

Screening test for inducible clindamycin resistance in *Streptococcus* spp. β-hemolytic group with disk diffusion (D-zone test) and broth

> Appropriate QC organisms should be tested daily for all antimicrobial agents routinely in‐ cluded in the antimicrobial battery until a laboratory achieves "satisfactory performance". CLSI makes the definition of "satisfactory performance" as obtaining unacceptable results in no more than 1 out of 20 or 3 out of 30 results obtained in consecutive test days for each antimicrobial agent/organism combination. Once this satisfactory performance is obtained, a laboratory can convert from daily QC testing to weekly QC testing. As long as all QC test results are within the acceptable limits, the laboratory can continue weekly testing, however on occasions when a modification in the test is made, consecutive QC testing is required (Table 2., adapted from reference 2).


tive action is necessary. On the other hand, if the reason causing for the error cannot be identified, the test must be carried out again the day the error is observed, preferably with a new working culture or subculture, but should also be monitored for a total of five consecu‐ tive test days. During five consecutive days, if all results are within the acceptable limits no additional corrective action is required. However, if any of the results are outside the accept‐ able limits, additional corrective action is required. At this point, a systematic error, rather than a random should be suspected and the components of AST should be thoroughly in‐ vestigated. The reasons include; wrong measurement, clerical errors, problems in the adjust‐ ment of turbidity, past expiration date materials, failure in providing proper growth conditions (temperature, atmosphere), improper storage of disks, contamination of QC strain, loss of characteristics, inoculum prepared from an old plate (> 24 hours), etc.. In order to start to routine QC testing, satisfactory performance for another 20 or 30 consecutive days

When an out-of-range QC results necessitates a corrective action, the factors listed in Table 3 should be considered for troubleshooting (Table 3., adapted from references 24 and 25).

Improper storage

>1 month) Contamination Nonviability

Contamination

of time

Transcription error

Use of expired materials

Inadequate maintenance (e.g., use of the same working culture for

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Use of damaged plates, panels, cards, tubes (e.g., cracked, leaking)

Inoculum prepared from differential or selective media containing anti-infective agents or other growth-inhibiting compounds

Improper disk placement (e.g., inadequate contact with the agar)

Inoculum suspensions were incorrectly prepared or adjusted Inoculum prepared from a plate incubated for the incorrect length

Changes in the organisms (e.g., mutation, loss of plasmid)

Use of a defective agar plate (too thick or too thin) Inadequate volume of broth in tubes or wells

Use of wrong disk/reagents, ancillary supplies

Incorrect reading or interpretation of test results

is required once the reason causing the error is detected and corrected.

**QC Strain** Use of the wrong QC strain

**Testing supplies** Improper storage or shipping conditions

**Testing process** Use of the wrong incubation temperature or conditions

**Equipment** Not functioning properly or out of calibration (e.g., pipettes)

**Table 3.** Factors Frequently Causing Out-of-range Results

**Table 2.** Required Quality Control Frequency after Modifications in the Test

For both, disk diffusion and MIC testing, addition of any new antimicrobial agent to the ex‐ isting panel requires 20 or 30 consecutive days of satisfactory testing before it can be tested on a weekly schedule.

#### **3.7. Corrective action**

Corrective action is defined as the "action to eliminate the cause of a detected nonconformi‐ ty or other undesirable situation" [28] and in regard to AST, is needed whenever any of the weekly QC results are not within the acceptable limits. The factors causing for the deviation in the results are various but can be divided in two as being results due to identifiable errors and results with no error identified [24, 25]. Identifiable errors, also named obvious errors, are easy to detect and also easy to correct. Most usual reasons causing for identifiable errors include; use of the wrong disk, use of the wrong QC strain, contamination of the strain or media, use of the wrong incubation temperature or conditions. If the reason causing the outof-range results is one of the identifiable errors, the test must be carried out again the day the error is observed. If results of the repeat test are in acceptable limits, no further correc‐ tive action is necessary. On the other hand, if the reason causing for the error cannot be identified, the test must be carried out again the day the error is observed, preferably with a new working culture or subculture, but should also be monitored for a total of five consecu‐ tive test days. During five consecutive days, if all results are within the acceptable limits no additional corrective action is required. However, if any of the results are outside the accept‐ able limits, additional corrective action is required. At this point, a systematic error, rather than a random should be suspected and the components of AST should be thoroughly in‐ vestigated. The reasons include; wrong measurement, clerical errors, problems in the adjust‐ ment of turbidity, past expiration date materials, failure in providing proper growth conditions (temperature, atmosphere), improper storage of disks, contamination of QC strain, loss of characteristics, inoculum prepared from an old plate (> 24 hours), etc.. In order to start to routine QC testing, satisfactory performance for another 20 or 30 consecutive days is required once the reason causing the error is detected and corrected.

When an out-of-range QC results necessitates a corrective action, the factors listed in Table 3 should be considered for troubleshooting (Table 3., adapted from references 24 and 25).


**Table 3.** Factors Frequently Causing Out-of-range Results

**Day(s)\* Modification in the Test**

\* Number of days of consecutive QC testing required

on a weekly schedule.

424 Latest Research into Quality Control

**3.7. Corrective action**

**Table 2.** Required Quality Control Frequency after Modifications in the Test

**1** Start to use new shipment or lot number of disks/MIC

**5** Start to use prepared agar plates (disk diffusion), broth or

**20 or 30** Use new method for MIC test (e.g., convert from visual

For both, disk diffusion and MIC testing, addition of any new antimicrobial agent to the ex‐ isting panel requires 20 or 30 consecutive days of satisfactory testing before it can be tested

Corrective action is defined as the "action to eliminate the cause of a detected nonconformi‐ ty or other undesirable situation" [28] and in regard to AST, is needed whenever any of the weekly QC results are not within the acceptable limits. The factors causing for the deviation in the results are various but can be divided in two as being results due to identifiable errors and results with no error identified [24, 25]. Identifiable errors, also named obvious errors, are easy to detect and also easy to correct. Most usual reasons causing for identifiable errors include; use of the wrong disk, use of the wrong QC strain, contamination of the strain or media, use of the wrong incubation temperature or conditions. If the reason causing the outof-range results is one of the identifiable errors, the test must be carried out again the day the error is observed. If results of the repeat test are in acceptable limits, no further correc‐

panels or prepared agar plates

Start to use disks from a new manufacturer

agar (MIC) from a new manufacturer

device which has its own QC protocol

overnight to rapid MIC test) Use new manufacturer of MIC test

Expand or reduce the dilution range in MIC testing Repair of instrument that affects the AST results

Convert inoculum preparation/standardization method from visual adjustment of turbidity to use a photometric

Update of the software which affects the AST results

reading to instrument reading of panel, convert from

Change method of measuring zones in disk diffusion test

Convert inoculum preparation/standardization method to

(e.g., start using an automated zone reader)

a method that is dependent on user technique

#### **3.8. Documentation of the quality control test results**

Results from all QC tests should be documented on a QC log sheet [23]. On this log sheet information regarding the following are required: the date, the technician who performed the test, antimicrobial agents used (potency, lot, expiration date, etc.), media used (lot, expi‐ ration date, etc.). Once the log sheet has been filled by the technician who performed and read the test, a second technician, or the supervisor, should check the results. Also, correc‐ tive actions taken, if any, and their outcomes should be noted.

**3.9. Organism - Antimicrobial susceptibility test result verification**

**Category Observed susceptibility result**

**II** I or R to carbapenems in *Enterobacteriaceae*

ble 3 (adapted from reference 2).

One of the most widely used supplemental QC measure is the use of susceptibility test re‐ sults to verify results generated on patient results. Species with "typical" antibiograms are useful in verification of the identification as well as the susceptibility results. CLSI suggests some results to be confirmed before they are reported, these mostly include rare resistance phenotypes. The rare resistance phenotypes are divided in three categories; Category I; not reported or only rarely reported to date, Category II; uncommon in most institutions, and Category III; may be common, but is generally considered of epidemiological concern. Since category I includes the least encountered and most significant results, it is highly important to detect these results before being reported unnoticed and to follow the necessary steps for the verification. Unusual resistance phenotypes which require confirmation are given in Ta‐

**I** NS to carbapenems, extended-spectrum cephalosporins or

*Streptococcus*

*gonorrhoeae*

group *Streptococcus*

fluoroquinolones in *H. influenzae*

NS to extended-spectrum cephalosporins, meropenem or minocycline, R to ampicillin or penicillin in *N. meningitidis*

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NS to daptomycin, ertapenem, meropenem, linezolid, or vancomycin, R to quinupristin-dalfopristin in viridans

I or R to trimethoprim-sulfamethoxazole in *S. maltophilia* R to amoxicillin-clavulanic acid, R to ampicillin without accompanying β-lactamase production in *H. influenzae*

NS to extended spectrum cephalosporins in *N.*

NS to linezolid or vancomycin in *S. pneumoniae* NS to ampicillin, penicillin, extended-spectrum cephalosporins, daptomycin, ertapenem, meropenem,

linezolid or vancomycin in β-hemolytic group

I or R to 3rd generation cephalosporins or fluoroquinolones in *Salmonella* and *Shigella* spp.

R to colistin/polymyxin in *A. baumannii* I or R to colistin/polymyxin in *P. aeruginosa*

A useful and simple way of monitoring QC results is to use the Shewhart diagram, in which the daily readings are plotted on a chart with upper and lower control limits marked [29]. It provides the visual assessment of the results but can also provide in depth information if a more formal mathematical approach is followed [20]. An example of presenting daily QC re‐ sults on a Shewhart diagram is given in Figure 1. The famous rules of Westgard and Klee [30] can be easily adopted to the QC of disk diffusion test in which the control diameters are treated as mean ±2 SD [20].

One QC result lies outside the limits (Westgard rule 12s): It is a warning, whether it's a ran‐ dom error or the beginning of an emerging problem. Routine test results for that day may be reported if there is no other evidence of problems in the current tests. It does not require corrective action by itself, unless the result is far out of range or there are other indications of a problem.

Two consecutive QC results are outside the limits in the same side of the mean of the range (Westgard rule 22s): Indicates an error in the test methodology (a systematic error), corrective action is required.

Ten consecutive QC results falling on one side of the mean (Westgard rule 10Ẍ): Results may be accepted but this likely indicates a systematic problem which should be acted on.

**Figure 1.** Example for daily disk diffusion QC results for *Escherichia coli* ATCC® 25922 vs. ampicillin plotted on a She‐ whart diagram (acceptable zone limits: 16 – 22 mm).

#### **3.9. Organism - Antimicrobial susceptibility test result verification**

**3.8. Documentation of the quality control test results**

tive actions taken, if any, and their outcomes should be noted.

treated as mean ±2 SD [20].

426 Latest Research into Quality Control

of a problem.

action is required.

whart diagram (acceptable zone limits: 16 – 22 mm).

Results from all QC tests should be documented on a QC log sheet [23]. On this log sheet information regarding the following are required: the date, the technician who performed the test, antimicrobial agents used (potency, lot, expiration date, etc.), media used (lot, expi‐ ration date, etc.). Once the log sheet has been filled by the technician who performed and read the test, a second technician, or the supervisor, should check the results. Also, correc‐

A useful and simple way of monitoring QC results is to use the Shewhart diagram, in which the daily readings are plotted on a chart with upper and lower control limits marked [29]. It provides the visual assessment of the results but can also provide in depth information if a more formal mathematical approach is followed [20]. An example of presenting daily QC re‐ sults on a Shewhart diagram is given in Figure 1. The famous rules of Westgard and Klee [30] can be easily adopted to the QC of disk diffusion test in which the control diameters are

One QC result lies outside the limits (Westgard rule 12s): It is a warning, whether it's a ran‐ dom error or the beginning of an emerging problem. Routine test results for that day may be reported if there is no other evidence of problems in the current tests. It does not require corrective action by itself, unless the result is far out of range or there are other indications

Two consecutive QC results are outside the limits in the same side of the mean of the range (Westgard rule 22s): Indicates an error in the test methodology (a systematic error), corrective

Ten consecutive QC results falling on one side of the mean (Westgard rule 10Ẍ): Results may

**Figure 1.** Example for daily disk diffusion QC results for *Escherichia coli* ATCC® 25922 vs. ampicillin plotted on a She‐

be accepted but this likely indicates a systematic problem which should be acted on.

One of the most widely used supplemental QC measure is the use of susceptibility test re‐ sults to verify results generated on patient results. Species with "typical" antibiograms are useful in verification of the identification as well as the susceptibility results. CLSI suggests some results to be confirmed before they are reported, these mostly include rare resistance phenotypes. The rare resistance phenotypes are divided in three categories; Category I; not reported or only rarely reported to date, Category II; uncommon in most institutions, and Category III; may be common, but is generally considered of epidemiological concern. Since category I includes the least encountered and most significant results, it is highly important to detect these results before being reported unnoticed and to follow the necessary steps for the verification. Unusual resistance phenotypes which require confirmation are given in Ta‐ ble 3 (adapted from reference 2).



**3.10. Real-time review of results**

spread of this unusual resistance.

among laboratory workers.

**3.12. External quality assessment**

laboratories, thus corrective action is delayed.

**3.13. Internal quality assessment**

**3.11. Education**

Accuracy of the susceptibility test results should be continously monitored. This is mostly accomplished by daily reviewing of the data that is being produced. Profiles which are like‐ ly, somewhat likely, somewhat unlikely and nearly impossible should be identified, wheth‐ er manually or with the help of a software programmed to recognize different patterns of susceptibility data [1]. Prompt recognition of unusual resistance or inconsistent susceptibili‐ ty helps the laboratory to timely confirm the susceptibility results. In order to confirm the results, first step is to exclude the transcriptional and reading errors and make sure of the purity of the inoculum which has been tested. If no errors are found in the previous steps, the identification of the organism should be confirmed and the susceptibility test be repeat‐ ed, preferably with another method. In cases where no errors are detected and the unusual resistance is confirmed, the clinician may be warned and measures can be taken to limit the

Quality Assurance in Antimicrobial Susceptibility Testing

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429

Education is an important component of the QA process. Having knowledge about the methods also provides the understanding of their limitations and pitfalls. A well-educated technician may timely recognize atypical results and is aware of the approach to follow for the resolution and avoidance of errors [20]. A very efficient way of training in-service per‐ sonnel is the end-point interpretation control [24, 25]. Laboratory workers, who perform AST, are provided with a set of selected disk diffusion plates and are asked to read the re‐ sults. The recorded results are then compared by an experienced reader, e.g., the laboratory director, and the individual performances of each technician is evaluated and if necessary, corrected. It significantly helps to minimize variation in the interpretation of zone sizes

In external quality assessment (EQA) programs, a central laboratory distributes test strains with known susceptibility profiles to all participant laboratories. Each participating labora‐ tory tests and reports the results to the central laboratory. Once all the results are returned from participants, the central laboratory evaluates the results and prepares a feedback re‐ port. The benefit of participating in such program is that each individual laboratory can as‐ sess ist own performance compared with other laboratories, at national and international levels, it functions as an educational tool, and also provides the evidence of performance re‐ quired by the accrediting bodies. On the other hand, the number of strains distributed in a year is relatively small, which brings the disadvantage of the rare errors going unnoticed [20]. Also, in contrast to internal QC, which is capable of acting on problems encountered on daily basis, it takes quite a while for the EQA feedback reports to be sent to the participating

Internal quality assessment (IQA) is a complementary activity to EQA in which routine tests are repeated on the same day as the original, but this time, with the identity of the specimen

**Table 4.** Unusual Resistance Phenotypes Which Require Confirmation

The general approach to be followed is, for all three categories, to confirm the identification of the organism and the AST. If the results are confirmed, the infection control should be informed about the case.

#### **3.10. Real-time review of results**

Accuracy of the susceptibility test results should be continously monitored. This is mostly accomplished by daily reviewing of the data that is being produced. Profiles which are like‐ ly, somewhat likely, somewhat unlikely and nearly impossible should be identified, wheth‐ er manually or with the help of a software programmed to recognize different patterns of susceptibility data [1]. Prompt recognition of unusual resistance or inconsistent susceptibili‐ ty helps the laboratory to timely confirm the susceptibility results. In order to confirm the results, first step is to exclude the transcriptional and reading errors and make sure of the purity of the inoculum which has been tested. If no errors are found in the previous steps, the identification of the organism should be confirmed and the susceptibility test be repeat‐ ed, preferably with another method. In cases where no errors are detected and the unusual resistance is confirmed, the clinician may be warned and measures can be taken to limit the spread of this unusual resistance.

#### **3.11. Education**

**Category Observed susceptibility result**

428 Latest Research into Quality Control

I to ampicillin, penicillin, I or R to rifampin, NS to

R to linezolid, NS to daptomycin for *Enterococcus* spp. NS to daptomycin, R to linezolid, I or R to quinupristindalfopristin, vancomycin MIC = 4 µg/mL or vancomycin

NS to daptomycin, I or R to quinupristin-dalfopristin or vancomycin, R to daptomycin in coagulase-negative

I or R to fluoroquinolone, imipenem, meropenem, quinupristin-dalfopristin, rifampin in *S. pneumoniae* I or R to quinupristin-dalfopristin in β-hemolytic group

I or R to extended spectrum cephalosporins in *E. coli*,

azithromycin in *N. meningitidis*

MIC ≥ 8 µg/mL for *S. aureus*

*Staphylococcus* spp.

*Streptococcus*

*meningitidis*

breakpoints

The general approach to be followed is, for all three categories, to confirm the identification of the organism and the AST. If the results are confirmed, the infection control should be

NS; nonsusceptible, I; intermediate, R; resistant

informed about the case.

**Table 4.** Unusual Resistance Phenotypes Which Require Confirmation

*Enterococcus* spp.

R to oxacillin in *S. aureus*

*Enterobacteriaceae*

*Klebsiella* spp. or *P. mirabilis*

carbapenem in *P. aeruginosa*

I or R to carbapenem in *A. baumannii*

R to amikacin, gentamicin, and tobramycin, or

I or R to fluoroquinolone in *N. gonorrhoeae*

I or R to chloramphenicol or fluoroquinolone in *N.*

R to vancomycin or high-level aminoglycoside in

R to amoxicillin, penicillin or extended spectrum cephalosporins in *S. pneumoniae* using nonmeningitis

**III** R to amikacin, gentamicin, and tobramycin in

Education is an important component of the QA process. Having knowledge about the methods also provides the understanding of their limitations and pitfalls. A well-educated technician may timely recognize atypical results and is aware of the approach to follow for the resolution and avoidance of errors [20]. A very efficient way of training in-service per‐ sonnel is the end-point interpretation control [24, 25]. Laboratory workers, who perform AST, are provided with a set of selected disk diffusion plates and are asked to read the re‐ sults. The recorded results are then compared by an experienced reader, e.g., the laboratory director, and the individual performances of each technician is evaluated and if necessary, corrected. It significantly helps to minimize variation in the interpretation of zone sizes among laboratory workers.

#### **3.12. External quality assessment**

In external quality assessment (EQA) programs, a central laboratory distributes test strains with known susceptibility profiles to all participant laboratories. Each participating labora‐ tory tests and reports the results to the central laboratory. Once all the results are returned from participants, the central laboratory evaluates the results and prepares a feedback re‐ port. The benefit of participating in such program is that each individual laboratory can as‐ sess ist own performance compared with other laboratories, at national and international levels, it functions as an educational tool, and also provides the evidence of performance re‐ quired by the accrediting bodies. On the other hand, the number of strains distributed in a year is relatively small, which brings the disadvantage of the rare errors going unnoticed [20]. Also, in contrast to internal QC, which is capable of acting on problems encountered on daily basis, it takes quite a while for the EQA feedback reports to be sent to the participating laboratories, thus corrective action is delayed.

#### **3.13. Internal quality assessment**

Internal quality assessment (IQA) is a complementary activity to EQA in which routine tests are repeated on the same day as the original, but this time, with the identity of the specimen blinded. After the reports are produced, the results are compared and discrepancies noted. This activity helps to monitor the precision and accuracy of the test procedure and may highlight problem areas not detected by other QC methods. It monitors not only the per‐ formance of the test and reagents, but also the performance of the persons carrying out the tests [20]. The EQA and the IQA are complementary activities, while IQA focuses on moni‐ toring a single laboratory on a daily basis, EQA compares the performance of different labo‐ ratories and is important for maintaining long-term accuracy of the AST methods employed [21].

laboratory test conditions are far different from *in vivo* conditions where the organism and the antimicrobial agent do actually interact. Factors, such as bacterial inoculum size, pH, cat‐ ion concentration and oxygen tension differ greatly depending on the site of infection [1]. In spite of all these limitations, the clinical microbiology laboratory should follow the most upto-date guidelines to serve the patients in the best possible way. With a well constructed QA program in operation, a laboratory should aim to ensure that the right test is carried out on the right specimen, and that the right result and right interpretation is delivered to the right

Quality Assurance in Antimicrobial Susceptibility Testing

http://dx.doi.org/10.5772/51998

431

[1] Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing. (2007). In: Forbes B. A., Sahm D. F., Weissfeld A. C. (ed.) Bailey & Scott's Diagnostic Micro‐

[2] CLSI. (2012). Performance Standards for Antimicrobial Susceptibility Testing; Twen‐ ty-Second Informational Supplement. CLSI document M100-S22. Wayne, PA: Clinical

[3] Fleming, A. (1924). A comparison of the activities of antiseptics on bacteria and on leucocytes. Proceedings of the Royal Society of London, Series B 96, 171-80.

[4] Fleming, A. (1929). On the antibacterial action of cultures of a penicillium, with spe‐ cial reference to their use in the isolatation of B. influenzae. British Journal of Experi‐

[5] Vincent, J. G., Vincent, H. V. (1944). Filter paper disc modification of the Oxford cup penicillin determination. Proceedings of the Society for Experimental Biology and

[6] Schmith, K., Reymann, F. E. (1940). Experimentelle og kliniske undersogelser over

[7] Waterworth, P. M. (1951). A comparative study of methods of testing sensitivity to antibiotics and of the factors influencing the results. Journal of Medical Laboratory

gonococcers folsomhed overfor sulfapyridin. Nordisk Medicin 8, 2493-9.

biology. 12th ed. St. Louis, MO: Mosby Elsevier; 187-214.

and Laboratory Standards Institute.

mental Pathology 110, 226-36.

Medicine 55, 162-4.

Technology 9, 65-85.

person at the right time.

Acibadem University, Istanbul, Turkey

**Author details**

Onur Karatuna

**References**

#### **3.14. Proficiency testing programs**

They are a type of EQA in which simulated patient specimens are sent to participating labo‐ ratories. Again, the reports are produced by each laboratory, and returned to the central lab‐ oratory for evaluation. In the United States, government mandates that clinical laboratories be accredited and licensed. The government and licensing agencies are using proficiency testing as an objective method for the accreditation of laboratories [21]. In 1988, the U.S. Congress passed the Clinical Laboratory Improvement Amendment (CLIA '88) which man‐ dated proficiency testing (PT) as a major part of the laboratory accreditation process [31]. The initial CLIA '88 proposal called for two PT specimens per year but final legislative rule, published in 2003, expanded this to study five samples three times per year. The definition of failure is defined as two of five incorrect results on two of the three consecutive PT sur‐ veys [32].

#### **4. Quality control of automated antimicrobial susceptibility test systems**

According to the work load and the resources a laboratory has, a laboratory can choose to use one of many types of commercial automated antimicrobial susceptibility test systems. Most of these systems use the principle of turbidimetric detection of bacterial growth in a broth medium by use of a photometer which periodically examines the test wells [26]. The most widely used systems in the world are VITEK 2 System (bioMérieux Vitek, Hazelwood, MO), BD Phoenix System (BD Diagnostic Systems, Sparks, MD), MicroScan WalkAway SI (Siemens Healthcare Diagnostics, Sacramento, CA) and TREK Sensititre (ARIS 2X, Trek Di‐ agnostic Systems, Cleveland, OH). Each device has its own QC procedure and commercial susceptibility testing devices are not addressed in CLSI standards. CLSI only describes methods regarding generic reference procedures, however these reference methods are used by the US Food and Drug Administration before clearence is given to a commercial system for marketing in the US to evaluate its performance.

#### **5. Conclusion**

Although great improvement has been done in AST methodology and automated suscepti‐ bility systems have been introduced which provide same-day results, it should be consid‐ ered that there are still many variables not covered by the standard methods. First of all, the laboratory test conditions are far different from *in vivo* conditions where the organism and the antimicrobial agent do actually interact. Factors, such as bacterial inoculum size, pH, cat‐ ion concentration and oxygen tension differ greatly depending on the site of infection [1]. In spite of all these limitations, the clinical microbiology laboratory should follow the most upto-date guidelines to serve the patients in the best possible way. With a well constructed QA program in operation, a laboratory should aim to ensure that the right test is carried out on the right specimen, and that the right result and right interpretation is delivered to the right person at the right time.

#### **Author details**

blinded. After the reports are produced, the results are compared and discrepancies noted. This activity helps to monitor the precision and accuracy of the test procedure and may highlight problem areas not detected by other QC methods. It monitors not only the per‐ formance of the test and reagents, but also the performance of the persons carrying out the tests [20]. The EQA and the IQA are complementary activities, while IQA focuses on moni‐ toring a single laboratory on a daily basis, EQA compares the performance of different labo‐ ratories and is important for maintaining long-term accuracy of the AST methods employed

They are a type of EQA in which simulated patient specimens are sent to participating labo‐ ratories. Again, the reports are produced by each laboratory, and returned to the central lab‐ oratory for evaluation. In the United States, government mandates that clinical laboratories be accredited and licensed. The government and licensing agencies are using proficiency testing as an objective method for the accreditation of laboratories [21]. In 1988, the U.S. Congress passed the Clinical Laboratory Improvement Amendment (CLIA '88) which man‐ dated proficiency testing (PT) as a major part of the laboratory accreditation process [31]. The initial CLIA '88 proposal called for two PT specimens per year but final legislative rule, published in 2003, expanded this to study five samples three times per year. The definition of failure is defined as two of five incorrect results on two of the three consecutive PT sur‐

**4. Quality control of automated antimicrobial susceptibility test systems**

According to the work load and the resources a laboratory has, a laboratory can choose to use one of many types of commercial automated antimicrobial susceptibility test systems. Most of these systems use the principle of turbidimetric detection of bacterial growth in a broth medium by use of a photometer which periodically examines the test wells [26]. The most widely used systems in the world are VITEK 2 System (bioMérieux Vitek, Hazelwood, MO), BD Phoenix System (BD Diagnostic Systems, Sparks, MD), MicroScan WalkAway SI (Siemens Healthcare Diagnostics, Sacramento, CA) and TREK Sensititre (ARIS 2X, Trek Di‐ agnostic Systems, Cleveland, OH). Each device has its own QC procedure and commercial susceptibility testing devices are not addressed in CLSI standards. CLSI only describes methods regarding generic reference procedures, however these reference methods are used by the US Food and Drug Administration before clearence is given to a commercial system

Although great improvement has been done in AST methodology and automated suscepti‐ bility systems have been introduced which provide same-day results, it should be consid‐ ered that there are still many variables not covered by the standard methods. First of all, the

[21].

veys [32].

**5. Conclusion**

**3.14. Proficiency testing programs**

430 Latest Research into Quality Control

for marketing in the US to evaluate its performance.

Onur Karatuna

Acibadem University, Istanbul, Turkey

#### **References**


[8] World Health Organization. (1961). Standardization of Methods for Conducting Mi‐ crobic Sensitivity Tests. Second Report of the Expert Committee on Antibiotics. WHO Technical Report Series, No. 210. WHO, Geneva.

[22] Washington J. A. (1988). Current problems in antimicrobial susceptibility testing. Di‐

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[23] Rankin I. D. (2005). Quality Assurance/Quality Control (QA/QC) In: Coyle M. B. (ed.) Manual of Antimicrobial Susceptibility Testing. 1st ed, Washington, D.C.: American

[24] CLSI. (2012). Performance Standards for Antimicrobial Disk Susceptibility Tests; Ap‐ proved Standard–Eleventh Edition. CLSI document M02-A11. Wayne, PA: Clinical

[25] CLSI. (2012). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Ninth Edition. CLSI document M07-A9.

[26] Marsik F. J. (2011). Antimicrobial Susceptibility Testing. In: Mahon C. R., Lehman D. C., Manuselis G. (ed.) Textbook of Diagnostic Microbiology. 4th ed. Maryland

[27] European Comittee on Antimicrobial Susceptibility Testing. (2012). EUCAST recom‐ mended strains for internal quality control. Version 2.1, valid from 2012-06-29. http:// www.eucast.org/fileadmin/src/media/PDFs/EUCAST\_files/Disk\_test\_documents/

[28] ISO. (2000). Quality Management Systems–Fundamentals and Vocabulary. ISO 9000.

[29] Shewhart W. A. (1931). Economic control of quality of the manufactured product.

[30] Westgard J. O., Barry P. L., Hunt M. R., Groth T. A. (1981). A multirule Shewhart chart for quality control in clinical chemistry. Clinical Chemistry 27, 493-501.

[31] US Department of Health and Human Services. (1992). Clinical Laboratory Improve‐ ment Amendments of 1988; Final Rules and Notice. 42 CFR Part 493. The Federal

[32] US Centers for Medicare & Medicaid Services (CMS). (2003). Medicare, Medicaid, and CLIA Programs: Laboratory Requirements Relating to Quality Systems and Cer‐

tain Personnel Qualifications. Final Rule. The Federal Register 16, 3640-714.

EUCAST\_QC\_tables\_2.1\_120629\_errata.pdf (accessed 30 July 2012).

Geneva: International Organization for Standardization.

agnostic Microbiology and Infectious Diseases 9, 135-8.

Wayne, PA: Clinical and Laboratory Standards Institute.

Society for Microbiology; 63-89.

and Laboratory Standards Institute.

Heights, MO: Saunders Elsevier; 276-314.

New York: Van Nostrand.

Register 57, 7188-288.


[22] Washington J. A. (1988). Current problems in antimicrobial susceptibility testing. Di‐ agnostic Microbiology and Infectious Diseases 9, 135-8.

[8] World Health Organization. (1961). Standardization of Methods for Conducting Mi‐ crobic Sensitivity Tests. Second Report of the Expert Committee on Antibiotics.

[9] Bauer, A. W., Kirby, W. M. M., Sherris, J. C., Turck, M. (1966). Antibiotic susceptibili‐ ty testing by a standardized single disk method. American Journal of Clinical Pathol‐

[10] Bergeron, M. G., Ouellette, M. (1998). Preventing antibiotic resistance through rapid genotypic identification of bacteria and of their antibiotic resistance genes in the clin‐

[11] National Committee for Clinical Laboratory Standards. (1975). Performance Stand‐ ards for Antimicrobial Disk Susceptibility Tests; Approved Standard M2-A7 ASM-2.

[12] Werkgroep Richtlijnen Gevoeligheidsbepalingen Report. (1990). Standaardisatie van

[13] Comité de l'Antibiogramme de la Société Française de Microbiologie. (1996). Clinical

[14] The Swedish Reference Group for Antibiotics. (1997). A revised system for antibiotic sensitivity testing. Scandinavian Journal of Infectious Diseases Suppl. 105.

[15] Deutsches Institut für Normung. (2000). Methoden zur Empfindlichkeitsprüfung von bakteriellen Krankheitserregern (auβer Mykobakterien) gegen Chemotherapeutika.

[16] Andrews, J. M. For the BSAC Working Party on Susceptibility Testing. (2000). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemo‐

[17] European Comittee on Antimicrobial Susceptibility Testing – EUCAST. (2012). http://

[18] Antimicrobial Susceptibility Testing. (2006). In: Winn W., Allen S., Janda W., Kone‐ man E., Procop G., Schreckenberger P., Woods G. (ed.) Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Baltimore, MD: Lippincott Williams &

[19] August M. J., Hindler J. A., Huber T. W., Sewell D. L. (1990). Cumitech 3A. Quality Control and Quality Assurance Practices in Clinical Microbiology. Coordinating ed.

[20] King A., Brown D. F. J. (2001). Quality assurance of antimicrobial susceptibility test‐ ing by disc diffusion. Journal of Antimicrobial Chemotherapy ,48, Suppl. S1, 71-6. [21] Westgard J. O., Klee G. G. (2006). Quality Management. In: Burtis C. A., Ashwood E. R., Bruns D. E. (ed.) Tietz Textbook of Clinical Chemistry and Molecular Diagnostics.

Weissfeld S. A. Washington, D.C.: American Society for Microbiology.

ical microbiology laboratory. Journal of Clinical Microbiology 36, 2169-72.

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ogy 45, 493-6.

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Wilkins; 945-1021.

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4th ed. St. Louis, MO: Elsevier Saunders; 485-529.

NCCLS, Villanova, PA.

Gevoeligheidsbepalingen. WRG, Bilthoven.

Microbiology and Infection 2, Suppl 1, S1-49.


**Chapter 20**

**The Investigation of Gene Regulation and Variation in**

Dynamic regulation of genes is an important part of the cell life cycle in health and disease. The regulation includes the variety and alteration of genome and gene expression, and the concept such as quality of genome will be useful to predict and assess the developmental stages of the cells, disease status and drug sensitivity. Recent technologies and worldwide sequencing projects have revealed 26,383 annotated genes in the 2.91-Gigabase human genome [1,2]. The main molecular functions of the annotated genes, as categorized by Gene Ontology (GO), are enzyme, signal transduction, nucleic acid binding, cell adhesion, chaperone, cytoskeletal structural protein, extracellular matrix, immunoglobulin, ion channel, motor, structural pro‐ tein of muscle, protooncogene, select calcium binding protein, intracellular transporter, and transporter [1,3]. Despite a wealth of knowledge, the function of 42% of the annotated genes re‐ mains unknown [1]. When the human genome sequence was published in 2001 [1], there were a predicted 39,114 genes, of which 59% were of unknown function. According to the Interna‐ tional Human Genome Sequencing Consortium, the number of identified genes is approxi‐ mately 32,000, of which 51% show a match within InterPro, a database that integrates diverse information about protein families, domains, and functional sites [2-5]. In 2001, InterPro com‐ bined sequence and pattern information from four databases (PRINTS, PROSITE, Pfam, Pro‐ site Profile); however, it now includes information from an additional eight databases (SMART, ProDom, PIRSF, SUPERFAMILY, PANTHER, CATH-Gene3D, TIGRFAM, and HA‐ MAP) [2,4-16]. In [2], the InterPro entries are collapsed into 12 broad categories: cellular proc‐ esses, metabolism, DNA replication/modification, transcription/translation, intracellular signaling, cell–cell communication, protein folding and degradation, transport, multifunction‐ al proteins, cytoskeletal/structural, defense and immunity, and miscellaneous function. The

> © 2012 Tanabe and Jee; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Tanabe and Jee; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Human Cancers and Other Diseases**

Additional information is available at the end of the chapter

Shihori Tanabe and Sun Ha Jee

http://dx.doi.org/10.5772/51271

**1. Introduction**

## **The Investigation of Gene Regulation and Variation in Human Cancers and Other Diseases**

Shihori Tanabe and Sun Ha Jee

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51271

#### **1. Introduction**

Dynamic regulation of genes is an important part of the cell life cycle in health and disease. The regulation includes the variety and alteration of genome and gene expression, and the concept such as quality of genome will be useful to predict and assess the developmental stages of the cells, disease status and drug sensitivity. Recent technologies and worldwide sequencing projects have revealed 26,383 annotated genes in the 2.91-Gigabase human genome [1,2]. The main molecular functions of the annotated genes, as categorized by Gene Ontology (GO), are enzyme, signal transduction, nucleic acid binding, cell adhesion, chaperone, cytoskeletal structural protein, extracellular matrix, immunoglobulin, ion channel, motor, structural pro‐ tein of muscle, protooncogene, select calcium binding protein, intracellular transporter, and transporter [1,3]. Despite a wealth of knowledge, the function of 42% of the annotated genes re‐ mains unknown [1]. When the human genome sequence was published in 2001 [1], there were a predicted 39,114 genes, of which 59% were of unknown function. According to the Interna‐ tional Human Genome Sequencing Consortium, the number of identified genes is approxi‐ mately 32,000, of which 51% show a match within InterPro, a database that integrates diverse information about protein families, domains, and functional sites [2-5]. In 2001, InterPro com‐ bined sequence and pattern information from four databases (PRINTS, PROSITE, Pfam, Pro‐ site Profile); however, it now includes information from an additional eight databases (SMART, ProDom, PIRSF, SUPERFAMILY, PANTHER, CATH-Gene3D, TIGRFAM, and HA‐ MAP) [2,4-16]. In [2], the InterPro entries are collapsed into 12 broad categories: cellular proc‐ esses, metabolism, DNA replication/modification, transcription/translation, intracellular signaling, cell–cell communication, protein folding and degradation, transport, multifunction‐ al proteins, cytoskeletal/structural, defense and immunity, and miscellaneous function. The

© 2012 Tanabe and Jee; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Tanabe and Jee; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

rate of single nucleotide polymorphism (SNP) variation has been reported as 1 in 1250 base pairs [1] and more than 1.4 million SNPs have been identified [2] (Table 1).

sifies genes by their functions using published experimental evidence and evolutionary rela‐ tionships to predict function even in the absence of direct experimental evidence [13]; CATH-Gene3D is a comprehensive database of protein domain assignments for sequences from the major sequence databases [14]; TIGRFAM is a collection of protein family defini‐ tions built to aid high-throughput annotation of specific protein functions [15]; and HAMAP is composed of two databases: the proteome database and the family database, and of an au‐ tomatic annotation pipeline mainly focused on microbial proteomes [16]. Hidden Markov

The Investigation of Gene Regulation and Variation in Human Cancers and Other Diseases

**Context URL Reference**

http://www.ebi.ac.uk/interpro/ [4], [5]

[6]

http://dx.doi.org/10.5772/51271

437

[8]

[10]

http://www.bioinf.manchester.ac.uk/ dbbrowser/PRINTS/index.php

http://prosite.expasy.org/ [7]

http://smart.embl.de/ [9]

http://prodom.prabi.fr/prodom/current/

http://pir.georgetown.edu/pirsf/ [11]

http://supfam.org/SUPERFAMILY/ [12]

http://www.pantherdb.org/ [13]

http://gene3d.biochem.ucl.ac.uk/ [14]

html/home.php

http://pfam.sanger.ac.uk/http:// pfam.janelia.org/http://pfam.sbc.su.se/

models are usually used for the database algorithm.

a collection of diagnostic protein family

alignments

UK, the USA and Sweden

uniProt

knowledge Database

proteins and domains

for all proteins and genomes

major sequence databases

integrative predictive models of protein families, domain and functional sites of multiple databases such as PRINTS, PROSITE, Pfam, SMART, ProDom, PIRSF, SUPERFAMILY, PANTHER, CATH-Gene3D, TIGRFAM, and HAMAP

"fingerprints" which are groups of conserved motifs, evident in multiple sequence

a protein domain database for functional characterization and annotation which consists of documentation entries describing protein domains, families and functional

a database of collection of protein families, each represented by multiple sequence

an online resource for the identification and annotation of protein domains and the

a comprehensive set of protein domain families automatically generated from the

the classification system which reflects evolutionary relationships of full-length

their functions using published scientific experimental evidence and evolutionary relationships to predict function even in the absence of direct experimental evidence

a comprehensive database of protein domain assignments for sequences from the

sites as well as associated patterns and profiles to identify them

alignments and hidden Markov models, available via servers in the

analysis of protein domain architectures, of which abbreviation

is Simple Modular Architecture Research Tool

a database of structural and functional annotation

the classification system which classifies genes by

**Database Name**

InterPro

PRINTS

PROSITE

Pfam

SMART

ProDom

PIRSF

SUPERFAMILY

PANTHER

CATH-Gene3D


**Table 1.** Genomic and gene characteristics revealed by the Human Genome Project.

Among the databases combined in InterPro (Table 2), PRINTS, PROSITE, and Pfam contain protein families in which the homology between each protein is predicted by the degree of sequence similarity [8]. The others—SMART, ProDom, PIRSF, SUPERFAMILY, PANTHER, CATH-Gene3D, TIGRFAM, and HAMAP [4-16]—have unique characteristics and URLs, and have been developed sharing information among each other and incorporating informa‐ tion from GO. In detail, PRINTS is a collection of diagnostic protein family "fingerprints", which are groups of conserved motifs, evident in multiple sequence alignments [6]; PRO‐ SITE is a protein domain database for functional characterization and annotation that con‐ sists of documentation entries describing protein domains, families, and functional sites as well as associated patterns and profiles to identify them [7]; Pfam contains collections of protein families, each represented by multiple sequence alignments and hidden Markov models, available via servers in the UK, the USA, and Sweden [8]; SMART (Simple Modular Architecture Research Tool) is an online resource for the identification and annotation of protein domains and the analysis of protein domain architectures [9]; ProDom is a compre‐ hensive set of protein domain families generated automatically from the UniProt database [10]; PIRSF is a classification system that reflects evolutionary relationships among fulllength proteins and domains [11]; SUPERFAMILY is a database of structural and functional annotation for all proteins and genomes [12]; PANTHER is a classification system that clas‐ sifies genes by their functions using published experimental evidence and evolutionary rela‐ tionships to predict function even in the absence of direct experimental evidence [13]; CATH-Gene3D is a comprehensive database of protein domain assignments for sequences from the major sequence databases [14]; TIGRFAM is a collection of protein family defini‐ tions built to aid high-throughput annotation of specific protein functions [15]; and HAMAP is composed of two databases: the proteome database and the family database, and of an au‐ tomatic annotation pipeline mainly focused on microbial proteomes [16]. Hidden Markov models are usually used for the database algorithm.

rate of single nucleotide polymorphism (SNP) variation has been reported as 1 in 1250 base

Size of the genome 2.91 Gbp [1] Number of annotated genes 26,383 [1]

select calcium binding protein, intracellular transporter, transporter

Number of identified genes approx. 32,000 [2]

Rate of SNP variation 1/1250 bp [1] SNPs identified more than 1.4 million [2]

Among the databases combined in InterPro (Table 2), PRINTS, PROSITE, and Pfam contain protein families in which the homology between each protein is predicted by the degree of sequence similarity [8]. The others—SMART, ProDom, PIRSF, SUPERFAMILY, PANTHER, CATH-Gene3D, TIGRFAM, and HAMAP [4-16]—have unique characteristics and URLs, and have been developed sharing information among each other and incorporating informa‐ tion from GO. In detail, PRINTS is a collection of diagnostic protein family "fingerprints", which are groups of conserved motifs, evident in multiple sequence alignments [6]; PRO‐ SITE is a protein domain database for functional characterization and annotation that con‐ sists of documentation entries describing protein domains, families, and functional sites as well as associated patterns and profiles to identify them [7]; Pfam contains collections of protein families, each represented by multiple sequence alignments and hidden Markov models, available via servers in the UK, the USA, and Sweden [8]; SMART (Simple Modular Architecture Research Tool) is an online resource for the identification and annotation of protein domains and the analysis of protein domain architectures [9]; ProDom is a compre‐ hensive set of protein domain families generated automatically from the UniProt database [10]; PIRSF is a classification system that reflects evolutionary relationships among fulllength proteins and domains [11]; SUPERFAMILY is a database of structural and functional annotation for all proteins and genomes [12]; PANTHER is a classification system that clas‐

enzyme, signal transduction, nucleic acid binding, cell adhesion, chaperone, cytoskeletal structural protein, extracellular matrix, immunoglobulin, ion channel, motor, structural protein of muscle, protooncogene,

42% [1]

39,114 [1]

59% [1]

51% [2]

[1]

pairs [1] and more than 1.4 million SNPs have been identified [2] (Table 1).

**Table 1.** Genomic and gene characteristics revealed by the Human Genome Project.

Main molecular functions of annotated genes

436 Latest Research into Quality Control

Percentage of annotated genes with unknown function

Number of hypothetical and annotated genes

Percentage of hypothetical and annotated genes with unknown function

> Percentage of matches with InterPro



Highly parallel identification of cancer-related genes using small hairpin RNA screening has revealed that the expression of known and putative oncogenes, such as *EGFR*, *KRAS*, *MYC*, *BCR-ABL*, *MYB*, *CRKL*, and *CDK4* that are essential for cancer proliferation, is altered in cancer cells [33]. Other genes such as *PTPN1*, *NF1*, *SMARCB1*, and *SMARCE1* have been identified as essential for the imatinib response of leukemia cells, and *TOPOIIA* expression is involved in re‐

sistance to etoposide, an anti-topoisomerase II agent, in small cell lung cancer [33-36].

*TP53* mutation

alteration in cell cycle mRNA expression

alteration in cytokeratin mRNA expression

alteration in kallikrein mRNA expression

mitochondrial DNA mutations

**Table 3.** Genomic markers of cancer.

**2.2. Genes related to cell proliferation**

**Marker Cancer Type Reference**

bladder, ovary, and brain cancers; sarcomas, lymphomas, and leukemias

*EGFR* mutation non-small cell lung cancer [21] *RAS* mutation colorectal tumors [22] DNA microsatellite alterations bladder cancer [23]

Cyclins, which regulate the cell cycle, play important roles in cell proliferation and the uncontrolled cell proliferation that is the most important factor in tumorigenesis [37]. Tu‐ mor cells accumulate mutations that result in constitutive mitogenic signaling and defec‐ tive responses to anti-mitogenic signals that contribute to unscheduled proliferation [38]. In cancer, unscheduled proliferation, genomic instability, and chromosomal instability are the three major factors in cell cycle dysregulation [38]. Regulation of the cell cycle is mainly conducted by complexes of cyclins and cyclin-dependent kinases [38]. Cyclin D1 in cell migration and proliferation is temporo-spatially separated by its biphasic expres‐ sion induced by thrombin, a G protein-coupled receptor agonist, which is mediated by nuclear factor of activated T cells c1 (*NFATC1*) and signal transducer and activator of transcription 3 (*STAT3*) [39]. Cyclin D1 regulates kinase activity and the G1–S phase tran‐

head and neck cancer [18] bladder cancer [20] lung cancer (small cell lung cancer and non-small cell lung cancer); breast, colon, esophagus, liver,

The Investigation of Gene Regulation and Variation in Human Cancers and Other Diseases

colon cancer [24]

squamous cell carcinoma [26]

prostate cancer [27]

bladder cancer, head and neck cancer, lung cancer [29] colorectal tumors [30], [32] pancreatic cancer [31]

[19]

http://dx.doi.org/10.5772/51271

439

**Table 2.** Database information.

#### **2. Gene regulation**

#### **2.1. Gene markers for cancer and cancer stem cells**

Several molecular markers of cancer have been identified [17]. Metastatic cancer cells can transfer into bodily fluids through the cellular epithelia, which enables the detection of can‐ cer markers in bodily fluids such as blood plasma, urine, or saliva [17]. The different types of cancer markers include genomic DNA point mutations, microsatellite alterations, promot‐ er hypermethylation, viral sequences, aberrant chromosomal copy number, chromosomal translocations, deletions, or loss of heterozygosity, telomere extension, alterations in RNA or protein expression, and mitochondrial DNA mutations [17].

Molecular markers of cancer include *TP53* (encoding p53), which has been shown to be mu‐ tated in head and neck, lung, colon, pancreatic, and bladder cancer [17,18]; colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissue, and hematopoietic tissue cancers [19]; and bladder cancer [20]. Mutation of the epidermal growth factor receptor (*EGFR*) gene is an important predictive/prognostic factor for EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer [21]. *RAS* oncogene mutations have been identified in colorectal tumors [22]. Microsatellites, which are tandem iterations of simple di-, tri-, or tetranucleo‐ tide repeats, have been reported to be unstable in some inherited diseases and in some types of cancer [23], including head and neck, lung, breast, and bladder cancer [17,23].

The expression levels of the cell cycle-related proteins p21 (*CDKN1A*), p53 (*TP53*), cyclin D1 (*CCND1*), and aurora kinase A (*AURKA*) may be used as prognostic markers to predict re‐ currence in stage II and stage III colon cancer [24]. In addition, markers of the epithelial– mesenchymal transition (EMT)–such as reduced expression of keratins, a switch from E-Cadherin to N-Cadherin, and enhanced migration in D492M cells—might be a useful mark‐ er in breast cancer [25]. Furthermore, expression of the stem cell markers cytokeratins 15 and 19 was altered in squamous cell carcinoma: cytokeratin 15 levels were decreased and the lo‐ calization of cytokeratin 19 was altered [26]. *KLK3*, which encodes prostate-specific antigen, a member of the kallikrein family of serine proteases, is a biomarker for prostate cancer de‐ tection and disease monitoring [27,28]. Mitochondrial DNA mutations have been associated with bladder, head and neck, lung, colorectal, and pancreatic cancer [29-32] (Table 3).

Highly parallel identification of cancer-related genes using small hairpin RNA screening has revealed that the expression of known and putative oncogenes, such as *EGFR*, *KRAS*, *MYC*, *BCR-ABL*, *MYB*, *CRKL*, and *CDK4* that are essential for cancer proliferation, is altered in cancer cells [33]. Other genes such as *PTPN1*, *NF1*, *SMARCB1*, and *SMARCE1* have been identified as essential for the imatinib response of leukemia cells, and *TOPOIIA* expression is involved in re‐ sistance to etoposide, an anti-topoisomerase II agent, in small cell lung cancer [33-36].


**Table 3.** Genomic markers of cancer.

**Database Name**

> a collection of protein family definitions built to aid in high-throughput annotation of specific protein functions

database, and of an automatic annotation pipeline

**2.1. Gene markers for cancer and cancer stem cells**

protein expression, and mitochondrial DNA mutations [17].

a system which composed of two databases, the proteome database and the family

TIGRFAM

HAMAP

**Table 2.** Database information.

438 Latest Research into Quality Control

**2. Gene regulation**

**Context URL Reference**

Several molecular markers of cancer have been identified [17]. Metastatic cancer cells can transfer into bodily fluids through the cellular epithelia, which enables the detection of can‐ cer markers in bodily fluids such as blood plasma, urine, or saliva [17]. The different types of cancer markers include genomic DNA point mutations, microsatellite alterations, promot‐ er hypermethylation, viral sequences, aberrant chromosomal copy number, chromosomal translocations, deletions, or loss of heterozygosity, telomere extension, alterations in RNA or

Molecular markers of cancer include *TP53* (encoding p53), which has been shown to be mu‐ tated in head and neck, lung, colon, pancreatic, and bladder cancer [17,18]; colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissue, and hematopoietic tissue cancers [19]; and bladder cancer [20]. Mutation of the epidermal growth factor receptor (*EGFR*) gene is an important predictive/prognostic factor for EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer [21]. *RAS* oncogene mutations have been identified in colorectal tumors [22]. Microsatellites, which are tandem iterations of simple di-, tri-, or tetranucleo‐ tide repeats, have been reported to be unstable in some inherited diseases and in some types

The expression levels of the cell cycle-related proteins p21 (*CDKN1A*), p53 (*TP53*), cyclin D1 (*CCND1*), and aurora kinase A (*AURKA*) may be used as prognostic markers to predict re‐ currence in stage II and stage III colon cancer [24]. In addition, markers of the epithelial– mesenchymal transition (EMT)–such as reduced expression of keratins, a switch from E-Cadherin to N-Cadherin, and enhanced migration in D492M cells—might be a useful mark‐ er in breast cancer [25]. Furthermore, expression of the stem cell markers cytokeratins 15 and 19 was altered in squamous cell carcinoma: cytokeratin 15 levels were decreased and the lo‐ calization of cytokeratin 19 was altered [26]. *KLK3*, which encodes prostate-specific antigen, a member of the kallikrein family of serine proteases, is a biomarker for prostate cancer de‐ tection and disease monitoring [27,28]. Mitochondrial DNA mutations have been associated

of cancer [23], including head and neck, lung, breast, and bladder cancer [17,23].

with bladder, head and neck, lung, colorectal, and pancreatic cancer [29-32] (Table 3).

http://www.jcvi.org/cgi-bin/tigrfams/

http://hamap.expasy.org/ [16]

[15]

index.cgi

#### **2.2. Genes related to cell proliferation**

Cyclins, which regulate the cell cycle, play important roles in cell proliferation and the uncontrolled cell proliferation that is the most important factor in tumorigenesis [37]. Tu‐ mor cells accumulate mutations that result in constitutive mitogenic signaling and defec‐ tive responses to anti-mitogenic signals that contribute to unscheduled proliferation [38]. In cancer, unscheduled proliferation, genomic instability, and chromosomal instability are the three major factors in cell cycle dysregulation [38]. Regulation of the cell cycle is mainly conducted by complexes of cyclins and cyclin-dependent kinases [38]. Cyclin D1 in cell migration and proliferation is temporo-spatially separated by its biphasic expres‐ sion induced by thrombin, a G protein-coupled receptor agonist, which is mediated by nuclear factor of activated T cells c1 (*NFATC1*) and signal transducer and activator of transcription 3 (*STAT3*) [39]. Cyclin D1 regulates kinase activity and the G1–S phase tran‐ sition in the cell cycle; deregulated cyclin D1 expression is well documented in breast, colon, and prostate cancers [39,40]. The expression of cyclin D1 is regulated by several factors including cytokines such as interleukin 3 and interleukin 6 *via* STAT3 and STAT5, or extracellular matrix factors such as collagen, fibronectin, and vitronectin, which activate focal adhesion kinase upon integrin clustering, and hepatocyte nuclear factor 6 [41]. Cyclin D1 is a crucial regulator of Wnt- and Notch-regulated development [41,42]. The binding of Wnt to its receptor, Frizzled, causes release of β-catenin to trans‐ locate from the cytoplasm to the nucleus, where it forms a complex with the ternary complex factor and/or the lymphoid enhancer-binding factor [41,43]. Cyclin D1 is in‐ duced by overexpression of β-catenin, which is a major component of adherens junc‐ tions that link the actin cytoskeleton to members of the cadherin family of transmembrane cell–cell adhesion receptors. It plays an important role in linking the cy‐ toplasmic side of cadherin-mediated cell–cell contacts to the actin cytoskeleton [43]. Betacatenin is upregulated in colorectal cancer, which is considered to trigger cyclin D1 gene expression followed by uncontrolled progression of the cell cycle [43]. In addition, β-cat‐ enin plays another role in signaling that involves transactivation, in complex with tran‐ scription factors of the lymphoid enhancing factor family in the nucleus [43]. The pathway involving β-catenin/LEF1 and elevation of cyclin D1 might be crucial for tu‐ morigenesis [43]. Inhibiting EglN2, a member of the EglN (also called PHD or HPH) family of prolyl hydroxylases that regulates the heterodimeric transcription factor hypo‐ xia-inducible factor (HIF), causes a decrease in the expression of its interaction partner cyclin D1 in cancer cells and impairs the cells' ability to proliferate *in vivo* [44].

encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis [49]. The expression of HOXA1, which plays an important role in proliferation, apoptosis, adhesion, invasion, the EMT, and anchorage-independent growth, was significantly increased in oral squamous cell carcino‐ ma compared with in healthy oral mucosa [49], and it might be a useful prognostic marker

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441

Wnt/β-catenin signaling controls skeletal development and differentiation [50]. The initiat‐ ing step of skeletal development is mesenchymal condensation, during which mesenchymal progenitor cells are at least bipotentiate [50]. Osteochondral progenitor cells differentiate in‐ to osteoblasts instead of chondrocytes when Wnt/β-catenin signaling is activated [50]. *In vi‐ tro* models using human pluripotent stem cell-derived neural progenitor cells have been used to examine whether G11778A-mutated mitochondrial DNA, which is associated with Leber's hereditary optic neuropathy, might be involved in the differentiation of neural pro‐ genitor cells into neurons, oligodendrocytes, and astrocytes [51]. The differentiation of neu‐ ral progenitor cells can be visualized by staining for the neuronal marker class III betatubulin [51]. Alternative splicing of exons play an important role in cellular differentiation and pathogenesis [52]. Alternative splicing in colorectal cancer and renal cell cancer samples has been analyzed by the Bioinformatics Exon Array Tool (BEAT, http://beat.ba.itb.cnr.it/) using an Affymetrix GeneChip Exon Array [52]. When the dataset was analyzed using GO terms, the cell differentiation (GO:0030154)-related gene delta-like 1 (Drosophila) (*DLL1*)

Cell proliferation and death are regulated by various molecules. Recently, microRNAs have been revealed to play important roles during death receptor-mediated apoptosis (programmed cell death) [53]. Transfection with miR-133b caused a proapoptotic effect on tumor necrosis factor alpha (TNFα)-stimulated HeLa cells [53]: the expression of apoptosis regulatory proteins such as transgelin 2 (TAGLN2), myosin, heavy chain 9, non-muscle (MYH9), cytoskeleton-associated protein 4 (CKAP4), polypyrimidine tract binding protein 1 (PTBP1), glutathione-S-transferase pi 1 (GSTP1), and copine III (CPNE3) were down-regulated compared with in control cells [53]. The BCL protein family plays a major role in regulation of the apoptotic cascade [54]. BCL2-associated protein (BAX) promotes apoptosis and delays disease progression, and has been associat‐ ed with longer disease-free survival in patients with a number of gastrointestinal can‐ cers, such as esophageal, stomach, small intestine, and colon cancer; moreover, high BCL6 expression is correlated with worse prognosis in patients with other gastrointesti‐ nal tumors, such as esophageal adenocarcinoma [54]. There are two major cell death pathways that transduce the effects of various death inducers: the extrinsic death path‐ way that is mediated through cell death receptors of the TNF receptor family, such as the Fas receptor; and the intrinsic death pathway that proceeds through mitochondria [55]. The expression of apoptosis signal-regulating kinase (ASK1), which plays an impor‐ tant role as a mitogen-activated protein kinase kinase kinase in apoptosis signaling, is in‐

for patients with this disease [49].

was found to be involved in colorectal cancer [52].

**2.4. Genes related to apoptosis**

Progression of the eukaryotic cell cycle is driven by cyclin-dependent protein kinases (CDKs), which are binding partner of cyclins. The CDK oscillator acts as the primary or‐ ganizer of the cell cycle [45]. Phosphorylation of cyclin-Cdk complexes is one of the pri‐ mary mechanisms of cell cycle regulation [46]. Cyclins are degraded by ubiquitinmediated proteolysis [46]. The ubiquitylation and degradation of cyclin 1 and cyclin 2 are mediated by the SCF complex, a multi-subunit ubiquitin ligase that contains Skp1, a member of the cullin family (Cdc53) and an F-box protein, as well as a RING-finger-con‐ taining protein [46]. CDKs including CDK1, CDK2, CDK4, CDK6, and CDK11 have vari‐ ous functions that have been investigated using loss-of-function, target validation, and gain-of-function mouse models [38]. CDK1 is a mitotic CDK, also known as cell division control protein 2 (CDC2). It is one of the master regulators of mitosis as it controls the centrosome cycle as well as mitotic onset; deficiency in CDK1 results in embryonic lethali‐ ty in the first cell divisions [38,47]. CDK2, CDK4, and CDK6 are interphase CDKs that are not essential for the mammalian cell cycle; they are, however, required for the prolifera‐ tion of specific cell types [38]. Deficiency in CDK2, CDK4, and CDK6 caused mid-gesta‐ tion embryonic lethality because of hematopoietic defects [38,47].

#### **2.3. Genes related to cell differentiation**

Inhibitor of differentiation 1 (Id1) is associated with the induction of cell proliferation and invasion [48], as well as the invasive features of cancer and the EMT [48]. The *HOX* genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis [49]. The expression of HOXA1, which plays an important role in proliferation, apoptosis, adhesion, invasion, the EMT, and anchorage-independent growth, was significantly increased in oral squamous cell carcino‐ ma compared with in healthy oral mucosa [49], and it might be a useful prognostic marker for patients with this disease [49].

Wnt/β-catenin signaling controls skeletal development and differentiation [50]. The initiat‐ ing step of skeletal development is mesenchymal condensation, during which mesenchymal progenitor cells are at least bipotentiate [50]. Osteochondral progenitor cells differentiate in‐ to osteoblasts instead of chondrocytes when Wnt/β-catenin signaling is activated [50]. *In vi‐ tro* models using human pluripotent stem cell-derived neural progenitor cells have been used to examine whether G11778A-mutated mitochondrial DNA, which is associated with Leber's hereditary optic neuropathy, might be involved in the differentiation of neural pro‐ genitor cells into neurons, oligodendrocytes, and astrocytes [51]. The differentiation of neu‐ ral progenitor cells can be visualized by staining for the neuronal marker class III betatubulin [51]. Alternative splicing of exons play an important role in cellular differentiation and pathogenesis [52]. Alternative splicing in colorectal cancer and renal cell cancer samples has been analyzed by the Bioinformatics Exon Array Tool (BEAT, http://beat.ba.itb.cnr.it/) using an Affymetrix GeneChip Exon Array [52]. When the dataset was analyzed using GO terms, the cell differentiation (GO:0030154)-related gene delta-like 1 (Drosophila) (*DLL1*) was found to be involved in colorectal cancer [52].

#### **2.4. Genes related to apoptosis**

sition in the cell cycle; deregulated cyclin D1 expression is well documented in breast, colon, and prostate cancers [39,40]. The expression of cyclin D1 is regulated by several factors including cytokines such as interleukin 3 and interleukin 6 *via* STAT3 and STAT5, or extracellular matrix factors such as collagen, fibronectin, and vitronectin, which activate focal adhesion kinase upon integrin clustering, and hepatocyte nuclear factor 6 [41]. Cyclin D1 is a crucial regulator of Wnt- and Notch-regulated development [41,42]. The binding of Wnt to its receptor, Frizzled, causes release of β-catenin to trans‐ locate from the cytoplasm to the nucleus, where it forms a complex with the ternary complex factor and/or the lymphoid enhancer-binding factor [41,43]. Cyclin D1 is in‐ duced by overexpression of β-catenin, which is a major component of adherens junc‐ tions that link the actin cytoskeleton to members of the cadherin family of transmembrane cell–cell adhesion receptors. It plays an important role in linking the cy‐ toplasmic side of cadherin-mediated cell–cell contacts to the actin cytoskeleton [43]. Betacatenin is upregulated in colorectal cancer, which is considered to trigger cyclin D1 gene expression followed by uncontrolled progression of the cell cycle [43]. In addition, β-cat‐ enin plays another role in signaling that involves transactivation, in complex with tran‐ scription factors of the lymphoid enhancing factor family in the nucleus [43]. The pathway involving β-catenin/LEF1 and elevation of cyclin D1 might be crucial for tu‐ morigenesis [43]. Inhibiting EglN2, a member of the EglN (also called PHD or HPH) family of prolyl hydroxylases that regulates the heterodimeric transcription factor hypo‐ xia-inducible factor (HIF), causes a decrease in the expression of its interaction partner

cyclin D1 in cancer cells and impairs the cells' ability to proliferate *in vivo* [44].

tion embryonic lethality because of hematopoietic defects [38,47].

**2.3. Genes related to cell differentiation**

440 Latest Research into Quality Control

Progression of the eukaryotic cell cycle is driven by cyclin-dependent protein kinases (CDKs), which are binding partner of cyclins. The CDK oscillator acts as the primary or‐ ganizer of the cell cycle [45]. Phosphorylation of cyclin-Cdk complexes is one of the pri‐ mary mechanisms of cell cycle regulation [46]. Cyclins are degraded by ubiquitinmediated proteolysis [46]. The ubiquitylation and degradation of cyclin 1 and cyclin 2 are mediated by the SCF complex, a multi-subunit ubiquitin ligase that contains Skp1, a member of the cullin family (Cdc53) and an F-box protein, as well as a RING-finger-con‐ taining protein [46]. CDKs including CDK1, CDK2, CDK4, CDK6, and CDK11 have vari‐ ous functions that have been investigated using loss-of-function, target validation, and gain-of-function mouse models [38]. CDK1 is a mitotic CDK, also known as cell division control protein 2 (CDC2). It is one of the master regulators of mitosis as it controls the centrosome cycle as well as mitotic onset; deficiency in CDK1 results in embryonic lethali‐ ty in the first cell divisions [38,47]. CDK2, CDK4, and CDK6 are interphase CDKs that are not essential for the mammalian cell cycle; they are, however, required for the prolifera‐ tion of specific cell types [38]. Deficiency in CDK2, CDK4, and CDK6 caused mid-gesta‐

Inhibitor of differentiation 1 (Id1) is associated with the induction of cell proliferation and invasion [48], as well as the invasive features of cancer and the EMT [48]. The *HOX* genes Cell proliferation and death are regulated by various molecules. Recently, microRNAs have been revealed to play important roles during death receptor-mediated apoptosis (programmed cell death) [53]. Transfection with miR-133b caused a proapoptotic effect on tumor necrosis factor alpha (TNFα)-stimulated HeLa cells [53]: the expression of apoptosis regulatory proteins such as transgelin 2 (TAGLN2), myosin, heavy chain 9, non-muscle (MYH9), cytoskeleton-associated protein 4 (CKAP4), polypyrimidine tract binding protein 1 (PTBP1), glutathione-S-transferase pi 1 (GSTP1), and copine III (CPNE3) were down-regulated compared with in control cells [53]. The BCL protein family plays a major role in regulation of the apoptotic cascade [54]. BCL2-associated protein (BAX) promotes apoptosis and delays disease progression, and has been associat‐ ed with longer disease-free survival in patients with a number of gastrointestinal can‐ cers, such as esophageal, stomach, small intestine, and colon cancer; moreover, high BCL6 expression is correlated with worse prognosis in patients with other gastrointesti‐ nal tumors, such as esophageal adenocarcinoma [54]. There are two major cell death pathways that transduce the effects of various death inducers: the extrinsic death path‐ way that is mediated through cell death receptors of the TNF receptor family, such as the Fas receptor; and the intrinsic death pathway that proceeds through mitochondria [55]. The expression of apoptosis signal-regulating kinase (ASK1), which plays an impor‐ tant role as a mitogen-activated protein kinase kinase kinase in apoptosis signaling, is in‐ creased in gastric cancer [56]. Furthermore, the levels of cyclin D1 and phosphorylated JNK were higher in gastric cancer than in non-tumor epithelium [56]. ASK1 may play a role in the development of gastric cancer [56].

consisting of TdT reaction buffer, TdT enzyme, and BrdUTP, then stained with PI to detect a fluorescein isothiocyanate-labeled anti-BrdU antibody [63]. Cell viability and proliferation as‐ says were used to validate internal tandem duplication mutations in *FLT3* as a therapeutic tar‐ get for human acute myeloid leukemia [64]. Cell viability and proliferation can be determined

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Despite extensive research efforts for several decades, the genetic basis of common human diseases such as cancers remains largely unknown [65]. Genome-wide association studies (GWAS) have emerged as an important tool for the discovery of genomic regions that har‐ bor genetic variants conferring risk for various cancers [66,67]. Family-based linkage studies and studies comprising tens of thousands of gene-based SNPs can also assay genetic varia‐ tion across the genome [68], but the National Institutes of Health guidelines for GWAS re‐ quire a sufficient density of genetic markers to capture a large proportion of the common variants in the study population, measured in enough individuals to provide sufficient pow‐ er to detect variants of modest effect [67]. The recent success of GWAS can be attributed to the convergence of new technologies that can genotype hundreds of thousands of SNPs in

GWAS have been conducted in the five of the most common cancer types: breast, prostate, colorectal, lung, and melanoma (Table 4) and have identified more than 20 novel disease lo‐ ci, confirming that susceptibility to these diseases is polygenic [70]. For many years, human genetics has been used to map rare mutations with large effect sizes in families or genetical‐ ly homogeneous populations, such as *BRCA1/BRCA2* mutations in Ashkenazi women with breast cancer and ovarian cancer [71]. A number of SNPs have now been associated with breast cancer; for example, a SNP in intron 2 of the *FGFR2* gene, which encodes a receptor tyrosine kinase that is amplified and overexpressed in 5–10% of breast tumors [72,73], and SNPs on chromosomes 16q and 5q. The locus on 16q contains a gene *TNRC9* and a hypothet‐ ical gene *LOC643714*. The function of TNRC9 is unknown but the presence of an HMG box motif suggests that it might act as a transcription factor. The 5q locus includes *MAP3K1*, which encodes a protein involved in signal transduction (but not previously known to be involved in cancer) and two other genes: *MGC33648* and *MIER3*. In addition, several of the breast cancer loci appear to be associated with specific subtypes of the disease. In particular, the *FGFR2* association is strongly associated with estrogen receptor-positive breast cancer, while the *TNRC9* SNP is associated with both estrogen receptor-positive and -negative breast cancer [74,75]. It is surprising that none of the strongest associations map to regions harboring estrogen/progesterone genes in women of European background, particularly be‐ cause a GWAS in Asian women reported a convincing association with markers near the es‐ trogen receptor alpha (*ESR1*) gene [76]. In prostate cancer, the first and most important region to emerge was 8q24. This region was first associated with prostate cancer through

using a Vi-cell XR automated cell viability analyzer (Beckman Coulter) [64].

**3. Genomic variation in disease**

hundreds or thousands of samples [66,69].

**3.1. Genome-wide association studies in cancer**

#### **2.5. Detection of cell proliferation or apoptosis**

Several methods have been suggested for the diagnosis of cancer [57]. Protein markers for cancer include prostate-specific antigen for prostate cancer, CA125 for ovarian cancer, carci‐ noembryonic antigen for colon cancer, human chorionic gonadotropin for trophoblastic can‐ cer, and a-fetoprotein for hepatocellular carcinoma and germ cell tumors [57]. Assays to detect telomerase activity in clinical samples include the TRAP (telomere repeat amplifica‐ tion protocol) assay, which involves protein extraction and subsequent primer-directed PCR amplification of telomere extensions [57].

Assays for the detection of kinases that regulate cell growth, proliferation, differentiation, and metabolism have been developed [58]. The assay technology includes fluorescence po‐ larization to detect protein phosphorylation, scintillation proximity to detect protein de‐ phosphorylation by phosphatases, fluorescence resonance energy transfer to detect protein cleavage or modification, immunosorbent assays to detect phosphorylation state, luciferasebased ATP detection to detect the kinase-dependent depletion of ATP, luminescent oxygen channeling to detect phosphorylation, time-resolved fluorescence resonance energy transfer to detect phosphopeptide formation, and enzyme fragment complementation to detect mo‐ lecular interactions with kinases [58,59]. Cell proliferation can also be determined by the tet‐ razolium hydroxide (XTT) cell proliferation assay, in which absorbance is measured by an ELISA reader under 490-nm-wavelength light (Biological Industries) [60].

Cell proliferation assays and apoptosis assays have been used to examine the effects of in‐ hibitors on cancer cells [61]. The cell proliferation of Neuro-2A cells, neuroblastoma cells, can be determined using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay reagent (Promega) [61]. A colony formation assay using Neuro-2A cells was used to deter‐ mine the effect of an inhibitor of GSK-3β [61]. In this experiment, colonies were allowed to form for 10 days, after which the cells were fixed with 70% ethanol and stained with 1% methylene blue. Apoptosis was then measured by flow cytometry using an Annexin V-allo‐ phycocyanin (APC) /propidium iodide (PI) detection kit (BD PharMingen) [61]. Apoptosis was also determined using 4'6-diamidino-2-phenylindole (DAPI) staining, observing apop‐ totic nuclear morphology, and immunoblotting with antibodies to β-catenin, X-linked inhib‐ itor of apoptosis, and BCL2 [61]. Cell cycle analysis using PI to quantify the proportions of cells in the G1/G0 or G2–M phases was used to examine cell cycle status [61].

Viable cells can be determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli‐ um bromide) colorimetric assays [62]. Absorbance at 570 nm is used to detect the incorporation of MTT. Apoptosis can also be determined by caspase activation using an anti-poly ADP-ri‐ bose polymerase (PARP) antibody [62]. Viable cells can also be determined using a 3-(4,5-di‐ methyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) kit (Promega) [63]. The terminal transferase dUTP nick end labeling (TUNEL) assay is com‐ monly used to detect apoptosis [63]. Harvested cells are resuspended in DNA labeling solution consisting of TdT reaction buffer, TdT enzyme, and BrdUTP, then stained with PI to detect a fluorescein isothiocyanate-labeled anti-BrdU antibody [63]. Cell viability and proliferation as‐ says were used to validate internal tandem duplication mutations in *FLT3* as a therapeutic tar‐ get for human acute myeloid leukemia [64]. Cell viability and proliferation can be determined using a Vi-cell XR automated cell viability analyzer (Beckman Coulter) [64].

#### **3. Genomic variation in disease**

creased in gastric cancer [56]. Furthermore, the levels of cyclin D1 and phosphorylated JNK were higher in gastric cancer than in non-tumor epithelium [56]. ASK1 may play a

Several methods have been suggested for the diagnosis of cancer [57]. Protein markers for cancer include prostate-specific antigen for prostate cancer, CA125 for ovarian cancer, carci‐ noembryonic antigen for colon cancer, human chorionic gonadotropin for trophoblastic can‐ cer, and a-fetoprotein for hepatocellular carcinoma and germ cell tumors [57]. Assays to detect telomerase activity in clinical samples include the TRAP (telomere repeat amplifica‐ tion protocol) assay, which involves protein extraction and subsequent primer-directed PCR

Assays for the detection of kinases that regulate cell growth, proliferation, differentiation, and metabolism have been developed [58]. The assay technology includes fluorescence po‐ larization to detect protein phosphorylation, scintillation proximity to detect protein de‐ phosphorylation by phosphatases, fluorescence resonance energy transfer to detect protein cleavage or modification, immunosorbent assays to detect phosphorylation state, luciferasebased ATP detection to detect the kinase-dependent depletion of ATP, luminescent oxygen channeling to detect phosphorylation, time-resolved fluorescence resonance energy transfer to detect phosphopeptide formation, and enzyme fragment complementation to detect mo‐ lecular interactions with kinases [58,59]. Cell proliferation can also be determined by the tet‐ razolium hydroxide (XTT) cell proliferation assay, in which absorbance is measured by an

Cell proliferation assays and apoptosis assays have been used to examine the effects of in‐ hibitors on cancer cells [61]. The cell proliferation of Neuro-2A cells, neuroblastoma cells, can be determined using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay reagent (Promega) [61]. A colony formation assay using Neuro-2A cells was used to deter‐ mine the effect of an inhibitor of GSK-3β [61]. In this experiment, colonies were allowed to form for 10 days, after which the cells were fixed with 70% ethanol and stained with 1% methylene blue. Apoptosis was then measured by flow cytometry using an Annexin V-allo‐ phycocyanin (APC) /propidium iodide (PI) detection kit (BD PharMingen) [61]. Apoptosis was also determined using 4'6-diamidino-2-phenylindole (DAPI) staining, observing apop‐ totic nuclear morphology, and immunoblotting with antibodies to β-catenin, X-linked inhib‐ itor of apoptosis, and BCL2 [61]. Cell cycle analysis using PI to quantify the proportions of

Viable cells can be determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli‐ um bromide) colorimetric assays [62]. Absorbance at 570 nm is used to detect the incorporation of MTT. Apoptosis can also be determined by caspase activation using an anti-poly ADP-ri‐ bose polymerase (PARP) antibody [62]. Viable cells can also be determined using a 3-(4,5-di‐ methyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) kit (Promega) [63]. The terminal transferase dUTP nick end labeling (TUNEL) assay is com‐ monly used to detect apoptosis [63]. Harvested cells are resuspended in DNA labeling solution

ELISA reader under 490-nm-wavelength light (Biological Industries) [60].

cells in the G1/G0 or G2–M phases was used to examine cell cycle status [61].

role in the development of gastric cancer [56].

442 Latest Research into Quality Control

**2.5. Detection of cell proliferation or apoptosis**

amplification of telomere extensions [57].

#### **3.1. Genome-wide association studies in cancer**

Despite extensive research efforts for several decades, the genetic basis of common human diseases such as cancers remains largely unknown [65]. Genome-wide association studies (GWAS) have emerged as an important tool for the discovery of genomic regions that har‐ bor genetic variants conferring risk for various cancers [66,67]. Family-based linkage studies and studies comprising tens of thousands of gene-based SNPs can also assay genetic varia‐ tion across the genome [68], but the National Institutes of Health guidelines for GWAS re‐ quire a sufficient density of genetic markers to capture a large proportion of the common variants in the study population, measured in enough individuals to provide sufficient pow‐ er to detect variants of modest effect [67]. The recent success of GWAS can be attributed to the convergence of new technologies that can genotype hundreds of thousands of SNPs in hundreds or thousands of samples [66,69].

GWAS have been conducted in the five of the most common cancer types: breast, prostate, colorectal, lung, and melanoma (Table 4) and have identified more than 20 novel disease lo‐ ci, confirming that susceptibility to these diseases is polygenic [70]. For many years, human genetics has been used to map rare mutations with large effect sizes in families or genetical‐ ly homogeneous populations, such as *BRCA1/BRCA2* mutations in Ashkenazi women with breast cancer and ovarian cancer [71]. A number of SNPs have now been associated with breast cancer; for example, a SNP in intron 2 of the *FGFR2* gene, which encodes a receptor tyrosine kinase that is amplified and overexpressed in 5–10% of breast tumors [72,73], and SNPs on chromosomes 16q and 5q. The locus on 16q contains a gene *TNRC9* and a hypothet‐ ical gene *LOC643714*. The function of TNRC9 is unknown but the presence of an HMG box motif suggests that it might act as a transcription factor. The 5q locus includes *MAP3K1*, which encodes a protein involved in signal transduction (but not previously known to be involved in cancer) and two other genes: *MGC33648* and *MIER3*. In addition, several of the breast cancer loci appear to be associated with specific subtypes of the disease. In particular, the *FGFR2* association is strongly associated with estrogen receptor-positive breast cancer, while the *TNRC9* SNP is associated with both estrogen receptor-positive and -negative breast cancer [74,75]. It is surprising that none of the strongest associations map to regions harboring estrogen/progesterone genes in women of European background, particularly be‐ cause a GWAS in Asian women reported a convincing association with markers near the es‐ trogen receptor alpha (*ESR1*) gene [76]. In prostate cancer, the first and most important region to emerge was 8q24. This region was first associated with prostate cancer through linkage studies by the deCode group, was followed up by association analyses [77], and has been confirmed in subsequent GWAS [78-81]. Another signal, on chromosome 10q13, points to a variant in the promoter of the *MSMB* gene, which encodes the PSP94 protein; this is now under intense investigation as a biomarker for prostate cancer [80,81].

**Cancer type**

**ReferenceYear Platform**

[91] 2012 Affymetrix

[92] 2012 Illumina

[93] 2011 Affymetrix

[94] 2011 Affymetrix

[95] 2011 Illumina

[96] 2010 Illumina

[97] 2010 Affymetrix

[98] 2010 Illumina

[99] 2010 Illumina

[100] 2010 Illumina

[101] 2009 Illumina

[102] 2009 Affymetrix

[103] 2008 Affymetrix

[104] 2008 Affymetrix

[105] 2007 Affymetrix

[106] 2007 Perlegen

[107] 2007 Illumina

**[SNP passing**

**Ethnic group**

**Initial sample size Replication sample size**

**groups**

**Cases Controls**

445

http://dx.doi.org/10.5772/51271

**Cases Controls Ethnic**

The Investigation of Gene Regulation and Variation in Human Cancers and Other Diseases

Chinese 1,950 - Chinese 4,160 -

Japanese 240 - Japanese 222 -

East Asian 2,062 2,066 East Asians 15,091 14,877

European 302 321 European 1,153 1,215

British 1,694 2,365 British 7,317 8,124

Swedish & Finnish 617 4,583 European 1,001 7,604

European 899 804 European 1,264 1,222

European 2,702 5,726 European 7,386 7,576

UK 3,659 4,897 European 12,576 12,223

1,145 1,142 8,625 9,657

30 30 - - -

1,193 1,166

Jewish

390 634 26,646 24,889

1,145 1,142 1,176 2,072

Framing-ham 1,345 - - - -

British 1,145 - British 4,335 -

Chinese 1,505 1,522 Chinese 1,554 1,576

Ashkenazi Jewish 249 299 Ashkenazi

European 1,145 1,142

**QC]**

[613,031]

[470,796]

[684,457]

[782,838]

[~296,114]

[285,984]

[592,163]

[285,984]

[582,886]

[528,252]

[528,173]

[200,220]

[492,900]

[70,897]

[205,586]

[528,173]

[up to 607,728]

In general, the susceptibility alleles discovered thus far are common—that is, with a fre‐ quency in one or more population of >10%, and each allele confers a small contribution to the overall risk of the disease. For nearly all regions conclusively identified by GWAS, the effect sizes per allele are estimated at <1.3. It was not anticipated that GWAS in certain can‐ cers would yield many novel regions when other cancers strongly associated with particular environmental exposures have yielded so few regions. For example, prostate cancer, breast cancer, and colon cancer have been associated with 29, 13, and 10 regions of the genome, respectively, while there are only three associated regions for lung cancer in smokers, and three for bladder cancer despite analysis of sufficiently large data sets [67]. Several GWAS for lung cancer have identified the same locus on 15q25, suggesting that this is an important susceptibility locus for this disease [82-87]. This locus contains the nicotinic acetylcholine re‐ ceptor subunit genes *CHRNA3* and *CHRNA5*, suggesting that susceptibility may be mediat‐ ed through smoking behavior [86,87].

GWAS represent an important advance in discovering genetic variants influencing disease but have important limitations. There is a high potential for false-positive results, they do not yield information on gene function, they are insensitive to rare and structural var‐ iants, they require large sample sizes, and incur possible biases because of case and con‐ trol selection and genotyping errors [88]. Clinicians and scientists must understand the unique aspects of these studies and be able to assess and interpret GWAS results for themselves and their patients. However, at present these studies mainly represent a val‐ uable discovery tool for examining genomic function and clarifying pathophysiological mechanisms. However, through GWAS, the identification of variants, genes, and path‐ ways involved in multiple cancers offers a potential route to new therapies, improved di‐ agnosis, and better disease prevention [65].



linkage studies by the deCode group, was followed up by association analyses [77], and has been confirmed in subsequent GWAS [78-81]. Another signal, on chromosome 10q13, points to a variant in the promoter of the *MSMB* gene, which encodes the PSP94 protein; this is

In general, the susceptibility alleles discovered thus far are common—that is, with a fre‐ quency in one or more population of >10%, and each allele confers a small contribution to the overall risk of the disease. For nearly all regions conclusively identified by GWAS, the effect sizes per allele are estimated at <1.3. It was not anticipated that GWAS in certain can‐ cers would yield many novel regions when other cancers strongly associated with particular environmental exposures have yielded so few regions. For example, prostate cancer, breast cancer, and colon cancer have been associated with 29, 13, and 10 regions of the genome, respectively, while there are only three associated regions for lung cancer in smokers, and three for bladder cancer despite analysis of sufficiently large data sets [67]. Several GWAS for lung cancer have identified the same locus on 15q25, suggesting that this is an important susceptibility locus for this disease [82-87]. This locus contains the nicotinic acetylcholine re‐ ceptor subunit genes *CHRNA3* and *CHRNA5*, suggesting that susceptibility may be mediat‐

GWAS represent an important advance in discovering genetic variants influencing disease but have important limitations. There is a high potential for false-positive results, they do not yield information on gene function, they are insensitive to rare and structural var‐ iants, they require large sample sizes, and incur possible biases because of case and con‐ trol selection and genotyping errors [88]. Clinicians and scientists must understand the unique aspects of these studies and be able to assess and interpret GWAS results for themselves and their patients. However, at present these studies mainly represent a val‐ uable discovery tool for examining genomic function and clarifying pathophysiological mechanisms. However, through GWAS, the identification of variants, genes, and path‐ ways involved in multiple cancers offers a potential route to new therapies, improved di‐

now under intense investigation as a biomarker for prostate cancer [80,81].

ed through smoking behavior [86,87].

444 Latest Research into Quality Control

agnosis, and better disease prevention [65].

**ReferenceYear Platform**

[90] 2012 Affymetrix

Breast cancer [89] 2012 Affymetrix

**[SNP passing**

**Ethnic group** **Initial sample size Replication sample size**

**groups**

**Cases Controls**

Han Chinese 1,297 1,585 Taiwan Chinese 1,066 1,065 Korean 5,038 6,869 Japanese 1,934 1,875

**Cases Controls Ethnic**

Korean 2,273 2,052 Korean 4,049 3,845

Chinese 2,918 2,324 Chinese 6,838 6,888

**QC]**

[555,525]

[690,947]

**Cancer type**


**Cancer type**

**ReferenceYear Platform**

[119] 2008 Illumina

[120] 2007 Affymetrix &

[121] 2007 Affymetrix

[122] 2007 Illumina

[123] 2007 Illumina

[125] 2010 Illumina

[126] 2010 Affymetrix

[127] 2008 Illumina

[128] 2008 Illumina

[129] 2008 Illumina

[130] 2007 Illumina

[131] 2007 Illumina

[132] 2007 Affymetrix &

[133] 2011 Affymetrix

[134] 2011 Illumina

Lung cancer

Colorectal cancer[124] 2011 Illumina

**[SNP passing**

**Ethnic group**

**Initial sample size Replication sample size**

**groups**

1,172 1,157 3,941 3,964

1,235 1,599 1,242 917

1,453 3,064 East Asia 1,210 2,445

1,172 1,157 3,124 3,142

**Cases Controls**

447

http://dx.doi.org/10.5772/51271

**Cases Controls Ethnic**

The Investigation of Gene Regulation and Variation in Human Cancers and Other Diseases

Framingham 1,345 - - -

European 2,906 3,416 European 8,161 9,101

European 3,334 4,628 European 14,851 15,569

Czech

1,902 1,929 4,878 4,914

981 1,002 16,476 15,351

922 927 17,872 17,526

940 965 7,473 5,984

930 960 7,334 5,246

1,257 1,336 6,223 6,443

Chinese 2,331 3,077 Chinese 6,313 6,409

White 327 European 587

4,121 794

7,344 815

German 371 1,263 German

**QC]**

[527,869]

Illumina [60,275]

[70,897]

[316,515]

[538,548]

[378,739]

[~550,000] (imputed)

[460,945]

[~548,586]

[541,628]

[547,647]

[547,647]

[547,647]

Illumina [99,632]

[906,703]

[307,260]


**Cancer type**

**ReferenceYear Platform**

[109] 2012 Affymetrix

[110] 2011 NR

[111] 2011 Illumina

[112] 2011 Illumina

[113] 2011 Affymetrix

[114] 2010 Affymetrix

[115] 2010 Illumina

[116] 2009 Illumina

[117] 2009 Illumina

[118] 2008 Illumina

Prostate cancer [108] 2012 Illumina

446 Latest Research into Quality Control

**[SNP passing**

**Ethnic group** **Initial sample size Replication sample size**

**groups**

4,723 4,792 - - -

Japanese Chinese African American

American Senegalese Ghanaian Barbadian

European 2,782 4,458 European 8,217 6,732

European 202 100 European 1,122 1,167

Caucasian 222 415 Caucasian 500 155

Japanese 1,583 3,386 Japanese 3,001 5,415

Icelandic 1,968 35,382 European 11,806 12,387

European 1,854 1,894 3,268 3,366

Chinese, Japanese, African American, Latino, and Hawaiian

7,140

1,275

86 271 246

**Cases Controls**

5,455

1,695

414 968 253

19,879 18,761

**Cases Controls Ethnic**

European 6,621 6,939 European

African American 3,425 3,290 African

European 1,854 1,894 European,

European 1,176 1,101 European 1,964 3,172

**QC]**

[509,916]

& Illumina [1,117,531] (imputed)

[2.6 million] (imputed)

[571,243]

[1,047,198]

[387,384]

[419,613]

[510,687]

[541,129]

[310,520]

[541,129]


**3.2. Genetic risk score in cancer and diabetes**

Type 2 diabetes mellitus and cancers are major health problems worldwide [150,151]. The recent increase in the prevalence of these diseases is largely attributable to environmental factors. However, convincing evidence shows that genetic factors may play an important role in these diseases [152,153]. Recent GWAS have led to the identification of a series of SNPs that are robustly associated with either the risk of diabetes or cancers [151,154-159]. For type 2 diabetes mellitus, common SNPs have been identified in the *PPARG*, *KCNJ11*, and *TCF7L2* genes, and have been widely replicated in populations of various ethnicities [160-162]. Other potential new loci include *HHEX*, *CDKAL1*, *CDKN2A/B*, *IGF2BP2*, *SLC30A8*, and *WFS1* [65,155-159,163,164]. A number of SNPs have been identified as associated with breast cancer risk, including *FGFR2*, *CASP8*, *ERBB4*, *TAB2*, *BARX2*, *TMEM45B*, *ESR1*,

The Investigation of Gene Regulation and Variation in Human Cancers and Other Diseases

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449

*FGFR2*, *TNRC9*, *MAP3K1*, *MGC33648*, *MIER3*, and *RAD51L1* [74,75,151] (Table 5).

confirm whether a GRS improves disease risk prediction.

ber of corresponding risk alleles (0, 1, or 2).

Combining multiple loci with modest effects into a global genetic risk score (GRS) might im‐ prove the identification of those at risk for common complex diseases such as type 2 diabe‐ tes and cancers [165-167]. Several studies have developed methods to predict the risk of certain diseases, such as coronary heart disease, type 2 diabetes, and breast cancer, aggregat‐ ing information from multiple SNPs into a single GRS [151,168,169]. For example, in the Atherosclerosis Risk in Communities study, the aggregation of multiple SNPs into a single GRS was responsible for improving the prediction of coronary heart disease incidence [168]. In a study that used a GRS to determine the risk of type 2 diabetes in US men and women, individuals in the highest quintile of GRS had a significantly increased risk of type 2 diabe‐ tes compared with those in the lowest quintile; however, the addition of a GRS to the con‐ ventional model consisting of lifestyle risk factors only increased the area under the curve by only 1% (AUC=0.78). In this instance, the GRS was determined to be useful only when combined with the body mass index or a family history of diabetes [169]. For breast cancer, a GRS was created using 14 SNPs previously associated with breast cancer, and was substan‐ tially more predictive of estrogen receptor-positive breast cancer than of estrogen receptornegative breast cancer, particularly for absolute risk [151]. Further studies are needed to

The GRS is calculated on the basis of reproducible tagging of SNP-associated loci reaching genome-wide levels of significance. The GRS can be created by two methods: a simple count method (count GRS) and a weighted method (weighted GRS) [169,170]. Both methods antic‐ ipate each SNP to be independently associated with risk. An additive genetic model is used for each SNP, applying a linear weighting of 0, 1, or 2 to genotypes containing 0, 1, or 2 risk alleles, respectively. This model is known to perform well even when the true genetic model is unknown or wrongly specified [171]. The count model assumes that each SNP in the pan‐ el contributes equally to the disease risk and is calculated by summing the values for each of the SNPs. The weighted GRS is calculated by multiplying each B-coefficient, the estimates resulting from an analysis carried out on variables that have been standardized, by the num‐

**Table 4.** Summary of GWAS for the five of the most common types of cancer.

#### **3.2. Genetic risk score in cancer and diabetes**

**Cancer type**

448 Latest Research into Quality Control

**ReferenceYear Platform**

[135] 2011 Illumina

[136] 2010 Affymetrix

[137] 2010 Affymetrix

[138] 2010 Illumina

[139] 2009 Illumina

[140] 2009 Illumina

[141] 2008 Illumina

[142] 2008 Illumina

[143] 2008 Illumina

[144] 2007 Affymetrix

[146] 2011 Illumina

[147] 2011 Illumina

[148] 2011 Illumina

[149] 2009 Illumina

Melanoma [145] 2011 Illumina

**[SNP passing**

**Ethnic group** **Initial sample size Replication sample size**

**groups**

377 377 511 1,007

1,154 1,138 2,724 3,694

1,926 2,522 2,513 4,752

4,848 smokers

**Cases Controls**

**Cases Controls Ethnic**

Italian 600 - Italian 317 -

Han Chinese 245 - Han Chinese 305 -

Korean 621 1,541 Korean 804 1,470

European 5,739 5,848 European 7,561 13,818

European 1,952 1,438 European 5,608 6,767

Italian 338 335 Norwegian 265 356

European 2,804 7,618 European 5,551 7,449

European 2,168 4,387 European 5,193 15,144

European 1,804 1,026 European 6,483 23,324

European 156 2,150 NR - -

European 1,539 3,917 European 2,312 1,867

10,995 smokers

**QC]**

[620,901] (pooled)

[265,996]

[246,758]

[542,050]

[515,922]

[511,919]

[317,498]

[306,207]

[310,023]

[~116,204] (pooled)

[594,997]

[5,480,804 (imputed)]

[818,977]

[491,227]

[~317,000]

**Table 4.** Summary of GWAS for the five of the most common types of cancer.

Type 2 diabetes mellitus and cancers are major health problems worldwide [150,151]. The recent increase in the prevalence of these diseases is largely attributable to environmental factors. However, convincing evidence shows that genetic factors may play an important role in these diseases [152,153]. Recent GWAS have led to the identification of a series of SNPs that are robustly associated with either the risk of diabetes or cancers [151,154-159]. For type 2 diabetes mellitus, common SNPs have been identified in the *PPARG*, *KCNJ11*, and *TCF7L2* genes, and have been widely replicated in populations of various ethnicities [160-162]. Other potential new loci include *HHEX*, *CDKAL1*, *CDKN2A/B*, *IGF2BP2*, *SLC30A8*, and *WFS1* [65,155-159,163,164]. A number of SNPs have been identified as associated with breast cancer risk, including *FGFR2*, *CASP8*, *ERBB4*, *TAB2*, *BARX2*, *TMEM45B*, *ESR1*, *FGFR2*, *TNRC9*, *MAP3K1*, *MGC33648*, *MIER3*, and *RAD51L1* [74,75,151] (Table 5).

Combining multiple loci with modest effects into a global genetic risk score (GRS) might im‐ prove the identification of those at risk for common complex diseases such as type 2 diabe‐ tes and cancers [165-167]. Several studies have developed methods to predict the risk of certain diseases, such as coronary heart disease, type 2 diabetes, and breast cancer, aggregat‐ ing information from multiple SNPs into a single GRS [151,168,169]. For example, in the Atherosclerosis Risk in Communities study, the aggregation of multiple SNPs into a single GRS was responsible for improving the prediction of coronary heart disease incidence [168]. In a study that used a GRS to determine the risk of type 2 diabetes in US men and women, individuals in the highest quintile of GRS had a significantly increased risk of type 2 diabe‐ tes compared with those in the lowest quintile; however, the addition of a GRS to the con‐ ventional model consisting of lifestyle risk factors only increased the area under the curve by only 1% (AUC=0.78). In this instance, the GRS was determined to be useful only when combined with the body mass index or a family history of diabetes [169]. For breast cancer, a GRS was created using 14 SNPs previously associated with breast cancer, and was substan‐ tially more predictive of estrogen receptor-positive breast cancer than of estrogen receptornegative breast cancer, particularly for absolute risk [151]. Further studies are needed to confirm whether a GRS improves disease risk prediction.

The GRS is calculated on the basis of reproducible tagging of SNP-associated loci reaching genome-wide levels of significance. The GRS can be created by two methods: a simple count method (count GRS) and a weighted method (weighted GRS) [169,170]. Both methods antic‐ ipate each SNP to be independently associated with risk. An additive genetic model is used for each SNP, applying a linear weighting of 0, 1, or 2 to genotypes containing 0, 1, or 2 risk alleles, respectively. This model is known to perform well even when the true genetic model is unknown or wrongly specified [171]. The count model assumes that each SNP in the pan‐ el contributes equally to the disease risk and is calculated by summing the values for each of the SNPs. The weighted GRS is calculated by multiplying each B-coefficient, the estimates resulting from an analysis carried out on variables that have been standardized, by the num‐ ber of corresponding risk alleles (0, 1, or 2).


**3.3. Cancer Cell Line Encyclopedia**

**4. Conclusion**

**Acknowledgements**

**Author details**

Shihori Tanabe1\* and Sun Ha Jee2

These databases will enable to overview genome quality.

the important factors for detecting the development of the disease.

ticular, we thank Jaeseong Jo for his great assistance.

\*Address all correspondence to: stanabe@nihs.go.jp

1 National Institute of Health Sciences, Tokyo, Japan

2 Institute for Health Promotion, Yonsei University, Seoul, Korea

The Cancer Cell Line Encyclopedia (CCLE) has made predictive modeling of anticancer drug sensitivity a realistic proposition, by determining genomic markers of drug sensitivity in cancer cells [172,173]. The CCLE contains information from 947 human cancer cell lines including data on gene expression, chromosomal copy number, and massively parallel se‐ quencing data. It has been used to identify genetic, lineage-specific, and gene expressionbased predictors of drug sensitivity [172]. This has revealed, for example, that the plasma cell lineage is correlated with sensitivity to IGF1 receptor inhibitors, aryl hydrocarbon recep‐ tor (*AHR*) expression is associated with MEK inhibitor efficacy in *NRAS*-mutant lines, and *SLFN11* expression is associated with sensitivity to topoisomerase inhibitors [172]. Genomic markers of drug sensitivity in cancer cells have also been systematically identified using the Genomics of Drug Sensitivity in Cancer database (http://www.cancerRxgene.org) [173].

The Investigation of Gene Regulation and Variation in Human Cancers and Other Diseases

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451

There are dramatic changes in the genomes of cancer cells, which vary according to cancer subtype. Integrative and wide investigations of cancer cell genomes have revealed muta‐ tions and alterations in gene expression that are associated with the disease. Databases that include abundant data related to gene and protein conformation, gene expression, and ge‐ nomic mutations enable the construction of dynamic cellular simulations and disease mod‐ els. New sequencing tools such as next-generation sequencing will reveal new horizons in the prediction of disease and drug sensitivity, which play an important role in personalized medicine. Appropriate translation of the abundance of information to clinical practice is one of most important future challenges for medicine. The quality of genome would be one of

The authors are grateful to all those who helped with preparation of the manuscript. In par‐

**Table 5.** Studies using a genetic risk score for cancers and diabetes, comprising SNPs identified in GWAS.

#### **3.3. Cancer Cell Line Encyclopedia**

The Cancer Cell Line Encyclopedia (CCLE) has made predictive modeling of anticancer drug sensitivity a realistic proposition, by determining genomic markers of drug sensitivity in cancer cells [172,173]. The CCLE contains information from 947 human cancer cell lines including data on gene expression, chromosomal copy number, and massively parallel se‐ quencing data. It has been used to identify genetic, lineage-specific, and gene expressionbased predictors of drug sensitivity [172]. This has revealed, for example, that the plasma cell lineage is correlated with sensitivity to IGF1 receptor inhibitors, aryl hydrocarbon recep‐ tor (*AHR*) expression is associated with MEK inhibitor efficacy in *NRAS*-mutant lines, and *SLFN11* expression is associated with sensitivity to topoisomerase inhibitors [172]. Genomic markers of drug sensitivity in cancer cells have also been systematically identified using the Genomics of Drug Sensitivity in Cancer database (http://www.cancerRxgene.org) [173]. These databases will enable to overview genome quality.

#### **4. Conclusion**

**Disease Reference Year Ethnic group Participants No. of SNPs Genes found from GWAS**

[169] 2009 European 2,809 diabetic

Breast cancer [151] 2010 UK 10,306

[165] 2008 Framingham 2,377 diabetic patients 18 NOTCH2 (rs10923931),

patients & 3,501 health controls

breast cancer patients & 10,393 controls

**Table 5.** Studies using a genetic risk score for cancers and diabetes, comprising SNPs identified in GWAS.

BCL11A (rs10490072), THADA (rs7578597), IGF2BP2 (rs1470579), PPARg (rs1801282), ADAMTS9 (rs4607103), CDKAL (rs7754840), VEGFA (rs9472138), JAZF1 (rs86475), SLC30A8 (rs13266634), CDKNA/2B (rs10811661), HHEX (rs1111875),

CDC123, CAMK1D (rs12779790), TCF7L2 (rs7903146), KCNJ11 (rs5219), INS (rs689), DCD (rs1153188), TSPAN8, LGR5 (rs7961581)

10 WFS1 (rs10010131),

14 FGFR2 (rs2981582),

TNRC9 (rs3803662), 2q35 (rs13387042), MAP3K1 (rs889312), 8q24 (rs13281615), 2p (rs4666451), 5pas (rs981782), CASP8 (rs104548), LSP1 (rs3817198), 5p (rs30099), TGFB1 (rs198/2073), ATM (rs1800054), TNRC9 (rs8051542), TNRC9 (rs12443621)

HHEX (rs1111875), CDKAL1 (rs7756992), IGF2BP2 (rs4402960), SLC30A8 (rs13266634), CDKN2A/B (rs10811661), TCF7L2 (rs12255372), PPARG (rs1801282), KCNJ11 (rs5219)

Type 2 diabetes

450 Latest Research into Quality Control

There are dramatic changes in the genomes of cancer cells, which vary according to cancer subtype. Integrative and wide investigations of cancer cell genomes have revealed muta‐ tions and alterations in gene expression that are associated with the disease. Databases that include abundant data related to gene and protein conformation, gene expression, and ge‐ nomic mutations enable the construction of dynamic cellular simulations and disease mod‐ els. New sequencing tools such as next-generation sequencing will reveal new horizons in the prediction of disease and drug sensitivity, which play an important role in personalized medicine. Appropriate translation of the abundance of information to clinical practice is one of most important future challenges for medicine. The quality of genome would be one of the important factors for detecting the development of the disease.

#### **Acknowledgements**

The authors are grateful to all those who helped with preparation of the manuscript. In par‐ ticular, we thank Jaeseong Jo for his great assistance.

#### **Author details**

Shihori Tanabe1\* and Sun Ha Jee2

\*Address all correspondence to: stanabe@nihs.go.jp

1 National Institute of Health Sciences, Tokyo, Japan

2 Institute for Health Promotion, Yonsei University, Seoul, Korea

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e374.


**Chapter 21**

**Quality Control Considerations for**

Lisa Duffy, Liangtao Zhang, Donald R. Love and

Additional information is available at the end of the chapter

ques that pool DNA from hundreds of different cells [1,3].

Alice M. George

**1. Introduction**

and overlapping cells [1,4].

http://dx.doi.org/10.5772/51266

**Fluorescence** *In Situ* **Hybridisation of**

**Paraffin-Embedded Pathology Specimens**

Paraffin FISH testing is the application of the fluorescence *in situ* hybridisation (FISH) meth‐ odology to formalin fixed paraffin embedded sections (FFPE), and has proven a powerful tool for both histopathologists and cytogeneticists. Pathologists use the method to confirm or exclude a histological diagnosis, to differentiate between tumour subtypes, or as a confir‐ matory tool where the tissue morphology is poor or the immunohistochemistry (IHC) stain‐ ing is uninformative [1]. Similarly, cytogeneticists find it useful when the tissue sample is insufficient or unsatisfactory for conventional culture methods, or when such methods fail to yield a result. The method can also be used to confirm abnormalities found in other tissue samples. Paraffin testing has a further advantage over conventional cytogenetic and molecu‐ lar testing methods, as it can localize the anomaly within specific cells or tissue areas, and this provides the ability to study anomalies at a single cell level [2,3], unlike DNA techni‐

Compared to FISH testing on conventional suspension samples (Figure 1), paraffin FISH can be labour intensive and highly variable due to differing fixation times between samples and referring histology labs, and the interpretation may be limited due to truncation of signal

> © 2012 Duffy et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 Duffy et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**in a Diagnostic Laboratory Environment**

## **Quality Control Considerations for Fluorescence** *In Situ* **Hybridisation of Paraffin-Embedded Pathology Specimens in a Diagnostic Laboratory Environment**

Lisa Duffy, Liangtao Zhang, Donald R. Love and Alice M. George

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51266

#### **1. Introduction**

Paraffin FISH testing is the application of the fluorescence *in situ* hybridisation (FISH) meth‐ odology to formalin fixed paraffin embedded sections (FFPE), and has proven a powerful tool for both histopathologists and cytogeneticists. Pathologists use the method to confirm or exclude a histological diagnosis, to differentiate between tumour subtypes, or as a confir‐ matory tool where the tissue morphology is poor or the immunohistochemistry (IHC) stain‐ ing is uninformative [1]. Similarly, cytogeneticists find it useful when the tissue sample is insufficient or unsatisfactory for conventional culture methods, or when such methods fail to yield a result. The method can also be used to confirm abnormalities found in other tissue samples. Paraffin testing has a further advantage over conventional cytogenetic and molecu‐ lar testing methods, as it can localize the anomaly within specific cells or tissue areas, and this provides the ability to study anomalies at a single cell level [2,3], unlike DNA techni‐ ques that pool DNA from hundreds of different cells [1,3].

Compared to FISH testing on conventional suspension samples (Figure 1), paraffin FISH can be labour intensive and highly variable due to differing fixation times between samples and referring histology labs, and the interpretation may be limited due to truncation of signal and overlapping cells [1,4].

© 2012 Duffy et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Duffy et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

pretreated, probed and co-denatured using the traditional FISH methodology [6,7]. However, one of the most crucial factors for paraffin analysis is the assessment of the correct target area before beginning the procedure – without this, an erroneous result may occur (Figure 2), which

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For this reason, robust internal and external quality control procedures are required for di‐ agnostic paraffin FISH testing and the exclusion of non-target tissue before analysis decreas‐ es the likelihood of an incorrect result due to an analysis error [1]. This protocol therefore aims to provide a guide to some of the considerations and troubleshooting that are necessa‐ ry when using the method for diagnostic medical testing. It is adapted from the method used by the Diagnostic Genetics Department, LabPlus at Auckland City Hospital, New Zea‐ land. There are a number of variations to the basic FISH method that can be used depending on the nature and number of samples being processed, and new technology has also been developed to automate the process (Xmatrix, Abbott Molecular). In this protocol however, we have suggested extra steps that are designed to help improve the quality of the testing procedure for diagnostic use. Probes used for diagnostic testing are commercially available and may be downloaded and gathered from the websites of companies such as Abbott Mo‐

One slide (2-5 micron thickness usually) is needed per probe or probe set, and if a haematoxy‐ lin and eosin (H+E) slide is not provided by pathologists, an extra slide must also go through the deparaffinisation steps before staining with the Shandon Rapid-Chrome™ Frozen Section Staining kit (alternatively the individual stain kit components can be made from powder).

**Figure 3.** Slide pretreatment steps for paraffin FISH. (A) Appearance of unstaind paraffin slides after aging in a 60ºC oven - note melted or "bubbled" appearance. (B) Unstained paraffin slides and after the pre-treatment steps.

may be costly to patients if it results in the appropriate treatment being with held [1].

lecular, Cytocell, Zytovision or Kreatech Diagnostics.

**2. Method**

**Figure 1.** A comparison of the paraffin pre-treatment process with the conventional FISH pre-treatment process on suspension semples.

For these reasons, it must be considered separately from the conventional suspension FISH method, and while it can be used as either a stand-alone technique, or an adjunct to conven‐ tional cytogenetics techniques [5], it must be noted that due to the use of interphase nuclei, a prior knowledge of the anomaly of interest is required.

**Figure 2.** Errors that occur during the paraffin pre-treatment process.

The basic premise of the method involves establishing the area of interest for testing on the H+E stained pathology slide, and transferring this area to an unstained paraffin slide, which is then pretreated, probed and co-denatured using the traditional FISH methodology [6,7]. However, one of the most crucial factors for paraffin analysis is the assessment of the correct target area before beginning the procedure – without this, an erroneous result may occur (Figure 2), which may be costly to patients if it results in the appropriate treatment being with held [1].

For this reason, robust internal and external quality control procedures are required for di‐ agnostic paraffin FISH testing and the exclusion of non-target tissue before analysis decreas‐ es the likelihood of an incorrect result due to an analysis error [1]. This protocol therefore aims to provide a guide to some of the considerations and troubleshooting that are necessa‐ ry when using the method for diagnostic medical testing. It is adapted from the method used by the Diagnostic Genetics Department, LabPlus at Auckland City Hospital, New Zea‐ land. There are a number of variations to the basic FISH method that can be used depending on the nature and number of samples being processed, and new technology has also been developed to automate the process (Xmatrix, Abbott Molecular). In this protocol however, we have suggested extra steps that are designed to help improve the quality of the testing procedure for diagnostic use. Probes used for diagnostic testing are commercially available and may be downloaded and gathered from the websites of companies such as Abbott Mo‐ lecular, Cytocell, Zytovision or Kreatech Diagnostics.

#### **2. Method**

**Figure 1.** A comparison of the paraffin pre-treatment process with the conventional FISH pre-treatment process on

For these reasons, it must be considered separately from the conventional suspension FISH method, and while it can be used as either a stand-alone technique, or an adjunct to conven‐ tional cytogenetics techniques [5], it must be noted that due to the use of interphase nuclei, a

The basic premise of the method involves establishing the area of interest for testing on the H+E stained pathology slide, and transferring this area to an unstained paraffin slide, which is then

prior knowledge of the anomaly of interest is required.

**Figure 2.** Errors that occur during the paraffin pre-treatment process.

suspension semples.

470 Latest Research into Quality Control

One slide (2-5 micron thickness usually) is needed per probe or probe set, and if a haematoxy‐ lin and eosin (H+E) slide is not provided by pathologists, an extra slide must also go through the deparaffinisation steps before staining with the Shandon Rapid-Chrome™ Frozen Section Staining kit (alternatively the individual stain kit components can be made from powder).

**Figure 3.** Slide pretreatment steps for paraffin FISH. (A) Appearance of unstaind paraffin slides after aging in a 60ºC oven - note melted or "bubbled" appearance. (B) Unstained paraffin slides and after the pre-treatment steps.


**c.** On completion, immerse slide/s with cover slip in deionised water to cool down and

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**d.** Wash briefly in a coplin jar of deionised water at room temperature and drain off

**a.** Add an appropriate amount (~15μl) of enzyme reagent (Invitrogen Tissue Pre-treat‐ ment Kit) to the slide/s, depending on the size of hybridisation area, and cover with a

**b.** Incubate slide/s for 15-45 minutes in a humidified chamber at 37°C (This time is de‐

**c.** Remove cover slip/s and wash briefly in a coplin jar of deionised water at room

**d.** Dehydrate slide/s for 2 minutes each in each of 70%, 80% and 100% ethanol solutions and air dry at room temperature. Please note that a different ethanol series is used for

**e.** Check the tissue morphology of the pre-treated slide looks the same as that of the H+E.

**f.** The pre-treated paraffin slide/s should then be carefully matched against the marked H&E slide/s, and the area for testing transferred to the pre-treated slide/s using a mark‐ er pen initially, followed by the diamond-tipped engraver. This means that the area can

**Figure 5.** Haemotoxylin and eosin (H+E) stained slides marked with the target area for analysis. This reduces the volume of probe necessary and ensures that non-target tissue is excluded as much as possible before the FISH

pendent on the type of tumours and length of formalin fixation).

the dehydration steps to avoid reagent contamination issues.

still be visualised after the post-wash steps.

gently remove the cover slip.

**4.** Enzyme Digestion (approx. 40 minutes).

excessive water.

square of parafilm.

temperature.

analysis procedure.


**Figure 4.** A haemotoxylin and eosin (H+E) stained slide with the target area for analysis marked by a pathologist.


**1.** Deparaffinisation (approx. 60 minutes); see Figure 3

**2.** Haemotoxylin and Eosin (H+E) slides; see Figure 4

solutions, followed by deionised water at room temperature.

fume hood, with intermittent shaking.

aging (Figure 3).

472 Latest Research into Quality Control

**a.** Leave slide/s on the hotplate/in the oven at approximately 65°C for 30-60 minutes for

**b.** Perform deparaffinization by placing slide/s in xylene for at least 10 minutes in the

**c.** Rehydrate slide/s by placing them for 2 minutes in each of 100%, 80%, and 70% ethanol

**Figure 4.** A haemotoxylin and eosin (H+E) stained slide with the target area for analysis marked by a pathologist.

Staining kit and mount the slide using Shandon Mount.

**c.** Check slides for stain quality under a light microscope.

the type of tissues and length of formalin fixation).

**d.** Take slide/s to pathologist for marking (Figure 5).

**3.** Heat Pre-treatment (approx. 30 minutes)

or pressure cooker.

**b.** Leave slides on the hotplate for at least 30 min to dry the mountant.

**a.** Take rehydrated slide/s and stain using the Shandon Rapid-Chrome™ Frozen Section

**a.** Add 35μl of heat pre-treatment solution (Invitrogen Tissue Pre-treatment Kit) to the slide/s, cover with a 22x22mm (or bigger sized cover slip) glass cover slip and seal with rubber cement. Alternatively slides can be heat-pre-treated in coplin Jar at 95°C

**b.** Heat slide/s on the thermal cycler for 15-60 minutes at 95°C (The time is dependent on


**Figure 5.** Haemotoxylin and eosin (H+E) stained slides marked with the target area for analysis. This reduces the volume of probe necessary and ensures that non-target tissue is excluded as much as possible before the FISH analysis procedure.

**7.** Post Hybridization Wash (5 Minutes)

**8.** Analysis and interpretation; see Figure 8.

terpretation of the FISH signal pattern.

ance between two observers.

**Figure 8.** Analysis principles for paraffin FISH slides.

**a.** Briefly soak slide/s in 2xSSC and gently remove rubber cement.

**c.** Place slide/s in 2xSSC/0.01% Tween 20 (or NP40) for 1 min.

**e.** Visualize FISHed-slide/s under fluorescence microscope.

**b.** Wash slide/s in 0.4xSSC/0.03% Tween 20 (or NP40) at 72°C for 2 min.

**d.** Briefly drain slide/s, apply DAPI counter stain and put cover slip on.

detection must be done according to the manufacturer's instructions.

When using indirectly labelled commercial probes that require antibody detection, signal

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**a.** With a pathologist's consultation, check the H+E slide on a transmitted light microscope to assess whether the sample contains a mixture of cell types, as this may affect the in‐

**b.** Check the paraffin FISH slide on a fluorescence microscope using the 10x objective to ensure the area marked on the slide approximately matches that on the H+E slide.

**c.** Using two observers, analyse a minimum of at least 8 representative sites within the marked region (a minimum of 4 different areas per observer), scoring only cells that show both the target and control loci. Analysis of areas of areas where the cells are not overlapped is preferable, and a third analyst is required where there is discord‐

**Figure 6.** Transfer of target area for analysis from the H+E slide to the pre-treated FISH slide prior to the probing steps.

**Figure 7.** Engraving of target area on to the pre-treated paraffin FISH slide. (A) Draw target area onto bottom of slide with fix-resistant pen. (B & C) Engrave marked area onto bottom of slide using diamond-tipped engraver to keep area visible after post-wash steps.

**5.** Probe preparation (approx. 10 minutes)

Use Ready-To-Use probes or refer to the probe preparation protocol outlined by the manufacturer.


When using indirectly labelled commercial probes that require antibody detection, signal detection must be done according to the manufacturer's instructions.

**8.** Analysis and interpretation; see Figure 8.

**Figure 6.** Transfer of target area for analysis from the H+E slide to the pre-treated FISH slide prior to the probing steps.

**Figure 7.** Engraving of target area on to the pre-treated paraffin FISH slide. (A) Draw target area onto bottom of slide with fix-resistant pen. (B & C) Engrave marked area onto bottom of slide using diamond-tipped engraver to keep area

Use Ready-To-Use probes or refer to the probe preparation protocol outlined by the

**a.** Apply an appropriate amount (2-10μl) of probe mix to the hybridization site marked on each slide, depending on the size of cover slip being used, and seal with rubber cement. Leave the slide/s in the incubator or in a drawer at room temperature for a few minutes

to allow the rubber cement to dry before placing them in the thermal cycler.

**b.** Denature slide/s together with probe mix for 10-20 min at 85°C or 5-10min at 95°C.

**c.** After co-denaturation, slide/s may be placed in a humidified box in the incubator at

visible after post-wash steps.

474 Latest Research into Quality Control

manufacturer.

**5.** Probe preparation (approx. 10 minutes)

**6.** Co-denaturation and hybridization (approx. 25 minutes)

37°C for at least 12-16 hours, usually no more than 72 hours.


**Figure 8.** Analysis principles for paraffin FISH slides.


### **3. Troubleshooting**

Problem: Unclear whether slides have been aged before arrival, as repeating this step may decrease the hybridization efficiency of the probe.

Solution: The ethanol series (in step 1) is necessary to rehydrate the tissue for the enzyme solution to act on, and may cause the tissue to become translucent, however it will become

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Problem: Scratching or loss of tissue during washing steps. Small tissue samples (e.g. core

Solution: As the tissue becomes soft during pre-treatment it may easily fall off or get scratch‐ ed; coplin jars of deionised water can be used to dip slides into rather than the more aggres‐ sive use of squirter bottles or running tap water (do not leave the pre treated slide in water for a long time, especially for a core biopsy or a tiny sample). The size of the tissue gives a good indication as to the fragility of the tissue, so this should be taken into account before beginning the pre treatment steps. Increasing the ageing step may also help to fix the tissue to the slide better, although it may also decrease the hybridization efficiency of the probe to the sample. Alternatively, skipping the heat pretreatment step and doing a reduced enzyme

biopsies) may become fragile during the pretreatment steps and fall off the slide.

Problem: The tissue does not look the same as the H+E slide after dehydration steps.

Solution: This can either be due to loss of tissue during pretreatment or different cuts through the tissue block. Untreated slides should be closely examined to find one that ap‐ pears to match the pretreated slide and a new H+E slide created using this slide. See also

Problem: Transfer of area is difficult due to a slight difference in the morphology of the tis‐ sue in different layers of the tissue section, or different orientation of tissue on pre-treated

Solution: If the morphology of tissue on the pre-treated slide looks different to that of the H +E slide, check it against the remaining untreated slides to see if it looks like tissue has been lost during the pre-treatment procedure. If tissue has been lost, simply start the procedure over again with a new slide. If the morphology of the tissue appears different between the untreated slides, ask a pathologist for help selecting an appropriate slide to pre-treat, and try to find two similar untreated slides. Pre-treat one and make the other into an H+E slide

Problem: There is more than one target area marked on slide – is more probe required?

required if the areas are greater than can be covered by a 13mm diameter cover slip.

Solution: Assess the size of the areas – if there are several small areas, the total volume of probe does not need to be increased, simply aliquot the volume of probe equally over the different areas and place a small cover slip over each. More than one aliquot of probe is only

white again once the slide is dehydrated.

treatment on the sample may combat this.

slide to that of the H+E slide.

to allow for more accurate marking.

steps for reducing the loss of tissue during pretreatment.

[Steps 3 and 4]

[Step 4]

[Step 4]

[Step 4 and 5]

[Step 1]

Solution: Although some waxes do not change in appearance, pre-aged slides generally have a bubbled or melted appearance of the wax compared to the smooth appearance of non-aged slides in general (N.B: some wax types do not change in appearance so this is a rule of thumb only).

Problem: The use of xylene to remove the wax from around the sample is not ideal as xylol is highly toxic.

[Step 1]

Solution: An alternative to xylene is HemoDe from Scientific Safety Solvents.

Problem: Finding that the wrong tissue was sent by the referring laboratory.

[Step 2]

Solution: Ask for a copy of the pathology report to be sent with all samples, and get patholo‐ gists to ring the referring laboratory to request the appropriate sample for testing.

Problem: Incomplete staining of the H+E slide causing correct target area to be missed by pathologist.

[Step 2]

Solution: Slides should be quality checked before taking them to a pathologist. Check the stain by eye to see if there are obvious colour differences across the slide – if one of the stains has been missed in an area it will appear either a dull purple (eosin missed) or a dull pink (haema‐ toxylin missed or there is a problem with the pH of the bluing reagent) compared to the rest of slide. If there are any doubts, ask a histopathology technologist for assistance.

Problem: Cover slip moves after the slide has been marked because mountant is not com‐ pletely hardened. This causes the target area to move.

[Step 2]

Solution: Leave the slides on the hotplate for a longer period of time, or change mountant to a faster drying version such as Entellan (Note: it is not possible to remove the Entellan with methanol after it has been cover slipped, hence why DPX is the preferred mountant).

The Rapid-Chrome™ Frozen Section Staining kit uses Shandon Mount; however alterna‐ tives such as Entellan are available.

Problem: Disappearance of tissue on slide during dehydration steps.

[Steps 3 and 4]

Solution: The ethanol series (in step 1) is necessary to rehydrate the tissue for the enzyme solution to act on, and may cause the tissue to become translucent, however it will become white again once the slide is dehydrated.

Problem: Scratching or loss of tissue during washing steps. Small tissue samples (e.g. core biopsies) may become fragile during the pretreatment steps and fall off the slide.

#### [Steps 3 and 4]

**3. Troubleshooting**

476 Latest Research into Quality Control

a rule of thumb only).

xylol is highly toxic.

[Step 1]

[Step 1]

[Step 2]

[Step 2]

[Step 2]

[Steps 3 and 4]

by pathologist.

decrease the hybridization efficiency of the probe.

Problem: Unclear whether slides have been aged before arrival, as repeating this step may

Solution: Although some waxes do not change in appearance, pre-aged slides generally have a bubbled or melted appearance of the wax compared to the smooth appearance of non-aged slides in general (N.B: some wax types do not change in appearance so this is

Problem: The use of xylene to remove the wax from around the sample is not ideal as

Solution: Ask for a copy of the pathology report to be sent with all samples, and get patholo‐

Problem: Incomplete staining of the H+E slide causing correct target area to be missed

Solution: Slides should be quality checked before taking them to a pathologist. Check the stain by eye to see if there are obvious colour differences across the slide – if one of the stains has been missed in an area it will appear either a dull purple (eosin missed) or a dull pink (haema‐ toxylin missed or there is a problem with the pH of the bluing reagent) compared to the rest of

Problem: Cover slip moves after the slide has been marked because mountant is not com‐

Solution: Leave the slides on the hotplate for a longer period of time, or change mountant to a faster drying version such as Entellan (Note: it is not possible to remove the Entellan with

The Rapid-Chrome™ Frozen Section Staining kit uses Shandon Mount; however alterna‐

methanol after it has been cover slipped, hence why DPX is the preferred mountant).

Problem: Disappearance of tissue on slide during dehydration steps.

Solution: An alternative to xylene is HemoDe from Scientific Safety Solvents. Problem: Finding that the wrong tissue was sent by the referring laboratory.

gists to ring the referring laboratory to request the appropriate sample for testing.

slide. If there are any doubts, ask a histopathology technologist for assistance.

pletely hardened. This causes the target area to move.

tives such as Entellan are available.

Solution: As the tissue becomes soft during pre-treatment it may easily fall off or get scratch‐ ed; coplin jars of deionised water can be used to dip slides into rather than the more aggres‐ sive use of squirter bottles or running tap water (do not leave the pre treated slide in water for a long time, especially for a core biopsy or a tiny sample). The size of the tissue gives a good indication as to the fragility of the tissue, so this should be taken into account before beginning the pre treatment steps. Increasing the ageing step may also help to fix the tissue to the slide better, although it may also decrease the hybridization efficiency of the probe to the sample. Alternatively, skipping the heat pretreatment step and doing a reduced enzyme treatment on the sample may combat this.

Problem: The tissue does not look the same as the H+E slide after dehydration steps.

#### [Step 4]

Solution: This can either be due to loss of tissue during pretreatment or different cuts through the tissue block. Untreated slides should be closely examined to find one that ap‐ pears to match the pretreated slide and a new H+E slide created using this slide. See also steps for reducing the loss of tissue during pretreatment.

Problem: Transfer of area is difficult due to a slight difference in the morphology of the tis‐ sue in different layers of the tissue section, or different orientation of tissue on pre-treated slide to that of the H+E slide.

#### [Step 4]

Solution: If the morphology of tissue on the pre-treated slide looks different to that of the H +E slide, check it against the remaining untreated slides to see if it looks like tissue has been lost during the pre-treatment procedure. If tissue has been lost, simply start the procedure over again with a new slide. If the morphology of the tissue appears different between the untreated slides, ask a pathologist for help selecting an appropriate slide to pre-treat, and try to find two similar untreated slides. Pre-treat one and make the other into an H+E slide to allow for more accurate marking.

Problem: There is more than one target area marked on slide – is more probe required?

#### [Step 4 and 5]

Solution: Assess the size of the areas – if there are several small areas, the total volume of probe does not need to be increased, simply aliquot the volume of probe equally over the different areas and place a small cover slip over each. More than one aliquot of probe is only required if the areas are greater than can be covered by a 13mm diameter cover slip.

Problem: The hybridisation buffer for a probe runs out.

[Step 5]

Solution: As hybridisation buffers are all fairly similar, it is fine to use the buffer of similar probe as a substitution. Alternatively, hybridization mix can be made up:

Poor signal quality may also be a result of incorrect post wash stringency. There is an alter‐ native wash technique that uses 50% formamide/2xSSC to increase the stringency of the wash. However, this is not always ideal, as it significantly increases the length of the post

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Solution: This is due to incorrect stringency of the post wash [1]. For a quick fix, slides can be rewashed using the quick wash procedure reported here, or alternatively washing at a

Solution: Only cells showing both the control and target loci should be scored (e.g. 2R2G), so if both the control probe and the probe for the region of interest are on the same chromo‐ some, it is most likely to be due to poor hybridisation of one of the probes. First check to see using single colour filters whether the signal colour is present but weak – if it is, repeat the pre-treatment and hybridisation steps again on the same slide (for a shorter time e.g. 15/15

Solution: In most cases, amplification with only a primary antibody is necessary, and further amplification can also increase the level of background on the slide(s). However if the signal is not bright enough, carefully remove the cover slip, rinse slide in 1xPBS (or SSC) and per‐ form further amplification steps with secondary or tertiary antibodies as many times as nec‐ essary. After adding each antibody, slides should be covered with parafilm and incubated in a humidified chamber at 37°C for 5 minutes before being washed in 4xSSC/0.05% Tween20

Solution: Look at the signal intensity on single colour filters – rubbish generally appears to be brighter and shinier compared to real signals, and background will appear fuzzy and in‐ distinct compared to real signal. High background may be due to the slides not being prop‐ erly sealed with rubber cement during the pretreatment steps, as this allows the solution to

Solution: High background may be due to insufficient removal of material during the pre‐ treatment steps. With high case numbers, solutions can become contaminated, therefore the

Problem: High levels of cross hybridization due to non-specific binding of probes.

higher temperature or use of a different post wash procedure can be tried [11].

Problem: Using an indirectly labeled probe and can't get a good signal quality.

for 2 minutes. Then mount with 8μl Vectashield antifade solution with DAPI.

Problem: Distinguishing between real signal and background or 'rubbish' on slide.

wash, and also uses formamide which is extremely toxic [11].

Problem: Cells only show one signal colour.

buffer: enzyme treatment).

evaporate and the tissue to dry out.

Problem: High background on the slides when analyzing.

[Step 8]

[Step 8]

[Step 8]

[Step 8]

[Step 8]

Hybridization mix

(10% dextran sulphate, 50% formamide in 2xSSC, 0.1% SDS, pH 7.0)


Problem: A thermal cycler is not available for use.

[Step 6]

Solution: Denaturation of the slide(s) can be done separately using 70% formamide/2xSSC, as it gives better quality denaturation although the downside is that it is highly toxic. The hybridi‐ sation steps can also be done adequately in a programmable system (e.g. Thermobyte).

Problem: The cover slip is hard to remove before the post wash steps.

[Step 7]

Solution: Place slide in 2xSSC solution and agitate gently after removing the rubber cement, and then remove cover slip. If the cover slip is still stuck to slide, slide the blade of a scalpel un‐ der one corner of the slide and lift gently before immersing the slide in a 2xSSC solution and ag‐ itating it gently. This may need to be repeated several times if the cover slip remains stuck.

Problem: Weak or patchy signal quality.

[Step 8]

Solution: This can be difficult to fix, as it primarily occurs as a result of poor handling and fixation of tissue prior to receiving the sample for FISH testing [8,9]. Different tissue samples may require the pretreatment times to be varied [10]. The heat pretreatment buffer prepares the tissue for the enzyme to act on and the enzyme degrades the cellular material away from the DNA, in order to allow the probe to anneal to the chromatin. Variation of either or both these times is effective, and the steps may be repeated on the probed slide to reduce the need for lengthy pretreatment times on a new slide. Bone samples such as trephines may show poor hybridization efficiency of the probe, and require hydrogen chloride treatment, unless the sample has already been decalcified prior to arrival.

Poor signal quality may also be a result of incorrect post wash stringency. There is an alter‐ native wash technique that uses 50% formamide/2xSSC to increase the stringency of the wash. However, this is not always ideal, as it significantly increases the length of the post wash, and also uses formamide which is extremely toxic [11].

Problem: High levels of cross hybridization due to non-specific binding of probes.

[Step 8]

Problem: The hybridisation buffer for a probe runs out.

with HCl then transfer to a 50ml Falcon tube.

**3.** Add 25μl Tween 20 and invert to mix.

Problem: Weak or patchy signal quality.

Problem: A thermal cycler is not available for use.

Solution: As hybridisation buffers are all fairly similar, it is fine to use the buffer of similar

**1.** Mix 12.5ml formamide, 2.5ml 20xSSC pH7.0 and 10ml MilliQ water. Adjust pH to 7.0

**2.** Add 2.5mg dextran sulphate and place on a roller mixer at room temperature for

**4.** Aliquot 500μl into sterile eppendorf tubes. Store at -20°C and use a fresh aliquot

Solution: Denaturation of the slide(s) can be done separately using 70% formamide/2xSSC, as it gives better quality denaturation although the downside is that it is highly toxic. The hybridi‐

Solution: Place slide in 2xSSC solution and agitate gently after removing the rubber cement, and then remove cover slip. If the cover slip is still stuck to slide, slide the blade of a scalpel un‐ der one corner of the slide and lift gently before immersing the slide in a 2xSSC solution and ag‐ itating it gently. This may need to be repeated several times if the cover slip remains stuck.

Solution: This can be difficult to fix, as it primarily occurs as a result of poor handling and fixation of tissue prior to receiving the sample for FISH testing [8,9]. Different tissue samples may require the pretreatment times to be varied [10]. The heat pretreatment buffer prepares the tissue for the enzyme to act on and the enzyme degrades the cellular material away from the DNA, in order to allow the probe to anneal to the chromatin. Variation of either or both these times is effective, and the steps may be repeated on the probed slide to reduce the need for lengthy pretreatment times on a new slide. Bone samples such as trephines may show poor hybridization efficiency of the probe, and require hydrogen chloride treatment,

sation steps can also be done adequately in a programmable system (e.g. Thermobyte).

Problem: The cover slip is hard to remove before the post wash steps.

unless the sample has already been decalcified prior to arrival.

probe as a substitution. Alternatively, hybridization mix can be made up:

(10% dextran sulphate, 50% formamide in 2xSSC, 0.1% SDS, pH 7.0)

[Step 5]

Hybridization mix

478 Latest Research into Quality Control

1-2 hours.

each time.

[Step 6]

[Step 7]

[Step 8]

Solution: This is due to incorrect stringency of the post wash [1]. For a quick fix, slides can be rewashed using the quick wash procedure reported here, or alternatively washing at a higher temperature or use of a different post wash procedure can be tried [11].

Problem: Cells only show one signal colour.

[Step 8]

Solution: Only cells showing both the control and target loci should be scored (e.g. 2R2G), so if both the control probe and the probe for the region of interest are on the same chromo‐ some, it is most likely to be due to poor hybridisation of one of the probes. First check to see using single colour filters whether the signal colour is present but weak – if it is, repeat the pre-treatment and hybridisation steps again on the same slide (for a shorter time e.g. 15/15 buffer: enzyme treatment).

Problem: Using an indirectly labeled probe and can't get a good signal quality.

[Step 8]

Solution: In most cases, amplification with only a primary antibody is necessary, and further amplification can also increase the level of background on the slide(s). However if the signal is not bright enough, carefully remove the cover slip, rinse slide in 1xPBS (or SSC) and per‐ form further amplification steps with secondary or tertiary antibodies as many times as nec‐ essary. After adding each antibody, slides should be covered with parafilm and incubated in a humidified chamber at 37°C for 5 minutes before being washed in 4xSSC/0.05% Tween20 for 2 minutes. Then mount with 8μl Vectashield antifade solution with DAPI.

Problem: Distinguishing between real signal and background or 'rubbish' on slide.

[Step 8]

Solution: Look at the signal intensity on single colour filters – rubbish generally appears to be brighter and shinier compared to real signals, and background will appear fuzzy and in‐ distinct compared to real signal. High background may be due to the slides not being prop‐ erly sealed with rubber cement during the pretreatment steps, as this allows the solution to evaporate and the tissue to dry out.

Problem: High background on the slides when analyzing.

[Step 8]

Solution: High background may be due to insufficient removal of material during the pre‐ treatment steps. With high case numbers, solutions can become contaminated, therefore the solutions in the pretreatment steps need to be changed regularly, and it pays to have an ad‐ ditional coplin jar of 100% ethanol to dip the slides into after the xylol step in order to re‐ duce contamination from the xylol solution. Alternatively, background may be due to the cover slip not being sealed properly during the pretreatment and co-denaturation steps, causing the tissue to dry out. By placing the slide in the incubator to allow the rubber ce‐ ment to dry before these steps, this effect can be reduced. The use of a glass coverslip rather than a plastic coverslip also helps, as plastic acts as an insulator, and therefore will hold the temperature and increase the drying of the tissue.

sible to get a pathologist to mark several smaller sites containing only target cells, as this re‐

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481

Solution: While this makes analysis difficult, switching to the 10x objective and moving the stage to a different position will reduce the likelihood of reanalyzing the same cells. Numer‐ ical scoring is also preferable in such a case, as it provides a reliable basis for interpretation.

Solution: Select good areas where the cells are not overlapping using the DAPI filter and use numerical scoring of individual cell signal patterns (this may mean increasing the number of sites examined if the cells are widely dispersed). If a gene rearrangement probe is being used, it may be sufficient just to report the presence or absence of a rearrangement without

Solution: If the target abnormality is either a gain (trisomy/tetrasomy) or loss (deletion) of a signal, it pays to establish thresholds using normal control slides to estimate the level of ar‐ tefactual gain or loss of signal, and to check the manufacturer's product information to see if splitting of the probe or non-target binding/polymorphisms are common with the probe. The variance in the signal patterns can also be checked – if the percentage of cells showing a 1R2G signal pattern is roughly equivalent to those showing a 2R1G signal, then it is reasona‐

Solution: Get a third analyst to score the sample. If two analysts have similar results, discard the third analysis, or if all three give different results, take an average of all three results to al‐ low robust interpretation. If the three results differ hugely, it is preferable to confirm the result with a secondary probe where possible, or request a repeat sample from another block. Where

Solution: Where the result is not straightforward use quantitative scoring and use appro‐ priate thresholds for interpretation. Paraffin FISH is not the most suitable method of de‐ tection for these cases, although methods that involve taking thicker slices of the section

the interpretation is still not clear, the case can be reported as inconclusive or failed.

Problem: A low level abnormality, multiple clones or mosaicism is suspected.

Problem: Target area marked is very small, so it is difficult to test a variety of areas.

Problem: The cells are highly dispersed or highly clustered, making analysis difficult.

Problem: Distinguishing between real loss and gain of signal compared to artefact.

ble to assume that it is due to artefactual truncation of signal.

Problem: There is discordance between analysts.

duces the risk of error before beginning the analysis.

[Step 8]

[Step 8]

[Step 8]

[Step 8]

[Step 8]

have been developed [12].

doing individual cell analysis.

The use of detergents in the post wash steps also helps to solubilize proteins, and if Tween20 is not effective, then NP-40 can also be used.

Problem: There is a mixed cell population in the marked target area (e.g. Tumour cells with non-target lymphocytes also present); see Figure 9.

**Figure 9.** The analysis of slides with mixed tissue populations.

#### [Step 8]

Solution: Check the H+E slide first before analysing the FISH slide to see whether there is clustering of cell types, or differences in morphology between the different cell types. Then scan the marked target area on the FISH slide using the 10x objective to find areas which appear to be targeted cells and switch to a higher objective for confirmation and then ana‐ lyse using appropriate filter. Consideration of accidental analysis of non-target cells must al‐ so be taken into account when interpreting such cases, therefore increasing the number of cells or sites analysed will increase the accuracy of the analysis. Alternatively, it may be pos‐ sible to get a pathologist to mark several smaller sites containing only target cells, as this re‐ duces the risk of error before beginning the analysis.

Problem: Target area marked is very small, so it is difficult to test a variety of areas.

#### [Step 8]

solutions in the pretreatment steps need to be changed regularly, and it pays to have an ad‐ ditional coplin jar of 100% ethanol to dip the slides into after the xylol step in order to re‐ duce contamination from the xylol solution. Alternatively, background may be due to the cover slip not being sealed properly during the pretreatment and co-denaturation steps, causing the tissue to dry out. By placing the slide in the incubator to allow the rubber ce‐ ment to dry before these steps, this effect can be reduced. The use of a glass coverslip rather than a plastic coverslip also helps, as plastic acts as an insulator, and therefore will hold the

The use of detergents in the post wash steps also helps to solubilize proteins, and if Tween20

Problem: There is a mixed cell population in the marked target area (e.g. Tumour cells with

Solution: Check the H+E slide first before analysing the FISH slide to see whether there is clustering of cell types, or differences in morphology between the different cell types. Then scan the marked target area on the FISH slide using the 10x objective to find areas which appear to be targeted cells and switch to a higher objective for confirmation and then ana‐ lyse using appropriate filter. Consideration of accidental analysis of non-target cells must al‐ so be taken into account when interpreting such cases, therefore increasing the number of cells or sites analysed will increase the accuracy of the analysis. Alternatively, it may be pos‐

temperature and increase the drying of the tissue.

non-target lymphocytes also present); see Figure 9.

**Figure 9.** The analysis of slides with mixed tissue populations.

[Step 8]

is not effective, then NP-40 can also be used.

480 Latest Research into Quality Control

Solution: While this makes analysis difficult, switching to the 10x objective and moving the stage to a different position will reduce the likelihood of reanalyzing the same cells. Numer‐ ical scoring is also preferable in such a case, as it provides a reliable basis for interpretation.

Problem: The cells are highly dispersed or highly clustered, making analysis difficult.

#### [Step 8]

Solution: Select good areas where the cells are not overlapping using the DAPI filter and use numerical scoring of individual cell signal patterns (this may mean increasing the number of sites examined if the cells are widely dispersed). If a gene rearrangement probe is being used, it may be sufficient just to report the presence or absence of a rearrangement without doing individual cell analysis.

Problem: Distinguishing between real loss and gain of signal compared to artefact.

#### [Step 8]

Solution: If the target abnormality is either a gain (trisomy/tetrasomy) or loss (deletion) of a signal, it pays to establish thresholds using normal control slides to estimate the level of ar‐ tefactual gain or loss of signal, and to check the manufacturer's product information to see if splitting of the probe or non-target binding/polymorphisms are common with the probe. The variance in the signal patterns can also be checked – if the percentage of cells showing a 1R2G signal pattern is roughly equivalent to those showing a 2R1G signal, then it is reasona‐ ble to assume that it is due to artefactual truncation of signal.

Problem: There is discordance between analysts.

#### [Step 8]

Solution: Get a third analyst to score the sample. If two analysts have similar results, discard the third analysis, or if all three give different results, take an average of all three results to al‐ low robust interpretation. If the three results differ hugely, it is preferable to confirm the result with a secondary probe where possible, or request a repeat sample from another block. Where the interpretation is still not clear, the case can be reported as inconclusive or failed.

Problem: A low level abnormality, multiple clones or mosaicism is suspected.

#### [Step 8]

Solution: Where the result is not straightforward use quantitative scoring and use appro‐ priate thresholds for interpretation. Paraffin FISH is not the most suitable method of de‐ tection for these cases, although methods that involve taking thicker slices of the section have been developed [12].

#### **4. Conclusion**

The role of pathologists is crucial to the analysis of paraffin FISH sections from the begin‐ ning of the process. They can help to eliminate very basic laboratory errors, such as identify‐ ing whether incorrect tissue has been sent prior to processing the slides, and can also help to identify the appropriate target tissue within the paraffin section prior to analysing the sam‐ ple, so that inappropriate tissues can be reduced or eliminated. When analysing products of conception, the fetal component can be very small compared to the maternal component, and without guidance of pathologists, an erroneous result may occur. Similarly, in breast cancer samples, it is important to eliminate areas of contained carcinoma (*in situ* compo‐ nents such as DCIS and LCIS) and lymphocytes, as these may result in false positive or neg‐ ative results, which can be deleterious if treatments such as Herceptin are then withheld from the patient. Some samples such as lymphomas or graft versus host disease may require extensive guidance from pathologists as knowledge of the disease characteristics will allow for highly targeted analysis. In follicular lymphoma, the follicles need to be identified so that centrocytes and centroblasts are targeted for analysis, and normal lymphocytes and re‐ active cells are avoided when analysing the sample (Swerdlow et al. 2008). For this reason, it is best to include a variety of areas to get a representative result. It should be noted that ex‐ ternal quality assurance programmes may differ in the number of sites required for analysis. Generally speaking, fewer sites are required, if initially the non-target tissue is eliminated.

analysis, as this will give an indication as to whether the sample is made up solely of target tissue, or whether it contains a mixture of target and non-target tissue that must be taken

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483

**Figure 11.** Artefactual considerations for paraffin FISH samples - truncation and overlapping of cells in specimen.

Due to both the potential for analysis of the incorrect target cells as outlined, and the artefac‐ tual variation that can arise when using the FISH technique [13], it is necessary to establish

into account when making the final interpretation.

**Figure 12.** The need for thresholds for paraffin FISH analysis.

**Figure 10.** Artefactual signal changes on suspension FISH slides.

Despite such assistance however, care must also be taken during the analysis of paraffin samples, as in many cases it is impossible to completely remove the non-target tissue from the area of interest. It is therefore important to check the H+E slide before beginning the analysis, as this will give an indication as to whether the sample is made up solely of target tissue, or whether it contains a mixture of target and non-target tissue that must be taken into account when making the final interpretation.

**4. Conclusion**

482 Latest Research into Quality Control

**Figure 10.** Artefactual signal changes on suspension FISH slides.

Despite such assistance however, care must also be taken during the analysis of paraffin samples, as in many cases it is impossible to completely remove the non-target tissue from the area of interest. It is therefore important to check the H+E slide before beginning the

The role of pathologists is crucial to the analysis of paraffin FISH sections from the begin‐ ning of the process. They can help to eliminate very basic laboratory errors, such as identify‐ ing whether incorrect tissue has been sent prior to processing the slides, and can also help to identify the appropriate target tissue within the paraffin section prior to analysing the sam‐ ple, so that inappropriate tissues can be reduced or eliminated. When analysing products of conception, the fetal component can be very small compared to the maternal component, and without guidance of pathologists, an erroneous result may occur. Similarly, in breast cancer samples, it is important to eliminate areas of contained carcinoma (*in situ* compo‐ nents such as DCIS and LCIS) and lymphocytes, as these may result in false positive or neg‐ ative results, which can be deleterious if treatments such as Herceptin are then withheld from the patient. Some samples such as lymphomas or graft versus host disease may require extensive guidance from pathologists as knowledge of the disease characteristics will allow for highly targeted analysis. In follicular lymphoma, the follicles need to be identified so that centrocytes and centroblasts are targeted for analysis, and normal lymphocytes and re‐ active cells are avoided when analysing the sample (Swerdlow et al. 2008). For this reason, it is best to include a variety of areas to get a representative result. It should be noted that ex‐ ternal quality assurance programmes may differ in the number of sites required for analysis. Generally speaking, fewer sites are required, if initially the non-target tissue is eliminated.

**Figure 11.** Artefactual considerations for paraffin FISH samples - truncation and overlapping of cells in specimen.

**Figure 12.** The need for thresholds for paraffin FISH analysis.

Due to both the potential for analysis of the incorrect target cells as outlined, and the artefac‐ tual variation that can arise when using the FISH technique [13], it is necessary to establish robust thresholds to guide the interpretation of results. Signal pattern changes can occur due to poor hybridization of probe, background 'rubbish-autofluorescence' or 'accidental over‐ lap' of red and green signals (Figure 10).

0.01% pepsin/HCl solution

2X SSC/0.1% Tween20

0.4xSSC/0.3%Tween20 solution 2xSSC/0.01% Tween20 solution

H-1200). Store in the dark at 4°C.

Centrifuge –Heraeus Biofuge Pico

Olympus BX60 fluorescence microscope

Incubator – Contherm Scientific NZ

Shandon Rapid-Chrome™ Frozen Section Staining kit

Coverslips (13mm diameter round, 22x22mm and 24 x 50mm)

Poly-lysine slides (with tissue sections of 2-5μm thickness)

Rubber cement – Weldtite Vulcanising Rubber Solution

Diamond pen or diamond-tipped engraver –Easy Marker Engraver (Taiwan)

Vectashield antifade mounting solution with 1.5μg/ml DAPI (Vector laboratories Cat #

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485

Fluorescence microscope - Zeiss Axio Imager.M1 microscope, Zeiss Axioplan microscope,

Purified H2O DNA probes

Xylol

Equipment

Atlas cooler box

Blotting paper

Eppendorf tubes

Glass coplin jars

Humidified box

Pipettes (2 ul and 100ul)

H&E slide Hotplate

Parafilm

Pipette tips

Fix-resistant marker pen

These can lead to the appearance of false or atypical signal patterns; therefore thresholds need to be established to distinguish between false positives and negatives. Paraffin anal‐ ysis requires higher thresholds than those for suspension cultures, as there is the addi‐ tional complication of overlap and truncation of cells [1,12], causing artefactual gain or loss of signals (Figures 11 and 12).

Thresholds are of particular importance when dealing with cases that show atypical, non-tar‐ get (e.g. unexpected loss or increase of copy number instead of a gene rearrangement) or low level abnormalities, or those where mosaicism or multiple clones appear to be present, as it is unclear in most cases as to how they may impact on patient treatment. While paraffin FISH is usually not the most appropriate way to deal with such cases, but when tissue is scarce or has already been processed, it can sometimes be the only option for testing. Numerical scoring of the tissue in such cases will give an indication of the major signal pattern(s) and the level of var‐ iation inherent in the tissue, particularly in tumours where there can be concurrent increase in the ploidy level, together with loss or gain of the target loci. This will allow a judgment to be made about whether the variation is likely to be artefactual or not, as false aneuploidies will show almost equivalent levels of loss between target and control loci.

Due to the potential complexities of paraffin analysis, the use of both cytogenetic and patholo‐ gy external quality control programs such as the College of American Pathologists (CAP) and Australasian Society of Cytogeneticists (ASoC) is recommended, as it allows quality issues to be addressed from both the cytogenetic and pathology perspectives. This provides a balanced perspective on the degree of analytical stringency that is required prior to releasing result.

### **Appendices**

*Materials* Reagents Biotin and Digoxygenin Bovine serum albumin (BSA) Deionised water Enzyme reagent (Invitrogen cat #00-8401) Ethanol (70%, 80% and 100%) Heat pre-treatment solution pH7.0 (Invitrogen cat #00-8401) Hybridisation buffer Non ionic detergent: NP40 (Vysis 30-80482). Store in -20°C. Phosphate Buffered Saline (PBS)

0.01% pepsin/HCl solution

Purified H2O

robust thresholds to guide the interpretation of results. Signal pattern changes can occur due to poor hybridization of probe, background 'rubbish-autofluorescence' or 'accidental over‐

These can lead to the appearance of false or atypical signal patterns; therefore thresholds need to be established to distinguish between false positives and negatives. Paraffin anal‐ ysis requires higher thresholds than those for suspension cultures, as there is the addi‐ tional complication of overlap and truncation of cells [1,12], causing artefactual gain or

Thresholds are of particular importance when dealing with cases that show atypical, non-tar‐ get (e.g. unexpected loss or increase of copy number instead of a gene rearrangement) or low level abnormalities, or those where mosaicism or multiple clones appear to be present, as it is unclear in most cases as to how they may impact on patient treatment. While paraffin FISH is usually not the most appropriate way to deal with such cases, but when tissue is scarce or has already been processed, it can sometimes be the only option for testing. Numerical scoring of the tissue in such cases will give an indication of the major signal pattern(s) and the level of var‐ iation inherent in the tissue, particularly in tumours where there can be concurrent increase in the ploidy level, together with loss or gain of the target loci. This will allow a judgment to be made about whether the variation is likely to be artefactual or not, as false aneuploidies will

Due to the potential complexities of paraffin analysis, the use of both cytogenetic and patholo‐ gy external quality control programs such as the College of American Pathologists (CAP) and Australasian Society of Cytogeneticists (ASoC) is recommended, as it allows quality issues to be addressed from both the cytogenetic and pathology perspectives. This provides a balanced perspective on the degree of analytical stringency that is required prior to releasing result.

show almost equivalent levels of loss between target and control loci.

lap' of red and green signals (Figure 10).

484 Latest Research into Quality Control

loss of signals (Figures 11 and 12).

**Appendices**

Biotin and Digoxygenin

Hybridisation buffer

Ethanol (70%, 80% and 100%)

Phosphate Buffered Saline (PBS)

Bovine serum albumin (BSA) Deionised water

Heat pre-treatment solution pH7.0 (Invitrogen cat #00-8401)

Non ionic detergent: NP40 (Vysis 30-80482). Store in -20°C.

Enzyme reagent (Invitrogen cat #00-8401)

*Materials* Reagents DNA probes

Shandon Rapid-Chrome™ Frozen Section Staining kit

2X SSC/0.1% Tween20

0.4xSSC/0.3%Tween20 solution

2xSSC/0.01% Tween20 solution

Vectashield antifade mounting solution with 1.5μg/ml DAPI (Vector laboratories Cat # H-1200). Store in the dark at 4°C.

Xylol

Equipment

Atlas cooler box

Blotting paper

Centrifuge –Heraeus Biofuge Pico

Coverslips (13mm diameter round, 22x22mm and 24 x 50mm)

Diamond pen or diamond-tipped engraver –Easy Marker Engraver (Taiwan)

Eppendorf tubes

Fix-resistant marker pen

Fluorescence microscope - Zeiss Axio Imager.M1 microscope, Zeiss Axioplan microscope, Olympus BX60 fluorescence microscope

Glass coplin jars

H&E slide

Hotplate

Humidified box

Incubator – Contherm Scientific NZ

Parafilm

Pipettes (2 ul and 100ul)

Pipette tips

Poly-lysine slides (with tissue sections of 2-5μm thickness)

Rubber cement – Weldtite Vulcanising Rubber Solution

Safety goggles Scalpel Scissors Slide drying racks Thermal cycler – MJC Research PTC- 100 and PTC-200 Peltier Thermal Cyclers Transmitted light microscope (Zeiss) Fine tweezers (2 pairs) Water bath – Grant Instruments (Cambridge) *Recipes*

Add 1ml of MilliQ water to 1.5mg lyophilised antibody for a final concentration of

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Ethanol 80% Mix ethanol absolute (molecular biology grade) and distilled water in a 4:1

Ethanol 70% Mix ethanol absolute (molecular biology grade) and distilled water in a 7:3

0.01N HCI. Add 1mL of 5N HCl to 499mLs of distilled water. Store at room temperature for

1xPBS. Ca++ and Mg++ free. Dissolve 8.0g sodium chloride, 0.2g potassium chloride, 2.89g Na2HPO4.12H20 and 0.2g KH2PO4 in order in 750ml of MilliQ water. Adjust the volume to 1

Pre-treatment reagents for paraffin embedded tissue – Zymed (Invitrogen) Spot-

20xSSC (7.0). Dissolve 175.3g sodium chloride and 88.2g trisodium citrate in 800ml MilliQ water. (or use SSC that comes with the Vysis kits; add 4 bottles to make 1L), pH to 7.0 and

4xSSC (pH7.0). Add 200ml of 20xSSC to 700ml MilliQ water. pH to 7.0 and adjust the final

2xSSC (pH7.0). Add 100ml 20xSSC (pH 7.0) to 800ml MilliQ water. pH to 7.0 and adjust the

1xSSC (pH 7.0). Add 50ml of 20xSSC (pH 7.0) to 950ml of milliQ water. Adjust the pH to 7.0,

0.4XSSC/0.3% NP40 (Quickwash buffer). Add 20ml of 20xSSC and 3ml of NP40 to 900ml MilliQ water. Adjust the pH to 7.0 and final volume to 1 litre. Store at room temperature.

Reagent A. 1 litre of heat pretreatment solution, pH 7.0 (Ready-To-Use).

adjust the final volume to 1 litre. Autoclave and store at room temperature.

4XSSC/0.05% Tween20. Add 500μl Tween20 to 1 litre of 4xSSC. Mix well.

Reagent B. 5 ml of enzyme pretreatment reagent (Ready-To-Use).

volume to 1 litre. Autoclave and store at room temperature.

2XSSC/0.1% NP40. Add 1mL of NP40 to 1L of 2XSSC (pH7.0)

autoclave and store at room temperature.

final volume to 1 litre. Autoclave and store at room temperature.

1.5mg/ml.

ratio (v/v).

ratio (v/v).

up to 1 year.

Hydrochloric acid (HCl)

Phosphate buffered saline (PBS)

light™HER2 CISH kit (84-0146)

Saline sodium citrate (SSC)

litre and autoclave. Store at room temperature.

Ethanol 100% Molecular biology grade.

0.2M HCl. Add 2.4ml of 5N HCl to 60mls of MilliQ water.

Biotin- and Avidin-conjugated antibodies

Store antibodies as 20μl aliquots at 4°C in sterile eppendorf tubes. Do not freeze.

Texas Red Avidin DCS (Biotek/Vector Laboratories Cat #A-2016).

Add 0.5ml of MilliQ water to 1mg lyophilised antibody for a final concentration of 2mg/ml.

Fluorescein Avidin DCS (Cell sorter grade), (Biotek/Vector Laboratories Cat #A-2011).

2mg/ml stock solution aliquotted at 20μl and stored in the dark at 4°C. Dilute 1:400 in 4xSSC/1% BSA immediately prior to use.

Biotinylated goat anti-avidin D (Biotek/Vector Laboratories Cat #BA0300).

Add 1ml of MilliQ water to 0.5mg lyophilised antibody for a final concentration of 0.5mg/ml.

Bovine Serum albumin (BSA)

1% BSA in 4xSSC. Dissolve 0.25g of BSA (Sigma A-7030) in 25ml 4xSSC pH 7.0. Store at 4°C for up to 1 month.

FITC– conjugated anti-digoxygenin antibodies

Store antibodies as 50μl aliquots at 4°C in sterile eppendorf tubes. Do not freeze.

Anti-digoxigenin-fluorescein, FAB fragments (Boehringer Mannheim Cat #1207741).

Add 1ml of MilliQ water to 200μg lyophilised antibody for a final concentration of 0.2mg/ml.

Rabbit fluorescein anti-sheep IgG(H+L) (Biotek/Vector Laboratories Cat #FI-6000).

Add 1ml of MilliQ water to 1.5mg lyophilised antibody for a final concentration of 1.5mg/ml.

Goat fluorescein anti-rabbit IgG(H+L) (Biotek/Vector Laboratories Cat #FI-1000).

Add 1ml of MilliQ water to 1.5mg lyophilised antibody for a final concentration of 1.5mg/ml.

Ethanol 100% Molecular biology grade.

Ethanol 80% Mix ethanol absolute (molecular biology grade) and distilled water in a 4:1 ratio (v/v).

Ethanol 70% Mix ethanol absolute (molecular biology grade) and distilled water in a 7:3 ratio (v/v).

Hydrochloric acid (HCl)

Safety goggles

486 Latest Research into Quality Control

Slide drying racks

Fine tweezers (2 pairs)

Transmitted light microscope (Zeiss)

Water bath – Grant Instruments (Cambridge)

Biotin- and Avidin-conjugated antibodies

4xSSC/1% BSA immediately prior to use.

FITC– conjugated anti-digoxygenin antibodies

Bovine Serum albumin (BSA)

Thermal cycler – MJC Research PTC- 100 and PTC-200 Peltier Thermal Cyclers

Store antibodies as 20μl aliquots at 4°C in sterile eppendorf tubes. Do not freeze.

Biotinylated goat anti-avidin D (Biotek/Vector Laboratories Cat #BA0300).

Add 0.5ml of MilliQ water to 1mg lyophilised antibody for a final concentration of 2mg/ml.

2mg/ml stock solution aliquotted at 20μl and stored in the dark at 4°C. Dilute 1:400 in

Add 1ml of MilliQ water to 0.5mg lyophilised antibody for a final concentration of

1% BSA in 4xSSC. Dissolve 0.25g of BSA (Sigma A-7030) in 25ml 4xSSC pH 7.0. Store at 4°C

Add 1ml of MilliQ water to 200μg lyophilised antibody for a final concentration of

Add 1ml of MilliQ water to 1.5mg lyophilised antibody for a final concentration of

Store antibodies as 50μl aliquots at 4°C in sterile eppendorf tubes. Do not freeze.

Anti-digoxigenin-fluorescein, FAB fragments (Boehringer Mannheim Cat #1207741).

Rabbit fluorescein anti-sheep IgG(H+L) (Biotek/Vector Laboratories Cat #FI-6000).

Goat fluorescein anti-rabbit IgG(H+L) (Biotek/Vector Laboratories Cat #FI-1000).

Fluorescein Avidin DCS (Cell sorter grade), (Biotek/Vector Laboratories Cat #A-2011).

Texas Red Avidin DCS (Biotek/Vector Laboratories Cat #A-2016).

Scalpel Scissors

*Recipes*

0.5mg/ml.

0.2mg/ml.

1.5mg/ml.

for up to 1 month.

0.2M HCl. Add 2.4ml of 5N HCl to 60mls of MilliQ water.

0.01N HCI. Add 1mL of 5N HCl to 499mLs of distilled water. Store at room temperature for up to 1 year.

Phosphate buffered saline (PBS)

1xPBS. Ca++ and Mg++ free. Dissolve 8.0g sodium chloride, 0.2g potassium chloride, 2.89g Na2HPO4.12H20 and 0.2g KH2PO4 in order in 750ml of MilliQ water. Adjust the volume to 1 litre and autoclave. Store at room temperature.

Pre-treatment reagents for paraffin embedded tissue – Zymed (Invitrogen) Spotlight™HER2 CISH kit (84-0146)

Reagent A. 1 litre of heat pretreatment solution, pH 7.0 (Ready-To-Use).

Reagent B. 5 ml of enzyme pretreatment reagent (Ready-To-Use).

Saline sodium citrate (SSC)

20xSSC (7.0). Dissolve 175.3g sodium chloride and 88.2g trisodium citrate in 800ml MilliQ water. (or use SSC that comes with the Vysis kits; add 4 bottles to make 1L), pH to 7.0 and adjust the final volume to 1 litre. Autoclave and store at room temperature.

4xSSC (pH7.0). Add 200ml of 20xSSC to 700ml MilliQ water. pH to 7.0 and adjust the final volume to 1 litre. Autoclave and store at room temperature.

4XSSC/0.05% Tween20. Add 500μl Tween20 to 1 litre of 4xSSC. Mix well.

2XSSC/0.1% NP40. Add 1mL of NP40 to 1L of 2XSSC (pH7.0)

2xSSC (pH7.0). Add 100ml 20xSSC (pH 7.0) to 800ml MilliQ water. pH to 7.0 and adjust the final volume to 1 litre. Autoclave and store at room temperature.

1xSSC (pH 7.0). Add 50ml of 20xSSC (pH 7.0) to 950ml of milliQ water. Adjust the pH to 7.0, autoclave and store at room temperature.

0.4XSSC/0.3% NP40 (Quickwash buffer). Add 20ml of 20xSSC and 3ml of NP40 to 900ml MilliQ water. Adjust the pH to 7.0 and final volume to 1 litre. Store at room temperature.

#### *Caution*

All reagents are potentially hazardous. Appropriate safety procedures must be followed when handling these materials. Avoid contact with skin and mucous membranes, and heat‐ ing of slides should be performed in a fume hood, as formalin fixed specimens may produce toxic fumes when heated during processing. For more information consult the Hazardous Substances Data Bank (HSDB) - http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB.

[6] Naeim, F., Rao, P. N., Song, S., & Grody, W. W. (2008). Principles of Molecular Tech‐ niques. *In: Naeim F, Rao PN, Song S, Grody WW, editors. Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches*, Elsevier Inc., Chapter 4,

Quality Control Considerations for Fluorescence *In Situ* Hybridisation of Paraffin-Embedded Pathology Specimens

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[7] Solovei, I., Grasser, F., & Lanctôt, C. (2007). FISH on Histological Sections. *Cold*

[8] Varella-Garcia, M. (2006). Stratification of non-small cell lung cancer patients for therapy with epidermal growth factor receptor inhibitors: the EGFR fluorescence in

[9] Kaeda, S. (2009). Molecular Pathology and Genetic Testing from the Perspective of a

[10] Srinivasan, S., Sedmak, D., & Jewell, S. (2002). Effect of fixatives and tissue process‐ ing on the content and integrity of nucleic acids. *Am. J. Path.*, 161(6), 1961-1971.

[11] Barch, M. J., Knutsen, T., & Spurbeck, J. (1997). Molecular Cytogenetics: Definitions, Clinical Aspects, and Protocols. *In: The AGT Cytogenetics Laboratory Manual* (Third

[12] Thompson, C., Le Boit, P. E., Nederlof, P. M., & Gray, J. W. (1994). Thick-section fluo‐ rescence in situ hybridization on formalin-fixed, paraffin embedded archival tissue

[13] Iourov, I. Y., Soloviev, I. V., Vorsanova, S. G., Monakhov, V. V., & Yurov, Y. B. (2005). An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics. *J. Histo. & Cyto.*, 53(3), 401-408.

[14] Swerdlow, S. H., Campo, E., Harris, N. L., Jaffe, E. S., Pileri, S. A., Stein, H., Thiele, J., & Vardiman, J. W. (2008). *WHO classification of tumours of Haematopoeitc and Lymphoid Tissues* (4th Edition), International Agency for Research on Cancer (IARC), 220-226.

*Spring Harb. Protoc.*, doi:10.1101/pdb.prot4729.

Commercial Laboratory. *Connection*, 7-11.

Edition), Lippincott-Raven, Chapter 13, 557-595.

provides a histogenetic profile. *Am. J. Path.*, 144(2), 237-243.

situ hybridization assay. *Diagnostic Pathology*, 1, 19 .

72-74.

Formamide: perform steps involving formamide in hood to avoid inhalation of fumes

Xylene: perform steps involving xylene in hood to avoid inhalation of fumes

Commercial probes and hybridisation buffer solutions: Wear gloves at all times, and when co-denaturing probes use a fume hood, as formamide may be present in probe mixtures and give off toxic fumes.

### **Author details**

Lisa Duffy1 , Liangtao Zhang1 , Donald R. Love1,2 and Alice M. George1\*

\*Address all correspondence to: AliceG@adhb.govt.nz

1 LabPlus, Auckland City Hospital, Auckland, New Zealand

2 School of Biological Sciences, The University of Auckland, New Zealand

#### **References**


[6] Naeim, F., Rao, P. N., Song, S., & Grody, W. W. (2008). Principles of Molecular Tech‐ niques. *In: Naeim F, Rao PN, Song S, Grody WW, editors. Hematopathology: Morphology, Immunophenotype, Cytogenetics and Molecular Approaches*, Elsevier Inc., Chapter 4, 72-74.

*Caution*

488 Latest Research into Quality Control

give off toxic fumes.

**Author details**

, Liangtao Zhang1

liams and Wilkens, 474.

*ics*, 3(1), 1-15.

141-151.

\*Address all correspondence to: AliceG@adhb.govt.nz

1 LabPlus, Auckland City Hospital, Auckland, New Zealand

Gene Rearrangements. *J. Med. Diag.*, 7(3), 346-351.

2 School of Biological Sciences, The University of Auckland, New Zealand

Lisa Duffy1

**References**

All reagents are potentially hazardous. Appropriate safety procedures must be followed when handling these materials. Avoid contact with skin and mucous membranes, and heat‐ ing of slides should be performed in a fume hood, as formalin fixed specimens may produce toxic fumes when heated during processing. For more information consult the Hazardous

Commercial probes and hybridisation buffer solutions: Wear gloves at all times, and when co-denaturing probes use a fume hood, as formamide may be present in probe mixtures and

, Donald R. Love1,2 and Alice M. George1\*

[1] Pfeifer, J. D. (2006). *Molecular Genetic Testing in Surgical Pathology*, Lippincott Wil‐

[2] Vorasanova, S. G., Yurov, Y. B., & Iourov, I. Y. (2010). Human interphase chromo‐ somes: a review of available molecular cytogenetic technologies. *Molecular Cytogenet‐*

[3] Maierhofer, C., Gangnus, R., Diebold, J., & Speicher, M. R. (2003). Multicolour decon‐ volution microscopy of thick biological specimens. *Am. J. Path.*, 162(2), 373-379. [4] Ventura, R. A., Martin-Subero, J. I., Jones, M., Mc Parland, J., Gesk, S., Mason, D. Y., & Siebert, R. (2006). FISH Analysis for the Detection of Lymphoma-Associated Chro‐ mosomal Abnormalities in Routine Paraffin-Embedded Tissue. *J. Med. Diagn.*, 8(2),

[5] George, T. I., Wrede, J. E., Bangs, C. D., Cherry, A. M., Warnke, R. A., & Arber, D. A. (2005). Low-Grade B-Cell Lymphomas With Plasmacytic Differentiation Lack PAX5

Substances Data Bank (HSDB) - http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB. Formamide: perform steps involving formamide in hood to avoid inhalation of fumes

Xylene: perform steps involving xylene in hood to avoid inhalation of fumes


**Chapter 22**

**Quality Control of Biomarkers: From the Samples to**

The recent advances in biotechnology and the improved understanding of disease's mecha‐ nisms and pathophsyology have strongly shifted the treatment paradigm of empiric knowl‐ edge to targeted therapy. Science has enhanced its ability to guide application of new and existing treatments with development, assay verification, biological validation and applica‐ tion of biomarkers; however, in order to be successful, it is needed a thorough understand‐ ing of the relationship between the choice of a biomarker and its influence on the treatment

Current biochemical and molecular biological knowledge states that genetic information flows from genomic DNA to mRNA transcripts, which are then translated to proteins; this class of molecules, which also include enzymes, directly influence the concentrations of their substrates and products, which are integrating parts in several tightly-controlled metabolic pathways. Finally, the existence and multiple interactions of these low-molecular weight

Metabolome, the link between phenotype and genotype, is the last comprehensive grouping for downstream products of the genome and contemplates the total complement of all the low-molecular weight molecules (metabolites) in a cell, tissue, or organism, required for growth, maintenance, or basal function in any given specific physiological state. [3] The po‐ tential size of the metabolome is arguable, as studies suggest more and more that an impor‐

The monitoring of metabolite changes has been the primary indicator of disease, and has made it possible to diagnose it in individuals. For that reason, the measurement of metabo‐

> © 2012 Ravagnani et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Ravagnani et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

metabolites within a cell, tissue, or organism, generates a phenotype. [2]

tant role is played by residing microflora and its metabolic products. [2]

**Data Interpretation**

http://dx.doi.org/10.5772/51555

**1. Introduction**

effects. [1]

F. G. Ravagnani, D. M. Saidemberg, A. L. C. Faria, S. B. Sartor, D. N. Oliveira and R. R. Catharino

Additional information is available at the end of the chapter

## **Quality Control of Biomarkers: From the Samples to Data Interpretation**

F. G. Ravagnani, D. M. Saidemberg, A. L. C. Faria,

S. B. Sartor, D. N. Oliveira and R. R. Catharino

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51555

#### **1. Introduction**

The recent advances in biotechnology and the improved understanding of disease's mecha‐ nisms and pathophsyology have strongly shifted the treatment paradigm of empiric knowl‐ edge to targeted therapy. Science has enhanced its ability to guide application of new and existing treatments with development, assay verification, biological validation and applica‐ tion of biomarkers; however, in order to be successful, it is needed a thorough understand‐ ing of the relationship between the choice of a biomarker and its influence on the treatment effects. [1]

Current biochemical and molecular biological knowledge states that genetic information flows from genomic DNA to mRNA transcripts, which are then translated to proteins; this class of molecules, which also include enzymes, directly influence the concentrations of their substrates and products, which are integrating parts in several tightly-controlled metabolic pathways. Finally, the existence and multiple interactions of these low-molecular weight metabolites within a cell, tissue, or organism, generates a phenotype. [2]

Metabolome, the link between phenotype and genotype, is the last comprehensive grouping for downstream products of the genome and contemplates the total complement of all the low-molecular weight molecules (metabolites) in a cell, tissue, or organism, required for growth, maintenance, or basal function in any given specific physiological state. [3] The po‐ tential size of the metabolome is arguable, as studies suggest more and more that an impor‐ tant role is played by residing microflora and its metabolic products. [2]

The monitoring of metabolite changes has been the primary indicator of disease, and has made it possible to diagnose it in individuals. For that reason, the measurement of metabo‐

© 2012 Ravagnani et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ravagnani et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lites has become an essential part of clinical practice. Employing a wide range of biological fluids, such as blood (including both plasma and serum), saliva, cerebrospinal fluid (CSF), synovial fluid, urine, semen, and tissue homogenates have ensured the widespread use of metabolites as a very powerful diagnostic tool. [4]

etry technique relies on the capacity of converting neutral molecules into gaseous ions, with or without fragmentation, which are then characterized by their mass to charge ratios (*m/z*) and relative abundances. The introduction of a sample into the system, which can be a gas chromatography or liquid chromatography system is necessary to allow the study of differ‐

Quality Control of Biomarkers: From the Samples to Data Interpretation

http://dx.doi.org/10.5772/51555

493

Historically, most studies with metabolites have been performed with a combination of high resolution capillary gas chromatography, combined with electron impact ionization mass spectrometry (GC-MS). This configuration allowed, for decades, the separation and identifi‐ cation of key micromolecules from complex mixtures, including fatty acids, amino acids, and organic acids in biofluids, generating diagnostic information for several metabolic dis‐

Despite its age, GC is still a very useful and informative technique that seems to be far away from retirement; however, there are some limitations in relation to the size and metabolite types that can be analyzed by this technique, and the extensive sample preparation for this purpose. This resulted in the use of nuclear magnetic resonance (NMR) as a tool for metabo‐ lite profiling; however, besides the richness of information about molecular structures ob‐ tained by this approach, NMR has low sensitivity, allowing just the most abundant compounds to be identified. In contraposition of GC-MS and NMR, the mass spectrometry with a high performance liquid chromatographic system (LC-MS), and the possibility of tan‐ dem mass spectrometry (LC-MS) as post-source fragmentation, especially after soft ioniza‐ tion techniques, offers the possibility of analyzing a wide range of polar and medium

polarity compounds with good quantification, sensibility and reproducibility [16].

Hollywood et al. (2006) [18] have summarized the main metabolomic strategies:

1. Metabolomic target analysis, which is a more restrict approach. For example, the metabo‐ lites originated from a particular enzymatic system after any kind of biotic or abiotic dis‐

2. Metabolite profiling, which is focused in a group of specific metabolites, for example, lip‐ ids associated to a determined metabolic pathway; or related with clinical and pharmaceuti‐ cal analyses, to map drug metabolism in an organism. This strategy can be also applied with

According to Birkemeier et al. (2005) [17], the metabolomic approaches are in dynamic de‐ velopment and a diversity of synonyms have been suggested, such as metabonomics, me‐ tabolite profiling (fingerprinting), among others. Several analytical platforms have been introduced, including spectroscopies using diverse electromagnetic wavelengths, like me‐ tabolite profiling with the use of infrared spectroscopy (IR), near infrared (NIR), or ultravio‐ let (UV), besides gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography with electrospray ionization mass spectrometry (LC-ESIMS), capillary elec‐ trophoresis with mass spectrometry (CE-MS) or liquid chromatography with nuclear mag‐ netic resonance (LC-NMR), and these are only a few examples of the technologies involved with metabolomic studies. There is not a single approach to analyze the wide range of chem‐ ically different biomolecules, but it is important to choose the technology that fits better to

ent structures and ionic forms.

your target molecules [17].

turbance.

orders in qualitative and quantitative pathways [15-16].

Despite significant advances in analytical technologies the past few years, the discovery of metabolomic biomarkers in biological fluids still remains a challenge. As discussed, metabo‐ lome plays an important role in biological systems, hence, are attractive candidates to un‐ derstand disease phenotypes. [5-6] It represents a diverse group of low-molecular weight structures including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thi‐ ols, carbohydrates and a few others. [7]

Biomarkers are defined as "characteristics that are objectively measured and evaluated as in‐ dicators of normal biological processes, pathogenic processes or pharmacological responses to therapeutic intervention". They can be categorized as biomarkers of exposure, biomark‐ ers of effect and biomarkers of susceptibility. [8] Those characteristics are informative for clin‐ ical outcome and can be broadly understood as prognostic or predictive biomarkers. [9-10]

Along the variety of chemical classes and physical properties that constitute metabolites, as well as the dynamic range of metabolite concentrations across large orders of magni‐ tude, it becomes clear why it is necessary to employ an extensive array of analytical tech‐ niques in metabolomic research, for it represents a comprehensive method for metabolite assessment. [11-12]

Enabling the parallel assessment of the levels of a broad number of endogenous and exoge‐ nous metabolites, it has been demonstrated to have great impact on investigation of physio‐ logical status, diseases diagnosis, biomarker discovery and identification of disrupted pathways due to disease or treatment. [13-14]

#### **2. Mass Spectrometry and Biomarkers**

#### **2.1. Mass spectrometry in metabolomics**

Nowadays, mass spectrometry is one of the most promising approaches for quantifying and qualifying known and unknown specific molecules within a very complex sample, and for elucidating the structure and chemical properties of different compounds. A mass spectrom‐ eter consists of three major components: (1) Ion Source: For producing gaseous ions from the substance being studied, some examples are electron impact (EI), chemical ionization (CI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photon ionization (APPI), thermospray ionization (TSI), among others; (2) Ana‐ lyzer: For resolving or separating ions according to their mass-to-charge ratios, some ana‐ lyzer examples are: quadrupole, time of flight, ion traps, Fourier transform ion cyclotron resonance, orbitrap, among others; (3) Detector system: For detecting the ions and recording the relative abundance of each of the resolved ionic species, for example: electron multiplier, microchannel plate detector, Daly detector, Faraday cup, among others. The mass spectrom‐ etry technique relies on the capacity of converting neutral molecules into gaseous ions, with or without fragmentation, which are then characterized by their mass to charge ratios (*m/z*) and relative abundances. The introduction of a sample into the system, which can be a gas chromatography or liquid chromatography system is necessary to allow the study of differ‐ ent structures and ionic forms.

lites has become an essential part of clinical practice. Employing a wide range of biological fluids, such as blood (including both plasma and serum), saliva, cerebrospinal fluid (CSF), synovial fluid, urine, semen, and tissue homogenates have ensured the widespread use of

Despite significant advances in analytical technologies the past few years, the discovery of metabolomic biomarkers in biological fluids still remains a challenge. As discussed, metabo‐ lome plays an important role in biological systems, hence, are attractive candidates to un‐ derstand disease phenotypes. [5-6] It represents a diverse group of low-molecular weight structures including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thi‐

Biomarkers are defined as "characteristics that are objectively measured and evaluated as in‐ dicators of normal biological processes, pathogenic processes or pharmacological responses to therapeutic intervention". They can be categorized as biomarkers of exposure, biomark‐ ers of effect and biomarkers of susceptibility. [8] Those characteristics are informative for clin‐ ical outcome and can be broadly understood as prognostic or predictive biomarkers. [9-10] Along the variety of chemical classes and physical properties that constitute metabolites, as well as the dynamic range of metabolite concentrations across large orders of magni‐ tude, it becomes clear why it is necessary to employ an extensive array of analytical tech‐ niques in metabolomic research, for it represents a comprehensive method for metabolite

Enabling the parallel assessment of the levels of a broad number of endogenous and exoge‐ nous metabolites, it has been demonstrated to have great impact on investigation of physio‐ logical status, diseases diagnosis, biomarker discovery and identification of disrupted

Nowadays, mass spectrometry is one of the most promising approaches for quantifying and qualifying known and unknown specific molecules within a very complex sample, and for elucidating the structure and chemical properties of different compounds. A mass spectrom‐ eter consists of three major components: (1) Ion Source: For producing gaseous ions from the substance being studied, some examples are electron impact (EI), chemical ionization (CI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photon ionization (APPI), thermospray ionization (TSI), among others; (2) Ana‐ lyzer: For resolving or separating ions according to their mass-to-charge ratios, some ana‐ lyzer examples are: quadrupole, time of flight, ion traps, Fourier transform ion cyclotron resonance, orbitrap, among others; (3) Detector system: For detecting the ions and recording the relative abundance of each of the resolved ionic species, for example: electron multiplier, microchannel plate detector, Daly detector, Faraday cup, among others. The mass spectrom‐

metabolites as a very powerful diagnostic tool. [4]

ols, carbohydrates and a few others. [7]

492 Latest Research into Quality Control

pathways due to disease or treatment. [13-14]

**2. Mass Spectrometry and Biomarkers**

**2.1. Mass spectrometry in metabolomics**

assessment. [11-12]

Historically, most studies with metabolites have been performed with a combination of high resolution capillary gas chromatography, combined with electron impact ionization mass spectrometry (GC-MS). This configuration allowed, for decades, the separation and identifi‐ cation of key micromolecules from complex mixtures, including fatty acids, amino acids, and organic acids in biofluids, generating diagnostic information for several metabolic dis‐ orders in qualitative and quantitative pathways [15-16].

Despite its age, GC is still a very useful and informative technique that seems to be far away from retirement; however, there are some limitations in relation to the size and metabolite types that can be analyzed by this technique, and the extensive sample preparation for this purpose. This resulted in the use of nuclear magnetic resonance (NMR) as a tool for metabo‐ lite profiling; however, besides the richness of information about molecular structures ob‐ tained by this approach, NMR has low sensitivity, allowing just the most abundant compounds to be identified. In contraposition of GC-MS and NMR, the mass spectrometry with a high performance liquid chromatographic system (LC-MS), and the possibility of tan‐ dem mass spectrometry (LC-MS) as post-source fragmentation, especially after soft ioniza‐ tion techniques, offers the possibility of analyzing a wide range of polar and medium polarity compounds with good quantification, sensibility and reproducibility [16].

According to Birkemeier et al. (2005) [17], the metabolomic approaches are in dynamic de‐ velopment and a diversity of synonyms have been suggested, such as metabonomics, me‐ tabolite profiling (fingerprinting), among others. Several analytical platforms have been introduced, including spectroscopies using diverse electromagnetic wavelengths, like me‐ tabolite profiling with the use of infrared spectroscopy (IR), near infrared (NIR), or ultravio‐ let (UV), besides gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography with electrospray ionization mass spectrometry (LC-ESIMS), capillary elec‐ trophoresis with mass spectrometry (CE-MS) or liquid chromatography with nuclear mag‐ netic resonance (LC-NMR), and these are only a few examples of the technologies involved with metabolomic studies. There is not a single approach to analyze the wide range of chem‐ ically different biomolecules, but it is important to choose the technology that fits better to your target molecules [17].

Hollywood et al. (2006) [18] have summarized the main metabolomic strategies:

1. Metabolomic target analysis, which is a more restrict approach. For example, the metabo‐ lites originated from a particular enzymatic system after any kind of biotic or abiotic dis‐ turbance.

2. Metabolite profiling, which is focused in a group of specific metabolites, for example, lip‐ ids associated to a determined metabolic pathway; or related with clinical and pharmaceuti‐ cal analyses, to map drug metabolism in an organism. This strategy can be also applied with other approaches, e.g.: *a.* "Metabolite fingerprinting", this approach is used in order to clas‐ sify samples based both in their biological relevance to the organism, and in their origin. The fingerprinting technology is fast, but not necessarily gives specific information about metab‐ olites. *b.* "Metabolite footprinting", exometabolome or secretome, this is similar approach to the fingerprinting, however the target now is a non-invasive analysis, in order to identify the extracellular metabolites. This technique is generally employed to the study of culture cells, with the advantage of not needing to extract the metabolites, and not having to inter‐ rupt the metabolism in a given moment before the analysis. Otherwise, this technique can be used for analysing the secretion of any organism, including the secretome of human em‐ bryos before *in vitro* fertilization, with the purpose of finding viable embryos and general disease biomarkers.

One of the most common applications for this new approach besides the well described pro‐ teomics application is the identification of membrane lipids, which have been successfully analyzed by different authors for several biological tissues. MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phos‐ phatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applica‐

Quality Control of Biomarkers: From the Samples to Data Interpretation

http://dx.doi.org/10.5772/51555

495

There are several possibilities for MALDI imaging technology; however applications to the study of small molecule biomarkers are becoming an interesting novel possibility for this ionization method, mainly when considering the development of new matrices which gen‐ erate low noise levels in the low *m/z* range of the spectra. Bnabdellah et al. (2009) [24] have described the detection and identification of 13 primary metabolites (AMP, ADP, ATP, UDP-GlcNAc, among others), directly from rat brain sections by chemical mass spectrome‐ try imaging. Matrix-assisted laser desorption/ionization tandem mass spectrometry (MAL‐

Metabolite distribution via imaging mass spectrometry (IMS) is an increasingly utilized tool in the field of neurochemistry. As most previous IMS studies analyzed the relative abundan‐ ces of larger metabolite species, it is important to expand its application to smaller mole‐ cules, such as neurotransmitters [25]. However, it has been pointed out two technical problems that must be resolved to achieve neurotransmitter imaging, the lower concentra‐ tions of bioactive molecules, compared with those of membrane lipids, require higher sensi‐ tivity and/or signal-to-noise (S/N) ratios in signal detection, and the rapid molecular turnover of the neurotransmitters; thus, tissue preparation procedures should be performed

Furthermore, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrome‐ try has attracted great interest for monitoring drug delivery and metabolism. Since this emerging technique enables simultaneous imaging of many types of metabolite molecules, MALDI-IMS can visualize and distinguish the parent drug and its metabolites. As another important advantage, changes in endogenous metabolites in response to drug administra‐

Another applications of MALDI and MALDI imaging to the study of small molecule bio‐ markers are the use of the method for detecting drug-related degradation products [27] analysis of drugs from intact biological samples and crude extracts, a method that can be ap‐ plied to rapid drug screening and precise identification of toxic substances in poisoning cas‐ es and *postmortem* examinations [28], the application of MALDI imaging mass spectrometry to the study of elevated nigral levels of dynorphin neuropeptides in L-DOPA-induced dys‐ kinesia in rat model of Parkinson's disease [29], it is also possible to point out the recent ad‐ vances in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury [30] and the using of proteomic or lipidomic signatures for discovery and spatial mapping of molecular disturbances within the microenvironment of chronic

DI-MS/MS) was combined with 9-aminoacridine as a powerful matrix in this study.

tions for this chemical visualization approach [23].

carefully to minimize *postmortem* changes [25].

tion can be mapped and evaluated in tissue sections [26].

wounds using MALDI imaging technology [31].

3. Metabolomics itself, which is the comprehensible analysis of the whole metabolome (all the mensurable metabolites), under a specific analysis condition. This term is frequently mistaken with metabonomics, a technique that focus in a wider profile of metabolites in‐ volved with different metabolic pathways interacting under the effect of some external stim‐ uli, including diseases, drugs, toxins, among other.

#### **2.2. MALDI AND MALDI-Imaging**

Matrix-assisted laser desorption/ionization (MALDI) is an ionization method with common applications to high mass biomolecules, being a key technique in mass spectrometry (MS), and more traditionally to the proteomics field. MALDI-MS is extremely sensitive, easy-toapply, and relatively tolerant to contaminants [19]. Its high-speed data acquisition and large-scale, off-line sample preparation has made it once again the focus for high-through‐ put proteomic analyses. These and other unique properties of MALDI offer new possibilities in applications such as rapid molecular profiling and imaging by MS [19].

More recently, there is a growing focus on the use of MALDI ionization system to the analy‐ sis of small molecules, however it is important to take into consideration that the coupling of LC-MALDI is a more delicate issue than the coupling of HPLC with other ionization sources such as ESI, because MALDI, based on desorption of molecules from a solid surface layer, is a priori not compatible with LC or CE [20]. A simple alternative to this limitation is the auto‐ matic deposition of fractions from a chromatographic separation on a MALDI-TOF target. More advanced techniques have been developed recently: electrospray deposition, electri‐ cally mediated deposition, rotating ball inlet, continuous vacuum deposition, and continu‐ ous off-line atmospheric-pressure deposition. The current interfacing improvements will surely expand the use of LC-MALDI in the metabolomic area [20,21].

Another good advantage of MALDI ionization is the possibility of obtaining tissue imag‐ ing. This is a new technology that allows the simultaneous investigation of the content and temporal/spatial distribution of molecules within a tissue section, enabling to find the exact localization of any biomarker of interest for the prediction of pathologies and for the discov‐ ery of future secondary complications originated from different metabolic disease [22].

One of the most common applications for this new approach besides the well described pro‐ teomics application is the identification of membrane lipids, which have been successfully analyzed by different authors for several biological tissues. MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phos‐ phatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applica‐ tions for this chemical visualization approach [23].

other approaches, e.g.: *a.* "Metabolite fingerprinting", this approach is used in order to clas‐ sify samples based both in their biological relevance to the organism, and in their origin. The fingerprinting technology is fast, but not necessarily gives specific information about metab‐ olites. *b.* "Metabolite footprinting", exometabolome or secretome, this is similar approach to the fingerprinting, however the target now is a non-invasive analysis, in order to identify the extracellular metabolites. This technique is generally employed to the study of culture cells, with the advantage of not needing to extract the metabolites, and not having to inter‐ rupt the metabolism in a given moment before the analysis. Otherwise, this technique can be used for analysing the secretion of any organism, including the secretome of human em‐ bryos before *in vitro* fertilization, with the purpose of finding viable embryos and general

3. Metabolomics itself, which is the comprehensible analysis of the whole metabolome (all the mensurable metabolites), under a specific analysis condition. This term is frequently mistaken with metabonomics, a technique that focus in a wider profile of metabolites in‐ volved with different metabolic pathways interacting under the effect of some external stim‐

Matrix-assisted laser desorption/ionization (MALDI) is an ionization method with common applications to high mass biomolecules, being a key technique in mass spectrometry (MS), and more traditionally to the proteomics field. MALDI-MS is extremely sensitive, easy-toapply, and relatively tolerant to contaminants [19]. Its high-speed data acquisition and large-scale, off-line sample preparation has made it once again the focus for high-through‐ put proteomic analyses. These and other unique properties of MALDI offer new possibilities

More recently, there is a growing focus on the use of MALDI ionization system to the analy‐ sis of small molecules, however it is important to take into consideration that the coupling of LC-MALDI is a more delicate issue than the coupling of HPLC with other ionization sources such as ESI, because MALDI, based on desorption of molecules from a solid surface layer, is a priori not compatible with LC or CE [20]. A simple alternative to this limitation is the auto‐ matic deposition of fractions from a chromatographic separation on a MALDI-TOF target. More advanced techniques have been developed recently: electrospray deposition, electri‐ cally mediated deposition, rotating ball inlet, continuous vacuum deposition, and continu‐ ous off-line atmospheric-pressure deposition. The current interfacing improvements will

Another good advantage of MALDI ionization is the possibility of obtaining tissue imag‐ ing. This is a new technology that allows the simultaneous investigation of the content and temporal/spatial distribution of molecules within a tissue section, enabling to find the exact localization of any biomarker of interest for the prediction of pathologies and for the discov‐ ery of future secondary complications originated from different metabolic disease [22].

in applications such as rapid molecular profiling and imaging by MS [19].

surely expand the use of LC-MALDI in the metabolomic area [20,21].

disease biomarkers.

494 Latest Research into Quality Control

uli, including diseases, drugs, toxins, among other.

**2.2. MALDI AND MALDI-Imaging**

There are several possibilities for MALDI imaging technology; however applications to the study of small molecule biomarkers are becoming an interesting novel possibility for this ionization method, mainly when considering the development of new matrices which gen‐ erate low noise levels in the low *m/z* range of the spectra. Bnabdellah et al. (2009) [24] have described the detection and identification of 13 primary metabolites (AMP, ADP, ATP, UDP-GlcNAc, among others), directly from rat brain sections by chemical mass spectrome‐ try imaging. Matrix-assisted laser desorption/ionization tandem mass spectrometry (MAL‐ DI-MS/MS) was combined with 9-aminoacridine as a powerful matrix in this study.

Metabolite distribution via imaging mass spectrometry (IMS) is an increasingly utilized tool in the field of neurochemistry. As most previous IMS studies analyzed the relative abundan‐ ces of larger metabolite species, it is important to expand its application to smaller mole‐ cules, such as neurotransmitters [25]. However, it has been pointed out two technical problems that must be resolved to achieve neurotransmitter imaging, the lower concentra‐ tions of bioactive molecules, compared with those of membrane lipids, require higher sensi‐ tivity and/or signal-to-noise (S/N) ratios in signal detection, and the rapid molecular turnover of the neurotransmitters; thus, tissue preparation procedures should be performed carefully to minimize *postmortem* changes [25].

Furthermore, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrome‐ try has attracted great interest for monitoring drug delivery and metabolism. Since this emerging technique enables simultaneous imaging of many types of metabolite molecules, MALDI-IMS can visualize and distinguish the parent drug and its metabolites. As another important advantage, changes in endogenous metabolites in response to drug administra‐ tion can be mapped and evaluated in tissue sections [26].

Another applications of MALDI and MALDI imaging to the study of small molecule bio‐ markers are the use of the method for detecting drug-related degradation products [27] analysis of drugs from intact biological samples and crude extracts, a method that can be ap‐ plied to rapid drug screening and precise identification of toxic substances in poisoning cas‐ es and *postmortem* examinations [28], the application of MALDI imaging mass spectrometry to the study of elevated nigral levels of dynorphin neuropeptides in L-DOPA-induced dys‐ kinesia in rat model of Parkinson's disease [29], it is also possible to point out the recent ad‐ vances in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury [30] and the using of proteomic or lipidomic signatures for discovery and spatial mapping of molecular disturbances within the microenvironment of chronic wounds using MALDI imaging technology [31].

#### **2.3. Orbitrap**

The orbitrap mass analyzer is a powerful and relatively new technology, which operates in the absence of any magnetic or *rf* fields. In this analyzer, ion stability is achieved only due to ions orbiting around an axial electrode. Orbiting ions also perform harmonic oscillations along the electrode with frequency proportional to (*m/z*)-1/2. These oscillations are detected using image current detection and are transformed into mass spectra using fast FT, similar‐ ly to FT-ICR [32]. In an orbitrap, ions are injected tangentially into the electric field be‐ tween the electrodes and trapped because their electrostatic attraction to the inner electrode is balanced by centrifugal forces. Thus, ions cycle around the central electrode in rings. In ad‐ dition, the ions also move back and forth along the axis of the central electrode. Therefore, ions of a specific mass-to-charge ratio move in rings which oscillate along the central spin‐ dle. The frequency of these harmonic oscillations is independent of the ion velocity and is in‐ versely proportional to the square root of the mass-to-charge ratio (*m/z*). The entire instrument operates in LC/MS mode (1 spectrum/s) with nominal mass resolving power of 60 000 and uses automatic gain control to provide high-accuracy mass measurements, within 2 ppm us‐ ing internal standards and within 5 ppm with external calibration. The maximum resolv‐ ing power exceeds 100 000 (Full Width at Half-Maximum – FWHM). Rapid, automated datadependent capabilities enable real-time acquisition of up to three high-mass accuracy MS/MS spectra per second [32,33].

Other recent applications of orbitrap mass analyzer in the search of biomarkers include: the analysis of serotonin and related compounds in urine and the identification of a potential biomarker for attention deficit hyperactivity/hyperkinetic disorder [37,38]; the quantitative profiling of phosphatidylethanol molecular species, which are a group of aberrant phospho‐ lipids formed in cell membranes in the presence of ethanol by the catalytic action of the en‐ zyme phospholipase D on phosphatidylcholine in human blood, by liquid chromatography high resolution mass spectrometry performed on an LTQ-Orbitrap XL hybrid mass spec‐ trometer equipped with an electrospray ionization source operated in negative ion mode [39]; frozen sections (12 μm thick) of an *ex vivo* tissue sample set comprising primary color‐ ectal adenocarcinoma samples and colorectal adenocarcinoma liver metastasis samples were analyzed by negative ion desorption electrospray ionization (DESI), with spatial resolution of 100 μm using a computer-controlled DESI imaging stage mounted on a high resolution orbitrap mass spectrometer. DESI-IMS data were found to predominantly feature complex lipids, including phosphatidyl-inositols, phophatidyl-ethanolamines, phosphatidyl-serines, phosphatidyl-ethanolamine plasmalogens, phosphatidic acids, phosphatidyl-glycerols, ce‐ ramides, sphingolipids, and sulfatides among others, were identified based on their exact mass and MS/MS fragmentation spectra [40]; among several other applications of this prom‐ ising technology to the discovery of important biomarkers in different biological systems, taking advantage of the high resolution and speed for LC-MS of this new analytical system.

Quality Control of Biomarkers: From the Samples to Data Interpretation

http://dx.doi.org/10.5772/51555

497

Gas chromatography (GC) can be understood as the chromatographic technique in which a gas is the mobile phase and, since 1952, when the first paper in this field was published, GC has always been considered simple, fast and applicable to the separation of many volatile materials, especially petrochemicals, for which distillation was the preferred method of sep‐ aration at that time. Now, GC is a very important technique, and global market for instru‐

Chromatography is the separation process of a mixture into individual components; through the separation process, each component in the sample can be identified (qualitative‐ ly) and measured (quantitatively). There are several kinds of chromatographic techniques with theirs corresponding instruments, and gas chromatography is one of those techniques. GC is used for compounds that are thermally stable and volatile - or that can become volati‐ lizable. Because of its simplicity, sensitivity and effectiveness in separating components, GC is one of the most important tools in chemistry. The principle of basic operation of this in‐ strument involves the evaporation of the sample in a heated inlet port (injector), separation of the components in a mixture employing a prepared column specially and detection of each component by a specific detector. At the end of the process, the amplified detector sig‐ nals are often recorded and evaluated by integrator software, calculating the analytical re‐ sults. The sample is introduced into a stream of inert gas, the carrier gas, and transported through the column by its flow. The column can be a packed column or a capillary column, depending on the properties of the sample. As the gas flow passes through the column, the components of the sample move in velocities that are influenced by the degree of interaction of each component with the stationary phase in the column. Consequently, the different

ments is estimated around to US\$ 1 billion or over 30,000 instruments annually [41].

**2.4. Gas Chromatography**

Some recent applications of this mass analyzer in the search of biomarkers include the ontissue digestion of proteins followed by detection of the resulting peptides, taking advant‐ age of the high resolution obtained. Trypsin was applied by a spraying device for MALDI imaging experiments in a LTQ-Orbitrap mass spectrometer. The mass accuracy under imag‐ ing conditions was better than 3 ppm RMS. This allowed for confident identification of tryp‐ tic peptides by comparison with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) measurements of an adjacent mouse brain section [34].

Another possible application for this mass analyzer is the monitoring of metabolites in hu‐ man urine, approximately 970 metabolite signals with repeatable peak areas could be puta‐ tively identified in human urine, by elemental composition assignment within a 3 ppm mass error. The ability of the methodology for the verification of non-molecular ions, which arise from adduct formation, and the possibility of distinguishing isomers could also be demon‐ strated. Careful examination of the raw data and the use of masses for predicted metabolites produced an extension of the metabolite list [35].

Orbitrap mass analyzer has been also successfully applied to the monitoring of environmen‐ tal contamination. The use of pharmaceuticals in livestock production is a potential source of surface water, groundwater and soil contamination. A rapid, versatile and selective multimethod was developed and validated for screening pharmaceuticals and fungicides com‐ pounds, in surface and groundwater, in one single full-scan MS method, using benchtop U-HPLC-Exactive Orbitrap MS at 50,000 (FWHM) resolution. It demonstrates that the ultrahigh resolution and reliable mass accuracy of Exactive Orbitrap MS permits the detection of pharmaceutical residues in a concentration range of 10-100 ng.L-1, applying a post-target screening approach, in the multi-method conditions [36].

Other recent applications of orbitrap mass analyzer in the search of biomarkers include: the analysis of serotonin and related compounds in urine and the identification of a potential biomarker for attention deficit hyperactivity/hyperkinetic disorder [37,38]; the quantitative profiling of phosphatidylethanol molecular species, which are a group of aberrant phospho‐ lipids formed in cell membranes in the presence of ethanol by the catalytic action of the en‐ zyme phospholipase D on phosphatidylcholine in human blood, by liquid chromatography high resolution mass spectrometry performed on an LTQ-Orbitrap XL hybrid mass spec‐ trometer equipped with an electrospray ionization source operated in negative ion mode [39]; frozen sections (12 μm thick) of an *ex vivo* tissue sample set comprising primary color‐ ectal adenocarcinoma samples and colorectal adenocarcinoma liver metastasis samples were analyzed by negative ion desorption electrospray ionization (DESI), with spatial resolution of 100 μm using a computer-controlled DESI imaging stage mounted on a high resolution orbitrap mass spectrometer. DESI-IMS data were found to predominantly feature complex lipids, including phosphatidyl-inositols, phophatidyl-ethanolamines, phosphatidyl-serines, phosphatidyl-ethanolamine plasmalogens, phosphatidic acids, phosphatidyl-glycerols, ce‐ ramides, sphingolipids, and sulfatides among others, were identified based on their exact mass and MS/MS fragmentation spectra [40]; among several other applications of this prom‐ ising technology to the discovery of important biomarkers in different biological systems, taking advantage of the high resolution and speed for LC-MS of this new analytical system.

#### **2.4. Gas Chromatography**

**2.3. Orbitrap**

496 Latest Research into Quality Control

spectra per second [32,33].

produced an extension of the metabolite list [35].

screening approach, in the multi-method conditions [36].

The orbitrap mass analyzer is a powerful and relatively new technology, which operates in the absence of any magnetic or *rf* fields. In this analyzer, ion stability is achieved only due to ions orbiting around an axial electrode. Orbiting ions also perform harmonic oscillations along the electrode with frequency proportional to (*m/z*)-1/2. These oscillations are detected using image current detection and are transformed into mass spectra using fast FT, similar‐ ly to FT-ICR [32]. In an orbitrap, ions are injected tangentially into the electric field be‐ tween the electrodes and trapped because their electrostatic attraction to the inner electrode is balanced by centrifugal forces. Thus, ions cycle around the central electrode in rings. In ad‐ dition, the ions also move back and forth along the axis of the central electrode. Therefore, ions of a specific mass-to-charge ratio move in rings which oscillate along the central spin‐ dle. The frequency of these harmonic oscillations is independent of the ion velocity and is in‐ versely proportional to the square root of the mass-to-charge ratio (*m/z*). The entire instrument operates in LC/MS mode (1 spectrum/s) with nominal mass resolving power of 60 000 and uses automatic gain control to provide high-accuracy mass measurements, within 2 ppm us‐ ing internal standards and within 5 ppm with external calibration. The maximum resolv‐ ing power exceeds 100 000 (Full Width at Half-Maximum – FWHM). Rapid, automated datadependent capabilities enable real-time acquisition of up to three high-mass accuracy MS/MS

Some recent applications of this mass analyzer in the search of biomarkers include the ontissue digestion of proteins followed by detection of the resulting peptides, taking advant‐ age of the high resolution obtained. Trypsin was applied by a spraying device for MALDI imaging experiments in a LTQ-Orbitrap mass spectrometer. The mass accuracy under imag‐ ing conditions was better than 3 ppm RMS. This allowed for confident identification of tryp‐ tic peptides by comparison with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) measurements of an adjacent mouse brain section [34].

Another possible application for this mass analyzer is the monitoring of metabolites in hu‐ man urine, approximately 970 metabolite signals with repeatable peak areas could be puta‐ tively identified in human urine, by elemental composition assignment within a 3 ppm mass error. The ability of the methodology for the verification of non-molecular ions, which arise from adduct formation, and the possibility of distinguishing isomers could also be demon‐ strated. Careful examination of the raw data and the use of masses for predicted metabolites

Orbitrap mass analyzer has been also successfully applied to the monitoring of environmen‐ tal contamination. The use of pharmaceuticals in livestock production is a potential source of surface water, groundwater and soil contamination. A rapid, versatile and selective multimethod was developed and validated for screening pharmaceuticals and fungicides com‐ pounds, in surface and groundwater, in one single full-scan MS method, using benchtop U-HPLC-Exactive Orbitrap MS at 50,000 (FWHM) resolution. It demonstrates that the ultrahigh resolution and reliable mass accuracy of Exactive Orbitrap MS permits the detection of pharmaceutical residues in a concentration range of 10-100 ng.L-1, applying a post-target Gas chromatography (GC) can be understood as the chromatographic technique in which a gas is the mobile phase and, since 1952, when the first paper in this field was published, GC has always been considered simple, fast and applicable to the separation of many volatile materials, especially petrochemicals, for which distillation was the preferred method of sep‐ aration at that time. Now, GC is a very important technique, and global market for instru‐ ments is estimated around to US\$ 1 billion or over 30,000 instruments annually [41].

Chromatography is the separation process of a mixture into individual components; through the separation process, each component in the sample can be identified (qualitative‐ ly) and measured (quantitatively). There are several kinds of chromatographic techniques with theirs corresponding instruments, and gas chromatography is one of those techniques. GC is used for compounds that are thermally stable and volatile - or that can become volati‐ lizable. Because of its simplicity, sensitivity and effectiveness in separating components, GC is one of the most important tools in chemistry. The principle of basic operation of this in‐ strument involves the evaporation of the sample in a heated inlet port (injector), separation of the components in a mixture employing a prepared column specially and detection of each component by a specific detector. At the end of the process, the amplified detector sig‐ nals are often recorded and evaluated by integrator software, calculating the analytical re‐ sults. The sample is introduced into a stream of inert gas, the carrier gas, and transported through the column by its flow. The column can be a packed column or a capillary column, depending on the properties of the sample. As the gas flow passes through the column, the components of the sample move in velocities that are influenced by the degree of interaction of each component with the stationary phase in the column. Consequently, the different components are separated. Since the processes are temperature-dependent, the column is usually contained in a thermostat-controlled oven. Once that the components are eluted from the column, they can be quantified by a suitable detector and/or be collected for further analysis. There are some types of detectors and the choice of the ones depends on the type of components that will be detected and measured. The most common detectors are: flame ion‐ ization detectors (FIDs), thermal conductivity detectors (TCDs), electron capture detectors (ECDs), alkali flame ionization detectors – also called nitrogen/phosphorous detectors (NPDs), flame photometric detectors (FPDs) and photo ionization detectors (PIDs). Several of these are further described in separate leaflets [41,42].

**3. Statistical and chemometrical analysis of biomarkers**

information through statistical experimental design (SED) [51,52,53].

to identify more robust biomarkers [54,55].

In metabolomics, as well as in other branches of science and technology, there is a steady trend towards the use of more variables (properties) to characterize observations (e.g., sam‐ ples, experiments, time points). Often, these measurements can be arranged into a data ta‐ ble, where each row constitutes an observation and the columns represent the variables or factors we have measured (e.g., wavelength, mass number, chemical shift, etc). This devel‐ opment generates huge and complex data tables, which are hard to summarize and over‐ view without appropriate tools. Recently, with development of "omics" technologies (metabolomics, proteomics, foodomics, genomics, etc), the adoption of chemometric meth‐ ods has been playing a very important role in planning and analyzing the obtained results. That includes efficient and robust methods for modeling and analysis of complex chemical or biological data tables that produce interpretable and reliable models capable of handling incomplete, noisy, and collinear data structures. These methods include principal compo‐ nent analysis (PCA) and partial least squares (PLS). It is also completely important to em‐ phasize that chemometrics also provides a straightforward way to collect relevant

Quality Control of Biomarkers: From the Samples to Data Interpretation

http://dx.doi.org/10.5772/51555

499

Multivariate statistical analysis such as Principal Components Analysis (PCA) is probably the most widely used technique for analyzing metabolomics. PCA technique is robust and objective and it is an appropriate way to reduce data sets containing high numbers of varia‐ bles. By reducing the number of original variables to a smaller number of independent vari‐ ables, this approach highlights fundamental differences between groups of variables. PCA has been extensively used in metabonomics literature. Despite apparent satisfying publish‐ ed results, the known large sensitivity of PCA to noise can suggest that improvements are expected with more robust methods to identify biomarkers in noisy data. Moreover, the tra‐ ditional use of PCA remains highly questionable: biomarkers are identified from the load‐ ings of the two first principal components, while the two first components do not necessarily contain the most relevant variations between altered and normal spectra. Some‐ times, the results of the initial unsupervised analysis are confirmed by a second supervised analysis. This one employs classification methods as Partial Least Squares (PLS), SIMCA and neural networks, allowing firstly to separate normal and altered spectra, and secondly

Other data analysis methods frequently employed for disease diagnosis and biomarker iden‐ tification in metabolomics are Univariate Testing, Soft independent modeling of class analo‐ gy (SIMCA), Linear discriminant analysis (LDA), Partial least squares discriminant analysis (PLS-DA), Orthogonal projection to latent structures discriminant analysis, (OPLS-DA), Neu‐ ral networks (NN), Self organizing maps (SOM) and Support vector machines (SVM). Regard‐ less of the chosen method, both statistical and biological validations are critical. Multivariate methods are of special importance to metabolomics since one biomarker often will not be suf‐ ficiently specific for a given condition by itself. There is a wide range of methods and it is nat‐ ural that this can seem confusing to the non-specialist. The literature has already shown in previous works that it is more important that the chosen method is used correctly than the methodology itself. The reason for this is that all methods are data-driven, and since the pa‐ rameter definition is through pre-processing, the contained features are static. Many statisti‐

GC is a widely used method for separating and analyzing organic compounds. There are a variety of applications for gas chromatography in every laboratory and in different process‐ es within several industries. In chemical, petrochemical and pharmaceutical industries we can have measurements of any kind of organic compounds, such as process control as well as product control. Also for environmental measurements: aromatic pollutants in air and water, detection and measurement of pesticides, etc. Beside the wide application of GC, there are a few examples of applications on which this analysis technique plays an impor‐ tant role [43,44,45,46].

The detection of reliable biomarkers is a major research activity within the field of proteo‐ mics and a growing trend on metabolomics. A biomarker can be a single molecule or set of molecules that can be used to differentiate between normal and diseased states and can be separated and detected by Gas Chromatography - Mass Spectrometry (GC/MS). This com‐ bined technique is used to identify the presence of different substances in a given sample.

Kuhara et al. (2011) [47] has used a GC/MS-based approach to investigate the metabolome in urine of patients whom had been previously diagnosed with citrin deficiency. In this nonin‐ vasive technique, urine metabolic profiling provided should assist in the rapid and more re‐ liable differential chemical diagnosis of citrin deficiency from other hyperammonemic syndromes.

Another application of GC/MS in biomarker analysis is its application on the studies of vola‐ tile organic compounds (VOCs). These compounds are exhaled in breath and provide valua‐ ble information about the human health status. The composition of the breath is variable and depends on the disease's characteristics; for example, a sweetened smell indicates dia‐ betes, while the odor of rotten eggs, which are caused by sulfur-containing compounds, sug‐ gests liver problems [48,49]. Rudnicka (2011) [50] employed solid phase micro-extraction technique and gas chromatography coupled to time of flight in mass spectrometry (GC– TOF/MS) for the analysis of VOCs on exhaled air from patients with lung cancer and healthy persons. The total number of identified compounds in breathing samples equal 55 and the compound that enables as an indication of lung cancer was isopropyl alcohol.

These studies show how highly important and relevant are the studies on the use of chroma‐ tographic techniques for biomarker analysis and identification. It shows a wide range of ap‐ plications in a field not yet fully developed, which still may be a very suitable area for new ideas and uses for the next couple decades.

### **3. Statistical and chemometrical analysis of biomarkers**

components are separated. Since the processes are temperature-dependent, the column is usually contained in a thermostat-controlled oven. Once that the components are eluted from the column, they can be quantified by a suitable detector and/or be collected for further analysis. There are some types of detectors and the choice of the ones depends on the type of components that will be detected and measured. The most common detectors are: flame ion‐ ization detectors (FIDs), thermal conductivity detectors (TCDs), electron capture detectors (ECDs), alkali flame ionization detectors – also called nitrogen/phosphorous detectors (NPDs), flame photometric detectors (FPDs) and photo ionization detectors (PIDs). Several

GC is a widely used method for separating and analyzing organic compounds. There are a variety of applications for gas chromatography in every laboratory and in different process‐ es within several industries. In chemical, petrochemical and pharmaceutical industries we can have measurements of any kind of organic compounds, such as process control as well as product control. Also for environmental measurements: aromatic pollutants in air and water, detection and measurement of pesticides, etc. Beside the wide application of GC, there are a few examples of applications on which this analysis technique plays an impor‐

The detection of reliable biomarkers is a major research activity within the field of proteo‐ mics and a growing trend on metabolomics. A biomarker can be a single molecule or set of molecules that can be used to differentiate between normal and diseased states and can be separated and detected by Gas Chromatography - Mass Spectrometry (GC/MS). This com‐ bined technique is used to identify the presence of different substances in a given sample.

Kuhara et al. (2011) [47] has used a GC/MS-based approach to investigate the metabolome in urine of patients whom had been previously diagnosed with citrin deficiency. In this nonin‐ vasive technique, urine metabolic profiling provided should assist in the rapid and more re‐ liable differential chemical diagnosis of citrin deficiency from other hyperammonemic

Another application of GC/MS in biomarker analysis is its application on the studies of vola‐ tile organic compounds (VOCs). These compounds are exhaled in breath and provide valua‐ ble information about the human health status. The composition of the breath is variable and depends on the disease's characteristics; for example, a sweetened smell indicates dia‐ betes, while the odor of rotten eggs, which are caused by sulfur-containing compounds, sug‐ gests liver problems [48,49]. Rudnicka (2011) [50] employed solid phase micro-extraction technique and gas chromatography coupled to time of flight in mass spectrometry (GC– TOF/MS) for the analysis of VOCs on exhaled air from patients with lung cancer and healthy persons. The total number of identified compounds in breathing samples equal 55 and the compound that enables as an indication of lung cancer was isopropyl alcohol.

These studies show how highly important and relevant are the studies on the use of chroma‐ tographic techniques for biomarker analysis and identification. It shows a wide range of ap‐ plications in a field not yet fully developed, which still may be a very suitable area for new

of these are further described in separate leaflets [41,42].

tant role [43,44,45,46].

498 Latest Research into Quality Control

syndromes.

ideas and uses for the next couple decades.

In metabolomics, as well as in other branches of science and technology, there is a steady trend towards the use of more variables (properties) to characterize observations (e.g., sam‐ ples, experiments, time points). Often, these measurements can be arranged into a data ta‐ ble, where each row constitutes an observation and the columns represent the variables or factors we have measured (e.g., wavelength, mass number, chemical shift, etc). This devel‐ opment generates huge and complex data tables, which are hard to summarize and over‐ view without appropriate tools. Recently, with development of "omics" technologies (metabolomics, proteomics, foodomics, genomics, etc), the adoption of chemometric meth‐ ods has been playing a very important role in planning and analyzing the obtained results. That includes efficient and robust methods for modeling and analysis of complex chemical or biological data tables that produce interpretable and reliable models capable of handling incomplete, noisy, and collinear data structures. These methods include principal compo‐ nent analysis (PCA) and partial least squares (PLS). It is also completely important to em‐ phasize that chemometrics also provides a straightforward way to collect relevant information through statistical experimental design (SED) [51,52,53].

Multivariate statistical analysis such as Principal Components Analysis (PCA) is probably the most widely used technique for analyzing metabolomics. PCA technique is robust and objective and it is an appropriate way to reduce data sets containing high numbers of varia‐ bles. By reducing the number of original variables to a smaller number of independent vari‐ ables, this approach highlights fundamental differences between groups of variables. PCA has been extensively used in metabonomics literature. Despite apparent satisfying publish‐ ed results, the known large sensitivity of PCA to noise can suggest that improvements are expected with more robust methods to identify biomarkers in noisy data. Moreover, the tra‐ ditional use of PCA remains highly questionable: biomarkers are identified from the load‐ ings of the two first principal components, while the two first components do not necessarily contain the most relevant variations between altered and normal spectra. Some‐ times, the results of the initial unsupervised analysis are confirmed by a second supervised analysis. This one employs classification methods as Partial Least Squares (PLS), SIMCA and neural networks, allowing firstly to separate normal and altered spectra, and secondly to identify more robust biomarkers [54,55].

Other data analysis methods frequently employed for disease diagnosis and biomarker iden‐ tification in metabolomics are Univariate Testing, Soft independent modeling of class analo‐ gy (SIMCA), Linear discriminant analysis (LDA), Partial least squares discriminant analysis (PLS-DA), Orthogonal projection to latent structures discriminant analysis, (OPLS-DA), Neu‐ ral networks (NN), Self organizing maps (SOM) and Support vector machines (SVM). Regard‐ less of the chosen method, both statistical and biological validations are critical. Multivariate methods are of special importance to metabolomics since one biomarker often will not be suf‐ ficiently specific for a given condition by itself. There is a wide range of methods and it is nat‐ ural that this can seem confusing to the non-specialist. The literature has already shown in previous works that it is more important that the chosen method is used correctly than the methodology itself. The reason for this is that all methods are data-driven, and since the pa‐ rameter definition is through pre-processing, the contained features are static. Many statisti‐ cal methods will highlight the same metabolites with similar classification ability. It is clear, however, that pre-processing and scaling of the data can lead to dramatically different re‐ sults, both with regard to chosen biomarkers and classification ability of the model [53].

**References**

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133-47.

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[1] Lee, J., et al. (2011). Proteomics and biomarkers in clinical trials for drug develop‐

Quality Control of Biomarkers: From the Samples to Data Interpretation

http://dx.doi.org/10.5772/51555

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[2] Roberts, L. D., Souza, A. L., Gerszten, R. E., & Clish, C. B. (2012). Targeted Metabolo‐

[3] Goodacre, R. (2003). Metabolic Profiling: Its Role in Biomarker Discovery and Gene

[4] Ryan, D., Robards, K., Prenzler, P. D., & Kendall, M. (2011). Recent and potential de‐

[5] Koulman, A., Lane, G. A., Harrison, S. J., & Volmer, D. A. (2009). From differentiat‐

[6] de la Luz-Hernández, K. R., Rojas-del Calvo, L., Rabasa-Legón, Y., Lage-Castellanos, A., Castillo-Vitlloch, A., Díaz, J., et al. (2008). Metabolic and proteomic study of NS0 myeloma cell line following the adaptation to protein-free medium. *J Proteomics*, 71,

[7] Ryan, D., Robards, K., Prenzler, P. D., & Kendall, M. (2011). Recent and potential de‐

[8] Atkinson, A. J. C. W., De Gruttola, V., De Mets, D. L., Downing, G. J., Hoth, D. F., Oates, J. A., et al. (2001). Biomarkers and Surrogate Endpoints: Preferred Definitions

[9] Simon, R., & Altman, D. G. (1994). Statistical aspects of prognostic factor studies in

[10] Sargent, D. J., Conley, B. A., Allegra, C., & Collette, L. (2005). Clinical Trial designs for predictive marker validation in cancer treatment trials. *J Clin Oncol*, 23, 2020-7. [11] Nicholson, J. K., & Lindon, J. C. (2008). Systems biology: metabonomics. *Nature*, 455,

[12] Arakaki, A. K., Skolnick, J., & McDonald, J. F. (2008). Marker metabolites can be ther‐

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[14] Schauer, N., Steinhauser, D., Strelkov, S., Schomburg, D., Allison, G., Moritz, T., Lundgren, K., Roessner-Tunali, U., Forbes, M. G., Willmitzer, L., Fernie, Ar., & Kop‐ ka, J. (2005). GC-MS libraries for the rapid identification of metabolites in complex

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### **4. Bibliography**

Aitio A, Apostoli P. Quality assurance in biomarker measurement. Toxicol Lett. 1995 May; 77(1-3):195-204.

Aitio A, Bernard A, Fowler BA, Nordberg G. Biological Monitoring and Biomerkers. Hand‐ book on the Toxicology of Metals. Third Edition. 2007; Chapter 4: 65-78.

Christians U, Klepacki J, Shokati T, Klawitter J, Klawitter J. Mass spectrometry-based multi‐ plexing for the analysis of biomarkers in drug development and clinical diagnostics – How much is too much ? Microchem. J. 2012, *doi:10.1016/j.microc.2012.02.001*

Hendriks MMWB, Eeuwijk FA, Jellema RH, Westerhuis JA, Reijmers TH, Hoefsloot HCJ, Smilde AK. Data-processing strategies for metabolomics studies. Trends in Analytical Chemistry. 2011; 30 (10): 1685-98.

Holland NT, Smith MT, Eskenazi B, Bastaki M. Biological sample collection and processing for molecular epidemiological studies. Mutat Res. 2003 Jun;543(3):217-34.

Lampe JW, Rock CL. Biomarkers and biological indicators of chance. Nutrition in the Pre‐ vention and Treatment of Disease. 2001; Chapter 10:139-153.

Lee JW, Hall M. Method validation of protein biomarkers in support of drug development or clinical diagnosis/prognosis. J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 1;877(13):1259-71.

Roux A, Lison D, Junot C, Heilier JF. Applications of liquid chromatography coupled to mass spectrometry-based metabolomics in clinical chemistry and toxicology: A review. Clin Biochem. Jan;44(1):119-35.

#### **Author details**

F. G. Ravagnani1,2, D. M. Saidemberg1 , A. L. C. Faria1 , S. B. Sartor1 , D. N. Oliveira1 and R. R. Catharino1\*

\*Address all correspondence to: rrc@fcm.unicamp.br

1*INNOVARE* Biomarkers Laboratory, Department of Clinical Pathology, School of Medical Sciences, University of Campinas, Brazil

2 Laboratory of Bioenergetics, Department of Clinical Pathology, School of Medical Sciences, University of Campinas, Brazil

#### **References**

cal methods will highlight the same metabolites with similar classification ability. It is clear, however, that pre-processing and scaling of the data can lead to dramatically different re‐ sults, both with regard to chosen biomarkers and classification ability of the model [53].

Aitio A, Apostoli P. Quality assurance in biomarker measurement. Toxicol Lett. 1995 May;

Aitio A, Bernard A, Fowler BA, Nordberg G. Biological Monitoring and Biomerkers. Hand‐

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## *Edited by Isin Akyar*

Quality control has an emerging importance in every field of life. Quality control is a process that is used to guarantee a certain level of quality in a product or service. It might include whatever actions a business deems necessary to provide for the control and verification of certain characteristics of a product or service. With the improvement of technology everyday we meet new and complicated devices and methods in different fields. Quality control should be performed in all of those new techniques. In this book "Latest Research Into Quality Control" our aim was to collect information about quality control in many different fields. The aim of this book is to share useful and practical knowledge about quality control in several fields with the people who want to improve their knowledge.

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Latest Research into Quality Control

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