We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists

3,800+

Open access books available

116,000+

International authors and editors

120M+

Downloads

Our authors are among the

Top 1%

most cited scientists

12.2%

Contributors from top 500 universities

Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI)

## Interested in publishing with us? Contact book.department@intechopen.com

Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com

## **Meet the author**

Dr Peter Mikuš (PM) received his M.Sc. in Analytical Chemistry in 1995 from the Faculty of Natural Sciences Comenius University in Bratislava, Slovak Republic. He received his Ph.D. in Analytical Chemistry from the same Faculty in 2002 under the supervision of Prof. Dr. Dusan Kaniansky, an excellent analyst and expert in the field of instrumental development of capillary electomi-

gration techniques. During his Ph.D. study, in 1997, PM joined academic and research center, namely Consiglio Nazionale delle Ricerche, Istituto di Cromatografia in Rome, Italy. Here, he made research for a period of three months under the supervision of Prof. Dr. Salvatore Fanali, an expert in the field of advanced methodology and application of chiral capillary electomigration methods. From 2002 he worked at the Faculty of Pharmacy Comenius University as a teacher and research scientist. PM received his Assoc. Prof. qualification in Pharmaceutical Chemistry from the Faculty of Pharmacy Comenius University in 2007. Currently, he is Assistant Professor, the Head of the Department of Pharmaceutical Analysis and Nuclear Pharmacy, Vice-dean for Foreign Relations and Development of the Faculty, and Departmental Erasmus Coordinator at the Faculty of Pharmacy Comenius University.

Chapter 1

**Preface VII**

**Introduction and Overview 1**

**Advanced Chiral Separation 5**

**and Electrophoresis 111**

**Acknowledgements 149**

**Conclusion 147**

**References 151**

**Index 187**

**Abbreviations 183**

**Advanced Sample Preparation 51**

**Advanced Combinations of Detection** 

Contents

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

### Contents

**Preface XI**



Published by InTech with the financial support from the Faculty of Pharmacy Comenius University.

### Preface

Although the technologies on chiral/enantiomer separation and stereoselective analysis have matured in the past ca. 20 years, the development of new, even more advanced chiral separation materials, mechanisms and methods still belong to the more challenging tasks in separation science and analytical chemistry. An analysis of recent trends indicates that capillary electrophoresis (CE) can show real advantages over chromatographic methods in ultratrace chiral determination of biologically active ionogenic compounds in complex matrices, including mostly biological ones. This is due to the extremely high separation efficiency of CE, as well as numerous new chiral selectors providing a wide range of selectivities for CE. Along with these tools, there are many applicable in-capillary electromigration effects in CE (countercurrent migration, stacking effects, etc.) enhancing significantly separability and, moreover, enabling effective sample preparation (preconcentration, purification, analyte derivatization). Other possible on-line combinations of CE, such as column coupled CE-CE techniques and implementation of nonelectrophoretic techniques (extraction, membrane filtration, flow injection, etc.) into CE, offer additional approaches for highly effective sample preparation and separation. Chiral CE matured to a highly flexible and compatible technique enabling its hyphenation with powerful detection systems allows for extremely sensitive detection (e.g., laser induced fluorescence) and/or structural characterization of analytes (e.g., mass spectrometry). Within the last decade, more and less conventional analytical on-line approaches have been effectively utilized in this field, and their practical potentialities have been demonstrated in many application examples in the literature.

In the present scientific monograph, three main aspects of chiral analysis of biologically active compounds are highlighted and supported by a theoretical description. This comprehensive integrated view on the topic is composed from the sections dealing with (i) progressive enantioseparation approaches and new enantioselective agents, (ii) in-capillary sample preparation (preconcentration, purification, derivatization) and (iii) detection possibilities related to enhanced sensitivity of quantitative determination and/or structural characterization of analysed chiral molecules. The section dealing with the chiral separations is inserted prior to the section dealing with the sample preparation in this book. This is logical, because achieving chiral resolution is a prerequisite in chiral research and the optimization of chiral resolution is a starting point within the development of a new chiral method. Then, a sample preparation and detection can be optimized, method validated, and finally, applied.

Although this book deals with the advanced chiral CE, it should be realized that this methodology could be understood more generally as the advanced CE modified with a selector (chiral as well as achiral) providing a considerably higher application potential. This generalization is justified realizing the parallels between the chiral and achiral selector-mediated separation systems in terms of (i) the implementations of the selectors and separation mechanisms, (ii) compatibility of sample preparation, separation and detection steps in the presence of the selector, and (iii) the application of the CE method modified with the selector. Therefore, the reader can advantageously use this book as a guide when proposing the strategy for the advanced chiral analysis, as well as achiral one supported by the complexing equilibria.

The author wishes that the readers obtain an integral view on the topic, some new knowledge, a good source of the relevant thematic reviews, as well as original research works on the topic, and hopes the readers gain inspiration for solving their own problems when reading this book.

The author would like to thank the book reviewers, excellent chemists and analysts, Prof. Dr. Ladislav Novotný, Assoc. Prof. Dr. Jozef Polonský, Prof. Dr. Emil Havránek and Prof. Dr. Milan Remko, for their valuable advice and suggestions on the manuscript during its preparation and before its final editing and publication.

> **Peter Mikuš** Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic

Introduction and Overview 1

It is well-established that in most cases of chiral drugs the pharmacological activity is restricted to one of the enantiomers (eutomer), whereas the other enantiomer (distomer) has either no effect or may show side effects - even being toxic [Ward T.J. & Ward K.D., 2010; Gilpin R.K. & Gilpin C.S., 2009; Bartos & Gorog, 2009; Gübitz & Schmid, 2008; Tzanavaras, 2010; Christodoulou, 2010; Zeng A.G. et al., 2010]. Information on the qualitative and quantitative composition of biologically active chiral compounds (enantiomers, diastereoisomers) in various real matrices, such as biological, pharmaceutical, environmental, food, beverage, etc., is required by control authorities and it is relevant in particular research areas, [see e.g., Hashim, 2010]. Enantioselective drug absorption, distribution, metabolism, elimination or liberation studies are included among the most advanced analytical problems being solved in pharmaceutical and biomedical research. This is due to (i) the multicomponent character of biological matrices (many potentially interfering compounds per sample), (ii) a very low concentration of the analyte(s) (pgng/mL) among the matrix constituents present in the sample in a wide concentration scale (pg-mg/mL), (iii) identical physicochemical properties of enantiomers in an achiral environment and in many cases (iv) limited/minute amounts of the sample [Maier et al., 2001; Camilleri, 1991; Bonato, 2003; Lin C.C. et al., 2003; Scriba, 2003, 2011; Van Eeckhaut &

Michotte, 2006; Hernández et al., 2008, 2010; Caslavska & Thormann, 2011].

Among high performance separation techniques, high performance liquid chromatography (HPLC) is the most matured, universal, robust, sensitive, selective, and therefore, the most frequently used technique (also) for the analysis of biomarkers, drugs and their metabolites in biological samples, as it can be seen from many application examples, [see e.g., Maier et al., 2001; Camilleri, 1991; Bonato, 2003; Lin C.C. et al., 2003; Scriba, 2003; Van Eeckhaut & Michotte, 2006; Hernández et al., 2008; Konieczna et al., 2010; Gatti & Gioia, 2008; El-Enany et al., 2007]. On the other hand, among the benefits of capillary electrophoresis (CE), pronounced especially in the chiral field, we can count its high separation efficiency, versatility and simplicity in the creation of (chiral) separation systems, short analysis time, good compatibility with aqueous samples and low consumption of chiral selector (low cost of enantioselective analyses) [Chankvetadze, 2007; Altria K. et al., 2006; Ward T.J. & Baker, 2008; Suntornsuk, 2010; Preinerstorfer et al., 2009; Bartos & Gorog, 2009; Ward T.J. & Ward K.D., 2010; Frost et al., 2010; Scriba, 2011]. Moreover, an analysis of recent trends indicates that CE can show real advantages over chromatographic methods, especially when a high resolution power, high sensitivity and low limit of detection/quantitation is ensured. CE meeting these criteria is directly applicable in the area of (chiral) analysis of low molecular ionic (and in some cases also neutral) compounds, such as drugs, their metabolites,

**1.2 Possibilities of capillary electrophoresis in chiral bioanalysis** 

**1.1 Demands in chiral bioanalysis**

**Introduction and Overview** 

Chapter title

**1** 

Author Name

## **1**

Chapter title

Author Name

### **Introduction and Overview**

### **1.1 Demands in chiral bioanalysis**

Although this book deals with the advanced chiral CE, it should be realized that this methodology could be understood more generally as the advanced CE modified with a selector (chiral as well as achiral) providing a considerably higher application potential. This generalization is justified realizing the parallels between the chiral and achiral selector-mediated separation systems in terms of (i) the implementations of the selectors and separation mechanisms, (ii) compatibility of sample preparation, separation and detection steps in the presence of the selector, and (iii) the application of the CE method modified with the selector. Therefore, the reader can advantageously use this book as a guide when proposing the strategy for the advanced chiral analysis, as

The author wishes that the readers obtain an integral view on the topic, some new knowledge, a good source of the relevant thematic reviews, as well as original research works on the topic, and hopes the readers gain inspiration for solving their own prob-

The author would like to thank the book reviewers, excellent chemists and analysts, Prof. Dr. Ladislav Novotný, Assoc. Prof. Dr. Jozef Polonský, Prof. Dr. Emil Havránek and Prof. Dr. Milan Remko, for their valuable advice and suggestions on the manu-

Faculty of Pharmacy, Comenius University, Bratislava,

**Peter Mikuš**

Slovak Republic

script during its preparation and before its final editing and publication.

well as achiral one supported by the complexing equilibria.

lems when reading this book.

It is well-established that in most cases of chiral drugs the pharmacological activity is restricted to one of the enantiomers (eutomer), whereas the other enantiomer (distomer) has either no effect or may show side effects - even being toxic [Ward T.J. & Ward K.D., 2010; Gilpin R.K. & Gilpin C.S., 2009; Bartos & Gorog, 2009; Gübitz & Schmid, 2008; Tzanavaras, 2010; Christodoulou, 2010; Zeng A.G. et al., 2010]. Information on the qualitative and quantitative composition of biologically active chiral compounds (enantiomers, diastereoisomers) in various real matrices, such as biological, pharmaceutical, environmental, food, beverage, etc., is required by control authorities and it is relevant in particular research areas, [see e.g., Hashim, 2010]. Enantioselective drug absorption, distribution, metabolism, elimination or liberation studies are included among the most advanced analytical problems being solved in pharmaceutical and biomedical research. This is due to (i) the multicomponent character of biological matrices (many potentially interfering compounds per sample), (ii) a very low concentration of the analyte(s) (pgng/mL) among the matrix constituents present in the sample in a wide concentration scale (pg-mg/mL), (iii) identical physicochemical properties of enantiomers in an achiral environment and in many cases (iv) limited/minute amounts of the sample [Maier et al., 2001; Camilleri, 1991; Bonato, 2003; Lin C.C. et al., 2003; Scriba, 2003, 2011; Van Eeckhaut & Michotte, 2006; Hernández et al., 2008, 2010; Caslavska & Thormann, 2011].

Introduction and Overview 1

### **1.2 Possibilities of capillary electrophoresis in chiral bioanalysis**

Among high performance separation techniques, high performance liquid chromatography (HPLC) is the most matured, universal, robust, sensitive, selective, and therefore, the most frequently used technique (also) for the analysis of biomarkers, drugs and their metabolites in biological samples, as it can be seen from many application examples, [see e.g., Maier et al., 2001; Camilleri, 1991; Bonato, 2003; Lin C.C. et al., 2003; Scriba, 2003; Van Eeckhaut & Michotte, 2006; Hernández et al., 2008; Konieczna et al., 2010; Gatti & Gioia, 2008; El-Enany et al., 2007]. On the other hand, among the benefits of capillary electrophoresis (CE), pronounced especially in the chiral field, we can count its high separation efficiency, versatility and simplicity in the creation of (chiral) separation systems, short analysis time, good compatibility with aqueous samples and low consumption of chiral selector (low cost of enantioselective analyses) [Chankvetadze, 2007; Altria K. et al., 2006; Ward T.J. & Baker, 2008; Suntornsuk, 2010; Preinerstorfer et al., 2009; Bartos & Gorog, 2009; Ward T.J. & Ward K.D., 2010; Frost et al., 2010; Scriba, 2011]. Moreover, an analysis of recent trends indicates that CE can show real advantages over chromatographic methods, especially when a high resolution power, high sensitivity and low limit of detection/quantitation is ensured. CE meeting these criteria is directly applicable in the area of (chiral) analysis of low molecular ionic (and in some cases also neutral) compounds, such as drugs, their metabolites,

**1.3 Aim and scope** 

2007].

representative application examples.

This scientific monograph deals with the theory and practice of the advanced chiral analysis of biologically active substances, beginning with the chiral separation, continuing with sample preparation and finishing with detection. The knowledge and findings from the review and research papers (involving also the author's works) included here give an integral and comprehensive view on the progressive performance of the chiral separations, analyses in complex matrices, pharmacokinetic and metabolic studies of drugs and analysis of biomarkers in various models and real matrices. The cited papers cover mainly the period from the year 2000 until now, although several former illustrative works are also included [see extensive reviews, e.g., Mikuš & Maráková, 2009; Scriba, 2003, 2011; Bonato, 2003; Lin C.C. et al., 2003; Hernández et al., 2008, 2010; Ward T.J. & Hamburg, 2004; Natishan, 2005; Van Eeckhaut & Michotte, 2006; Ha P.T. et al., 2006; Gübitz & Schmid, 2006, 2007; Caslavska & Thormann, 2011]. Mikuš and Maráková [Mikuš & Maráková, 2009] recently provided a review on the advanced capillary electrophoresis for the chiral analysis of drugs, metabolites and biomarkers in biological samples discussing chiral, sample preparation and detection aspects supported by the application examples. Other extensive review papers by Bonato [Bonato, 2003], Caslavska and Thormann [Caslavska & Thormann, 2011] and Scriba [Scriba, 2011] cover recent advances in the determination of enantiomeric drugs and their metabolites in biological matrices (e.g., biological fluids, tissues, microsomal preparations), as well as pharmaceuticals by CE mediated microanalysis and provide, besides many examples, also a detailed background on this topic. Other beneficial review papers in this area include refs. by Lin et al. [Lin C.C. et al., 2003] discussing recent progress in pharmacokinetic applications of CE, Scriba [Scriba, 2003] giving a view on pharmaceutical and biomedical applications of chiral CE and capillary electrochromatography (CEC), Hernández et al. [Hernández et al., 2008, 2010] giving an update on sensitive chiral analysis by CE in a variety of real samples including complex biological matrices. Several other review papers dealing with pharmaceutical and biomedical applications of chiral electromigration methods have also appeared in recent years [Van Eeckhaut & Michotte, 2006; Ward T.J. & Hamburg, 2004; Natishan, 2005; Ha et al., 2006; Gübitz & Schmid, 2006,

Introduction and Overview 3

The aim of this scientific monograph is to demonstrate comprehensively the current position of CE in the area of advanced chiral analysis of biologically active substances in samples with complex matrices (mainly biological). Therefore, the aim is not only to illustrate this by various practical applications, but, especially, to highlight and critically evaluate the progressive of the analytical approaches employed/applied in such examples. These, included in the present book, cover new findings in (i) chiral CE separation approaches (progressive arrangements of separation systems, new chiral selectors), (ii) preconcentration, purification and derivatization pretreatment of complex samples (on-line combinations of various sample preparation techniques with chiral CE) and (iii) detection monitoring of qualitative and quantitative composition of separated electrophoretic zones in complex samples (sensitive detection and/or structural evaluation of analytes). Such advanced approaches, playing a key role in the automatization and miniaturization of analytical procedures along with providing maximum analytical information, are comprehensively described in terms of basic theory, advantages and limitations, and documented by

biomarkers, etc., present in complex matrices such as biological samples [Mikuš & Maráková, 2009; Bonato, 2003; Lin C.C. et al., 2003; Scriba, 2003, 2011; Van Eeckhaut & Michotte, 2006; Hernández et al., 2008, 2010; Kraly et al., 2006; Caslavska & Thormann, 2011; Kitagawa & Otsuka, 2011].

The high resolution power and low limit of detection/quantitation are provided in CE itself by (i) an extremely high peak efficiency and (ii) wide range of various applicable electromigration effects and electrophoretic experimental modes enhancing selectivity and/or decreasing limit of detection (LOD) [Mikuš & Maráková, 2009]. Among such effects/modes, the (countercurrent) movement of analytes/selectors via electroosmotic flow (EOF), countercurrent migration of charged analyte and oppositely charged selector, incapillary stacking effects for the analyte preconcentration, removing of undesired compounds by electrokinetic injection of the sample and/or by electronic switching in online coupled electrophoretic systems are of the highest importance [Lin C.H. & Kaneta, 2004; Hernández et al., 2008, 2010; Chankvetadze, 1997; Chankvetadze et al., 2001; Scriba 2002; Simpson et al., 2008; Kaniansky & Marák, 1990; Danková et al., 1999; Fanali et al., 2000; Breadmore et al., 2009; Malá et al., 2009; Mikuš & Maráková, 2010].

CE matured to a highly flexible and compatible technique also enables (iii) on-line combinations of CE with nonelectrophoretic techniques (e.g., extraction, membrane filtration, microdialysis, flow injection, etc.) offering additional approaches for the highly effective sample preparation (especially sample clean-up, but also preconcentration) and separation [Breadmore et al., 2009; Chen Y. et al., 2008; Wu X.Z., 2003; Kataoka, 2003; Lü W.J. et al., 2009; de Jong et al., 2006; Mikuš & Maráková, 2010].

The utilization of unique methodological effects and modes mentioned in (ii) and (iii) can significantly enhance analytical potential and the practical use of conventional (singlecolumn) CE, solving its weakest points, such as a poor sensitivity and high concentration LOD, high risk of capillary overloading by major sample matrix constituents and peak overlapping, by numerous matrix constituents. In this way, the need for off-line sample preparation (isolation and concentration of analytes), especially when complex matrices are used (such as proteinic blood derived samples, ionic urine samples, tissue homogenates etc.), can be overcome.

Possibilities to combine CE with various detection techniques are comparable with chromatographic techniques. The high flexibility and compatibility of CE can be demonstrated by on-column and end-column coupling (hyphenation) with powerful detection systems covering demands on extremely sensitive detection (e.g., laser induced fluorescence, LIF), as well as structural characterization of analytes (e.g., mass spectrometry, MS) [Hernández et al., 2008, 2010; Swinney & Bornhop, 2000; Hempel G., 2000; Kok et al., 1998]. Such hyphenation is an essential part of advanced CE methods applied in modern highly demanding analytical research [Mikuš & Maráková, 2009].

#### **1.3 Aim and scope**

2 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

biomarkers, etc., present in complex matrices such as biological samples [Mikuš & Maráková, 2009; Bonato, 2003; Lin C.C. et al., 2003; Scriba, 2003, 2011; Van Eeckhaut & Michotte, 2006; Hernández et al., 2008, 2010; Kraly et al., 2006; Caslavska & Thormann, 2011;

The high resolution power and low limit of detection/quantitation are provided in CE itself by (i) an extremely high peak efficiency and (ii) wide range of various applicable electromigration effects and electrophoretic experimental modes enhancing selectivity and/or decreasing limit of detection (LOD) [Mikuš & Maráková, 2009]. Among such effects/modes, the (countercurrent) movement of analytes/selectors via electroosmotic flow (EOF), countercurrent migration of charged analyte and oppositely charged selector, incapillary stacking effects for the analyte preconcentration, removing of undesired compounds by electrokinetic injection of the sample and/or by electronic switching in online coupled electrophoretic systems are of the highest importance [Lin C.H. & Kaneta, 2004; Hernández et al., 2008, 2010; Chankvetadze, 1997; Chankvetadze et al., 2001; Scriba 2002; Simpson et al., 2008; Kaniansky & Marák, 1990; Danková et al., 1999; Fanali et al., 2000;

CE matured to a highly flexible and compatible technique also enables (iii) on-line combinations of CE with nonelectrophoretic techniques (e.g., extraction, membrane filtration, microdialysis, flow injection, etc.) offering additional approaches for the highly effective sample preparation (especially sample clean-up, but also preconcentration) and separation [Breadmore et al., 2009; Chen Y. et al., 2008; Wu X.Z., 2003; Kataoka, 2003; Lü

The utilization of unique methodological effects and modes mentioned in (ii) and (iii) can significantly enhance analytical potential and the practical use of conventional (singlecolumn) CE, solving its weakest points, such as a poor sensitivity and high concentration LOD, high risk of capillary overloading by major sample matrix constituents and peak overlapping, by numerous matrix constituents. In this way, the need for off-line sample preparation (isolation and concentration of analytes), especially when complex matrices are used (such as proteinic blood derived samples, ionic urine samples, tissue homogenates

Possibilities to combine CE with various detection techniques are comparable with chromatographic techniques. The high flexibility and compatibility of CE can be demonstrated by on-column and end-column coupling (hyphenation) with powerful detection systems covering demands on extremely sensitive detection (e.g., laser induced fluorescence, LIF), as well as structural characterization of analytes (e.g., mass spectrometry, MS) [Hernández et al., 2008, 2010; Swinney & Bornhop, 2000; Hempel G., 2000; Kok et al., 1998]. Such hyphenation is an essential part of advanced CE methods applied in modern

Breadmore et al., 2009; Malá et al., 2009; Mikuš & Maráková, 2010].

W.J. et al., 2009; de Jong et al., 2006; Mikuš & Maráková, 2010].

highly demanding analytical research [Mikuš & Maráková, 2009].

Kitagawa & Otsuka, 2011].

etc.), can be overcome.

This scientific monograph deals with the theory and practice of the advanced chiral analysis of biologically active substances, beginning with the chiral separation, continuing with sample preparation and finishing with detection. The knowledge and findings from the review and research papers (involving also the author's works) included here give an integral and comprehensive view on the progressive performance of the chiral separations, analyses in complex matrices, pharmacokinetic and metabolic studies of drugs and analysis of biomarkers in various models and real matrices. The cited papers cover mainly the period from the year 2000 until now, although several former illustrative works are also included [see extensive reviews, e.g., Mikuš & Maráková, 2009; Scriba, 2003, 2011; Bonato, 2003; Lin C.C. et al., 2003; Hernández et al., 2008, 2010; Ward T.J. & Hamburg, 2004; Natishan, 2005; Van Eeckhaut & Michotte, 2006; Ha P.T. et al., 2006; Gübitz & Schmid, 2006, 2007; Caslavska & Thormann, 2011]. Mikuš and Maráková [Mikuš & Maráková, 2009] recently provided a review on the advanced capillary electrophoresis for the chiral analysis of drugs, metabolites and biomarkers in biological samples discussing chiral, sample preparation and detection aspects supported by the application examples. Other extensive review papers by Bonato [Bonato, 2003], Caslavska and Thormann [Caslavska & Thormann, 2011] and Scriba [Scriba, 2011] cover recent advances in the determination of enantiomeric drugs and their metabolites in biological matrices (e.g., biological fluids, tissues, microsomal preparations), as well as pharmaceuticals by CE mediated microanalysis and provide, besides many examples, also a detailed background on this topic. Other beneficial review papers in this area include refs. by Lin et al. [Lin C.C. et al., 2003] discussing recent progress in pharmacokinetic applications of CE, Scriba [Scriba, 2003] giving a view on pharmaceutical and biomedical applications of chiral CE and capillary electrochromatography (CEC), Hernández et al. [Hernández et al., 2008, 2010] giving an update on sensitive chiral analysis by CE in a variety of real samples including complex biological matrices. Several other review papers dealing with pharmaceutical and biomedical applications of chiral electromigration methods have also appeared in recent years [Van Eeckhaut & Michotte, 2006; Ward T.J. & Hamburg, 2004; Natishan, 2005; Ha et al., 2006; Gübitz & Schmid, 2006, 2007].

The aim of this scientific monograph is to demonstrate comprehensively the current position of CE in the area of advanced chiral analysis of biologically active substances in samples with complex matrices (mainly biological). Therefore, the aim is not only to illustrate this by various practical applications, but, especially, to highlight and critically evaluate the progressive of the analytical approaches employed/applied in such examples. These, included in the present book, cover new findings in (i) chiral CE separation approaches (progressive arrangements of separation systems, new chiral selectors), (ii) preconcentration, purification and derivatization pretreatment of complex samples (on-line combinations of various sample preparation techniques with chiral CE) and (iii) detection monitoring of qualitative and quantitative composition of separated electrophoretic zones in complex samples (sensitive detection and/or structural evaluation of analytes). Such advanced approaches, playing a key role in the automatization and miniaturization of analytical procedures along with providing maximum analytical information, are comprehensively described in terms of basic theory, advantages and limitations, and documented by representative application examples.

**2**

**Advanced Chiral Separation** 

**2.1 Chiral electromigration modes and enantioselective agents - introduction** 

additives are applied in enantioseparations [Huo & Kok, 2008].

Kang J., 2006; Zhao S. et al., 2006a, 2006b].

techniques, but also in MEEKC and MCE.

in biological samples are discussed in the text and tabulated.

Chiral separations by CE can be performed either indirectly, using a chiral derivatization agent forming irreversible diastereomeric pairs which can be resolved under achiral conditions, or directly, using chiral selectors as additives to the electrolyte, where reversible diastereomeric associates, enantiomer-chiral selector, are created that can be subsequently transformed into mobility differences of the individual stereoisomers [Chankvetadze & Blaschke, 2001; Rizzi, 2001]. In capillary electrochromatography (CEC), a hybrid CE / HPLC technique (i.e., CE with stationary phase), chiral stationary phases or chiral mobile phase

Several disadvantages of the indirect enantioseparation approach, such as (i) the need of a functional group which can be derivatized, (ii) the derivatization reagent has to be of high enantiomeric purity, (iii) the derivatization represents an additional time consuming step with a risk of racemization under the reaction conditions, result in it being rarely used. Therefore, it is not surprising that only a few new chiral derivatization procedures, employing new chiral derivatization reagents, have been developed recently [Cheng J. &

More attractive and therefore much more frequently used are direct enantioseparations representing elegant and simple solutions in the majority of problems in chiral analysis. See, for instance, recent (2000-2011) chiral separations of drugs, their metabolites and biomarkers in various (mostly biological) samples listed in Tables 2.1 and 3.1 of this book (these tables are divided according to the manner of a sample preparation step, i.e., off- or on-line). In this chapter and Table 2.1 a chiral separation step is accompanied by a conventional off-line sample pretreatment and the chiral separation mechanism itself is highlighted. The latest fundamental reviews on chiral separations are given by Ward T.J. and Ward K.D. [Ward T.J. & Ward K.D., 2010] and Scriba [Scriba, 2011]. The papers by Gübitz and Schmid [Gübitz & Schmid, 2000a, 2007, 2008], Eeckhaut and Michotte [Van Eeckhaut & Michotte, 2006] and Preinerstorfer et al. [Preinerstorfer et al., 2009] provide detailed overviews on the different classes of chiral selectors, including newly introduced ones, that are used in common CE

The following subsections summarize (i) basic electromigration modes and their possibilities in chiral separations, as well as (ii) basic characteristics of different groups of chiral selectors - giving a view on their complexing abilities (types of useful analytes) and advantages and limitations when introduced into CE. Recent applications in the enantioseparation of drugs

### **Advanced Chiral Separation**

### **2.1 Chiral electromigration modes and enantioselective agents - introduction**

Chiral separations by CE can be performed either indirectly, using a chiral derivatization agent forming irreversible diastereomeric pairs which can be resolved under achiral conditions, or directly, using chiral selectors as additives to the electrolyte, where reversible diastereomeric associates, enantiomer-chiral selector, are created that can be subsequently transformed into mobility differences of the individual stereoisomers [Chankvetadze & Blaschke, 2001; Rizzi, 2001]. In capillary electrochromatography (CEC), a hybrid CE / HPLC technique (i.e., CE with stationary phase), chiral stationary phases or chiral mobile phase additives are applied in enantioseparations [Huo & Kok, 2008].

Several disadvantages of the indirect enantioseparation approach, such as (i) the need of a functional group which can be derivatized, (ii) the derivatization reagent has to be of high enantiomeric purity, (iii) the derivatization represents an additional time consuming step with a risk of racemization under the reaction conditions, result in it being rarely used. Therefore, it is not surprising that only a few new chiral derivatization procedures, employing new chiral derivatization reagents, have been developed recently [Cheng J. & Kang J., 2006; Zhao S. et al., 2006a, 2006b].

More attractive and therefore much more frequently used are direct enantioseparations representing elegant and simple solutions in the majority of problems in chiral analysis. See, for instance, recent (2000-2011) chiral separations of drugs, their metabolites and biomarkers in various (mostly biological) samples listed in Tables 2.1 and 3.1 of this book (these tables are divided according to the manner of a sample preparation step, i.e., off- or on-line). In this chapter and Table 2.1 a chiral separation step is accompanied by a conventional off-line sample pretreatment and the chiral separation mechanism itself is highlighted. The latest fundamental reviews on chiral separations are given by Ward T.J. and Ward K.D. [Ward T.J. & Ward K.D., 2010] and Scriba [Scriba, 2011]. The papers by Gübitz and Schmid [Gübitz & Schmid, 2000a, 2007, 2008], Eeckhaut and Michotte [Van Eeckhaut & Michotte, 2006] and Preinerstorfer et al. [Preinerstorfer et al., 2009] provide detailed overviews on the different classes of chiral selectors, including newly introduced ones, that are used in common CE techniques, but also in MEEKC and MCE.

The following subsections summarize (i) basic electromigration modes and their possibilities in chiral separations, as well as (ii) basic characteristics of different groups of chiral selectors - giving a view on their complexing abilities (types of useful analytes) and advantages and limitations when introduced into CE. Recent applications in the enantioseparation of drugs in biological samples are discussed in the text and tabulated.

**Analyte Separation**

**Chiral** 

**pH**

**Biological** 

**Sample** 

**Detection LOQ Application**

**Ref.** 

**preparation** 

**methodb**

Human SPE UV Spiked

samples

Li Y. et al.,

2010

**sample** 

**method** 

Amino acids,

CEC


5.5-

derivatives

8.5

plasma

CSP


PP UV (high

4.1 and 1.5

Real

El-Hady &

El-Maali,

2008

detection

cell)

Human LLE UV 10 ng/mL

(LOD)

samples

Spiked

Nojavan &

Fakhari,

2011

sensitivity

ng/mL

samples

(LOD)

plasma

mexiletine

Catechin isomers CDEKC

Cetirizine and

Polymeric

maltodextrin

 2.0

plasma

EKC

hydroxyzine

Dioxopromethazine

CDEKC

and Polymeric maltodextrin


Human

LLE ECL 4.0 x 10-6 M

Spiked

Li X. et al.,

2009

Awadallah

(LOD)

ng/mL

UV 4.4-11.2

Clinical

Andersen

ng/mL samples

et al., 2003

samples et al., 2003

samples

Spiked

urine

hydrochloride

Advanced Chiral Separation 7

Ofloxacin CDEKC M-

desmethyl-Citalopram,

CDEKC S-


> 2.5

Plasma LPME

(19-31x)

acetonitrile

citalopram

Primaquine,

EKC Maltodextrin

 3.0

liver of rats,

plasma

al fraction of

Mitochondri

LLE DAD 40-100

Spiked

Bortocan &

ng/mL

samples

Bonato,

2004

carboxyprimaquine

Hydroxychloroquine

CDEKC S-


> 9.0

Liver

LLE DAD 129 ng/mL

Spiked

Cardoso et

samples

al., 2006

homogenate,

plasma

HP--CD

and its metabolites

Propaphenone and

CDEKC S-


> 2.0

> Serum LLE UV 10-12

ng/mL

samples

Thormann,

2006

Spiked

Afshar &

(LOD)

methanol

its metabolites


Caco-cells LLE, EK UV 11.4-10.8

**selectora**

### **2.2 Electromigration techniques in chiral separations**

Effective chiral separations can be performed by a wide range of electromigration techniques that provide a great variety of applicable separation mechanisms and, by that, a high application potential both analytically and preparatively. For the basic instrumental scheme of CE see Figure 2.1.

The latest review on advances of enantioseparations in CE is given by Lu and Chen [Lu H.A. & Chen G.N., 2011] and Scriba [Scriba, 2011]. Gübitz and Schmid [Gübitz & Schmid, 2000a, 2007, 2008] show recent progress in chiral separation principles in various CE techniques, namely capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), isotachophoresis (ITP), isoelectric focusing (IEF), capillary electrokinetic chromatography (EKC) and capillary electrochromatography (CEC). The authors included into their latest review [Gübitz & Schmid, 2008] microchip CE (MCE). Among the most recent reviews also belong refs. by Gebauer et al. [Gebauer et al., 2009, 2011], Silva [Silva, 2009] and Ryan et al. [Ryan et al., 2009] describing recent advances in the methodology, optimization and application of ITP, micellar EKC (MEKC) and microemulsion EKC (MEEKC), respectively. Preinerstorfer et al. [Preinerstorfer et al., 2009] included, besides common CE techniques, also MEEKC and MCE.

Figure 2.1. Instrumental scheme of capillary electrophoresis system.

Effective chiral separations can be performed by a wide range of electromigration techniques that provide a great variety of applicable separation mechanisms and, by that, a high application potential both analytically and preparatively. For the basic instrumental

The latest review on advances of enantioseparations in CE is given by Lu and Chen [Lu H.A. & Chen G.N., 2011] and Scriba [Scriba, 2011]. Gübitz and Schmid [Gübitz & Schmid, 2000a, 2007, 2008] show recent progress in chiral separation principles in various CE techniques, namely capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), isotachophoresis (ITP), isoelectric focusing (IEF), capillary electrokinetic chromatography (EKC) and capillary electrochromatography (CEC). The authors included into their latest review [Gübitz & Schmid, 2008] microchip CE (MCE). Among the most recent reviews also belong refs. by Gebauer et al. [Gebauer et al., 2009, 2011], Silva [Silva, 2009] and Ryan et al. [Ryan et al., 2009] describing recent advances in the methodology, optimization and application of ITP, micellar EKC (MEKC) and microemulsion EKC (MEEKC), respectively. Preinerstorfer et al. [Preinerstorfer et al., 2009] included, besides

**2.2 Electromigration techniques in chiral separations** 

common CE techniques, also MEEKC and MCE.

Figure 2.1. Instrumental scheme of capillary electrophoresis system.

scheme of CE see Figure 2.1.



Hydroxychloroqui

CDEKC S-


9.0 Urine LPME DAD 10-21

ng/mL netic study

et al., 2007

de Gaitani et

al., 2003

Kavran-Belin

et al., 2005

Pharmacoki

de Oliveira

HP--CD

ne and its

metabotites

5-sulfoxide

Baclofen CDEKC S-

Disopyramide CDEKC S-

Amlodipine CDEKC HP-

Ibuprofen and its

CDEKC HTM-


SPE UV 110 ng/mL

(plasma),

netic study

Karazniewicz,

2007

1.0-1.1x103

(urine)

(LOD)

samples

(LOD) samples

Spiked

Chou et al.,

2008

Clinical Hou, J. et al.,

2007

ng/mL

plasma

metabolites

Cetirizine CDEKC S-

Advanced Chiral Separation 9

Warfarin MEKC Polysodium

Ketamine,

CDEKC S-


LLE DAD 10 ng/mL

(LOD) samples

et al., 2005

Clinical

Theurillat

(horse)

norketamine

Amphetamine

CDEKC S-


ng/mL

samples

Spiked

Rudaz et al.,

2005

(LOD)

ng/mL

samples

Spiked

Servais et al.,

2006

derivates

Salbutamol NACE

HDAS-


(CDEKC)



6.0 Plasma SPE MS 100 ng/mL

Tioridazine CDEKC HP-


3.0 Plasma LLE DAD 250 ng/mL Spiked

samples

Spiked

 S--CD



(LOD)

M (LOD)


samples

samples

Pharmacoki

Glowka &

samples

Spiked

Fang L. et al.,

2006

Wang R. et

al., 2007


Ketoprofen CDEKC HTM--CD

5.0 Serum LLE UV 500-1000 ng/mL Pharmacoki

Glowka & Karzniewicz, 2004a Indobufen CDEKC HTM--CD

5.0 Serum LLE UV 200 ng/mL

Glowka & Karzniewicz, 2004b Apomorfine CDEKC HP--CD 3.0 Caco-cells Direct injection DAD 0.5x10-6 M Spiked samples Ha et al., 2004a Lactic acid CDEKC HP--CD 7.0 Plasma PP (10x) DAD 15-20x10-6 M

Pharmacoki

netic study

LIF 0.3x10-6 M Clinical

Quan et al.,

2005

samples

samples

samples

Pharmacoki

Esteban et al.,

2004

Mandrioli

et al., 2004

Fan et al.,

2004

Spiked samples Tan et al., 2005 Serine CDEKC HP--CD + D(+)-

Neuronal cells (rat's brain) Microdialysis

Methadone CDEKC S--CD 5.0 Serum LLE, EK UV - Clinical

CDEKC CM--CD 2.5 Plasma SPE (37,5x) DAD 5 ng/mL Clinical

Mirtazapine and

its metabolites

Anisodamine CDEKC CM--CD 2.5 Plasma

Salbutamol NACE

HDAS--CD

(CDEKC)

CDEKC HDAS--CD

Metamphetamine

and related

compounds

t-tramadol, t-Odemethyltramadol

Labetalol CDEKC ODAS--CD,

HDAS--CD

CDEKC DM--CD +

CM--CD

CDEKC CE--CD 2.5 Urine LPME, EH DAD 62.5 ng/mL

Deprenyl

metabolites

Mirtazapine and

its metabolites

CDEKC SBE--CD 2.5 Plasma, urine LLE UV 1.25 ng/mL

2.5 Plasma SPE DAD - Spiked

2.7 Urine SPE UV 0.1-0.5x10-6

M

Pharmacoki

de Santana et

al., 2008

netic study

study

Pharmacoki

netic study

samples

Metabolic

Szökö et al.,

2004

Liu H.C. et al., 2004

Goel et al.,

2004

(rabbits)

6.0 Urine SPE DAD 375 ng/mL

1.7 Urine LLE MS 5 ng/mL Spiked

LLE, EK UV 40-60

ng/mL

Spiked

Servais et al.,

2004

Iio et al., 2005

samples

samples

netic study

, AD

 glucose 10.0

netic study


Ofloxacin and its

CDEKC SB-


injection


LIF 100-250

Metabolic

Horstkötter &

Blaschke,

2001

Cherkaoui

samples et al., 2001

Metabolic

Penget al.,

2002

ng/mL

study

metabolites

Methadone and its

CDEKC CM-

metabolites

Benzoporfyrine

MEKC Sodium

9.2 Serum,

PP, SPE LIF 2180-3500

ng/mL

LIF 100 ng/mL Pharmacoki

netic study

netic study

et al., 2001

Behn et al.,

2001

Soetebeer

study

microsomes

cholate

derivate mono and

diacid

Tramadol CDEKC CM-


> 10.0

> Urine Direct

injection

3.0 Plasma LLE LIF 1.56 ng/mL Pharmacoki

+ M--CD

Carvedilol CDEKC succinyl-


CD +

M-

CDEKC HP-


> 9.65

Plasma LLE LIF 0.5 ng/mL

(LOD) samples

Blaschke,

2000

Clinical

Müller &

CM--CD,

S--CD

Table 2.1. Chiral CE determinations of biologically active compounds in various biological matrices employing conventional (off-

aline) sample preparation. Mixed selector systems are indicated by a plus sign. Charge of ionizable chiral selectors is obvious from pH (next column).

ITP = isotachophoresis, EKC = electrokinetic chromatography, MEKC = micellar electrokinetic chromatography, CDEKC =

cyclodextrin mediated electrokinetic chromatography, CDMEKC = cyclodextrin mediated micellar electrokinetic chromatography,

NACE = non-aqueous capillary electrophoresis, MCE = electrophoresis on microchip, CEC = capillary electrochromatography, S-


CD = sulphated-


sulfobuthylether-




CD, AD = analyte derivatization, EH = enzymatic hydrolysis, HD = hydrodynamic injection, EK = electrokinetic injection, FESS =

field-enhanced sample stacking, LVSS = large volume sample stacking, SPE = solid-phase extraction, LLE = liquid-liquid extraction,

LPME = liquid-phase microextraction, PP = protein precipitation, DAD = diode array detection, UV-ultraviolet (absorbance

detection), ECL = electrochemiluminiscent detection, LIF = laser induced fluorescent detection, MS = mass spectrometry, LOQ =

limit of quantification, LOD = limit of detection.












CD = dimethyl-

CD, CM-

CD = carboxymethyl-





CD, S-

CD = sulphated-







Chloroquine,

deethylchloroquine

Advanced Chiral Separation 11



CD, AD = analyte derivatization, EH = enzymatic hydrolysis, HD = hydrodynamic injection, EK = electrokinetic injection, FESS = field-enhanced sample stacking, LVSS = large volume sample stacking, SPE = solid-phase extraction, LLE = liquid-liquid extraction, LPME = liquid-phase microextraction, PP = protein precipitation, DAD = diode array detection, UV-ultraviolet (absorbance

detection), ECL = electrochemiluminiscent detection, LIF = laser induced fluorescent detection, MS = mass spectrometry, LOQ =

limit of quantification, LOD = limit of detection.

10 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

3,4-metylendioxy-

3,4-metylendioxy-

Hydroxymebendazole,

CDEKC S--CD

4.2

Plasma LLE UV 10-40

ng/mL

samples

(LOD)

hydroxyaminomebendazole, aminomebendazole

Cinchona alkaloids CDEKC HDM--CD

Ketamine and its

CDEKC S--CD

2.5

Plasma,

urine

(multiple

isomer)

Methadone CDEKC S--CD 2.5

Methadone CDEKC HS--CD 4.5

and -CD

Mirtazapine and its

CEC Vancomycin

6.0

CSP

CDEKC S--CD 7.0

Plasma,

saliva

metabolites

Albendazole

sulfoxide

Phenprocoumone CDEKC

Baclofen CDEKC

Clenbuterol CDEKC DM--CD 2.5

Tramadol and its

CDEKC SBE--CD 4.0

metabolites


Plasma PP, AD LIF 10 ng/mL

Plasma SPE MS 740 ng/mL

Plasma LLE MS - Metabolic

(LOD)

Spiked

samples

study

samples

Spiked

Chiang et al.,

2001

Toussaint

et al., 2001

Rudaz et al.,

2000


Urine Direct

injection

Oral fluids LLE DAD 7.6-8.1

Urine SPE DAD 1000-2000

Plasma LLE-EK, PP-

HD MS 0.5 ng/mL (LLE-EK),

250 ng/mL

(PP-HD)

Clinical

Martins et al.,

2008

ng/mL

ng/mL

LLE UV, LIF 100 ng/mL

(LOD)

LIF 200 ng/mL

samples

Clinical

Chankvetadz

samples

e et al., 2001b

samples

Clinical

Prost et al.,

2002

samples

Spiked

Aturki et al.,

2007

metabolites

5.0

Urine SPE (10x) DAD 100 ng/mL

Clinical

Tsimachidis

et al., 2008

Theurillat

et al., 2007

samples

study

Clinical

Schappler

et al., 2008

samples

LLE DAD - Metabolic

 metamphetamine and its metabolites CDMEK

 C


2.3

Urine LLE LIF - Spiked

samples

Clinical

Theurillat &

Thormann,

2008

Huang Y.S.

et al., 2003

sodium

cholate

### **2.2.1 Capillary electrophoresis**

The unique properties of CE in terms of enantioresolution, due to a combination of extremely high separation efficiency (N) and various electomigration effects, are comprehensively summarized by Chankvetadze [Chankvetadze, 2007] and generally described by the Equation 2.1 [Giddings, 1969]:

$$R\_S = \frac{1}{4} \sqrt{N} \frac{\Delta \mu}{\mu\_{av}} \tag{2.1}$$

In fact, enantiomeric separations performed by CE may be included in an EKC mode because the discrimination of the enantiomers of a chiral compound is due to their different interactions with a chiral selector, that is, enantiomers are distributed in a different way between the bulk solution and the chiral selector according to a chromatographic (interaction) mechanism. So the electrophoretic and chromatographic principles are acting simultaneously in EKC (notice that is principally true not only for chiral but also achiral separations modified by a selector). Therefore, in this monograph we consider all the enantiomeric separations performed in the zone electrophoretic mode to be EKC separations with the exception of chiral ITP and chiral IEF separations (no alternative terms are

Advanced Chiral Separation 13

Figure 2.2. Separation principles in capillary electrophoresis: (a) zone electrophoresis (ZE), where B is background electrolyte, (b) isotachophoresis (ITP), where L is leading electrolyte and T is terminating electrolyte, with different electrophoretic mobilities of these electrolytes, (c) isoelectric focusing (IEF), where A-H are ampholytic electrolytes, with

Thanks to a great variety of applicable chiral selectors with different physico chemical properties and complexing abilities (see section 2.3), chiral CE separation systems with high performance variability can be created. Here, several basic enantioresolution mechanisms

Inclusion (host-guest) complexation {cyclodextrins (CDs), crown ethers (CWEs)},

different pI values of these electrolytes. Reprinted from ref. [Boček, 1987].

**2.2.1.1 Interactions in enantioseparations and their manipulation** 

can be recognized that are based on:

Ligand-exchange (metal complexes),

introduced in the literature).

where *av* is the effective averaged mobility {*av=1/2(1+2*)} and is the mobility difference (*=1-<sup>2</sup>*).

For enantioresolutions by CE the effective mobilities of the enantiomers have to be different (*1≠<sup>2</sup>*). This occurs due to (i) a difference in the complex formation constants of the enantiomer-chiral selector complexes (*K1≠K2*) and (ii) a difference in the mobility of the enantiomer-chiral selector complexes (*c1≠c2*), as well as the mobility of the free enantiomer and the enantiomer-selector complex (*f≠c1, f≠c2*), as it can be seen from the mobility difference () model (Equation 2.2) developed for two enantiomers (*1, 2*) and the concentration of selector (C) by Wren and Rowe [Wren & Rowe, 1992, 1993]:

$$
\Delta \mu = \mu\_1 - \mu\_2 = \frac{\mu\_f + \mu\_{c1} K\_1 [C]}{1 + K\_1 [C]} - \frac{\mu\_f + \mu\_{c2} K\_2 [C]}{1 + K\_2 [C]} \tag{2.2}
$$

It is apparent from this equation that CE offers many possibilities to manipulate enantioresolution via electromigration and complexing effects. This is also discussed in detail in the following subsections.

Besides electromigration and complexing effects, flow counterbalancing [Chankvetadze et al., 1999], as a combination of the bulk flow moving with the opposite migration of both a chiral selector and a chiral analyte, is another interesting possibility to effectively manipulate enantioresolution that will be briefly mentioned later.

The great advantages of CE in terms of the arrangement of the chiral separation system flexibly and simply are: (i) creation of continuous (CZE) and discontinuous / gradient (ITP, IEF) electrolyte systems providing a high variety of separation mechanisms. For basic CE modes see Figure 2.2. Here, interesting separation, as well as preseparation, possibilities are given by the differences in arrangement and diffusion properties of electrophoretic zones. (ii) Implementation of chiral selector(s) or, in other words, chiral pseudostationary phase(s), merely by their dissolving in such separation systems, creating a proper chiral separation environment. An extremely high resolution power of chiral CE can be amplified further by a large excess of a chiral selector dissolved in the electrolyte solution compared to the separation techniques with immobilized chiral selectors (CEC, HPLC) [Chankvetadze & Blaschke, 2001].

The unique properties of CE in terms of enantioresolution, due to a combination of extremely high separation efficiency (N) and various electomigration effects, are comprehensively summarized by Chankvetadze [Chankvetadze, 2007] and generally

*RS N*

*SNR*

*c1≠*

concentration of selector (C) by Wren and Rowe [Wren & Rowe, 1992, 1993]:

manipulate enantioresolution that will be briefly mentioned later.

*f≠c1, f≠*

 <sup>4</sup> 1

> *av=1/2(1+2*)} and

For enantioresolutions by CE the effective mobilities of the enantiomers have to be different

*<sup>2</sup>*). This occurs due to (i) a difference in the complex formation constants of the enantiomer-chiral selector complexes (*K1≠K2*) and (ii) a difference in the mobility of the

*<sup>f</sup> <sup>c</sup> K C <sup>f</sup> <sup>c</sup>*

*cfCKcf*

It is apparent from this equation that CE offers many possibilities to manipulate enantioresolution via electromigration and complexing effects. This is also discussed in

Besides electromigration and complexing effects, flow counterbalancing [Chankvetadze et al., 1999], as a combination of the bulk flow moving with the opposite migration of both a chiral selector and a chiral analyte, is another interesting possibility to effectively

The great advantages of CE in terms of the arrangement of the chiral separation system flexibly and simply are: (i) creation of continuous (CZE) and discontinuous / gradient (ITP, IEF) electrolyte systems providing a high variety of separation mechanisms. For basic CE modes see Figure 2.2. Here, interesting separation, as well as preseparation, possibilities are given by the differences in arrangement and diffusion properties of electrophoretic zones. (ii) Implementation of chiral selector(s) or, in other words, chiral pseudostationary phase(s), merely by their dissolving in such separation systems, creating a proper chiral separation environment. An extremely high resolution power of chiral CE can be amplified further by a large excess of a chiral selector dissolved in the electrolyte solution compared to the separation techniques with immobilized chiral selectors (CEC, HPLC) [Chankvetadze &

1 1 <sup>1</sup> <sup>2</sup> 1 1

11

*K C*

*CK*

1

) model (Equation 2.2) developed for two enantiomers (*1, 2*) and the

*av*

2.1

is the mobility difference

2.2

*c2*), as well as the mobility of the free enantiomer

*K C*

2 2

22

2

*CK*

*c2*), as it can be seen from the mobility

*K C*

*CK*

**2.2.1 Capillary electrophoresis** 

where 

difference (

Blaschke, 2001].

(*=1-<sup>2</sup>*).

(*1≠*

described by the Equation 2.1 [Giddings, 1969]:

*av* is the effective averaged mobility {

enantiomer-chiral selector complexes (

detail in the following subsections.

and the enantiomer-selector complex (

21

 In fact, enantiomeric separations performed by CE may be included in an EKC mode because the discrimination of the enantiomers of a chiral compound is due to their different interactions with a chiral selector, that is, enantiomers are distributed in a different way between the bulk solution and the chiral selector according to a chromatographic (interaction) mechanism. So the electrophoretic and chromatographic principles are acting simultaneously in EKC (notice that is principally true not only for chiral but also achiral separations modified by a selector). Therefore, in this monograph we consider all the enantiomeric separations performed in the zone electrophoretic mode to be EKC separations with the exception of chiral ITP and chiral IEF separations (no alternative terms are introduced in the literature).


Figure 2.2. Separation principles in capillary electrophoresis: (a) zone electrophoresis (ZE), where B is background electrolyte, (b) isotachophoresis (ITP), where L is leading electrolyte and T is terminating electrolyte, with different electrophoretic mobilities of these electrolytes, (c) isoelectric focusing (IEF), where A-H are ampholytic electrolytes, with different pI values of these electrolytes. Reprinted from ref. [Boček, 1987].

#### **2.2.1.1 Interactions in enantioseparations and their manipulation**

Thanks to a great variety of applicable chiral selectors with different physico chemical properties and complexing abilities (see section 2.3), chiral CE separation systems with high performance variability can be created. Here, several basic enantioresolution mechanisms can be recognized that are based on:


conditions

Advanced Chiral Separation 15

pH 9.6. ES1: 6

I-deoxy-6I-monomethylamino-

charged at pH 5.2, ES3: 6I-deoxy-6I-trimethylammonium-

CD uncharged at pH 9.6, ES5: 6I-deoxy-6I-trimethylammonium-

Reprinted from ref. [Mikuš & Kaniansky, 2007].

R – enantioresolution (for a given ES).


a

Table 2.2. Enantioresolutions of 2,4-dinitrophenyl (DNP) labelled amino acids under different complexing and acid-base

**Analyte Electrolyte system** 

DNP-D,L-amino acid R [ES2] R [ES3] R [ES4] R [ES6] R [ES7]

DNP-D,L-glutamic acid 2.6 1.0 1.6 0.4 1.7

DNP-D,L-methioninesulfone 0.9 0.0 1.6 0.0 1.3

DNP-D,L-methionine sulfoxide 0.9 0.3 1.1 0.0 0.9


DNP-D,L-norvaline 2.7 1.7 2.4 0.8 2.2

DNP-D,L-citrulline 1.3 0.0 1.3 0.0 1.0

DNP-D,L-methionine 2.1 3.2 4.3 0.9 3.3

DNP-D,L-norleucine 5.1 6.5 6.0 1.3 4.5

conditionsa 6I-deoxy-6I-monomethylamino-R

DNP-D,L-ethionine 3.1 5.2 5.9 0.8 4.2

DNP-D,L-isoleucine 7.2 6.0 8.6 1.7 5.3

DNP-D,L-leucine 11.8 9.9 12.1 2.2 7.6

DNP-D,L-

aElectrolyte systems (ESs) were prepared at two different pH values: (i) 100 mM morpholinoethanesulfonic acid + 10 mM histidine

+ 0.2% methylhydroxyethylcellulose (w/v) + 20 mM cyclodextrin derivative, pH 5.2, (ii) 50 mM H3BO3 + 100 mM 1,3-

bis(tris(hydroxymethyl)-methylamino)propane + 0.2% methylhydroxyethylcellulose (w/v) + 20 mM cyclodextrin (CD) derivative,






Thus, the separations of enantiomeric couples with a wide range of polarities, charges and sizes can be easily accomplished [Gübitz & Schmid, 2000a, 2007, 2008; Preinerstorfer et al., 2009; Gebauer et al., 2009, 2011; Silva, 2009; Ryan et al., 2009], see examples in section 2.4, Table 2.1 and Table 3.1.

On the other hand, very subtle differences/modifications of the structure within the same group of chiral selectors also can provide significant differences in (enantio)selectivity, see Table 2.2 (notice differences in CE enantioresolutions under the same conditions, but different chiral selector – differing in one methyl group in their molecules). This demonstrates another powerful tool to manipulate (enantio)selectivity from the complex forming point of view in CE enantioseparations.

Figure 2.3. Influence of pH and concentration of chiral selector on the resolution of pheniramine enantiomers demonstrating the effectivity of charged chiral selector and countercurrent separation mechanism in EKC enantioseparation. (a) The concentration dependences at 0.5, 2.5 and 5.0 mg/mL concentrations of CE--CD (●) and native -CD (○) were obtained at pH 4.5 (20 mM -aminocaproic acid - acetic acid BGE); (b) the pH dependences were obtained at 5 mg/mL concentrations of the CDs and the glycine- or aminocaproic acid – acetic acid BGEs with pH 3.2-3.8 or 4.5, respectively. 0.2% (w/v) methyl-hydroxyethylcellulose served as an EOF suppressor in BGE. The driving current was stabilized at 100-120 A. CE--CD = carboxyethyl--cyclodextrin. Reprinted from ref. [Mikuš et al., 2005a].


aElectrolyte systems (ESs) were prepared at two different pH values: (i) 100 mM morpholinoethanesulfonic acid + 10 mM histidine + 0.2% methylhydroxyethylcellulose (w/v) + 20 mM cyclodextrin derivative, pH 5.2, (ii) 50 mM H3BO3 + 100 mM 1,3- pH 9.6. ES1: 6 bis(tris(hydroxymethyl)-methylamino)propane + 0.2% methylhydroxyethylcellulose (w/v) + 20 mM cyclodextrin (CD) derivative, I-deoxy-6I-monomethylamino--CD positively charged at pH 5.2, ES2: 6I-deoxy-6I-dimethylamino--CD positively charged at pH 5.2, ES3: 6I-deoxy-6I-trimethylammonium--CD positively charged at pH 5.2, ES4: 6I-deoxy-6I-monomethylamino-- CD uncharged at pH 9.6, ES5: 6I-deoxy-6I-trimethylammonium--CD positively charged at pH 9.6. R – enantioresolution (for a given ES).

Reprinted from ref. [Mikuš & Kaniansky, 2007].

14 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Thus, the separations of enantiomeric couples with a wide range of polarities, charges and sizes can be easily accomplished [Gübitz & Schmid, 2000a, 2007, 2008; Preinerstorfer et al., 2009; Gebauer et al., 2009, 2011; Silva, 2009; Ryan et al., 2009], see examples in section 2.4,

On the other hand, very subtle differences/modifications of the structure within the same group of chiral selectors also can provide significant differences in (enantio)selectivity, see Table 2.2 (notice differences in CE enantioresolutions under the same conditions, but different chiral selector – differing in one methyl group in their molecules). This demonstrates another powerful tool to manipulate (enantio)selectivity from the complex

Figure 2.3. Influence of pH and concentration of chiral selector on the resolution of pheniramine enantiomers demonstrating the effectivity of charged chiral selector and countercurrent separation mechanism in EKC enantioseparation. (a) The concentration dependences at 0.5, 2.5 and 5.0 mg/mL concentrations of CE--CD (●) and native -CD (○) were obtained at pH 4.5 (20 mM -aminocaproic acid - acetic acid BGE); (b) the pH dependences were obtained at 5 mg/mL concentrations of the CDs and the glycine- or aminocaproic acid – acetic acid BGEs with pH 3.2-3.8 or 4.5, respectively. 0.2% (w/v) methyl-hydroxyethylcellulose served as an EOF suppressor in BGE. The driving current was stabilized at 100-120 A. CE--CD = carboxyethyl--cyclodextrin. Reprinted from ref.

Affinity interactions (proteinic biopolymers, macrocyclic antibiotics),

Micelle / microemulsion solubilization (micelles, micelle polymers, oils),

Polymeric complexation (saccharidic biopolymers),

forming point of view in CE enantioseparations.

Table 2.1 and Table 3.1.

[Mikuš et al., 2005a].

Ion-pairing (ionic compounds in non-aqueous media).

*Non-aqueous media*. The elimination of the aqueous media in non-aqueous CE (NACE) can provide additional selectivity with respect to that obtained in aqueous CE, and favours the analysis of solutes with poor water solubility [Karbaum & Jira, 1999; Valkó et al., 1996; Wang F. & Khaledi, 1996]. In the same manner, non-aqueous solvents show several advantages regarding solubility of chiral selectors and reduce unwanted interactions with the capillary wall. Different forms of chemical equilibria in aqueous and non-aqueous systems can lead to different selectivities as a result of the fact that weak interactions which are disrupted by water can become effective in non-aqueous systems (see e.g., ion-pair formation, 2.3.7). Moreover, in non-aqueous solvents, less Joule heating is produced and

Advanced Chiral Separation 17

Figure 2.5. Influence of complementary complexing agents on the CE separation of a mixture of DNP-amino acid racemates. Electrolyte system with pH 5.2 and 20 mM 6I-deoxy-6I-monomethylamino--CD (as in Table 2.2) without any other coselector (a), with addition of 2 mM -CD (b). Peak labelling: 3 = DNP-DL-glutamic acid, 10 = DNP-DLmethioninesulfone, 11 = DNP-DL-methionine sulfoxide, 13 = DNP-DL--amino-n-butyric acid, 15 = DNP-DL-norvaline, 16 = DNP-DL-citruline, 20 = DNP-DL-methionine, 22 = DNP-DL-norleucine, 25 = DNP-DL-ethionine, 30 = DNP-DL--amino-caprylic acid. Reproduced

since higher voltage can be applied, retention times are shorter.

from [Mikuš et al., 2001].

*Aqueous media.* In CE, the improved separation enantioselectivity of charged solutes can be observed, in many cases, with oppositely charged chiral selectors compared to neutral ones (**Figure 2.3**). Higher stability of the formed complexes is one of the factors responsible for this enhanced enantioselectivity, as it was demonstrated by CE [Vespalec & Boček, 2000; Wenz et al., 2008] as well as nuclear magnetic resonance (NMR) measurements [Kitae et al., 1998], and as it is described in terms of complex formation mechanisms with particular chiral selectors in section 2.3. In aqueous media the complexing ability of ionizable compounds can be tuned by the pH of the buffer (changing the size of the effective charge) in this way creating optimal CE separating conditions (**Figure 2.3b**). Due to an enhanced enantioresolution power of such systems, very low amounts of charged chiral selectors are often sufficient for the successful CE enantioseparations (**Figure 2.3a**), and in some cases even micromolar concentrations are sufficient [Gübitz & Schmid, 2000a; Blanco & Valverde, 2003]. cases

Figure 2.4. Schematic representation of MEEKC separation. MEKC separation has principally the same experimental arrangement but no oil droplets are present in micelle cores. Hydrophobic analytes are distributed preferably into droplet (MEEKC) or micelle core (MEKC). Reproduced from [Altria K.D. et al., 2003].

*Amphiphilic media*. CE is usually carried out in aqueous background electrolytes (BGEs) and therefore it is useful for the separation of hydrophilic solutes and samples of aqueous nature. On the other hand, the formation of stable complexes (associates) of hydrophobic analytes (that are many of natural biologically active compounds) can be accomplished in aqueous solutions using amphiphilic pseudostationary phases with proper hydrophobic bounding sites. Thus, typically, chiral micelles or chiral mixed micelles (in MEKC) and microemulsions (in MEEKC) help solving additional problems in chiral CE, such as enantioseparation of hydrophobic analytes in aqueous buffers [Preinerstorfer et al., 2009; Silva, 2009; Ryan et al., 2009; Kahle & Foley, 2007a], see **Figure 2.4**. In this field, chiral micelle polymers appeared recently as a very attractive alternative to the conventional micelle systems offering significant benefits not only in separation (fast complexing kinetics), but also detection (especially MS) schemes (see 2.3.8). Such amphiphilic systems are beneficial for the analyses of water-based samples, such as body fluids, creating a powerful alternative to HPLC-MS.

*Aqueous media.* In CE, the improved separation enantioselectivity of charged solutes can be observed, in many cases, with oppositely charged chiral selectors compared to neutral ones (**Figure 2.3**). Higher stability of the formed complexes is one of the factors responsible for this enhanced enantioselectivity, as it was demonstrated by CE [Vespalec & Boček, 2000; Wenz et al., 2008] as well as nuclear magnetic resonance (NMR) measurements [Kitae et al., 1998], and as it is described in terms of complex formation mechanisms with particular chiral selectors in section 2.3. In aqueous media the complexing ability of ionizable compounds can be tuned by the pH of the buffer (changing the size of the effective charge) in this way creating optimal CE separating conditions (**Figure 2.3b**). Due to an enhanced enantioresolution power of such systems, very low amounts of charged chiral selectors are often sufficient for the successful CE enantioseparations (**Figure 2.3a**), and in some cases even micromolar concentrations are sufficient [Gübitz & Schmid, 2000a; Blanco & Valverde,

formed

Figure 2.4. Schematic representation of MEEKC separation. MEKC separation has principally the same experimental arrangement but no oil droplets are present in micelle cores. Hydrophobic analytes are distributed preferably into droplet (MEEKC) or micelle

*Amphiphilic media*. CE is usually carried out in aqueous background electrolytes (BGEs) and therefore it is useful for the separation of hydrophilic solutes and samples of aqueous nature. On the other hand, the formation of stable complexes (associates) of hydrophobic analytes (that are many of natural biologically active compounds) can be accomplished in aqueous solutions using amphiphilic pseudostationary phases with proper hydrophobic bounding sites. Thus, typically, chiral micelles or chiral mixed micelles (in MEKC) and microemulsions (in MEEKC) help solving additional problems in chiral CE, such as enantioseparation of hydrophobic analytes in aqueous buffers [Preinerstorfer et al., 2009; Silva, 2009; Ryan et al., 2009; Kahle & Foley, 2007a], see **Figure 2.4**. In this field, chiral micelle polymers appeared recently as a very attractive alternative to the conventional micelle systems offering significant benefits not only in separation (fast complexing kinetics), but also detection (especially MS) schemes (see 2.3.8). Such amphiphilic systems are beneficial for the analyses of water-based samples, such as body fluids, creating a

core (MEKC). Reproduced from [Altria K.D. et al., 2003].

powerful alternative to HPLC-MS.

2003].

*Non-aqueous media*. The elimination of the aqueous media in non-aqueous CE (NACE) can provide additional selectivity with respect to that obtained in aqueous CE, and favours the analysis of solutes with poor water solubility [Karbaum & Jira, 1999; Valkó et al., 1996; Wang F. & Khaledi, 1996]. In the same manner, non-aqueous solvents show several advantages regarding solubility of chiral selectors and reduce unwanted interactions with the capillary wall. Different forms of chemical equilibria in aqueous and non-aqueous systems can lead to different selectivities as a result of the fact that weak interactions which are disrupted by water can become effective in non-aqueous systems (see e.g., ion-pair formation, 2.3.7). Moreover, in non-aqueous solvents, less Joule heating is produced and since higher voltage can be applied, retention times are shorter.

Figure 2.5. Influence of complementary complexing agents on the CE separation of a mixture of DNP-amino acid racemates. Electrolyte system with pH 5.2 and 20 mM 6I-deoxy-6I-monomethylamino--CD (as in Table 2.2) without any other coselector (a), with addition of 2 mM -CD (b). Peak labelling: 3 = DNP-DL-glutamic acid, 10 = DNP-DLmethioninesulfone, 11 = DNP-DL-methionine sulfoxide, 13 = DNP-DL--amino-n-butyric acid, 15 = DNP-DL-norvaline, 16 = DNP-DL-citruline, 20 = DNP-DL-methionine, 22 = DNP-DL-norleucine, 25 = DNP-DL-ethionine, 30 = DNP-DL--amino-caprylic acid. Reproduced from [Mikuš et al., 2001]. 

Figure 2.6. Schematic of the separation principle of CDMEKC showing multiple complexing equilibria. The detector window is assumed to be positioned near the negative electrode. Reproduced from [Terabe, 1992]. negative

Figure 2.7. Representation of the electrophoretic mobilities of the analytes, chiral selector and mixed micelles in (A) buffer containing vancomycin (relative migration times: tvancomycin<teof<tacid) and (B) buffer containing vancomycin and SDS (relative migration times: teof<tacid<tvancomycin<tSDS). (C) Shows the equilibria of acid analytes (between the bulk solution and the free vancomycin or mixed micelle). Reproduced from ref. [Rundlett & Armstrong,

Advanced Chiral Separation 19

In chromatographic techniques the selectivity of enantioseparations is entirely defined by the chiral recognition, i.e., by the difference between the affinities of the enantiomers towards the chiral selector. Therefore, the selectivity of enantioseparations in common chromatographic techniques may, in the best case, approach the thermodynamic selectivity of the chiral recognition, but will never exceed it. One major consequence of the mobility contribution in separations in CE is that the apparent separation selectivity may exceed the thermodynamic selectivity of the recognition [Chankvetadze, 2007]. This belongs among unique features of electromigration methods, being not present in chromatographic methods, which can be advantageously utilized in enantiomeric separations. See the reviews on fundamental aspects of chiral electromigration techniques discussing the general aspects of migration models and the enantiomer migration order [Scriba, 2003; Chankvetadze, 1997; Chankvetadze & Blaschke, 2001]. A high enantioresolution power of CE, given by an extremely high separation (peak) efficiency, can be therefore further enhanced by electromigration effects based on increasing mobility difference between free and complexed forms of the enantiomer, as proposed by Wren and Rowe [Wren & Rowe, 1992, 1993], see Equation 2.2 in section 2.2.1. A contribution of intrinsic mobility of the chiral

**2.2.1.2 Electromigration effects in enantioseparations and their manipulation** 

1995].

*Combinations of selectors*. The possibility of various chiral selectors being easily combined with one another, as well as with achiral additive(s) (introducing multiple complexing equilibria), increases the chance of successfully separating not only particular enantiomeric pairs (via enhanced chiral recognition), but also multicomponent mixtures of enantiomeric pairs (via enhanced molecular recognition) [Mikuš & Kaniansky, 2007; Mikuš et al., 2001; Carlavilla et al., 2006], see Figure 2.5. MEKC systems based on mixed micelles (micelle plus another selector, e.g., CD), introduced by Terabe et al. [Nishi, H. et al., 1991], can provide new and interesting possibilities in (enantio)recognition in comparison with single type micelle systems. For the scheme of the separation principle of CDMEKC showing multiple complexing equilibria see Figure 2.6. Rundlett and Armstrong [Rundlett & Armstrong, 1995] proposed another chiral system based on mixed micelles with vancomycin where the authors illustrated the presence of the mixed micelle as a qualitatively new chiral selector (Figure 2.7). As a special case, dual selector systems can be presented, composed from two different chiral selectors being inactive in enantiorecognition when used alone, acting via synergistic effect and providing unique enantioseparation possibilities in CE [Gübitz & Schmid, 2008; Lurie, 1997; Fillet et al., 2000].

The possibility of combining different chiral systems in on-line coupled CE techniques (i.e., different chiral selectors in different CE techniques) can be utilized for a further significant enhancing of enantioresolution in comparison to single column application [Fanali et al., 2000].

Figure 2.6. Schematic of the separation principle of CDMEKC showing multiple complexing equilibria. The detector window is assumed to be positioned near the negative electrode.

*Combinations of selectors*. The possibility of various chiral selectors being easily combined with one another, as well as with achiral additive(s) (introducing multiple complexing equilibria), increases the chance of successfully separating not only particular enantiomeric pairs (via enhanced chiral recognition), but also multicomponent mixtures of enantiomeric pairs (via enhanced molecular recognition) [Mikuš & Kaniansky, 2007; Mikuš et al., 2001; Carlavilla et al., 2006], see Figure 2.5. MEKC systems based on mixed micelles (micelle plus another selector, e.g., CD), introduced by Terabe et al. [Nishi, H. et al., 1991], can provide new and interesting possibilities in (enantio)recognition in comparison with single type micelle systems. For the scheme of the separation principle of CDMEKC showing multiple complexing equilibria see Figure 2.6. Rundlett and Armstrong [Rundlett & Armstrong, 1995] proposed another chiral system based on mixed micelles with vancomycin where the authors illustrated the presence of the mixed micelle as a qualitatively new chiral selector (Figure 2.7). As a special case, dual selector systems can be presented, composed from two different chiral selectors being inactive in enantiorecognition when used alone, acting via synergistic effect and providing unique enantioseparation possibilities in CE [Gübitz &

The possibility of combining different chiral systems in on-line coupled CE techniques (i.e., different chiral selectors in different CE techniques) can be utilized for a further significant enhancing of enantioresolution in comparison to single column application [Fanali et al.,

Reproduced from [Terabe, 1992].

Schmid, 2008; Lurie, 1997; Fillet et al., 2000].

2000].

Figure 2.7. Representation of the electrophoretic mobilities of the analytes, chiral selector and mixed micelles in (A) buffer containing vancomycin (relative migration times: tvancomycin<teof<tacid) and (B) buffer containing vancomycin and SDS (relative migration times: teof<tacid<tvancomycin<tSDS). (C) Shows the equilibria of acid analytes (between the bulk solution and the free vancomycin or mixed micelle). Reproduced from ref. [Rundlett & Armstrong, 1995].

### **2.2.1.2 Electromigration effects in enantioseparations and their manipulation**

In chromatographic techniques the selectivity of enantioseparations is entirely defined by the chiral recognition, i.e., by the difference between the affinities of the enantiomers towards the chiral selector. Therefore, the selectivity of enantioseparations in common chromatographic techniques may, in the best case, approach the thermodynamic selectivity of the chiral recognition, but will never exceed it. One major consequence of the mobility contribution in separations in CE is that the apparent separation selectivity may exceed the thermodynamic selectivity of the recognition [Chankvetadze, 2007]. This belongs among unique features of electromigration methods, being not present in chromatographic methods, which can be advantageously utilized in enantiomeric separations. See the reviews on fundamental aspects of chiral electromigration techniques discussing the general aspects of migration models and the enantiomer migration order [Scriba, 2003; Chankvetadze, 1997; Chankvetadze & Blaschke, 2001]. A high enantioresolution power of CE, given by an extremely high separation (peak) efficiency, can be therefore further enhanced by electromigration effects based on increasing mobility difference between free and complexed forms of the enantiomer, as proposed by Wren and Rowe [Wren & Rowe, 1992, 1993], see Equation 2.2 in section 2.2.1. A contribution of intrinsic mobility of the chiral exceed

mobility of ionizable chiral selectors can be effectively tuned by the pH of the buffer creating very efficient chiral countercurrent migration CE systems, see the results from the relevant CE measurements in Figure 2.3b and Table 2.2. Enhanced effectivity of such systems is reflected in considerably decreased amounts of charged chiral selectors necessary for the successful CE enantioseparations (Figure 2.3a). Thanks to the many new charged chiral selectors (especially CDs and micelles) the possibilities to create new, effective countercurrent separation systems increase, see examples in section 2.4, Table 2.1 and Table 3.1. Besides enhanced enantioresolution, this migration mode is extremely useful also for hyphenated detection systems, eliminating detection interferences of chiral selectors due to

Advanced Chiral Separation 21

Figure 2.10. Scheme of a carrier molecule-based CE system. A = analyte, S = selector.

*Carrier molecule-based migration systems*. In addition, charged chiral selectors (or charged chiral pseudostationary phases) spread the application range of CE separating uncharged enantiomers according to their distribution between moving selector and solution phase, making CE (e.g., MEKC, CD-EKC) a universal separation technique like HPLC [Mikuš et al., 2005b; Zandkarimi et al., 2009], see Figure 2.8b and Figure 2.10 and examples in section 2.4.

*EOF supported migration systems.* Great variability of direct, countercurrent and carrier migration modes in CE, producing electrophoretic systems of different separation selectivities, is given not only by possible combinations of one or more charged and uncharged, as well as chiral and achiral additives, but also by the EOF modifying velocity and direction of movement of the species (analytes, selectors) present in the separation system, and by combinations of both electrophoretic and electroosmotic migration effects (Figures 2.4, 2.6, 2.7). These effects can also be utilized, besides enhancing enantioresolution and/or speeding

The benefits of the advanced migration modes can be pronounced not only in chiral resolution, but they can also simultaneously take effect in achiral resolution [Mikuš et al., 2001, 2006a; Marák et al., 2007; Mikuš & Kaniansky, 2007]. In biomedical analyses, they can be useful in simultaneous separation of structurally related analytes, e.g., chiral drugs and their metabolites, chiral drugs in multicomponent matrices, etc., see examples in section 2.4, Table 2.1 and Table 3.1. In this way, enhanced achiral resolution can also minimize requirements on sample preparation (purification) isolating the enantiomers of the interest

analysis, for the manipulation of the enantiomer migration order [Scriba, 2003].

from the matrix constituents during electrophoretic run [Mikuš et al., 2006a].

their migration from the detector site (see chapter 4).

selector to changes of effective mobility of the charged as well as electroneutral compounds in CE is illustrated in simplified form in Figure 2.8. Moreover, the EOF mobility can additionally influence overall mobility of analytes according to the principles of additivity of particular mobility terms. From these facts the enormous potential of CE to manipulate the separability is apparent, including chiral compounds.

Figure 2.8. Influencing of the effective mobility of: (a) ionic enantiomers R, S, (b) neutral enantiomers R, S, by a charged chiral selector C+. Inside the diagrams, the arrows indicate mobility contributions while the cut-outs indicate complex stability contributions. As some of the possible examples, diagrams (a) illustrate the main role of mobility differences between complexes while diagrams (b) illustrate the main role of complex stability differences between complexes for obtaining differences in effective mobilities of R and S enantiomers, R,ef, S,ef.

Figure 2.9. Scheme of a countercurrent migration CE system. A = analyte, S = selector.

*Countercurrent migration systems*. The electromigration effects enhancing enantioresolution can be implemented into CE via countercurrent migration of charged chiral selector and oppositely charged analyte enantiomers, see Figure 2.8a and Figure 2.9. Electrophoretic

selector to changes of effective mobility of the charged as well as electroneutral compounds in CE is illustrated in simplified form in Figure 2.8. Moreover, the EOF mobility can additionally influence overall mobility of analytes according to the principles of additivity of particular mobility terms. From these facts the enormous potential of CE to manipulate

Figure 2.8. Influencing of the effective mobility of: (a) ionic enantiomers R, S, (b) neutral enantiomers R, S, by a charged chiral selector C+. Inside the diagrams, the arrows indicate mobility contributions while the cut-outs indicate complex stability contributions. As some of the possible examples, diagrams (a) illustrate the main role of mobility differences between complexes while diagrams (b) illustrate the main role of complex stability differences between complexes for obtaining differences in effective mobilities of R and

Figure 2.9. Scheme of a countercurrent migration CE system. A = analyte, S = selector.

*Countercurrent migration systems*. The electromigration effects enhancing enantioresolution can be implemented into CE via countercurrent migration of charged chiral selector and oppositely charged analyte enantiomers, see Figure 2.8a and Figure 2.9. Electrophoretic

the separability is apparent, including chiral compounds.

S enantiomers, R,ef, S,ef.

mobility of ionizable chiral selectors can be effectively tuned by the pH of the buffer creating very efficient chiral countercurrent migration CE systems, see the results from the relevant CE measurements in Figure 2.3b and Table 2.2. Enhanced effectivity of such systems is reflected in considerably decreased amounts of charged chiral selectors necessary for the successful CE enantioseparations (Figure 2.3a). Thanks to the many new charged chiral selectors (especially CDs and micelles) the possibilities to create new, effective countercurrent separation systems increase, see examples in section 2.4, Table 2.1 and Table 3.1. Besides enhanced enantioresolution, this migration mode is extremely useful also for hyphenated detection systems, eliminating detection interferences of chiral selectors due to their migration from the detector site (see chapter 4). 

Figure 2.10. Scheme of a carrier molecule-based CE system. A = analyte, S = selector.

*Carrier molecule-based migration systems*. In addition, charged chiral selectors (or charged chiral pseudostationary phases) spread the application range of CE separating uncharged enantiomers according to their distribution between moving selector and solution phase, making CE (e.g., MEKC, CD-EKC) a universal separation technique like HPLC [Mikuš et al., 2005b; Zandkarimi et al., 2009], see Figure 2.8b and Figure 2.10 and examples in section 2.4.

*EOF supported migration systems.* Great variability of direct, countercurrent and carrier migration modes in CE, producing electrophoretic systems of different separation selectivities, is given not only by possible combinations of one or more charged and uncharged, as well as chiral and achiral additives, but also by the EOF modifying velocity and direction of movement of the species (analytes, selectors) present in the separation system, and by combinations of both electrophoretic and electroosmotic migration effects (Figures 2.4, 2.6, 2.7). These effects can also be utilized, besides enhancing enantioresolution and/or speeding analysis, for the manipulation of the enantiomer migration order [Scriba, 2003].

The benefits of the advanced migration modes can be pronounced not only in chiral resolution, but they can also simultaneously take effect in achiral resolution [Mikuš et al., 2001, 2006a; Marák et al., 2007; Mikuš & Kaniansky, 2007]. In biomedical analyses, they can be useful in simultaneous separation of structurally related analytes, e.g., chiral drugs and their metabolites, chiral drugs in multicomponent matrices, etc., see examples in section 2.4, Table 2.1 and Table 3.1. In this way, enhanced achiral resolution can also minimize requirements on sample preparation (purification) isolating the enantiomers of the interest from the matrix constituents during electrophoretic run [Mikuš et al., 2006a].

where *E* is the electric field strength, *D* is the average effective diffusion coefficient of two analytes (*1*, *2*), and *t* is the electrophoretic migration time. The advantages of the flow counterbalancing technique include the following: (i) enormous, in principle unlimited, enhancement of the apparent separation factor may be achieved in zonal discontinuous separations as shown in section 2.4. (ii) This technique allows easy discontinuous zonal separation of a binary mixture into a continuous separation with stepwise migration of the sample components from the inlet towards the outlet vial. (iii) FCCE may be used for micropreparative purposes and offers significantly higher sample capacity compared to discontinuous separations. Other potential advantages of mobility counterbalancing

Advanced Chiral Separation 23

The mobility counterbalancing technique is certainly not limited to binary mixtures and it can easily be applied in a stepwise mode for the separation of multicomponent samples. Counterbalancing of analyte electrophoretic mobility by pressure has been tried by Culbertson and Jorgenson [Culbertson & Jorgenson, 1994] for the enhancement of the detection sensitivity in achiral CE. Later, the same technique was used for the separation of isotopomers of phenylalanine [Culbertson & Jorgenson, 1999]. Several FCCE modes have been developed and applied for enantioseparations so far, see examples in section 2.4.

Capillary electrochromatography (CEC) combines electrophoretic and chromatographic separation mechanisms that can be beneficial in highly effective enantioresolutions, for the recent advances in this field see the review by Lu and Chen [Lu H.A. & Chen G.N., 2011]. For the CEC separation principle see Figure 2.12. Chiral stationary phases (CSPs) known from HPLC may be used in CEC. CSPs are based on immobilization of chiral selector onto/into appropriate polymeric matrix (e.g., polysiloxanes, modified silica structures, methacrylates). CDs, proteins, polysaccharides, macrocyclic antibiotics are the most often used chiral molecules for the chiral stationary phases, offering in this rigid state modified enantioselectivity in comparison with their free (mobile) forms. In addition to those, new CSPs, such as ionic liquids functionalized -cyclodextrin-and carbosilane dendrimer-bonded chiral stationary phases, are synthesized for producing modified enantioselectivity, [see e.g., Zhou Z. et al., 2010; Shou et al., 2008]. Moreover, the polymeric structure of these stationary phases can additionally influence CEC enantioresolution. An overview of previous developments in chiral CEC is given in former review articles [Gübitz & Schmid, 2000b;

techniques are discussed in ref. [Chankvetadze et al., 1999].

2008; Lämmerhofer et al., 2000; Fanali et al., 2001; Kang et al., 2002].

**2.2.2 Capillary electrochromatography** 

### **2.2.1.3 Counter-flow in enantioseparations and its manipulation**

Another promising mode of chiral CE separations is flow counterbalanced capillary electrophoresis (FCCE). The difference between countercurrent [Chankvetadze et al., 1994] and flow counterbalancing CE [Chankvetadze et al., 1999] techniques is that in the latter case a chiral selector and a chiral analyte do not migrate in the opposite directions to each other, but the bulk flow moves with a defined velocity in the opposite direction to the effective mobility of the analyte zone. The principle of this technique is schematically shown in Figure 2.11 [Chankvetadze et al., 1999].

Figure 2.11. A schematic representation of the flow counterbalanced separation principle in CE: (a) without counterbalanced flow; (b) with counterbalanced flow; (c) resulting mobilities. Reproduced from ref. [Chankvetadze et al., 1999].

In FCCE, the sample is driven forward by electromigration and then backward by a pressure-induced flow. The pressure/vacuum, the EOF, hydrodynamic pressure (levelling of the inlet and outlet vials), etc., may be used as a driving force for countermobilities in this technique [Chankvetadze et al., 1999]. The samples travel back and forth in the capillary until sufficient separation is obtained [Zhao J. et al., 1999]. In the mode of FCCE as proposed by Culbertson and Jorgenson [Culbertson & Jorgenson, 1994], the electric field and the pressure are applied alternatively, but not simultaneously as the driving forces. In another mode of FCCE, the counterbalancing driving force, such as pressure, may be applied to the separation chamber continuously during the entire time of electrokinetic separation [Chankvetadze et al., 1999]. An enormous increase of apparent separation factor in chiral and achiral CE separations may be achieved using this technique. <sup>2</sup>of CSPs

The potential advantage of FCCE can be seen from the Equation 2.3 [Zhao J. et al., 1999]:

$$R\_S = (\mu\_1 - \mu\_2) \frac{E \sqrt{t}}{4 \sqrt{2D}}\tag{2.3}$$

Another promising mode of chiral CE separations is flow counterbalanced capillary electrophoresis (FCCE). The difference between countercurrent [Chankvetadze et al., 1994] and flow counterbalancing CE [Chankvetadze et al., 1999] techniques is that in the latter case a chiral selector and a chiral analyte do not migrate in the opposite directions to each other, but the bulk flow moves with a defined velocity in the opposite direction to the effective mobility of the analyte zone. The principle of this technique is schematically shown

Figure 2.11. A schematic representation of the flow counterbalanced separation principle in CE: (a) without counterbalanced flow; (b) with counterbalanced flow; (c) resulting

In FCCE, the sample is driven forward by electromigration and then backward by a pressure-induced flow. The pressure/vacuum, the EOF, hydrodynamic pressure (levelling of the inlet and outlet vials), etc., may be used as a driving force for countermobilities in this technique [Chankvetadze et al., 1999]. The samples travel back and forth in the capillary until sufficient separation is obtained [Zhao J. et al., 1999]. In the mode of FCCE as proposed by Culbertson and Jorgenson [Culbertson & Jorgenson, 1994], the electric field and the pressure are applied alternatively, but not simultaneously as the driving forces. In another mode of FCCE, the counterbalancing driving force, such as pressure, may be applied to the separation chamber continuously during the entire time of electrokinetic separation [Chankvetadze et al., 1999]. An enormous increase of apparent separation factor in chiral

The potential advantage of FCCE can be seen from the Equation 2.3 [Zhao J. et al., 1999]:

 *<sup>D</sup> tE RS* <sup>24</sup> 

<sup>42</sup>

1 2 *Et*

<sup>21</sup> 2.3

mobilities. Reproduced from ref. [Chankvetadze et al., 1999].

and achiral CE separations may be achieved using this technique.

**2.2.1.3 Counter-flow in enantioseparations and its manipulation** 

in Figure 2.11 [Chankvetadze et al., 1999].

where *E* is the electric field strength, *D* is the average effective diffusion coefficient of two analytes (*1*, *2*), and *t* is the electrophoretic migration time. The advantages of the flow counterbalancing technique include the following: (i) enormous, in principle unlimited, enhancement of the apparent separation factor may be achieved in zonal discontinuous separations as shown in section 2.4. (ii) This technique allows easy discontinuous zonal separation of a binary mixture into a continuous separation with stepwise migration of the sample components from the inlet towards the outlet vial. (iii) FCCE may be used for micropreparative purposes and offers significantly higher sample capacity compared to discontinuous separations. Other potential advantages of mobility counterbalancing techniques are discussed in ref. [Chankvetadze et al., 1999].

The mobility counterbalancing technique is certainly not limited to binary mixtures and it can easily be applied in a stepwise mode for the separation of multicomponent samples. Counterbalancing of analyte electrophoretic mobility by pressure has been tried by Culbertson and Jorgenson [Culbertson & Jorgenson, 1994] for the enhancement of the detection sensitivity in achiral CE. Later, the same technique was used for the separation of isotopomers of phenylalanine [Culbertson & Jorgenson, 1999]. Several FCCE modes have been developed and applied for enantioseparations so far, see examples in section 2.4.

### **2.2.2 Capillary electrochromatography**

Capillary electrochromatography (CEC) combines electrophoretic and chromatographic separation mechanisms that can be beneficial in highly effective enantioresolutions, for the recent advances in this field see the review by Lu and Chen [Lu H.A. & Chen G.N., 2011]. For the CEC separation principle see Figure 2.12. Chiral stationary phases (CSPs) known from HPLC may be used in CEC. CSPs are based on immobilization of chiral selector onto/into appropriate polymeric matrix (e.g., polysiloxanes, modified silica structures, methacrylates). CDs, proteins, polysaccharides, macrocyclic antibiotics are the most often used chiral molecules for the chiral stationary phases, offering in this rigid state modified enantioselectivity in comparison with their free (mobile) forms. In addition to those, new CSPs, such as ionic liquids functionalized -cyclodextrin-and carbosilane dendrimer-bonded chiral stationary phases, are synthesized for producing modified enantioselectivity, [see e.g., Zhou Z. et al., 2010; Shou et al., 2008]. Moreover, the polymeric structure of these stationary phases can additionally influence CEC enantioresolution. An overview of previous developments in chiral CEC is given in former review articles [Gübitz & Schmid, 2000b; 2008; Lämmerhofer et al., 2000; Fanali et al., 2001; Kang et al., 2002].

Figure 2.12. Separation principle of capillary electrochromatography.

Chiral CEC stationary phases included in capillary wall coatings, particle packings or monolytes (as an example see Figure 2.13) are beneficial in situations with special requirements on separation buffer (aqueous, non-aqueous), stability and solubility of compounds (analytes, selectors, additives, etc.) in separation system, and some on-line detection modes (e.g., avoiding a contamination of detector by selector, often in the case of UV absorbance and mass spectrometry) [Huo Y. & Kok, 2008]. Depending on a chiral selector embedded into the monolith, the resulting chiral monolith can provide significant differences in the chiral selectivity, as illustrated in Figure 2.14. Imprinted chiral phases can offer an enhanced specificity of chiral CEC analyses [Nilsson et al., 2004; Turiel & Martin-Esteban, 2004].

Figure 2.14. Separation of D,L-phenylalanine by four chiral monolithic CSPs. (A) Fabricated with NH2--CD; (B) fabricated with -CD; (C) fabricated with Asp--CD; (D) fabricated with HP--CD. Temperature: 20°C; voltage: −10 kV; injection:−2 kV, 2 s; mobile phase:

Advanced Chiral Separation 25

Compared to HPLC, where a conical flow profile caused by hydrodynamic flow leads to band broadening, in CEC a rather plug-like profile generated by the EOF (see Figure 2.12) results in higher peak efficiency. However, there are also several disadvantages in CEC such as the complicated packing procedures, formation of air bubbles in the case of packed capillaries due to Joule heating, lower reproducibility of migration times due to fluctuation of EOF with different packings and sample matrices [Gübitz & Schmid, 2000, 2008; Lämmerhofer et al., 2000; Fanali et al., 2001; Kang et al., 2002]. Pretreatment of samples with complex matrices is necessary before CEC analysis to maintain separation reproducibility (cleaning of CEC columns is much more difficult and less efficient than CE columns), see

Microfluidic devices, such as microchips (Figure 2.15), can provide several additional advantages over electromigration techniques performed in capillary format [Li O.L. et al., 2008]. The heat dissipation is much better in chip format compared with that in a capillary and therefore, higher electric fields can be applied across microchip channels. This fact enables, along with a considerably reduced length of channels, significant shortening of separation time, see an example in Figure 2.16. Sample and reagent consumption is markedly reduced in microchannels, hence, the chiral MCE can provide the unique possibility of ultraspeed enantiomeric separations of microscale sample amounts. Both electrophoretic [Gong & Hauser, 2006; Piehl et al., 2004; Belder et al., 2006; Belder, 2006] and

phosphate, 5mM, pH= 6.5. Reprinted from ref. [Li Y. et al., 2010].

electrochromatographic modes are applicable [Weng X. et al., 2006].

examples in section 2.4 and Table 2.1.

**2.2.3 Microchip capillary electrophoresis** 

Figure 2.13. Surface-structure of enantioselective silica-based monolithic cation-exchange capillary column with aminophosphonic acid-derived chiral selector. Reproduced from ref. [Preinerstorfer et al., 2006].

Chiral CEC stationary phases included in capillary wall coatings, particle packings or monolytes (as an example see Figure 2.13) are beneficial in situations with special requirements on separation buffer (aqueous, non-aqueous), stability and solubility of compounds (analytes, selectors, additives, etc.) in separation system, and some on-line detection modes (e.g., avoiding a contamination of detector by selector, often in the case of UV absorbance and mass spectrometry) [Huo Y. & Kok, 2008]. Depending on a chiral selector embedded into the monolith, the resulting chiral monolith can provide significant differences in the chiral selectivity, as illustrated in Figure 2.14. Imprinted chiral phases can offer an enhanced specificity of chiral CEC analyses [Nilsson et al., 2004; Turiel & Martin-

Figure 2.13. Surface-structure of enantioselective silica-based monolithic cation-exchange capillary column with aminophosphonic acid-derived chiral selector. Reproduced from ref.

Figure 2.12. Separation principle of capillary electrochromatography.

Esteban, 2004].

[Preinerstorfer et al., 2006].

Figure 2.14. Separation of D,L-phenylalanine by four chiral monolithic CSPs. (A) Fabricated with NH2--CD; (B) fabricated with -CD; (C) fabricated with Asp--CD; (D) fabricated with HP--CD. Temperature: 20°C; voltage: −10 kV; injection:−2 kV, 2 s; mobile phase: phosphate, 5mM, pH= 6.5. Reprinted from ref. [Li Y. et al., 2010].

Compared to HPLC, where a conical flow profile caused by hydrodynamic flow leads to band broadening, in CEC a rather plug-like profile generated by the EOF (see Figure 2.12) results in higher peak efficiency. However, there are also several disadvantages in CEC such as the complicated packing procedures, formation of air bubbles in the case of packed capillaries due to Joule heating, lower reproducibility of migration times due to fluctuation of EOF with different packings and sample matrices [Gübitz & Schmid, 2000, 2008; Lämmerhofer et al., 2000; Fanali et al., 2001; Kang et al., 2002]. Pretreatment of samples with complex matrices is necessary before CEC analysis to maintain separation reproducibility (cleaning of CEC columns is much more difficult and less efficient than CE columns), see examples in section 2.4 and Table 2.1.

### **2.2.3 Microchip capillary electrophoresis**

Microfluidic devices, such as microchips (Figure 2.15), can provide several additional advantages over electromigration techniques performed in capillary format [Li O.L. et al., 2008]. The heat dissipation is much better in chip format compared with that in a capillary and therefore, higher electric fields can be applied across microchip channels. This fact enables, along with a considerably reduced length of channels, significant shortening of separation time, see an example in Figure 2.16. Sample and reagent consumption is markedly reduced in microchannels, hence, the chiral MCE can provide the unique possibility of ultraspeed enantiomeric separations of microscale sample amounts. Both electrophoretic [Gong & Hauser, 2006; Piehl et al., 2004; Belder et al., 2006; Belder, 2006] and electrochromatographic modes are applicable [Weng X. et al., 2006].

Figure 2.17. Comparison of chiral separations obtained in microchip electrophoresis (MCE) (a) and in classical capillary electrophoresis (CE) (b). For both the experiments the same electrolyte was used, while the column length was limited to 7 cm in MCE, a column of 40

Advanced Chiral Separation 27

In practice, however, the resolution achievable in MCE devices is often lower compared to that obtainable in classical CE utilizing considerably longer separation capillaries. This is shown in Figure 2.17, where the chiral separation of an FITC-labelled amine obtained at typical conditions in MCE and in classical CE is compared. In order to obtain sufficient resolution in chiral MCE, different strategies have been used [Belder, 2006], such as (i) enhancing the enantioselectivity of the system as much as possible (changing the type and amount of chiral selector, adding coselector, etc.), (ii) using folded separation channels, the column length can be extended without enlarging the compact footprint of the device, as shown in Figure 2.18, (iii) using coated channels, internal coatings improve separation performance by the suppression of both analyte wall interaction and electroosmosis; the impact of channel coating with poly(vinyl alcohol) on a chiral separation in MCE is shown in Figure 2.19 for the separation of an FITC-labelled amine. The use/combination of the above-mentioned tools applicable in MCE gives a good chance for real-time process control and for multidimensional separations, and makes MCE a powerful tool in real chiral

Figure 2.18. Channel layouts enabling long separation channels on a small device.

cm effective length was used in CE. Reproduced from ref. [Belder, 2006].

applications (pharmaceutical, biomedical, etc.).

Reproduced from ref. [Belder, 2006].

Figure 2.15. MCE. Experimental arrangement of microchip electrophoresis (left). Arrangement in the left single-channel chip: (1) sample, (2) run buffer, (3) sample waste and (4) buffer waste. The electrophoretic microchip – real detail (right). Arrangement in the right single-channel chip: reservoirs (sample, buffer, waste) and separation channel. For a chiral MCE, separation channel can be filled with chiral electrolyte (CE mode), or chiral/achiral electrolyte with chiral/achiral stationary phase (CEC mode). Reproduced (left) from [Kim M.S. et al., 2005].

Figure 2.16. Subsecond chiral separation of DNS-tryptophan. Electrolyte: 2% HS-CD, 25 mM triethylammonium phosphate buffer pH 2.5. A high field strength up to 2600 V/cm and short separation length of several millimetres were employed. Reproduced from ref. [Belder, 2006].

Figure 2.15. MCE. Experimental arrangement of microchip electrophoresis (left). Arrangement in the left single-channel chip: (1) sample, (2) run buffer, (3) sample waste and (4) buffer waste. The electrophoretic microchip – real detail (right). Arrangement in the right single-channel chip: reservoirs (sample, buffer, waste) and separation channel. For a chiral MCE, separation channel can be filled with chiral electrolyte (CE mode), or chiral/achiral electrolyte with chiral/achiral stationary phase (CEC mode). Reproduced (left) from [Kim

Figure 2.16. Subsecond chiral separation of DNS-tryptophan. Electrolyte: 2% HS-CD, 25 mM triethylammonium phosphate buffer pH 2.5. A high field strength up to 2600 V/cm and short separation length of several millimetres were employed. Reproduced from ref. [Belder,

M.S. et al., 2005].

2006].

Figure 2.17. Comparison of chiral separations obtained in microchip electrophoresis (MCE) (a) and in classical capillary electrophoresis (CE) (b). For both the experiments the same electrolyte was used, while the column length was limited to 7 cm in MCE, a column of 40 cm effective length was used in CE. Reproduced from ref. [Belder, 2006].

In practice, however, the resolution achievable in MCE devices is often lower compared to that obtainable in classical CE utilizing considerably longer separation capillaries. This is shown in Figure 2.17, where the chiral separation of an FITC-labelled amine obtained at typical conditions in MCE and in classical CE is compared. In order to obtain sufficient resolution in chiral MCE, different strategies have been used [Belder, 2006], such as (i) enhancing the enantioselectivity of the system as much as possible (changing the type and amount of chiral selector, adding coselector, etc.), (ii) using folded separation channels, the column length can be extended without enlarging the compact footprint of the device, as shown in Figure 2.18, (iii) using coated channels, internal coatings improve separation performance by the suppression of both analyte wall interaction and electroosmosis; the impact of channel coating with poly(vinyl alcohol) on a chiral separation in MCE is shown in Figure 2.19 for the separation of an FITC-labelled amine. The use/combination of the above-mentioned tools applicable in MCE gives a good chance for real-time process control and for multidimensional separations, and makes MCE a powerful tool in real chiral applications (pharmaceutical, biomedical, etc.).

Figure 2.18. Channel layouts enabling long separation channels on a small device. Reproduced from ref. [Belder, 2006].

discussed below) it is not surprising that CDs are dominating chiral selectors also in enantioselective analysis of many drugs in biological samples, as it is apparent from Table

Advanced Chiral Separation 29

Figure 2.20. Cyclodextrins. (a) Chemical structure of -CD. (b) Space filling model of -CD.

+--CD-(R)-AcLeu complex. The complexes were derived from the molecular mechanics

The basic mechanism of chiral discrimination using CDs is based on the inclusion of the analyte (guest), or at least its hydrophobic part, into the relatively hydrophobic cavity of CD (host), see an example in Figure 2.21. Here different sterical arrangements of chiral compounds in chiral CD cavity results in differences in stability of the formed complexes.

– molecular dynamics (MM-MD) calculations. Reproduced from ref. [Kitae et al., 1998].

+--CD-(S)-AcLeu complex, (b)

(c) -CD toroid structure showing spatial arrangement.

Figure 2.21. Structures of inclusion CD complexes. (a) per-NH3

2.1 and Table 3.1.

per-NH3

Figure 2.19. Influence of PVA-channel coating on chiral resolution of FITC-labelled (R)- (-) and (S)-(+)-1-cyclohexylethylamine in MCE. The effective separation lengths were 7 cm for the uncoated channel (a) and 7 cm (b) and 3.4 cm (c) for the PVA-coated microchip. Buffer: 40 mM CHES, 6.25 mM HP--CD, pH 9.2. Reproduced from ref. [Belder, 2006].

For examples of practical applications of the chiral MCE, see section 2.4 and Table 3.1. A brief comparison of CE and MCE, and clinical applications of MCE are reviewed in ref. [Li, S.F.Y. & Kricka L.J., 2006]. Chiral separations in microfluidic devices are nicely reviewed by Belder [2006].

#### **2.3 Chiral selectors as complexing agents, advantages and limitations**

Conventional as well as new chiral selectors suitable for CE are described in the following subsections. From this description, showing complexing properties (i.e., mechanism of chiral discrimination), advantages and limitations of various classes of chiral selectors, the high flexibility of chiral CE for the separation of a wide range of structurally different compounds is apparent.

### **2.3.1 Cyclodextrins**

Cyclodextrins (CDs), cyclic oligosaccharides with (typically) 6-8 D-glucose units in the macrocycle (-, -, -CDs, see Figure 2.20, although recently -CD was also introduced into CE [Wistuba et al., 2006]), are the most often employed chiral additives for the CE enantiomeric separations of low molecular organic compounds due to their outstanding broad selectivity spectra and other beneficial properties, such as UV transparency, availability, wide application range (polar, nonpolar, charged, uncharged analytes), and reasonable solubility in water [Chankvetadze, 2008; Scriba, 2008; Juvancz et al., 2008; Cserhati, 2008; Fanali, 2009]. Moreover, a fast complex forming kinetics with CDs is beneficial for highly efficient CE enantioseparations. From these reasons (and other facts

Figure 2.19. Influence of PVA-channel coating on chiral resolution of FITC-labelled (R)- (-) and (S)-(+)-1-cyclohexylethylamine in MCE. The effective separation lengths were 7 cm for the uncoated channel (a) and 7 cm (b) and 3.4 cm (c) for the PVA-coated microchip. Buffer:

For examples of practical applications of the chiral MCE, see section 2.4 and Table 3.1. A brief comparison of CE and MCE, and clinical applications of MCE are reviewed in ref. [Li, S.F.Y. & Kricka L.J., 2006]. Chiral separations in microfluidic devices are nicely reviewed by

Conventional as well as new chiral selectors suitable for CE are described in the following subsections. From this description, showing complexing properties (i.e., mechanism of chiral discrimination), advantages and limitations of various classes of chiral selectors, the high flexibility of chiral CE for the separation of a wide range of structurally different compounds

Cyclodextrins (CDs), cyclic oligosaccharides with (typically) 6-8 D-glucose units in the macrocycle (-, -, -CDs, see Figure 2.20, although recently -CD was also introduced into CE [Wistuba et al., 2006]), are the most often employed chiral additives for the CE enantiomeric separations of low molecular organic compounds due to their outstanding broad selectivity spectra and other beneficial properties, such as UV transparency, availability, wide application range (polar, nonpolar, charged, uncharged analytes), and reasonable solubility in water [Chankvetadze, 2008; Scriba, 2008; Juvancz et al., 2008; Cserhati, 2008; Fanali, 2009]. Moreover, a fast complex forming kinetics with CDs is beneficial for highly efficient CE enantioseparations. From these reasons (and other facts

40 mM CHES, 6.25 mM HP--CD, pH 9.2. Reproduced from ref. [Belder, 2006].

**2.3 Chiral selectors as complexing agents, advantages and limitations** 

Belder [2006].

is apparent.

**2.3.1 Cyclodextrins** 

discussed below) it is not surprising that CDs are dominating chiral selectors also in enantioselective analysis of many drugs in biological samples, as it is apparent from Table 2.1 and Table 3.1.

Figure 2.20. Cyclodextrins. (a) Chemical structure of -CD. (b) Space filling model of -CD. (c) -CD toroid structure showing spatial arrangement.

Figure 2.21. Structures of inclusion CD complexes. (a) per-NH3 +--CD-(S)-AcLeu complex, (b) per-NH3 +--CD-(R)-AcLeu complex. The complexes were derived from the molecular mechanics – molecular dynamics (MM-MD) calculations. Reproduced from ref. [Kitae et al., 1998].

The basic mechanism of chiral discrimination using CDs is based on the inclusion of the analyte (guest), or at least its hydrophobic part, into the relatively hydrophobic cavity of CD (host), see an example in Figure 2.21. Here different sterical arrangements of chiral compounds in chiral CD cavity results in differences in stability of the formed complexes.

Neutral CDs (e.g., replacing hydroxyl groups with alkyl or hydroxyalkyl groups) can offer modified depth and flexibility of the cavity, as well as the free cross-section of its smaller opening, leading to better accommodation of the guest and increased stability of the resulting inclusion complex [Vespalec & Boček, 2000; Blanco & Valverde, 2003], as an example see Figure 2.23. This can improve solubility of CDs and their complexes, as well as enantiomeric recognition in comparison with their native forms as illustrated on many biological samples (urine, plasma, rat brain), such as 2-hydroxypropyl--CD vs. lorazepam and its chiral 3O-glucuronides, methyl-O--CD vs. isoproterenol [Hadviger et al., 1996], hydroxypropyl--CD vs. amlodipine [Mikuš et al., 2008a], 2-hydroxypropyl--CD and 2 hydroxypropyl--CD vs. 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) or cyanobenz[*f*]isoindole (CBI) derivatives of serine [Quan et al., 2005; Zhao S. L. et al., 2005a],

Advanced Chiral Separation 31

Figure 2.23. Modification of the structure and character of -CD after its derivatization. (a) native -CD, (b) per-O-methyl--CD. In the MOLCAD (molecular computer-aided design) structures the hydrophilic parts are coloured with blue while the hydrophobic parts are

The improved chiral recognition was observed many times with charged CDs compared to neutral ones, see Figure 2.24 {compare NMR traces (b) and (d)}, as a result of the enhanced stability of host-guest complexes due to the additional strong electrostatic (Coulombic)

coloured with yellow. Reprinted from [csi.chemie.tu-darmstadt.de].

for details see Table 2.1 and Table 3.1.

Compounds containing an aromatic system in their molecules, including many drugs, are usually well-suited for inclusion into the CD cavity and hence, a good enantiorecognition between enantiomers is often easily achieved [Vespalec & Boček, 2000; Mikuš et al., 2002; Thiele et al., 2009; Denmark, 2011; Palcut & Rabara, 2009].

The hydroxyl groups present on the rim of the CD can be easily modified by chemical reactions with various functional groups creating a great amount of derivatized CDs with the desired properties, especially (i) complex forming ability, (ii) solubility, (iii) migration and (iv) detection capabilities. Therefore, it is not surprising that even in the field of newly developed chiral selectors the novel CD derivatives prevail significantly [Preinerstorfer et al., 2009]. The preparation of selectively substituted derivatives [Gübitz & Schmid, 2000a; Nzeadibe & Vigh, 2007; Cucinotta et al., 2010] is preferred as this eliminates the creation of mixtures of CDs having different substitution patterns, and, by that, different complexing / electromigration properties, see Figure 2.22, that can cause difficulties with separation reproducibility in CE [Vespalec & Boček, 1999; Mikuš et al., 1999; Mikuš & Kaniansky, 2007]. Several different groups of CD derivatives can be distinguished, namely (i) neutral CDs, (ii) negatively and positively charged CDs, (iii) amphoteric CDs and (iv) polymerized CDs.

Figure 2.22. Electropherograms showing different electromigration properties of the -CD aminoderivates. The sample constituents were separated according to differences in their actual ionic mobilities. There are clearly visible differences in migration velocities of the CD derivatives with different substitution degree (peaks 2, 3, and diM--CD) in the electropherogram. The concentration of the monoaminoderivative, 6I-deoxy-6Idimethylamino--CD, the major constituent in the analysed preparative, was ca. 1.7 mM. Tentative peak assignments: 1 = a migration region of the alkali and alkaline earth metal cations and alkyl- and arylamines; 2 = triamino--CD derivatives; 3 = diamino--CD derivatives; diM--CD = 6I-deoxy-6I-dimethylamino--CD; 6 = unidentified constituents. Contactless conductivity detection was used in this CE experiment to monitor the nonabsorbing analytes. Reproduced from ref. [Mikuš et al., 1999].

Compounds containing an aromatic system in their molecules, including many drugs, are usually well-suited for inclusion into the CD cavity and hence, a good enantiorecognition between enantiomers is often easily achieved [Vespalec & Boček, 2000; Mikuš et al., 2002;

The hydroxyl groups present on the rim of the CD can be easily modified by chemical reactions with various functional groups creating a great amount of derivatized CDs with the desired properties, especially (i) complex forming ability, (ii) solubility, (iii) migration and (iv) detection capabilities. Therefore, it is not surprising that even in the field of newly developed chiral selectors the novel CD derivatives prevail significantly [Preinerstorfer et al., 2009]. The preparation of selectively substituted derivatives [Gübitz & Schmid, 2000a; Nzeadibe & Vigh, 2007; Cucinotta et al., 2010] is preferred as this eliminates the creation of mixtures of CDs having different substitution patterns, and, by that, different complexing / electromigration properties, see Figure 2.22, that can cause difficulties with separation reproducibility in CE [Vespalec & Boček, 1999; Mikuš et al., 1999; Mikuš & Kaniansky, 2007]. Several different groups of CD derivatives can be distinguished, namely (i) neutral CDs, (ii) negatively and positively charged CDs, (iii) amphoteric CDs and (iv) polymerized CDs.

Figure 2.22. Electropherograms showing different electromigration properties of the -CD aminoderivates. The sample constituents were separated according to differences in their actual ionic mobilities. There are clearly visible differences in migration velocities of the CD derivatives with different substitution degree (peaks 2, 3, and diM--CD) in the electropherogram. The concentration of the monoaminoderivative, 6I-deoxy-6Idimethylamino--CD, the major constituent in the analysed preparative, was ca. 1.7 mM. Tentative peak assignments: 1 = a migration region of the alkali and alkaline earth metal cations and alkyl- and arylamines; 2 = triamino--CD derivatives; 3 = diamino--CD derivatives; diM--CD = 6I-deoxy-6I-dimethylamino--CD; 6 = unidentified constituents. Contactless conductivity detection was used in this CE experiment to monitor the non-

absorbing analytes. Reproduced from ref. [Mikuš et al., 1999].

Thiele et al., 2009; Denmark, 2011; Palcut & Rabara, 2009].

Neutral CDs (e.g., replacing hydroxyl groups with alkyl or hydroxyalkyl groups) can offer modified depth and flexibility of the cavity, as well as the free cross-section of its smaller opening, leading to better accommodation of the guest and increased stability of the resulting inclusion complex [Vespalec & Boček, 2000; Blanco & Valverde, 2003], as an example see Figure 2.23. This can improve solubility of CDs and their complexes, as well as enantiomeric recognition in comparison with their native forms as illustrated on many biological samples (urine, plasma, rat brain), such as 2-hydroxypropyl--CD vs. lorazepam and its chiral 3O-glucuronides, methyl-O--CD vs. isoproterenol [Hadviger et al., 1996], hydroxypropyl--CD vs. amlodipine [Mikuš et al., 2008a], 2-hydroxypropyl--CD and 2 hydroxypropyl--CD vs. 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) or cyanobenz[*f*]isoindole (CBI) derivatives of serine [Quan et al., 2005; Zhao S. L. et al., 2005a], for details see Table 2.1 and Table 3.1.

Figure 2.23. Modification of the structure and character of -CD after its derivatization. (a) native -CD, (b) per-O-methyl--CD. In the MOLCAD (molecular computer-aided design) structures the hydrophilic parts are coloured with blue while the hydrophobic parts are coloured with yellow. Reprinted from [csi.chemie.tu-darmstadt.de].

The improved chiral recognition was observed many times with charged CDs compared to neutral ones, see Figure 2.24 {compare NMR traces (b) and (d)}, as a result of the enhanced stability of host-guest complexes due to the additional strong electrostatic (Coulombic) interactions of oppositely charged functional groups of complexing partners [Vespalec & Boček, 2000; Wenz et al., 2008; Kitae et al., 1998]. For an example of the CD complex stabilized by the Coulombic interaction see Figure 2.25. The complex stability depends on the size of charge that is important especially for the effective use of ionizable CD derivatives in CE (having carboxyl or amino groups in macrocycle), as it was demonstrated (mainly from the electromigration point of view) in section 2.2. Substitution pattern is another tool for a quite significant tuning of the enantiorecognition ability of charged CDs [Mikuš & Kaniansky, 2007; Kitae et al., 1998], as illustrated in Figure 2.24 {compare NMR traces (b) and (c)}. The ionic character of CD derivatives is also responsible for their excellent water solubility. Enhanced enantiomeric recognition as well as solubility of these CDs and their complexes in comparison with their native forms was illustrated on many biological samples (urine, squirrel brain, human plasma), including carboxyethyl--CD vs. H1 antihistaminic drugs (dioxopromethazine, dimethindene, pheniramine) [Mikuš et al., 2006a, 2008b; Marák et al., 2007], a highly sulfated--CD vs. CBI derivatives of serine, glutamate and aspartate [Kirschner et al., 2007], a highly sulfated--CD vs. CBI derivatized baclofen [Kavran-Belin et al., 2005], a highly sulfated--CD vs. a racemic antiarrhythmic drug (disopyramide) [Fang, L. et al., 2006], for details see Table 2.1 and Table 3.1. CD per-NH3

Figure 2.25. Illustration of the enhanced chiral recognition due to Coulomb interaction as an attractive force. (a) ROESY spectrum of the per-NH3+--CD-(S)-AcLeu system in D2O at pD

Advanced Chiral Separation 33

(8x10-3 M) and (S)-AcLeu (4x10-3 M) in N2-saturated D2O. The mixing time for the ROESY measurement was 250 ms. (b) A plausible structure of the per-NH3+--CD-(S)-AcLeu complex deduced from 1H NMR spectroscopy. (c) 1H NMR signals of the CH3 protons at the acetyl group of *N*-acetylated leucine, AcLeu, (2x10-3 M) in the absence (lower) and the presence of protonated heptakis(6-amino-6-deoxy)--cyclodextrin, per-NH3+--CD, (8x10-3 M) (upper) in D2O at pD 6.0 and 25°C. Reproduced from ref. [Kitae et al., 1998].

Chiral crown ethers (CWEs) are cyclic polyethers that form stereoselectively inclusion complexes with primary amines (**Figure 2.26**). The CWE complexation differs from CD complexation by the inclusion of the hydrophilic part of the analyte (e.g., protonated amino

> O O O

O N H H <sup>+</sup> H

O- -O

R

O


O


O

Figure 2.26. Chemical structure of the complex of crown ether with primary amine.

+--CD

+-

6.0 and 25°C. The spectrum was measured for the solution of a mixture of per-NH3

**2.3.2 Crown ethers** 

group) in the cavity [Kuhn, 1999].

O

O

O-

O

Figure 2.24. 1H NMR spectra of (±)-AcTrp (2x10-3 M) in D2O at pD 6.0 in the absence and the presence of native -CD, and selectively substituted -CD derivatives (8x10-3 M). Here, only per-NH3+--CD exhibited enantiorecognition capability for (±)-AcTrp (split NMR signals). Reproduced from ref. [Kitae et al., 1998].

Polymerized CDs are mostly prepared by cross-linking the CDs with bifunctional reagents like diepoxydes and diisocyanates [Fenyvesi, 1988]. Associated analytes have more reduced mobility (close to zero) that can lead, for some analytes, to their better CE enantioseparation in comparison with the use of the native monomeric CDs. Moreover, improved solubility and rigid structure of CD cavites in polymer can additionally influence (enantio)recognition and utilization [Fenyvesi, 1988; Ingelse et al., 1995; Ševčík et al., 1996]. For example, polymeric -CD exhibited excellent molecular recognition for multicomponent mixtures of amino acid enantiomers [Mikuš et al., 2001].

interactions of oppositely charged functional groups of complexing partners [Vespalec & Boček, 2000; Wenz et al., 2008; Kitae et al., 1998]. For an example of the CD complex stabilized by the Coulombic interaction see Figure 2.25. The complex stability depends on the size of charge that is important especially for the effective use of ionizable CD derivatives in CE (having carboxyl or amino groups in macrocycle), as it was demonstrated (mainly from the electromigration point of view) in section 2.2. Substitution pattern is another tool for a quite significant tuning of the enantiorecognition ability of charged CDs [Mikuš & Kaniansky, 2007; Kitae et al., 1998], as illustrated in Figure 2.24 {compare NMR traces (b) and (c)}. The ionic character of CD derivatives is also responsible for their excellent water solubility. Enhanced enantiomeric recognition as well as solubility of these CDs and their complexes in comparison with their native forms was illustrated on many biological samples (urine, squirrel brain, human plasma), including carboxyethyl--CD vs. H1 antihistaminic drugs (dioxopromethazine, dimethindene, pheniramine) [Mikuš et al., 2006a, 2008b; Marák et al., 2007], a highly sulfated--CD vs. CBI derivatives of serine, glutamate and aspartate [Kirschner et al., 2007], a highly sulfated--CD vs. CBI derivatized baclofen [Kavran-Belin et al., 2005], a highly sulfated--CD vs. a racemic antiarrhythmic drug

(disopyramide) [Fang, L. et al., 2006], for details see Table 2.1 and Table 3.1.

Figure 2.24. 1H NMR spectra of (±)-AcTrp (2x10-3 M) in D2O at pD 6.0 in the absence and the presence of native -CD, and selectively substituted -CD derivatives (8x10-3 M). Here, only per-NH3+--CD exhibited enantiorecognition capability for (±)-AcTrp (split NMR signals).

Polymerized CDs are mostly prepared by cross-linking the CDs with bifunctional reagents like diepoxydes and diisocyanates [Fenyvesi, 1988]. Associated analytes have more reduced mobility (close to zero) that can lead, for some analytes, to their better CE enantioseparation in comparison with the use of the native monomeric CDs. Moreover, improved solubility and rigid structure of CD cavites in polymer can additionally influence (enantio)recognition and utilization [Fenyvesi, 1988; Ingelse et al., 1995; Ševčík et al., 1996]. For example, polymeric -CD exhibited excellent molecular recognition for multicomponent mixtures of

Reproduced from ref. [Kitae et al., 1998].

amino acid enantiomers [Mikuš et al., 2001].

Figure 2.25. Illustration of the enhanced chiral recognition due to Coulomb interaction as an attractive force. (a) ROESY spectrum of the per-NH3+--CD-(S)-AcLeu system in D2O at pD 6.0 and 25°C. The spectrum was measured for the solution of a mixture of per-NH3+--CD (8x10-3 M) and (S)-AcLeu (4x10-3 M) in N2-saturated D2O. The mixing time for the ROESY measurement was 250 ms. (b) A plausible structure of the per-NH3+--CD-(S)-AcLeu complex deduced from 1H NMR spectroscopy. (c) 1H NMR signals of the CH3 protons at the acetyl group of *N*-acetylated leucine, AcLeu, (2x10-3 M) in the absence (lower) and the presence of protonated heptakis(6-amino-6-deoxy)--cyclodextrin, per-NH3+--CD, (8x10-3 M) (upper) in D2O at pD 6.0 and 25°C. Reproduced from ref. [Kitae et al., 1998].

### **2.3.2 Crown ethers**

Chiral crown ethers (CWEs) are cyclic polyethers that form stereoselectively inclusion complexes with primary amines (**Figure 2.26**). The CWE complexation differs from CD complexation by the inclusion of the hydrophilic part of the analyte (e.g., protonated amino group) in the cavity [Kuhn, 1999].

Figure 2.26. Chemical structure of the complex of crown ether with primary amine.

**2.3.4 Proteins** 

Tanaka & Terabe, 1995; Armstrong et al., 1994a].

Reprinted from [ks.uiuc.edu].

**2.3.5 Macrocyclic antibiotics** 

problematic selectors such as CDs and their derivatives.

Proteins provide strong and highly selective interactions, so called affinity interactions, with the analytes. Stereoselectivity is further influenced by the tertiary structure of proteins. Different analytes can selectively bind to different binding sites of proteins. Proteins can be used, depending on their ionization state, for neutral, basic or acidic analytes. However, there are several limitations associated with the use of proteins in CE enantioseparations, mainly low obtainable separation efficiency, adsorption of proteins to the capillary wall and high UV absorption. To eliminate these disturbing effects, capillary coating and/or partial filling techniques and/or indirect detection have to be used [Vespalec & Boček, 2000;

Advanced Chiral Separation 35

Figure 2.28. 3D structure of avidin. A typical arrangement of avidin complex (avidin-biotin).

Various proteins, e.g., bovine serum albumin, 1-acid glycoprotein, ovomucoid, avidin (Figure 2.28), transferring, pepsin, penicillin G acylase, have been successfully employed in CE enantioseparations of amino acids and drugs. [Haginaka, 2000; Hödl et al., 2006; Gotti et al., 2006; Martínez-Gómez et al., 2007]. Although this group of selectors is frequently used in model CE enantioseparations, it is less often applied in pharmaceutical analysis, see e.g., ref. by Martinez-Gomez et al. [Martínez-Gómez et al., 2007] devoted to the **e**nantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector. However, no chiral application to biological samples has been referred from 2000, probably because of achieving the goal with less

Macrocyclic antibiotics, introduced by Armstrong et al. [Armstrong et al., 1994b], possess several asymmetric centres and many functional groups and structures (like hydrophobic pocket) allowing multiple interactions (affinity, inclusion, etc.) with the analytes and

18-Crown-6-tetracarboxylic acid (18C6H14) found widespread application as a chiral selector in CE for the chiral separation of amino acids, dipeptides, sympathomimetics and various drugs containing primary amino groups [Kuhn, 1999]. Wang et al. [Wang C.Y. et al., 2003] demonstrated applicability of a new CWE, (S,S)-1,7-bis(4-benzyl-5-hydroxy-2-oxo-3 azapentyl)-1,7-diaza-12-crown-4, for the chiral CE separations. However, there are several limitations with CWEs. The separation electrolyte may not contain cations, such as potassium or ammonium ions, because they compete with the enantiomers for the CWE cavity. This increases claims on the purity and composition of electrolyte systems, and limits some applications. Therefore, according to our best knowledge, there is only one application of CWE to the chiral analysis of biological sample from 2000 [Cho et al., 2004]. 18C6H14 was applied for enantioseparation of gemifloxacin in urine (for detail see Table 3.1).

### **2.3.3 Polysaccharides/oligosacharides**

A variety of linear neutral and charged carbohydrates were found to be applicable as selectors for chiral CE separations. The low UV absorption and often high attainable separation efficiency make these selectors attractive for CE [Riekkola et al., 1997]. The enantiorecognition capability of various neutral oligo- polysacharides (maltodextrins, dextrins, dextrans) is significantly influenced by their higher molecular structures, like helical hydrophobic cavities or pores of polymeric networks [Nishi, H. et al., 1996a]. Two applications to biological samples from 2000 [Bortocan & Bonato, 2004; Nojavan & Fakhari, 2011] show enantioselective separation and determination of primaquine and its metabolite, carboxyprimaquine, in rat liver mitochondrial fraction and plasma samples, and cetirizine and hydroxyzine in human plasma, by maltodextrin as chiral selector (for detail see Table 2.1).

Figure 2.27. Chemical structure of HS-Cys. Reprinted from ref. [Park et al., 2004].

Electrostatic interactions provide additional stereoselectivity effects in the case of ionic polysacharides [Nishi H., 1997] (heparin, dextran sulphate, chondroitin sulphate, hyaluronate, chitosans, aminoglycosides, colominic acid, highly sulfated cyclosophoraoses) suitable for a variety of basic drugs such as -blockers and sympathomimetics [Nishi H., 1997; Nishi, H.S. et al., 1996b; Du et al., 2002; Park et al., 2004]. For example, recently Park et al. [Park et al., 2004] synthesized highly sulfated cyclosophoraoses (HS-Cys) (Figure 2.27) from a family of neutral cyclosophoraoses isolated from *Rhizobium leguminosarum* and applied them as chiral selectors for the resolution of -blocker and sympathomimetic enantiomers. Contrary to the sulfated compounds, the neutral cyclosophoraoses themselves showed no chiral recognition ability for these drugs investigated.

### **2.3.4 Proteins**

34 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

18-Crown-6-tetracarboxylic acid (18C6H14) found widespread application as a chiral selector in CE for the chiral separation of amino acids, dipeptides, sympathomimetics and various drugs containing primary amino groups [Kuhn, 1999]. Wang et al. [Wang C.Y. et al., 2003] demonstrated applicability of a new CWE, (S,S)-1,7-bis(4-benzyl-5-hydroxy-2-oxo-3 azapentyl)-1,7-diaza-12-crown-4, for the chiral CE separations. However, there are several limitations with CWEs. The separation electrolyte may not contain cations, such as potassium or ammonium ions, because they compete with the enantiomers for the CWE cavity. This increases claims on the purity and composition of electrolyte systems, and limits some applications. Therefore, according to our best knowledge, there is only one application of CWE to the chiral analysis of biological sample from 2000 [Cho et al., 2004]. 18C6H14 was

A variety of linear neutral and charged carbohydrates were found to be applicable as selectors for chiral CE separations. The low UV absorption and often high attainable separation efficiency make these selectors attractive for CE [Riekkola et al., 1997]. The enantiorecognition capability of various neutral oligo- polysacharides (maltodextrins, dextrins, dextrans) is significantly influenced by their higher molecular structures, like helical hydrophobic cavities or pores of polymeric networks [Nishi, H. et al., 1996a]. Two applications to biological samples from 2000 [Bortocan & Bonato, 2004; Nojavan & Fakhari, 2011] show enantioselective separation and determination of primaquine and its metabolite, carboxyprimaquine, in rat liver mitochondrial fraction and plasma samples, and cetirizine and hydroxyzine in human

applied for enantioseparation of gemifloxacin in urine (for detail see Table 3.1).

plasma, by maltodextrin as chiral selector (for detail see Table 2.1).

showed no chiral recognition ability for these drugs investigated.

Figure 2.27. Chemical structure of HS-Cys. Reprinted from ref. [Park et al., 2004].

Electrostatic interactions provide additional stereoselectivity effects in the case of ionic polysacharides [Nishi H., 1997] (heparin, dextran sulphate, chondroitin sulphate, hyaluronate, chitosans, aminoglycosides, colominic acid, highly sulfated cyclosophoraoses) suitable for a variety of basic drugs such as -blockers and sympathomimetics [Nishi H., 1997; Nishi, H.S. et al., 1996b; Du et al., 2002; Park et al., 2004]. For example, recently Park et al. [Park et al., 2004] synthesized highly sulfated cyclosophoraoses (HS-Cys) (Figure 2.27) from a family of neutral cyclosophoraoses isolated from *Rhizobium leguminosarum* and applied them as chiral selectors for the resolution of -blocker and sympathomimetic enantiomers. Contrary to the sulfated compounds, the neutral cyclosophoraoses themselves

**2.3.3 Polysaccharides/oligosacharides** 

Proteins provide strong and highly selective interactions, so called affinity interactions, with the analytes. Stereoselectivity is further influenced by the tertiary structure of proteins. Different analytes can selectively bind to different binding sites of proteins. Proteins can be used, depending on their ionization state, for neutral, basic or acidic analytes. However, there are several limitations associated with the use of proteins in CE enantioseparations, mainly low obtainable separation efficiency, adsorption of proteins to the capillary wall and high UV absorption. To eliminate these disturbing effects, capillary coating and/or partial filling techniques and/or indirect detection have to be used [Vespalec & Boček, 2000; Tanaka & Terabe, 1995; Armstrong et al., 1994a].

Figure 2.28. 3D structure of avidin. A typical arrangement of avidin complex (avidin-biotin). Reprinted from [ks.uiuc.edu].

Various proteins, e.g., bovine serum albumin, 1-acid glycoprotein, ovomucoid, avidin (Figure 2.28), transferring, pepsin, penicillin G acylase, have been successfully employed in CE enantioseparations of amino acids and drugs. [Haginaka, 2000; Hödl et al., 2006; Gotti et al., 2006; Martínez-Gómez et al., 2007]. Although this group of selectors is frequently used in model CE enantioseparations, it is less often applied in pharmaceutical analysis, see e.g., ref. by Martinez-Gomez et al. [Martínez-Gómez et al., 2007] devoted to the **e**nantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector. However, no chiral application to biological samples has been referred from 2000, probably because of achieving the goal with less problematic selectors such as CDs and their derivatives.

### **2.3.5 Macrocyclic antibiotics**

Macrocyclic antibiotics, introduced by Armstrong et al. [Armstrong et al., 1994b], possess several asymmetric centres and many functional groups and structures (like hydrophobic pocket) allowing multiple interactions (affinity, inclusion, etc.) with the analytes and

tartrate-boric acid complex chiral selector in situ synthesis and its application in chiral non-

Advanced Chiral Separation 37

Figure 2.30. Chemical structures of SPD–borate complex and analytes. Reprinted from ref.

The limited stability of these selectors, their UV absorption (detection difficulties) and rather slow ligand-exchange kinetics (poor enantioresolution) [Blanco & Valverde, 2003] could be the main reasons for a rare utilization of these selectors in chiral CE. Recently, tartaric acid–Cu(II) complex has been used as a selector for the chiral separation of drugs with amino alcohol structure [Hödl et al., 2007], however, it has not been employed in real bioanalyses so far.

Ion-pairing reagents {e.g., (+)-*S*-Camphor-10-sulfonic acid; 2*R*,3*S*,4*R*,5*S* (−) 2,3,4,6-di-*O*isopropylidene-2-keto-l-gulonic acid; 1*S*,4*R*(+) ketopinic acid, see examples in Figure 2.31}, are applicable in CE usually in non-aqueous medium as the essential interactions (hydrogen bondings and dipole–dipole interactions) are less effective in aqueous medium [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. Exceptions are the use of chiral counterions as supporting agents for separations with coselectors (e.g., CDs) in aqueous

Figure 2.31. Chemical structures of ion-pairing reagents. (S)-(+)-ketopinic acid (left), (+)-(S)-

camphor-10-sulfonic acid (right). Reprinted from [sigmaaldrich.com].

from [Kodama et al., 2005].

**2.3.7 Ion-pairing reagents** 

organic mixtures [Kodama et al., 2003].

aqueous capillary electrophoresis is presented by Wang et al. [Wang L.J. et al., 2011].

providing extremely high enantiorecognition capability. Since these compounds have strong UV absorption, partial filling methods [Desiderio et al., 1997] and countercurrent approaches [Oswald & Ward, 1999] have been applied to overcome detection problems. Moreover, capillaries have to be coated for the elimination of adsorption of antibiotics at the capillary wall [Wang Z. et al., 2007].

Figure 2.29. Chemical structure of vancomycin.

Several classes of antibiotics have been introduced as chiral selectors showing different enantiorecognition capabilities towards amino acids, drugs, etc.: ansamycins (rifamycin B, rifamycin SV), glycopeptides (vancomycin, ristocetin, teicoplanin, avoparcin), aminoglycosides (streptomycin sulfate, kanamycin sulfate, fradiomycin sulfate), macrolides (erythromycin), polypeptides, [see e.g., Wang Z. et al., 2007; Hou J.G. et al., 2003; Ha P.T.T. et al., 2004b]. For the chemical structure of vancomycin see Figure 2.29.

Lack of recent CE applications to biological samples can be explained by the same arguments used for proteins. On the other hand, applications in this field were accomplished by antibiotics immobilized in CEC stationary phases (see section 2.2.2 and Table 2.1).

### **2.3.6 Ligand-exchange selectors**

Ligand-exchange selectors {usually chelate complexes (hemicomplexes) of amino acids with metal cations, e.g., histidine-copper(II)} can form ternary complexes with appropriate analytes (compounds containing aminocarboxyl or hydroxycarboxyl groups like amino acids and hydroxyl acids) having different stability for the two enantiomers [Gassmann et al., 1985]. Rizkov et al. [Rizkov et al., 2010] demonstrated beta-Amino alcohol selectors for enantioselective separation of amino acids by ligand-exchange capillary zone electrophoresis in a low molecular weight organogel. An approach related to the ligandexchange of metal complexes is the formation of mixed borate–diol ternary complexes [Kodama et al., 2006]. For example, recently the use of (5S)-pinandiol (SPD) as the chiral selector in the presence of borate as a central ion and SDS has been described (**Figure 2.30**) [Kodama et al., 2005]. Three diols (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol and 3-benzyloxy-1,2-propanediol) were successfully resolved by this technique. Di-n-amyl L-

providing extremely high enantiorecognition capability. Since these compounds have strong UV absorption, partial filling methods [Desiderio et al., 1997] and countercurrent approaches [Oswald & Ward, 1999] have been applied to overcome detection problems. Moreover, capillaries have to be coated for the elimination of adsorption of antibiotics at the

Several classes of antibiotics have been introduced as chiral selectors showing different enantiorecognition capabilities towards amino acids, drugs, etc.: ansamycins (rifamycin B, rifamycin SV), glycopeptides (vancomycin, ristocetin, teicoplanin, avoparcin), aminoglycosides (streptomycin sulfate, kanamycin sulfate, fradiomycin sulfate), macrolides (erythromycin), polypeptides, [see e.g., Wang Z. et al., 2007; Hou J.G. et al., 2003; Ha P.T.T.

O

NH

Cl

CH3

CH3

O

OH

NH2

HO

HO

OH

NH

O

O

NH

NH2

NH2

O

O

O O O

Cl

NH

CH3

CH3

O

O

O

HO

O

NH

OH

COOH

Lack of recent CE applications to biological samples can be explained by the same arguments used for proteins. On the other hand, applications in this field were accomplished by

Ligand-exchange selectors {usually chelate complexes (hemicomplexes) of amino acids with metal cations, e.g., histidine-copper(II)} can form ternary complexes with appropriate analytes (compounds containing aminocarboxyl or hydroxycarboxyl groups like amino acids and hydroxyl acids) having different stability for the two enantiomers [Gassmann et al., 1985]. Rizkov et al. [Rizkov et al., 2010] demonstrated beta-Amino alcohol selectors for enantioselective separation of amino acids by ligand-exchange capillary zone electrophoresis in a low molecular weight organogel. An approach related to the ligandexchange of metal complexes is the formation of mixed borate–diol ternary complexes [Kodama et al., 2006]. For example, recently the use of (5S)-pinandiol (SPD) as the chiral selector in the presence of borate as a central ion and SDS has been described (**Figure 2.30**) [Kodama et al., 2005]. Three diols (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol and 3-benzyloxy-1,2-propanediol) were successfully resolved by this technique. Di-n-amyl L-

et al., 2004b]. For the chemical structure of vancomycin see Figure 2.29.

antibiotics immobilized in CEC stationary phases (see section 2.2.2 and Table 2.1).

capillary wall [Wang Z. et al., 2007].

Figure 2.29. Chemical structure of vancomycin.

HO

HO

NH

CH3

NH

HO

**2.3.6 Ligand-exchange selectors** 

tartrate-boric acid complex chiral selector in situ synthesis and its application in chiral nonaqueous capillary electrophoresis is presented by Wang et al. [Wang L.J. et al., 2011].

Figure 2.30. Chemical structures of SPD–borate complex and analytes. Reprinted from ref. from [Kodama et al., 2005].

The limited stability of these selectors, their UV absorption (detection difficulties) and rather slow ligand-exchange kinetics (poor enantioresolution) [Blanco & Valverde, 2003] could be the main reasons for a rare utilization of these selectors in chiral CE. Recently, tartaric acid–Cu(II) complex has been used as a selector for the chiral separation of drugs with amino alcohol structure [Hödl et al., 2007], however, it has not been employed in real bioanalyses so far. &

### **2.3.7 Ion-pairing reagents**

Ion-pairing reagents {e.g., (+)-*S*-Camphor-10-sulfonic acid; 2*R*,3*S*,4*R*,5*S* (−) 2,3,4,6-di-*O*isopropylidene-2-keto-l-gulonic acid; 1*S*,4*R*(+) ketopinic acid, see examples in Figure 2.31}, are applicable in CE usually in non-aqueous medium as the essential interactions (hydrogen bondings and dipole–dipole interactions) are less effective in aqueous medium [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. Exceptions are the use of chiral counterions as supporting agents for separations with coselectors (e.g., CDs) in aqueous organic mixtures [Kodama et al., 2003].

Figure 2.31. Chemical structures of ion-pairing reagents. (S)-(+)-ketopinic acid (left), (+)-(S) camphor-10-sulfonic acid (right). Reprinted from [sigmaaldrich.com].

concentration range applicable (no critical micelle concentration) and therefore, higher obtainable signal to noise ratio (S/N). Further, the covalent bonds between surfactant monomers are stable (less background noise from ionized surfactant monomers of low molecular weights in mass spectrometer) and there are no disruptions in micelle formation (wider range of buffer additives applicable, e.g., organic modifiers). Micelle polymers have lower surface activity, association kinetics is fast due to a shallower and faster penetration of the solute into the compact polymeric structure (enhanced efficiency) [Riekkola et al., 1997; Palmer & McCarney, 2004b; Palmer, 2007; Zhao, S. et al., 2007; Akbay et al., 2005; Hou, J. G. et al., 2006; Rizvi et al., 2007]. Many of these properties are beneficial for enhancing chiral, as well as achiral, recognition {a broad range of structurally diverse racemic compounds such as phenylethylamines, -blockers, 2-(2-chlorophenoxy) propionic acid, benzoin derivatives, derivatized amino acids and benzodiazepines} and for improving detection (very pronounced in MS) when compared with traditional micelle systems [Rizvi et al., 2007]. Therefore, the utilization of these selectors tends to rise up as it is apparent from the following recent applications in biological samples (human urine, plasma), such as poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate vs. pseudoephedrine [Rizvi et al., 2007], polysodium-*N*-undecenoyl-L,L-leucyl-valinate vs. warfarin [Hou, J. et al., 2007], for

Advanced Chiral Separation 39

Figure 2.33. Structure of monomer and micelle polymer of alkenoxy surfactants. Reprinted

details see Table 2.1.

from ref. [Rizvi et al., 2004].

The ion-pairing mechanism under non-aqueous conditions took part in the enantioseparation of many drugs such as -blockers [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. It cooperated also in bioanalytical applications separating enantiomers of salbutamol with heptakis-(2,3-di-O-acetyl-6-O-sulfo)--CD in urine samples under non-aqueous EKC conditions [Servais et al., 2004, 2006] (for detail see Table 2.1).

### **2.3.8 Amphiphilic molecules, micelles, micelle polymers and microemulsions**

Amphiphilic molecules, composed of a polar head group and a hydrophobic tail, can form micelles above their critical micelle concentration (Figure 2.32). Micelles are aggregates (usually spherical) with a surface created by heads and core by tails. A typical recognition of analytes is based on the formation of associates with the micelles (less polar parts of analytes tend to enter the micelle core) having different stability for the two analytes of different hydrophobicity, or in other words, differences in their partition coefficients between the micelle phase and the electrolyte bulk phase. Chiral recognition is given by modifying partition coefficients of chiral analytes due to different steric effects between chiral analytes and chiral core or chiral surface of micelles. Micelle pseudostationary phases were introduced into CE by Terabe et al. [Terabe et al., 1984]. Different classes of charged as well as neutral amphiphilic molecules, namely bile salts, saponines, long-chain *N*-alkyl-lamino acids, *N*-alkanoyl-L-amino acids, *N*-dodecoxycarbonyl amino acids, alkylglycosides, alkylglucosides, were applied as chiral selectors in aqueous solutions for a wide range of substances of a hydrophilic or hydrophobic nature, see the specialized review article [Palmer & McCarney, 2004a]. For example, sodium cholate was used for the enantiomeric separation of mono and diacid benzoporfyrine derivates in serum and microsome samples [Peng et al., 2002] (for details see Table 2.1).

Micelle polymers {e.g., poly- sodium *N*-undecenoxy carbonyl-L-leucinate/isoleucinate (Figure 2.33), 3-[(3-dehydroabietamidopropyl)dimethyl-ammonio]-1-propane-sulfonate, undecyl-L-valine} can offer additional benefits in comparison to conventional micelles, such as elimination of intrinsic micelle equilibrium (enhanced chiral recognition), wide {e.g.,

The ion-pairing mechanism under non-aqueous conditions took part in the enantioseparation of many drugs such as -blockers [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. It cooperated also in bioanalytical applications separating enantiomers of salbutamol with heptakis-(2,3-di-O-acetyl-6-O-sulfo)--CD in urine samples under non-aqueous EKC conditions [Servais et al., 2004, 2006] (for detail see Table 2.1).

Amphiphilic molecules, composed of a polar head group and a hydrophobic tail, can form micelles above their critical micelle concentration (Figure 2.32). Micelles are aggregates (usually spherical) with a surface created by heads and core by tails. A typical recognition of analytes is based on the formation of associates with the micelles (less polar parts of analytes tend to enter the micelle core) having different stability for the two analytes of different hydrophobicity, or in other words, differences in their partition coefficients between the micelle phase and the electrolyte bulk phase. Chiral recognition is given by modifying partition coefficients of chiral analytes due to different steric effects between chiral analytes and chiral core or chiral surface of micelles. Micelle pseudostationary phases were introduced into CE by Terabe et al. [Terabe et al., 1984]. Different classes of charged as well as neutral amphiphilic molecules, namely bile salts, saponines, long-chain *N*-alkyl-lamino acids, *N*-alkanoyl-L-amino acids, *N*-dodecoxycarbonyl amino acids, alkylglycosides, alkylglucosides, were applied as chiral selectors in aqueous solutions for a wide range of substances of a hydrophilic or hydrophobic nature, see the specialized review article [Palmer & McCarney, 2004a]. For example, sodium cholate was used for the enantiomeric separation of mono and diacid benzoporfyrine derivates in serum and microsome samples

Figure 2.32. Mechanism of formation of micelle associate with neutral molecule. Reprinted

Micelle polymers {e.g., poly- sodium *N*-undecenoxy carbonyl-L-leucinate/isoleucinate (Figure 2.33), 3-[(3-dehydroabietamidopropyl)dimethyl-ammonio]-1-propane-sulfonate, undecyl-L-valine} can offer additional benefits in comparison to conventional micelles, such as elimination of intrinsic micelle equilibrium (enhanced chiral recognition), wide

**2.3.8 Amphiphilic molecules, micelles, micelle polymers and microemulsions** 

[Peng et al., 2002] (for details see Table 2.1).

from ref. [Terabe et al., 1984].

concentration range applicable (no critical micelle concentration) and therefore, higher obtainable signal to noise ratio (S/N). Further, the covalent bonds between surfactant monomers are stable (less background noise from ionized surfactant monomers of low molecular weights in mass spectrometer) and there are no disruptions in micelle formation (wider range of buffer additives applicable, e.g., organic modifiers). Micelle polymers have lower surface activity, association kinetics is fast due to a shallower and faster penetration of the solute into the compact polymeric structure (enhanced efficiency) [Riekkola et al., 1997; Palmer & McCarney, 2004b; Palmer, 2007; Zhao, S. et al., 2007; Akbay et al., 2005; Hou, J. G. et al., 2006; Rizvi et al., 2007]. Many of these properties are beneficial for enhancing chiral, as well as achiral, recognition {a broad range of structurally diverse racemic compounds such as phenylethylamines, -blockers, 2-(2-chlorophenoxy) propionic acid, benzoin derivatives, derivatized amino acids and benzodiazepines} and for improving detection (very pronounced in MS) when compared with traditional micelle systems [Rizvi et al., 2007]. Therefore, the utilization of these selectors tends to rise up as it is apparent from the following recent applications in biological samples (human urine, plasma), such as poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate vs. pseudoephedrine [Rizvi et al., 2007], polysodium-*N*-undecenoyl-L,L-leucyl-valinate vs. warfarin [Hou, J. et al., 2007], for details see Table 2.1.

Figure 2.33. Structure of monomer and micelle polymer of alkenoxy surfactants. Reprinted from ref. [Rizvi et al., 2004].

Microemulsions, spherical aggregates of nanometre size consisting of an oil core covered/stabilized by amphiphilic molecules in water solution (Figure 2.36), are optically transparent and thermodynamically stable phases that offer a new alternative to micelles for chiral separations aimed at hydrophobic molecules. Chiral microemulsions can utilize one chiral entity (either surfactant, cosurfactant or oil are chiral) or more than one chiral entity (e.g., both surfactant and cosurfactant are chiral) [Ryan et al., 2009; Kahle & Foley, 2006]. Only a few applications to chiral separations have been described (none yet in biological samples), including chiral oils, chiral amphiphilic molecules, as well as CDs as chiral additives [Marsh et al., 2004; McEvoy et al., 2007]. Kahle and Foley [Kahle & Foley, 2007a; 2007b, 2007c] investigated one-, two- and three-chiral-component microemulsions, composed of the chiral surfactant dodecoxycarbonylvaline, the cosurfactant 2-hexanol and one of the chiral oils dibutyl or diethyl tartrate for MEEKC. Six basic drugs, viz. four ephedrine derivatives and two -blockers, were used as test solutes. For most analytes, enantioselectivity, as well as resolution, were higher with the best dual-chirality system compared with the best one-chiral-component microemulsion due to synergistic effects.

Advanced Chiral Separation 41

Figure 2.36. Schematic representation of an oil-in-water microemulsion droplet. Adapted

Several alternative chiral selectors, compared to those described in sections 2.3.1-2.3.8, have been employed in chiral CE, including calixarenes (macrocyclic compounds consisting of benzene rings linked by methylene groups and chiral substituent, see Figure 2.37), cyclosphoraoses (unbranched cyclic -D-glucans), hemispherodextrins (capped CDs), tergurides (ergot alkaloids), amphiphilic aminosaccharides, cyclopeptides, guanosine gels, aptamers (single-stranded RNA or DNA oligonucleotides 15 to 60 base in length that bind with high affinity to specific molecular targets), e.g., anti-arginine 1-RNA aptamer, dendrimers (repetitively branched cascade molecules, see Figure 2.38), etc. [Van Eeckhaut & Michotte, 2006; Gübitz & Schmid, 2004, 2008; Riekkola et al., 1997; Mokhtari et al., 2011, Chen Y.M. et al. 1998]. These selectors can solve particular/specific problems in chiral

from ref. [Ryan et al., 2009].

**2.3.9 Alternative chiral selectors** 

Figure 2.34. Molecular structures of the vesicle-forming amphiphilic molecules. Sodium *N*- [4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n-*dodecyloxybenzoyl]-Lisoleucinate (SDLIL). Reprinted from ref. [Mohanty & Dey, 2006].

The use of vesicle-forming amphiphilic molecules (e.g., sodium N-[4-n-dodecyloxybenzoyl]- L-leucinate) [Mohanty & Dey, 2006] and ionic liquids (i.e., salts in the liquid state, organic salts with low melting points soluble in both polar and nonpolar solvents, e.g., undecenoxycarbonyl-L-pyrrolidinol bromide) [Rizvi & Shamsi, 2006] can be counted among the recent trends in chiral CE with new potentialities for biologically active chiral compounds. For example, two vesicle-forming single-tailed amino acid derivatized surfactants sodium *N*-[4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n*dodecyloxybenzoyl]-L-isoleucinate (SDLIL) (Figure 2.34) have been synthesized and used as a pseudo-stationary phase in MEKC for various model chiral analytes, namely atropisomers (±)-1,1'-bi-2-naphthol, (±)-1,1'-binaphthyl-2,2'-diamine, (±)-1,1'-binaphthyl-2,2' diylhydrogen phosphate and Tröger's base and chiral compound benzoin [Mohanty & Dey, 2006]. Results of these studies have suggested formation of vesicles in aqueous solutions. Microenvironment of the vesicle determined the depth of penetration of the analytes into vesicle, and, in this way, it influenced the separation selectivity. The application of novel ionic liquid-like surfactants and their polymers for chiral separation of acidic analytes in MEKC was reported first by Rizvi and Shamsi in 2006 [Rizvi & Shamsi, 2006]. The two model acidic analytes, (*rac*)-α-bromophenylacetic acid and (*rac*)-2-(2 chlorophenoxy)propanoic acid, were successfully separated with the amino alcohol-derived chiral ionic liquids (given in Figure 2.35) and their polymers at 25 mM surfactant concentration.

Figure 2.35. Amino alcohol-derived chiral ionic liquids for capillary electrophoresis. Reprinted from ref. [Rizvi & Shamsi, 2006].

Figure 2.34. Molecular structures of the vesicle-forming amphiphilic molecules. Sodium *N*- [4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n-*dodecyloxybenzoyl]-L-

The use of vesicle-forming amphiphilic molecules (e.g., sodium N-[4-n-dodecyloxybenzoyl]- L-leucinate) [Mohanty & Dey, 2006] and ionic liquids (i.e., salts in the liquid state, organic salts with low melting points soluble in both polar and nonpolar solvents, e.g., undecenoxycarbonyl-L-pyrrolidinol bromide) [Rizvi & Shamsi, 2006] can be counted among the recent trends in chiral CE with new potentialities for biologically active chiral compounds. For example, two vesicle-forming single-tailed amino acid derivatized surfactants sodium *N*-[4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n*dodecyloxybenzoyl]-L-isoleucinate (SDLIL) (Figure 2.34) have been synthesized and used as a pseudo-stationary phase in MEKC for various model chiral analytes, namely atropisomers (±)-1,1'-bi-2-naphthol, (±)-1,1'-binaphthyl-2,2'-diamine, (±)-1,1'-binaphthyl-2,2' diylhydrogen phosphate and Tröger's base and chiral compound benzoin [Mohanty & Dey, 2006]. Results of these studies have suggested formation of vesicles in aqueous solutions. Microenvironment of the vesicle determined the depth of penetration of the analytes into vesicle, and, in this way, it influenced the separation selectivity. The application of novel ionic liquid-like surfactants and their polymers for chiral separation of acidic analytes in MEKC was reported first by Rizvi and Shamsi in 2006 [Rizvi & Shamsi, 2006]. The two model acidic analytes, (*rac*)-α-bromophenylacetic acid and (*rac*)-2-(2 chlorophenoxy)propanoic acid, were successfully separated with the amino alcohol-derived chiral ionic liquids (given in Figure 2.35) and their polymers at 25 mM surfactant

potentialities

Figure 2.35. Amino alcohol-derived chiral ionic liquids for capillary electrophoresis.

isoleucinate (SDLIL). Reprinted from ref. [Mohanty & Dey, 2006].

concentration.

Reprinted from ref. [Rizvi & Shamsi, 2006].

Microemulsions, spherical aggregates of nanometre size consisting of an oil core covered/stabilized by amphiphilic molecules in water solution (Figure 2.36), are optically transparent and thermodynamically stable phases that offer a new alternative to micelles for chiral separations aimed at hydrophobic molecules. Chiral microemulsions can utilize one chiral entity (either surfactant, cosurfactant or oil are chiral) or more than one chiral entity (e.g., both surfactant and cosurfactant are chiral) [Ryan et al., 2009; Kahle & Foley, 2006]. Only a few applications to chiral separations have been described (none yet in biological samples), including chiral oils, chiral amphiphilic molecules, as well as CDs as chiral additives [Marsh et al., 2004; McEvoy et al., 2007]. Kahle and Foley [Kahle & Foley, 2007a; 2007b, 2007c] investigated one-, two- and three-chiral-component microemulsions, composed of the chiral surfactant dodecoxycarbonylvaline, the cosurfactant 2-hexanol and one of the chiral oils dibutyl or diethyl tartrate for MEEKC. Six basic drugs, viz. four ephedrine derivatives and two -blockers, were used as test solutes. For most analytes,

enantioselectivity, as well as resolution, were higher with the best dual-chirality system compared with the best one-chiral-component microemulsion due to synergistic effects.

Figure 2.36. Schematic representation of an oil-in-water microemulsion droplet. Adapted from ref. [Ryan et al., 2009].

### **2.3.9 Alternative chiral selectors**

Several alternative chiral selectors, compared to those described in sections 2.3.1-2.3.8, have been employed in chiral CE, including calixarenes (macrocyclic compounds consisting of benzene rings linked by methylene groups and chiral substituent, see Figure 2.37), cyclosphoraoses (unbranched cyclic -D-glucans), hemispherodextrins (capped CDs), tergurides (ergot alkaloids), amphiphilic aminosaccharides, cyclopeptides, guanosine gels, aptamers (single-stranded RNA or DNA oligonucleotides 15 to 60 base in length that bind with high affinity to specific molecular targets), e.g., anti-arginine 1-RNA aptamer, dendrimers (repetitively branched cascade molecules, see Figure 2.38), etc. [Van Eeckhaut & Michotte, 2006; Gübitz & Schmid, 2004, 2008; Riekkola et al., 1997; Mokhtari et al., 2011, Chen Y.M. et al. 1998]. These selectors can solve particular/specific problems in chiral

*New chiral selectors.* A sensitive MEKC-MS method using poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate was developed for enantioselective analysis of pseudoephedrine in human urine [Rizvi et al., 2007]. The permanent charge of this micelle chiral selector allows for setting separation conditions (electrolyte pH) suitable for obtaining the highest detection sensitivity maintaining sufficient enantioresolution. Other new chiral selectors and new chiral stationary phases can be also found in some applications given for the advanced

Advanced Chiral Separation 43

*Countercurrent migration.* Rudaz et al. [Rudaz et al., 2005] used the countercurrent migration of the negatively charged highly sulfated--CD to achieve a highly effective enantioseparation of amphetamine derivatives in plasma. This strategy allowed using a low concentration (0.15%) of the chiral selector for the complete enantioseparation of seven

Anionic CD, the heptakis(2,6-diacethyl-6-sulfato)--CD, migrating in the opposite direction to the analytes, was highly effective in low concentrations (0.85 mM) for the simultaneous chiral CD-EKC-MS separation of methamphetamine, 3,4-methylenedioxyamphetamine and

Figure 2.39. Electropherograms of extracts of blank plasma and plasma of spiked concentration of 4 M racemic drug. Conditions: detection buffer, 5 mM Ru(bpy)3 2+ in 100 mM (pH 6.5) phosphate buffer; separation buffer, 3 mg/mL of S--CD in 40 mM (pH 4.5) acetate buffer;

The CD-EKC method based on a combination of discontinuous buffer (separation buffer differed from detection solution, essential for chemiluminiscence detection) with low concentration (3 mg/mL) of anionic highly sulfated--CD was used for the baseline

applied voltage, 22 kV; injection time, 5 s. Reprinted from ref. [Fang, L. et al., 2006].

electromigration modes and mechanisms below.

amphetamine derivatives in analysis times of less than 6 min.

amphetamine in clinical human urine samples [Iio et al., 2005].

separations, however, their use is less universal in most cases as they can be expensive and their availability is limited. This could also be the reason why no application in chiral bioanalysis has been demonstrated so far.

Figure 2.37. Chiral calixarene. Reprinted from [rsc.org].

Figure 2.38. The chemical structures of the chiral dendrimers with axial chirality. Reprinted from ref. [Chen, Y.M. et al., 1998].

### **2.4 Applications of chiral separation systems in bioanalysis**

The following examples illustrate successful EKC enantioseparations of drugs in biological samples utilizing some of the progressive chiral principles mentioned in section 2.2. and/or new chiral selectors mentioned in section 2.3. CEC and MCE applications in this field are also included.

separations, however, their use is less universal in most cases as they can be expensive and their availability is limited. This could also be the reason why no application in chiral

Figure 2.38. The chemical structures of the chiral dendrimers with axial chirality. Reprinted

The following examples illustrate successful EKC enantioseparations of drugs in biological samples utilizing some of the progressive chiral principles mentioned in section 2.2. and/or new chiral selectors mentioned in section 2.3. CEC and MCE applications in this field are

**2.4 Applications of chiral separation systems in bioanalysis** 

bioanalysis has been demonstrated so far.

Figure 2.37. Chiral calixarene. Reprinted from [rsc.org].

from ref. [Chen, Y.M. et al., 1998].

also included.

*New chiral selectors.* A sensitive MEKC-MS method using poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate was developed for enantioselective analysis of pseudoephedrine in human urine [Rizvi et al., 2007]. The permanent charge of this micelle chiral selector allows for setting separation conditions (electrolyte pH) suitable for obtaining the highest detection sensitivity maintaining sufficient enantioresolution. Other new chiral selectors and new chiral stationary phases can be also found in some applications given for the advanced electromigration modes and mechanisms below.

*Countercurrent migration.* Rudaz et al. [Rudaz et al., 2005] used the countercurrent migration of the negatively charged highly sulfated--CD to achieve a highly effective enantioseparation of amphetamine derivatives in plasma. This strategy allowed using a low concentration (0.15%) of the chiral selector for the complete enantioseparation of seven amphetamine derivatives in analysis times of less than 6 min.

Anionic CD, the heptakis(2,6-diacethyl-6-sulfato)--CD, migrating in the opposite direction to the analytes, was highly effective in low concentrations (0.85 mM) for the simultaneous chiral CD-EKC-MS separation of methamphetamine, 3,4-methylenedioxyamphetamine and amphetamine in clinical human urine samples [Iio et al., 2005].

Figure 2.39. Electropherograms of extracts of blank plasma and plasma of spiked concentration of 4 M racemic drug. Conditions: detection buffer, 5 mM Ru(bpy)3 2+ in 100 mM (pH 6.5) phosphate buffer; separation buffer, 3 mg/mL of S--CD in 40 mM (pH 4.5) acetate buffer; applied voltage, 22 kV; injection time, 5 s. Reprinted from ref. [Fang, L. et al., 2006].

The CD-EKC method based on a combination of discontinuous buffer (separation buffer differed from detection solution, essential for chemiluminiscence detection) with low concentration (3 mg/mL) of anionic highly sulfated--CD was used for the baseline

the chiral selector enabling optimum separating conditions for the trace chiral analytes in

Advanced Chiral Separation 45

*Charged chiral pseudophases/carrier systems.* The enantioseparations of cyanobenz[*f*]isoindole serine (CBI) derivatives of glutamate and aspartate in squirrel brain samples were accomplished with a highly sulfated--CD as chiral selector at low pH and reverse polarity

*Mixed selector systems.* Aspartate enantiomers (NDA derivatives) were separated in the samples prepared from tissue of the central nervous system of *A. californica* using mixed

Figure 2.41. Separation of CBI-Ser enantiomers using different running buffers: (a) 100mM borate (pH 9.5), 30mM CD and 60mM SDC; (b) 100mM borate (pH 9.5) and 30mM -CD; (c) 100mM borate (pH 10.0), 60mM SDC. Capillary was 50 m i.d.×50 cm effective length. Voltage applied was 15 kV. Ser enantiomer concentration was 2.0 M. Reprinted from ref.

The CD-MEKC separation of CBI-D/L-serine enantiomers was achieved by using a dual chiral selector system consisting of -CD and chiral micelles formed by deoxycholic acid (SCD) [Zhao, S.L. et al., 2005b]. The essential role of the mixed chiral selector system for the

concentrated biological matrices [Mikuš et al., 2006a], see Figure 2.40.

micelles composed from -CD and SDS [Miao et al., 2005].

[Kirschner et al., 2007].

[Zhao, S.L. et al., 2005b].

enantioseparation of an oppositely charged racemic antiarrhythmic drug, disopyramide, in spiked plasma samples, see Figure 2.39 [Fang, L. et al., 2006].

Naphthalene-2,3-dicarboxaldehyde (NDA) derivatized baclofen was separated in human plasma using the CD-EKC-LIF method based on a highly sulfated--CD [Kavran-Belin et al., 2005]. The anionic CD migrated in the opposite direction toward negatively charged baclofen (pH 9.5) transported by EOF toward the cathode.

The NACE-MS with countercurrent migration of heptakis(2,3-di-*O*-acetyl-6-*O-*sulfo)--CD present in acidified methanol medium was used for the separation and determination of low levels of the enantiomers of a basic chiral drug (salbutamol) in biological samples (human urine) [Servais et al., 2006].

Figure 2.40. Electropherograms showing the effect of the charge of ionizable chiral selector, CE--CD, on the EKC separation of PHM enantiomers in a partially pretreated urine matrix. The separations were carried out with the uncharged CE--CD (a, b) and with the negatively charged CE--CD (c). Separating conditions: (a, b) EKC electrolyte system consisting of a 25mM glycine-acetic acid buffer, pH 3.2, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 0 (a) and 15 (b) mg/mL concentration of CE--CD, (c) EKC electrolyte system consisting of a 25mM aminocaproic acid-acetic acid buffer, pH 4.5, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 2.5 mg/mL concentration of CE- -CD. Sample and analyte: (a, b) PHM was present in the sample (20-times diluted urine) at a 150 ng/mL concentration. (c) PHM was present in the sample (4-times diluted urine) at a 30 ng/mL concentration. Reprinted from ref. [Mikuš et al., 2006a].

Negatively charged carboxyethyl--CD considerably improved enantioresolution of several oppositely charged H1-antihistamines (pheniramine and its metabolite, dimethindene, dioxopromethazine) in comparison with native CDs [Mikuš et al., 2006a]. Countercurrent migration and enhanced complexation were responsible not only for improved chiral separation of the drugs and the metabolite, but also achiral separation allowing resolution of the drug enantiomers from their metabolic products, as well as from the sample matrix constituents when metabolic study of pheniramine in urine samples was carried out [Marák et al., 2007]. A high effectivity of the separation was demonstrated by a low concentration of

enantioseparation of an oppositely charged racemic antiarrhythmic drug, disopyramide, in

Naphthalene-2,3-dicarboxaldehyde (NDA) derivatized baclofen was separated in human plasma using the CD-EKC-LIF method based on a highly sulfated--CD [Kavran-Belin et al., 2005]. The anionic CD migrated in the opposite direction toward negatively charged

The NACE-MS with countercurrent migration of heptakis(2,3-di-*O*-acetyl-6-*O-*sulfo)--CD present in acidified methanol medium was used for the separation and determination of low levels of the enantiomers of a basic chiral drug (salbutamol) in biological samples (human

Figure 2.40. Electropherograms showing the effect of the charge of ionizable chiral selector, CE--CD, on the EKC separation of PHM enantiomers in a partially pretreated urine matrix. The separations were carried out with the uncharged CE--CD (a, b) and with the negatively charged CE--CD (c). Separating conditions: (a, b) EKC electrolyte system consisting of a 25mM glycine-acetic acid buffer, pH 3.2, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 0 (a) and 15 (b) mg/mL concentration of CE--CD, (c) EKC electrolyte system consisting of a 25mM aminocaproic acid-acetic acid buffer, pH 4.5, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 2.5 mg/mL concentration of CE- -CD. Sample and analyte: (a, b) PHM was present in the sample (20-times diluted urine) at a 150 ng/mL concentration. (c) PHM was present in the sample (4-times diluted urine) at a

(c)

Negatively charged carboxyethyl--CD considerably improved enantioresolution of several oppositely charged H1-antihistamines (pheniramine and its metabolite, dimethindene, dioxopromethazine) in comparison with native CDs [Mikuš et al., 2006a]. Countercurrent migration and enhanced complexation were responsible not only for improved chiral separation of the drugs and the metabolite, but also achiral separation allowing resolution of the drug enantiomers from their metabolic products, as well as from the sample matrix constituents when metabolic study of pheniramine in urine samples was carried out [Marák et al., 2007]. A high effectivity of the separation was demonstrated by a low concentration of

30 ng/mL concentration. Reprinted from ref. [Mikuš et al., 2006a].

spiked plasma samples, see Figure 2.39 [Fang, L. et al., 2006].

baclofen (pH 9.5) transported by EOF toward the cathode.

urine) [Servais et al., 2006].

the chiral selector enabling optimum separating conditions for the trace chiral analytes in concentrated biological matrices [Mikuš et al., 2006a], see Figure 2.40.

*Charged chiral pseudophases/carrier systems.* The enantioseparations of cyanobenz[*f*]isoindole serine (CBI) derivatives of glutamate and aspartate in squirrel brain samples were accomplished with a highly sulfated--CD as chiral selector at low pH and reverse polarity [Kirschner et al., 2007].

*Mixed selector systems.* Aspartate enantiomers (NDA derivatives) were separated in the samples prepared from tissue of the central nervous system of *A. californica* using mixed micelles composed from -CD and SDS [Miao et al., 2005].

Figure 2.41. Separation of CBI-Ser enantiomers using different running buffers: (a) 100mM borate (pH 9.5), 30mM CD and 60mM SDC; (b) 100mM borate (pH 9.5) and 30mM -CD; (c) 100mM borate (pH 10.0), 60mM SDC. Capillary was 50 m i.d.×50 cm effective length. Voltage applied was 15 kV. Ser enantiomer concentration was 2.0 M. Reprinted from ref. [Zhao, S.L. et al., 2005b].

The CD-MEKC separation of CBI-D/L-serine enantiomers was achieved by using a dual chiral selector system consisting of -CD and chiral micelles formed by deoxycholic acid (SCD) [Zhao, S.L. et al., 2005b]. The essential role of the mixed chiral selector system for the

methyltrimethylammonium with a selectivity of 1.0078 was almost baseline separated in 3.5

Advanced Chiral Separation 47

*CEC.* CEC separations are carried out mainly in packed capillary columns. Immobilization provides an effective solution especially for the chiral selectors with problematic detection properties, such as proteins, macrocyclic antibiotics, etc., that was demonstrated on many model examples, such as vancomycin CSP vs. warfarin and various -blockers (atenolol, metoprolol, pindolol, oxprenolol, alprenolol, propranolol, carteolol, talinolol) vs. MS

Figure 2.43. Electrochromatogram of blank urine sample spiked with 10 g/mL of racemic MRT and 8-OH-M and 20 g/mL of racemic DMR. Both samples were subjected to the SPE procedure. Electrochromatographic conditions: stationary phase composition, vancomycin-CSP, mobile phase, 100 mM ammonium acetate buffer (pH 6)/H2O/MeOH/ACN (5:15:30:50, by vol.). Other CEC conditions: capillary column, 75 m i.d., 33 cm total length, 23 cm packed length, 24.5 cm effective length; applied voltage, 25 kV; capillary temperature, 20°C; pressurized column at both ends with 10 bar; injection by pressure at 10 bar, 0.5 min, followed by a plug of mobile phase at 10 bar, 0.2 min. (1) MRT, (2) 8-OH-M and (3) DMR.

Various new CSPs are also employed in model analyses. A new chiral capillary electrophoresis column coated by carbosilane dendrimers with peripheral Si-Cl groups and beta-cyclodextrin was prepared by Shou et al. [Shou et al., 2008]. The separation characteristics of stability and longevity of coated capillaries modified by carbosilane dendrimers were excellent. The varying of separation efficiency was less than 5%, after running for one month. The optimum conditions contained the running voltage of 12 kV, the UV detector wavelength of 214 nm, the sample injected time of 7 s and the phosphate buffer solution concentration of 40 mM. Chlortrimeton, hydrochloric promethazine and benzedrine were selected as the separation model targets. Especially, G2P columns could be used as separate chlortrimeton enantiomers effectively. Under these conditions, the column

efficiency was 2.5 x 105 plates/m with a resolution of 1.43 and baseline separation.

Other examples of the CEC application are illustrated on biological samples. For example, CEC based on vancomycin CSP offered an effective solution not only for the enantioseparation of

h [Zhao J. & Jorgenson, 1999].

detection [Zheng J. & Shamsi, 2006; Zheng J. et al., 2006].

Reprinted from ref. [Aturki et al., 2007].

enantioresolution in comparison with the capability of individual chiral selectors is demonstrated in Figure 2.41, compare electropherograms (a-c). In addition, an effectivity of the mixed chiral selector system in the enantioseparation of CBI-D/L-serine enantiomers in real biological matrices was demonstrated in this work. In this way, for the first time, peaks corresponding to L-serine and D-serine were completely separated in *Aplysia* ganglian (a sea mollusc widely used as a neuronal model) homogenates.

The evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary electrophoresis is presented by Chen et al. [Chen J. et al., 2010].

*Flow counterbalanced systems.* Several FCCE modes have been developed and applied for enantioseparations so far, like microfluidic temperature gradient focusing [Balss et al., 2004], electric field gradient focusing [Koegler & Ivory, 1996a, 1996b, Ivory, 2000, Huang, Z. & Ivory, 1999]. Notice that these techniques have a great potential not only in enantioseparations, but also in a sample pretreatment (focusing, see chapter 3).

Figure 2.42. Effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM-CD). Reproduced from ref. [Chankvetadze et al., 1999].

The effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM--CD) was studied by Chankvetadze et al. [Chankvetadze et al., 1999] and remarkable results are illustrated in Figure 2.42. Applications of counterpressure techniques for the enantioseparations in complex ionic matrices, however, have not been demonstrated. isotopic model

Synchronous cyclic capillary electrophoresis (SCCE) was proposed by Jorgenson's group [Zhao J. et al., 1999] as a technique which allows overcoming the dispersion problems in FCCE caused by the parabolic counterflow profile. This technique was applied for isotopic and chiral separations. In the third cycle, the chiral compound (-hydroxybenzyl)

enantioresolution in comparison with the capability of individual chiral selectors is demonstrated in Figure 2.41, compare electropherograms (a-c). In addition, an effectivity of the mixed chiral selector system in the enantioseparation of CBI-D/L-serine enantiomers in real biological matrices was demonstrated in this work. In this way, for the first time, peaks corresponding to L-serine and D-serine were completely separated in *Aplysia* ganglian (a sea

The evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary electrophoresis is presented by Chen et al. [Chen J. et al.,

*Flow counterbalanced systems.* Several FCCE modes have been developed and applied for enantioseparations so far, like microfluidic temperature gradient focusing [Balss et al., 2004], electric field gradient focusing [Koegler & Ivory, 1996a, 1996b, Ivory, 2000, Huang, Z. & Ivory, 1999]. Notice that these techniques have a great potential not only in

Figure 2.42. Effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM-CD). Reproduced from ref. [Chankvetadze et al., 1999].

The effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM--CD) was studied by Chankvetadze et al. [Chankvetadze et al., 1999] and remarkable results are illustrated in Figure 2.42. Applications of counterpressure techniques for the enantioseparations in complex ionic matrices, however, have not been demonstrated.

Synchronous cyclic capillary electrophoresis (SCCE) was proposed by Jorgenson's group [Zhao J. et al., 1999] as a technique which allows overcoming the dispersion problems in FCCE caused by the parabolic counterflow profile. This technique was applied for isotopic and chiral separations. In the third cycle, the chiral compound (-hydroxybenzyl)

enantioseparations, but also in a sample pretreatment (focusing, see chapter 3).

mollusc widely used as a neuronal model) homogenates.

2010].

methyltrimethylammonium with a selectivity of 1.0078 was almost baseline separated in 3.5 h [Zhao J. & Jorgenson, 1999].

*CEC.* CEC separations are carried out mainly in packed capillary columns. Immobilization provides an effective solution especially for the chiral selectors with problematic detection properties, such as proteins, macrocyclic antibiotics, etc., that was demonstrated on many model examples, such as vancomycin CSP vs. warfarin and various -blockers (atenolol, metoprolol, pindolol, oxprenolol, alprenolol, propranolol, carteolol, talinolol) vs. MS detection [Zheng J. & Shamsi, 2006; Zheng J. et al., 2006].

Figure 2.43. Electrochromatogram of blank urine sample spiked with 10 g/mL of racemic MRT and 8-OH-M and 20 g/mL of racemic DMR. Both samples were subjected to the SPE procedure. Electrochromatographic conditions: stationary phase composition, vancomycin-CSP, mobile phase, 100 mM ammonium acetate buffer (pH 6)/H2O/MeOH/ACN (5:15:30:50, by vol.). Other CEC conditions: capillary column, 75 m i.d., 33 cm total length, 23 cm packed length, 24.5 cm effective length; applied voltage, 25 kV; capillary temperature, 20°C; pressurized column at both ends with 10 bar; injection by pressure at 10 bar, 0.5 min, followed by a plug of mobile phase at 10 bar, 0.2 min. (1) MRT, (2) 8-OH-M and (3) DMR. Reprinted from ref. [Aturki et al., 2007].

Various new CSPs are also employed in model analyses. A new chiral capillary electrophoresis column coated by carbosilane dendrimers with peripheral Si-Cl groups and beta-cyclodextrin was prepared by Shou et al. [Shou et al., 2008]. The separation characteristics of stability and longevity of coated capillaries modified by carbosilane dendrimers were excellent. The varying of separation efficiency was less than 5%, after running for one month. The optimum conditions contained the running voltage of 12 kV, the UV detector wavelength of 214 nm, the sample injected time of 7 s and the phosphate buffer solution concentration of 40 mM. Chlortrimeton, hydrochloric promethazine and benzedrine were selected as the separation model targets. Especially, G2P columns could be used as separate chlortrimeton enantiomers effectively. Under these conditions, the column efficiency was 2.5 x 105 plates/m with a resolution of 1.43 and baseline separation.

Other examples of the CEC application are illustrated on biological samples. For example, CEC based on vancomycin CSP offered an effective solution not only for the enantioseparation of mirtazapine (MRT) and its metabolites {8-hydroxymirtazapine (8-OH-M) and *N*desmethylmirtazapine (DMR)} in urine samples (pretreated by off-line solid-phase extraction), but also for their reliable UV absorbance detection [Aturki et al., 2007], see Figure 2.43.

Figure 2.44. Separation of mexiletine hydrochloride in human plasma sample by Asp-CD fabricated monolithic column. (A) Plasma sample; (B) free plasma. 1 and 2, the enantiomers' peaks of mexiletine hydrochloride. Mobile phase: phosphate/trolamine buffer, 5mM, pH= 4.5; temperature: 20°C; voltage: −10 kV; injection: −2 kV, 2 s. Reprinted from ref. [Li Y. et al., 2010].

Figure 2.45. Enantiomeric separation of noradrenaline with CM--CD by formation of sandwich complexes with 18-crown-6 at pH 6.5 and 2.5. Separation voltage, 3 kV; detection potential, 1500 mV; injection, 1 kV (3 s); concentration of standard, 10-4 *M*. Reprinted from

Advanced Chiral Separation 49

A combination of an advanced enantioseparation mechanism (dual chiral selector system) with advanced CE format (MCE) was demonstrated by Schwarz and Hauser [Schwarz & Hauser, 2001]. The separation of the enantiomers of noradrenaline in the presence of 18 crown-6 is illustrated in Figure 2.45. The crown ether on its own has a small effect on the migration, but cannot lead to a chiral separation (Figure 2.45b). In combination with the carboxymethylated -cyclodextrin (Figure 2.45c), a chiral separation which is better than the one achieved by only carboxymethylated -cyclodextrin is obtained. The resolution factors (*R*) were determined as 1.30 and 1.35 (5.2 mg/mL CM--CD, pH=6.7) with and without the addition of 12 m*M* 18-crown-6, respectively. Moreover, such an advanced system is capable of also separating multicomponent mixtures of enantiomers, as presented in Figure 2.46.

ref. [Schwarz & Hauser, 2001].

Very attractive applications due to rapid separations and good enantioresolutions are represented by the chiral monolithic based CEC. A monolithic column fabricated with Asp- CD was applied to the separation of mexiletine hydrochloride enantiomers in a human plasma sample. Because of complex matrices, as little as possible trolamine was added to the phosphate buffer to decrease absorption between the sample and the solid-phase. Figure 2.44 shows the electropherogram of mexiletine hydrochloride extracted from the plasma sample. From Figure 2.44, two enantiomers were separated by baseline, the migration times were 3.46 and 3.84 min, respectively. Some components in plasma were also determined, but they did not interfere with the determination of the enantiomers. Thus, the method was applied to the complex matrix sample, and the column had good selectivity.

*MCE.* MCE with (+)-18-crown-6-tetracarboxylic acid as a chiral selector was applied for the countercurrent chiral separation of gemifloxacin in urine samples after an appropriate sample pretreatment (see in 3.2.2) [Cho et al., 2004]. A successful chiral separation was achieved in ca. 3 min, with an extremely small amount of the chiral selector (50 M), and minute amounts of both sample as well as BGE. This demonstrated significant potentialities of electromigration methods in highly effective ultraspeed microscale chiral analyses of drugs in complex matrices.

mirtazapine (MRT) and its metabolites {8-hydroxymirtazapine (8-OH-M) and *N*desmethylmirtazapine (DMR)} in urine samples (pretreated by off-line solid-phase extraction),

Figure 2.44. Separation of mexiletine hydrochloride in human plasma sample by Asp-CD fabricated monolithic column. (A) Plasma sample; (B) free plasma. 1 and 2, the enantiomers' peaks of mexiletine hydrochloride. Mobile phase: phosphate/trolamine buffer, 5mM, pH= 4.5; temperature: 20°C; voltage: −10 kV; injection: −2 kV, 2 s. Reprinted from ref. [Li Y. et al., 2010].

Very attractive applications due to rapid separations and good enantioresolutions are represented by the chiral monolithic based CEC. A monolithic column fabricated with Asp- CD was applied to the separation of mexiletine hydrochloride enantiomers in a human plasma sample. Because of complex matrices, as little as possible trolamine was added to the phosphate buffer to decrease absorption between the sample and the solid-phase. Figure 2.44 shows the electropherogram of mexiletine hydrochloride extracted from the plasma sample. From Figure 2.44, two enantiomers were separated by baseline, the migration times were 3.46 and 3.84 min, respectively. Some components in plasma were also determined, but they did not interfere with the determination of the enantiomers. Thus, the method was

*MCE.* MCE with (+)-18-crown-6-tetracarboxylic acid as a chiral selector was applied for the countercurrent chiral separation of gemifloxacin in urine samples after an appropriate sample pretreatment (see in 3.2.2) [Cho et al., 2004]. A successful chiral separation was achieved in ca. 3 min, with an extremely small amount of the chiral selector (50 M), and minute amounts of both sample as well as BGE. This demonstrated significant potentialities of electromigration methods in highly effective ultraspeed microscale chiral analyses of

applied to the complex matrix sample, and the column had good selectivity.

drugs in complex matrices.

but also for their reliable UV absorbance detection [Aturki et al., 2007], see Figure 2.43.

Figure 2.45. Enantiomeric separation of noradrenaline with CM--CD by formation of sandwich complexes with 18-crown-6 at pH 6.5 and 2.5. Separation voltage, 3 kV; detection potential, 1500 mV; injection, 1 kV (3 s); concentration of standard, 10-4 *M*. Reprinted from ref. [Schwarz & Hauser, 2001].

A combination of an advanced enantioseparation mechanism (dual chiral selector system) with advanced CE format (MCE) was demonstrated by Schwarz and Hauser [Schwarz & Hauser, 2001]. The separation of the enantiomers of noradrenaline in the presence of 18 crown-6 is illustrated in Figure 2.45. The crown ether on its own has a small effect on the migration, but cannot lead to a chiral separation (Figure 2.45b). In combination with the carboxymethylated -cyclodextrin (Figure 2.45c), a chiral separation which is better than the one achieved by only carboxymethylated -cyclodextrin is obtained. The resolution factors (*R*) were determined as 1.30 and 1.35 (5.2 mg/mL CM--CD, pH=6.7) with and without the addition of 12 m*M* 18-crown-6, respectively. Moreover, such an advanced system is capable of also separating multicomponent mixtures of enantiomers, as presented in Figure 2.46.

**3** 

**Advanced Sample Preparation** 

species advantages

**3.1 On-line sample preparation techniques in capillary electrophoresis - introduction**  The importance of sample preparation is related to high demands on (i) the sensitivity of quantitative determination of trace analytes, (ii) the selectivity of monitoring of analytes in multicomponent sample matrix and (iii) the automatization and miniaturization of analysis

*Sensitivity.* One of the most pronounced limitations of capillary electromigration methods when compared to more traditional liquid-phase separation techniques, such as HPLC, is a poor concentration sensitivity of photometric detectors, which are the most popular among on-capillary CE detectors. The reached LODs are by two orders inferior in comparison to the HPLC technique because of a short optical pathlength and small sample injection volume. Two basic approaches can be distinguished among many efforts that have been made to improve the sensitivity of detection in CE. Either more sensitive detection schemes can displace the UV detection mode [Hernández et al., 2008, 2010; Hempel, 2000] or an increased analyte mass can be cumulated in its zone prior to detection utilizing a proper sample

*Selectivity.* To achieve adequate separation selectivity, the analysis of real/complex samples in CE usually requires efficient sample treatment to remove interfering solutes, inorganic and organic salts, and particulate matter (as it is reviewed in this section). At the same time, the creation of a chiral environment is necessary for the separation of enantiomeric species by the electrokinetic chromatography (EKC) principle accomplished in zone electrophoretic mode simply by the addition of chiral selector molecules. In the same way, chiral selectivity can be implemented in isotachophoretic mode. Chiral separation principles in CE have been described in many review papers [Chankvetadze, 1997; Chankvetadze & Blaschke, 2001; Gübitz & Schmid, 2007, 2008; Preinerstorfer et al., 2009; Fanali, 2002; Fanali et al., 1998b; Ossicini & Fanali, 1997]. In addition to those, capillary electrochromatography (CEC) separation principles cannot be omitted for review, e.g., those by Schurig [Wistuba & Schurig, 2000a, 2000b]. On-line sample preparation and enantioseparation mechanisms should create a harmonized system, especially in chiral EKC and ITP where the chiral selector is not immobilized as it is in CEC, and all possible mutual negative interferences

*Automatization and miniaturization.* As one moves toward small sample volumes, sample handling and preparation steps become more difficult and the concentration step is therefore preferably done on-line instead of off-line. The microfluidic devices, such as microchips, can provide several additional advantages over electromigration techniques performed in capillary format. The heat dissipation is much better in chip format compared

[Mikuš & Maráková, 2010].

preparation, as it is reviewed in this section.

have to be carefully eliminated.

Figure 2.46. Rapid enantiomer MCE separation and amperometric detection of a mixture of adrenaline, noradrenaline and dopamine employing sandwich complexes. Buffer: (a) 50 mM MES, pH=6.7; (b) 20 mM phosphate, pH=3.0, 5.2 mg/mL CM--CD, 12 mM 18-crown-6. Separation voltage, 3 kV; detection potential, (a) 1300 mV, (b) 1500 mV; injection, 1 kV (3 s); concentration of standards, 10-4 M. Peak marks: dopamine hydrochloride, D, adrenaline, A, noradrenaline, NA. Reprinted from ref. [Schwarz & Hauser, 2001].

### **Advanced Sample Preparation**

### **3.1 On-line sample preparation techniques in capillary electrophoresis - introduction**  The importance of sample preparation is related to high demands on (i) the sensitivity of

50 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Figure 2.46. Rapid enantiomer MCE separation and amperometric detection of a mixture of adrenaline, noradrenaline and dopamine employing sandwich complexes. Buffer: (a) 50 mM MES, pH=6.7; (b) 20 mM phosphate, pH=3.0, 5.2 mg/mL CM--CD, 12 mM 18-crown-6. Separation voltage, 3 kV; detection potential, (a) 1300 mV, (b) 1500 mV; injection, 1 kV (3 s); concentration of standards, 10-4 M. Peak marks: dopamine hydrochloride, D, adrenaline, A,

noradrenaline, NA. Reprinted from ref. [Schwarz & Hauser, 2001].

quantitative determination of trace analytes, (ii) the selectivity of monitoring of analytes in multicomponent sample matrix and (iii) the automatization and miniaturization of analysis [Mikuš & Maráková, 2010].

*Sensitivity.* One of the most pronounced limitations of capillary electromigration methods when compared to more traditional liquid-phase separation techniques, such as HPLC, is a poor concentration sensitivity of photometric detectors, which are the most popular among on-capillary CE detectors. The reached LODs are by two orders inferior in comparison to the HPLC technique because of a short optical pathlength and small sample injection volume. Two basic approaches can be distinguished among many efforts that have been made to improve the sensitivity of detection in CE. Either more sensitive detection schemes can displace the UV detection mode [Hernández et al., 2008, 2010; Hempel, 2000] or an increased analyte mass can be cumulated in its zone prior to detection utilizing a proper sample preparation, as it is reviewed in this section.

*Selectivity.* To achieve adequate separation selectivity, the analysis of real/complex samples in CE usually requires efficient sample treatment to remove interfering solutes, inorganic and organic salts, and particulate matter (as it is reviewed in this section). At the same time, the creation of a chiral environment is necessary for the separation of enantiomeric species by the electrokinetic chromatography (EKC) principle accomplished in zone electrophoretic mode simply by the addition of chiral selector molecules. In the same way, chiral selectivity can be implemented in isotachophoretic mode. Chiral separation principles in CE have been described in many review papers [Chankvetadze, 1997; Chankvetadze & Blaschke, 2001; Gübitz & Schmid, 2007, 2008; Preinerstorfer et al., 2009; Fanali, 2002; Fanali et al., 1998b; Ossicini & Fanali, 1997]. In addition to those, capillary electrochromatography (CEC) separation principles cannot be omitted for review, e.g., those by Schurig [Wistuba & Schurig, 2000a, 2000b]. On-line sample preparation and enantioseparation mechanisms should create a harmonized system, especially in chiral EKC and ITP where the chiral selector is not immobilized as it is in CEC, and all possible mutual negative interferences have to be carefully eliminated. demands because CE. utilizing additional

*Automatization and miniaturization.* As one moves toward small sample volumes, sample handling and preparation steps become more difficult and the concentration step is therefore preferably done on-line instead of off-line. The microfluidic devices, such as microchips, can provide several additional advantages over electromigration techniques performed in capillary format. The heat dissipation is much better in chip format compared

**3.2 On-line sample preparation techniques based on electrophoretic principles** 

chiral analysis by CE.

preconcentration).

Several reviews have been published recently, focusing on the on-line sample preconcentration CE techniques based on electrophoretic principles leading to the compression of a long sample plug into a narrow band with a high concentration of analytes, so called stacking techniques [Lin C.H. & Kaneta, 2004; Gebauer et al., 2009, 2011; Silva, 2009; Ryan et al., 2009; Malá et al., 2007, 2011; Urbánek et al., 2003]. Among the latest detailed review papers are those by Lin and Kaneta [Lin C.H. & Kaneta, 2004], Simpson et al. [Simpson et al., 2008], Breadmore [Breadmore, 2007], Breadmore et al. [Breadmore et al., 2009] and Malá et al. [Malá et al., 2011], including research articles gathered in the period 2000-2011. The papers provide fundamentals and applications of basic types of on-line sample preconcentration techniques. McKibbin and Terabe [Britz-McKibbin & Terabe, 2003] emphasized on-line preconcentration strategies for trace analysis of metabolites by CE. Ruiz and Marina [Ruiz & Marina, 2006] reviewed sensitive

Advanced Sample Preparation 53

The stacking procedures, described in section 3.2.1, are modifications of the basic zone electrophoretic and/or isotachophoretic and/or isoelectric focusing separation modes (Figure 2.2). The stacking procedures are based on increasing analyte mass in its zone during the electromigration process via electromigration effects, enhancing sensitivity in this way. In all the cases, the key requirements are that there is an electrophoretic component in the preconcentration mechanism and that the analytes concentrate on a boundary through a change in velocity. Then we can recognize (i) field-strength-induced changes in velocity (field-enhanced sample stacking [Kim J.B. & Terabe, 2003; Quirino & Terabe, 2000; Chien & Burgi, 1992; Weiss et al., 2001], isotachophoresis and transient isotachophoresis [Beckers & Boček, 2000; Schwer et al., 1993; Shihabi, 2002]), and (ii) chemically induced changes in velocity (dynamic pH junction [Britz-McKibbin & Chen, 2000; Aebersold & Morrison, 1990; Kim J.B. et al., 2003], sweeping [Kitagawa et al., 2006; Palmer et al., 2001; Quirino & Terabe, 1998; Quirino & Terabe, 1999; Quirino et al., 2000]), see Table 3.2. In addition to these techniques, the counter-flow gradient focusing [Shackman & Ross, 2007], electrocapture [Horáková et al., 2007] and many others can be considered as the techniques based on a combination of field-strength and chemically induced changes in velocity offering new and interesting possibilities in on-line sample preparation (mainly

Some of the stacking techniques (and their combinations) can provide, besides (i) the preconcentration, other benefits, such as (ii) an effective sample purification isolating solute (group of solutes) from undesired matrix constituents [Simpson et al., 2008] or they can be combined with (iii) the chemical reaction of the analyte(s) [Ptolemy et al., 2005, 2006], in this way simplifying the overall analytical procedure. On the other hand, great attention must be paid to the selection of the type of chiral selector as its charge can interfere with a sample

preparation technique that employs electrophoretic principles.

to that in a capillary and therefore higher electric fields can be applied across channels of the microchip. This fact enables, along with a considerably reduced length of channels, significant shortening of separation time. The sample and reagent consumption is markedly reduced in microchannels. Hence, the chiral MCE can provide a unique possibility of ultraspeed enantiomeric separations of microscale sample amounts. On the other hand, the efficiency is often weak due to the shape of the chips and the quality of the injection [Guihen et al., 2009]. Until now, electrophoresis, rather than chromatography, has been the primary principle applied in microchip separations for several reasons as follows: (i) the materials typically used for microchips can barely withstand the high pressures applied for chromatographic separations; (ii) the packing of the chromatographic particles into the channels without voids can be difficult, (iii) the performance of chromatography decreases with decreased column length [Preinerstorfer et al., 2009]. Nevertheless, both electrophoretic [Gong & Hauser, 2006; Piehl et al., 2004; Belder et al., 2006], as well as electrochromatographic, modes [Weng, et al. 2006] are applicable in microchip format. Nowadays, the on-line coupling of sample treatment systems to CE or MCE are of great interest because it allows the automatization of the analytical process (from sample preparation to data treatment), which is a current trend in analytical chemistry.

Thus, the importance of on-line sample preparation is pronounced when ultratrace analytes (ng/mL and less) are determined in minute amounts (l, g and less) of samples with complex matrices (variable in qualitative and quantitative composition). This is a common situation in the analyses of drugs, their metabolites and biomarkers in biological samples where preconcentration of analytes, elimination of matrix interferents, and minimized sample handling is necessary to obtain relevant analytical results. Mikuš and Maráková [Mikuš & Maráková, 2010] recently provided a review on the chiral capillary electrophoresis with on-line sample preparation. The latest panorama of sample preparation methods for animal/human and plant samples given by Chen et al. [Chen Y., 2008] has been composed from almost 500 references, highlighting some promising methods which have fast developed over recent years and giving a somewhat brief introduction on most of the well-developed methods, including on-line stacking methods in CE. Wu [Wu X.Z., 2003] illustrated new approaches to sample preparation for CE and Kataoka [Kataoka, 2003] highlighted their utilization in clinical and pharmaceutical analysis. In addition to these rather general reviews, specialized reviews are included at the beginning of each following subsection. These highlight the role of electromigration effects and interactions in on-line sample preparation, and summarize basic electrophoretic (stacking) and non electrophoretic (mostly chromatographic and extraction) on-line sample preparation techniques aimed at preconcentration and purification of complex samples in chiral pharmaceutical and biomedical research. Application examples are listed in Table 3.1 showing the chiral pharmaceutical and biomedical analyses supported by on-line sample preparation procedures described in this chapter. On the other hand, conventional off-line sample preparation techniques, such as solid-phase extraction, liquid-phase extraction, solid-liquid extraction and dialysis (included in the above mentioned reviews), are not discussed here.

to that in a capillary and therefore higher electric fields can be applied across channels of the microchip. This fact enables, along with a considerably reduced length of channels, significant shortening of separation time. The sample and reagent consumption is markedly reduced in microchannels. Hence, the chiral MCE can provide a unique possibility of ultraspeed enantiomeric separations of microscale sample amounts. On the other hand, the efficiency is often weak due to the shape of the chips and the quality of the injection [Guihen et al., 2009]. Until now, electrophoresis, rather than chromatography, has been the primary principle applied in microchip separations for several reasons as follows: (i) the materials typically used for microchips can barely withstand the high pressures applied for chromatographic separations; (ii) the packing of the chromatographic particles into the channels without voids can be difficult, (iii) the performance of chromatography decreases with decreased column length [Preinerstorfer et al., 2009]. Nevertheless, both electrophoretic [Gong & Hauser, 2006; Piehl et al., 2004; Belder et al., 2006], as well as electrochromatographic, modes [Weng, et al. 2006] are applicable in microchip format. Nowadays, the on-line coupling of sample treatment systems to CE or MCE are of great interest because it allows the automatization of the analytical process (from sample preparation to data treatment), which is a current trend in

Thus, the importance of on-line sample preparation is pronounced when ultratrace analytes (ng/mL and less) are determined in minute amounts (l, g and less) of samples with complex matrices (variable in qualitative and quantitative composition). This is a common situation in the analyses of drugs, their metabolites and biomarkers in biological samples where preconcentration of analytes, elimination of matrix interferents, and minimized sample handling is necessary to obtain relevant analytical results. Mikuš and Maráková [Mikuš & Maráková, 2010] recently provided a review on the chiral capillary electrophoresis with on-line sample preparation. The latest panorama of sample preparation methods for animal/human and plant samples given by Chen et al. [Chen Y., 2008] has been composed from almost 500 references, highlighting some promising methods which have fast developed over recent years and giving a somewhat brief introduction on most of the well-developed methods, including on-line stacking methods in CE. Wu [Wu X.Z., 2003] illustrated new approaches to sample preparation for CE and Kataoka [Kataoka, 2003] highlighted their utilization in clinical and pharmaceutical analysis. In addition to these rather general reviews, specialized reviews are included at the beginning of each following subsection. These highlight the role of electromigration effects and interactions in on-line sample preparation, and summarize basic electrophoretic (stacking) and non electrophoretic (mostly chromatographic and extraction) on-line sample preparation techniques aimed at preconcentration and purification of complex samples in chiral pharmaceutical and biomedical research. Application examples are listed in Table 3.1 showing the chiral pharmaceutical and biomedical analyses supported by on-line sample preparation procedures described in this chapter. On the other hand, conventional off-line sample preparation techniques, such as solid-phase extraction, liquid-phase extraction, solid-liquid extraction and dialysis

(included in the above mentioned reviews), are not discussed here.

analytical chemistry.

### **3.2 On-line sample preparation techniques based on electrophoretic principles**

Several reviews have been published recently, focusing on the on-line sample preconcentration CE techniques based on electrophoretic principles leading to the compression of a long sample plug into a narrow band with a high concentration of analytes, so called stacking techniques [Lin C.H. & Kaneta, 2004; Gebauer et al., 2009, 2011; Silva, 2009; Ryan et al., 2009; Malá et al., 2007, 2011; Urbánek et al., 2003]. Among the latest detailed review papers are those by Lin and Kaneta [Lin C.H. & Kaneta, 2004], Simpson et al. [Simpson et al., 2008], Breadmore [Breadmore, 2007], Breadmore et al. [Breadmore et al., 2009] and Malá et al. [Malá et al., 2011], including research articles gathered in the period 2000-2011. The papers provide fundamentals and applications of basic types of on-line sample preconcentration techniques. McKibbin and Terabe [Britz-McKibbin & Terabe, 2003] emphasized on-line preconcentration strategies for trace analysis of metabolites by CE. Ruiz and Marina [Ruiz & Marina, 2006] reviewed sensitive chiral analysis by CE. to

The stacking procedures, described in section 3.2.1, are modifications of the basic zone electrophoretic and/or isotachophoretic and/or isoelectric focusing separation modes (Figure 2.2). The stacking procedures are based on increasing analyte mass in its zone during the electromigration process via electromigration effects, enhancing sensitivity in this way. In all the cases, the key requirements are that there is an electrophoretic component in the preconcentration mechanism and that the analytes concentrate on a boundary through a change in velocity. Then we can recognize (i) field-strength-induced changes in velocity (field-enhanced sample stacking [Kim J.B. & Terabe, 2003; Quirino & Terabe, 2000; Chien & Burgi, 1992; Weiss et al., 2001], isotachophoresis and transient isotachophoresis [Beckers & Boček, 2000; Schwer et al., 1993; Shihabi, 2002]), and (ii) chemically induced changes in velocity (dynamic pH junction [Britz-McKibbin & Chen, 2000; Aebersold & Morrison, 1990; Kim J.B. et al., 2003], sweeping [Kitagawa et al., 2006; Palmer et al., 2001; Quirino & Terabe, 1998; Quirino & Terabe, 1999; Quirino et al., 2000]), see Table 3.2. In addition to these techniques, the counter-flow gradient focusing [Shackman & Ross, 2007], electrocapture [Horáková et al., 2007] and many others can be considered as the techniques based on a combination of field-strength and chemically induced changes in velocity offering new and interesting possibilities in on-line sample preparation (mainly preconcentration).

Some of the stacking techniques (and their combinations) can provide, besides (i) the preconcentration, other benefits, such as (ii) an effective sample purification isolating solute (group of solutes) from undesired matrix constituents [Simpson et al., 2008] or they can be combined with (iii) the chemical reaction of the analyte(s) [Ptolemy et al., 2005, 2006], in this way simplifying the overall analytical procedure. On the other hand, great attention must be paid to the selection of the type of chiral selector as its charge can interfere with a sample preparation technique that employs electrophoretic principles.


New

Plasma HP-


UV, MS, Spiked real

Grard et al.,

2002

Thormann,

2006

Hedeland

Baldacci &

samples

preconcentration (180x) 3 ng/mL

EH/off, SPE/off clean-up, UV, ESI-MS Metabolic study

adrenoreceptors

antagonists

Lorazepam and its

Urine HP-


micelles SWP/on preconcentration

EK + tITP/on

DAD, 0.2 %

Enantiomeric

S-timolol,

1R,2S-0.033%

pharmaceuticals

purity testing of et al., 2007

ephedrine

LIF, 10-10 M Biomedical

study et al., 2007

Kirschner

preconcentration

metabolites

S-timolol, 1R,2S-

ephedrine solution Infusion Ketopinic acid and

diisoproylidenek

etogulonic acid

(NACE)

S-


preconcentration

derivatization

0.2x10


study

UV, 2 and

Biomedical

Ptolemy et

al., 2005

+ preconcentration, DPJ/on

preconcentration (100x)

EH, SPCD/on derivatization

UV, 0.4-

Biomedical

Ptolemy et

al., 2006a

study

Spiked samples

Balss et al.,

2004

0.6x10-6 M

DAD

(baclofen)

DAD,

Enantiomeric

Marák et al.,

2008

2.5 ng/mL

UV,

0.6x10-9 M model samples

DAD,

Spiked real

samples

3-5 ng/mL

et al., 1999

Fang, H.F.

et al., 2006b

Petersson

pharmaceuticals

Spiked real and

purity testing of

acid), LIF

(glutamic

+ preconcentration


(1200x)

Bacterial OPA/NAC HD + SPCD/on

CBI-amino acids Squirrel brain

samples

Muramic acid and

diaminopimelic acid cultures

Amino acids Bacterial

Advanced Sample Preparation 55

cultures

urine

DL-Glutamic acid,

baclofen

Dexbrompheniramin

Pharmaceutical

CE-


preconcentration (~102)

+ clean-up

preparations

Terbutaline Plasma, water

DM-


preconcentration (7000x)


preconcentration

matrices

derivatives

Ephedrine Urine

e

OPA/NAC,

CD



Analyte Sample Chiral selectora

Fenoprofen and

amino acid derivates

Cimaterol,

clenbuterol,

terbutaline

Amlodipine Plasma and

HPCD

EME/ preconcentration

3 ng/mL Nojavan &

Fakhari, 2010

Choi et al.,

2009

(124x)

SDME/in preconcentration

UV,

Spiked real

samples

0.5 ng/mL

DAD,

Spiked real

Li H. et al.,

2008

Mikuš et al.,

2006a

samples

Spiked real

samples and

metabolic study

0.92 ng/mL

UV, 1.1-4.8

ng/mL

(~103) + clean-up

urine

Human urine (+)-(18-crown-6)-

tetracarboxylic

acid

Trihexyphenidyl Serum CMCD LLE/off clean-up, FESS/on

preconcentration (490x)

preconcentration (~102)

+ clean-up

Human urine CE--CD ITP-EKC; ITP/on

Primary amine

drugs

(amphetamine)

Pheniramine and its

metabolites,

dimethindene,

dioxopromethazine

Pheniramine and its

Human urine CE--CD ITP-EKC; ITP/on

preconcentration (~102)

DAD, 5.2-

Metabolic study

Marák et al.,

2007

6.8 ng/mL

DAD, 9.3-

Pharmacokinetic

Mikuš et al.,

2008a

10.4 ng/mL

DAD,

Spiked real

Denola et al.,

2007

samples

<300

ng/mL

UV, LIF,

Spiked real

Cho et al.,

2004

samples

3-4x10-6 M

study

+ clean-up

preconcentration (~102)

+ clean-up

preconcentration (100-1000x)

Serum -CD PP/off clean-up, LVSS/on

Amlodipine Human urine HP--CD ITP-EKC; ITP/on

metabolites

Metoxamine,

carvedilol,

terbutaline,

metaproterenol

Gemifloxacin Urine, saliva CWE EK clean-up, MCE with

coupled channels/on cleanup

River water vancomycin LVSEP-ASEI/on

Human urine -CD t-ITP and FESI/on

Sample preparation: t

main purposeb

preconcentration (~103)

preconcentration (250x)

ype and

Detection

Application Ref.

and LOD

UV, 0.38-

Spiked real

Wang Z.Y.

et al., 2011

et al., 2011

samples

1 ng/mL Huang L.

2.10 ng/mL


FMOC-carnitine Synthetic

HDM-

CD FI/on derivatization DAD,

5x10-6 M

Enantiomeric

Mardones

model samples

FESS/on preconcentration, derivatization/off LIF, 0.5x10-9 M Spiked model samples Mechref & El Rassi, 1997

DAD,

purity testing in et al., 1999

samples

Fenoxy acid

Water matrices Octyl-


maltopyranoside

CD LLE/off clean-up, FESS/on

preconcentration 25-75

ng/mL

analysis

al., 1998

Forensic Tagliaro et

herbicides

Ephedrine,

Hair

amphetamine and

related compounds

blockers Serum CM

CD

3-carboxyadipic acid Minerals Vancomycin FESS/on preconcentration

Sulindac and its

Advanced Sample Preparation 57

Plasma DM-


UV,

Pharmakokineti

c study et al., 2006

1-3x10-7 M

LIF,

Environmental

Asami &

Imura, 2006

Britz-McKibbin

analysis

Spiked real

samples,

biomedical

et al., 2003b

study

Spiked real

Ibrahim et

al., 2007

samples

2x10-9 M

LIF,

4x10-9 M

preconcentration (500x)


preconcentration

preconcentration -CD, SDS -

metabolites

Glufosinate River water

sample

Flavins Bacterial cell


SWP/on preconcentration

(60x)

extracts,

plasma and

urine

Propiconazol Grape HP-

Triadimenol Methanol

HS-


CD


SRMP/on preconcentration

(10x) 0.8-3.8

ng/mL

matrices

micelles


SPE/off clean-up, SWP/on

DAD,

ng/mL

DAD,

Spiked model

Otsuka et al.,

2003

samples

preconcentration (100x) 90-100

preconcentration (5-25x)

10-50

Spiked real

Huang L.

samples et al., 2008

ng/mL

DAD,

Environmental

Castro-

analysis

Pu

y

ana et al.,

2008

Chen, Y.L.


(1000x) 10

PP/off clean-up, FESI/on UV,


(1

*R*, 2

pseudoephedrine,

(1

*R*, 2

(1

*S*, 2

pseudoephedrine,

(*S*)-(+)-

methamphetamine

Aspartate Tissue samples

CD

Microdialysis/on clean-up,

LIF,

Biomedical

Thompson

et al., 1999

study

9x10-7 M

(Daspartate)

Derivatization/on

from rats

D-serin + other

Tissue

CD, HP-

Microdialysis/on clean-up,

LIF,

Biomedical

O'Brien et al.,

2003

study

270x10-9 M;

LIF (sheath

flow

detection

cell),

21x10-9 M

Amperomet

Pharmacokinetic

Hadwiger

et al., 1996

study

ric

detection,

0.6 ng/mL

UV,

Pálmarsdóttir

et al., 1996

2.5x10-10 M

Isoproterenol Intravenous

M--CD

Microdialysis/on clean-up,

pH-mediated sample

stacking/on preconcentration

dialysed

samples

Bambuterol Plasma SLM/on + MLC/on clean-up,

double stacking/on

preconcentration (40 000x)

SLM/on + MLC/on clean-up,

UV,

Spiked real and

Pálmarsdóttir

et al., 1995

model samples

5x10-9 M

double stacking/on

preconcentration (400x)

Terbutaline, but also

Plasma,

Alkyl- or

hydroxyalkyl--

CDs

synthetic

samples

brompheniramine,

propranolol,

ephedrine

Derivatization/on

CD, HP--CD

homogenates

neurotransmitters

*S*)-

*S*)-ephedrine,

*R*)-

Urine, serum -CD

LLE/on clean-up, FESI/on

DAD, 0.15

Spiked real

Fang, H.F.

et al., 2006a

samples

to 0.25

ng/mL

preconcentration (3800x)


 of

a Or derivatization agent creating diastereomeric products. 

b An on- or off-line mode is given behind slash, preconcentration factor or amount of removed interfering compounds are given in brackets

 ITP = isotachophoresis, tITP = transient isotachophoresis, EKC = electrokinetic chromatography, CFGF = counter-flow gradient focusing, CWE = crown ether, NACE = non-aqueous capillary electrophoresis, MCE = electrophoresis on microchip, SPCD = incapillary sample preconcentration with chemical derivatization, OPA/NAC= *ortho*-phthalaldehyde/ *N*-acetyl l-cysteine, S-CD = sulphated--CD, HS--CD = highly sulphated--CD, M--CD = methyl--CD, DM--CD = dimethyl--CD, CM--CD = carboxymethyl--CD, CE--CD = carboxyethyl--CD, HP--CD = hydroxypropyl--CD, HP--CD = hydroxypropyl--CD, HDM-- CD = heptakisdimethyl--CD, EH = enzymatic hydrolysis, HD = hydrodynamic injection, EK = electrokinetic injection, FI= flow injection, DPJ= dynamic pH junction, SRMP=stacking with reverse migrating phase, FESS = field-enhanced sample stacking, LVSS = large volume sample stacking, FESI= field-enhanced sample injection, SPE = solid-phase extraction, LLE = liquid-liquid extraction, LPME = liquid-phase microextraction, LVSEP-ASEI=large volume sample stacking with EOF as a pump plus anion-selective exhaustive injection, PP = protein precipitation, CME = centrifuge microextraction, SDME = single drop microextraction, SLM = supported liquid membrane technique, DAD = diode array detection, UV-ultraviolet (absorbance detection), LIF = laser induced fluorescent detection, MS = mass spectrometry, LOD = limit of detection, SDS = sodium dodecylsulphate, FMOC = 9-fluorenylmethyl chloroformate, EME=electro membrane extraction. 

Technique Principle Characteristic features

FESI

LVSS,

NSM,

solution zone is concentrated at the boundar

of a high-conductivit

conductivity ratio of these two zones (c

Gseparation solution / Gsample solution). Water plu

injected prior to the injection of the anal

FESI.

stacked

g is

preconcentration and purificati

NSM ~10-fold; LVSS ~100-fold; FESI >1000-fold

Limitations. Limited applications {charged analytes,

separations only cations or anions in one run, samples with

a low-conductivity matrix (can be overcome b

mediation)}. Requirements {removin

(LVSS), EOF suppression}. Reproducibility with FESI (injecting

amount is biased by electrophoretic mobility, time depletion of

the analyte in the sample solution).

ges. Flexible. Directl

significant matrix ion (acting as a leader). Applicable for low-

conductivity (diluted) samples

ion (inserted in capillar

y applicable to samples with a

with the aid of added leading

y or in sample). Direct anal

Precipitation agent (e.g., acetonitrile) serves simultaneousl

g ion (transient pseudoisotachophoresis). Sample

purification aspect (neutral and oppositel

compounds do not interfere). Preconcentration: more than

Limitations. Preconcentration on

run. Analysis of neutral compounds is not possible.

ly cations or anions in one

three orders.

y char

ged

y as

soluble supernatants of precipitated proteinic samples.

ysis of water

tITP Temporary ITP action bein

Advanced Sample Preparation 59

zone electrophoretic regime. Anal

of each separated zone is ad

determined b

justed to that

y the concentration of the ion in

terminatin

the foregoing neighbour zone (canalyte ion ~ c

leading ion) resulting in the preconcentration of

diluted sample zones.

zones of electrolyte solution. The concentration

(terminating ion) electrophoretic mobility

between the highest (leading ion) and lowest

g replaced later by

Advanta

yte mi

grates

y pH

g of sample matrix

electrokinetical injection (FESI) with simultaneous

on aspect. Preconcentrations:

amount of charged analyte from the sample with

yte in

~

zone. Concentration effect is related to

y separation solution

y

Advantages. Eas

y to perform b

concentrations or electrical conductivities of the sample and

separation solutions. Possibility to inject a selectivel

y simpl

y optimizing

y lar

ge

Analyte from a low-conductivity sample

FESS:

Electrophoretic (stacking)


*N*-acetyl l-cysteine, S-CD =

Tryptophan Urine CD ITP-EKC; ITP/on preconcentration + clean-up (~99%) UV, 1.5 ng/mL Spiked real samples Danková et al., 1999 Isoxyzolylpenicilines

Milk HPCD

LLE/off clean-up, LVSS/on preconcentration DAD, 2 ng/mL Food analysis Zhu et al., 2003 Clenbuterol Acetic acid matrices DMCD

tITP/on preconcentration UV, 10-6 M Spiked model samples Toussaint et al., 2000 Table 3.1. Chiral CE determinations of biologically active compounds in various biological matrices employing advanced (on-line) sample preparation.

a Or derivatization agent creating diastereomeric products.

brackets

b An on- or off-line mode is given behind slash, preconcentration factor or amount of removed interfering compounds are given in

ITP = isotachophoresis, tITP = transient isotachophoresis, EKC = electrokinetic chromatography, CFGF = counter-flow gradient

focusing, CWE = crown ether, NACE = non-aqueous capillary electrophoresis, MCE = electrophoresis on microchip, SPCD = in-

sulphated--CD, HS--CD = highly sulphated--CD, M--CD = methyl--CD, DM--CD = dimethyl--CD, CM--CD =

carboxymethyl--CD, CE--CD = carboxyethyl--CD, HP--CD = hydroxypropyl--CD, HP--CD = hydroxypropyl--CD, HDM--

CD = heptakisdimethyl--CD, EH = enzymatic hydrolysis, HD = hydrodynamic injection, EK = electrokinetic injection, FI= flow

injection, DPJ= dynamic pH junction, SRMP=stacking with reverse migrating phase, FESS = field-enhanced sample stacking, LVSS =

large volume sample stacking, FESI= field-enhanced sample injection, SPE = solid-phase extraction, LLE = liquid-liquid extraction,

LPME = liquid-phase microextraction, LVSEP-ASEI=large volume sample stacking with EOF as a pump plus anion-selective

exhaustive injection, PP = protein precipitation, CME = centrifuge microextraction, SDME = single drop microextraction, SLM =

supported liquid membrane technique, DAD = diode array detection, UV-ultraviolet (absorbance detection), LIF = laser induced

fluorescent detection, MS = mass spectrometry, LOD = limit of detection, SDS = sodium dodecylsulphate, FMOC = 9-fluorenylmethyl

chloroformate, EME=electro membrane extraction.

capillary sample preconcentration with chemical derivatization, OPA/NAC= *ortho*-phthalaldehyde/


EOF

microchannel, or an

Electroosmotic velocit

surface vs. liquid.

y other fluid conduit.

y is dependent of the

charge in the electrical double layer, solid

vs. analytes vs. selector)

separabilit

Limitations. An EOF velocit

and char

systems is lower than the s

very pronounced in e.g., CEC s

in the hydrodynamically closed electrophoretic systems.

Chromatography The separation is

analytes between the stationar

phase accordin

mechanism differentiates mi

of the anal

chemical principles.

gration velocities

that eliminates an

ytes. Separations are based on

these analytes with the stationary phase. This

based on a distribution of the

Advanta

ges. An alternative separation mechanism to the

system. The stationary phase serves as an immobilized selector

y interferences of the selector with the

detection. CEC represents a single column chromato

implementation with EOF as a driving force.

Limitations. The chromato

graphic s

concentration of the analytes. Therefore, a chomato

pretreated sample must be further treated before the

terms of the instrumentation as well as the compactibilit

the electrophoretic and chromato

phase vs. electrolyte).

graphic s

ystems (mobile

y of

chromatographic techniques with electrophoresis is difficult in

electrophoretic separation. A coupling of the pressure driven

ystems do not provide a

graphically

graphic

electrophoretic one that enables to increase selectivity of the

y and mobile

g to the different interactions of

Nonelectrophoretic

Advanced Sample Preparation 61

y depends of a solid surface area

ge, therefore a precise control of the EOF velocit

be difficult. Hence, the reproducibility of the EOF based

ystems with suppressed EOF. It is

ystems. EOF cannot be applied

y can

separation efficiency.

implementation of the countercurrent migration effects (EOF

for an enhancement of the

y. A flat EOF profile is favourable for a hi

g

h

charged compounds in one run is possible too. EOF enables an

porous material, capillary tube, membrane,

liquid induced by an applied potential across a

on their char

ge. Therefore anal

possible, and separation of neutral, positivel

ysis of neutral compounds is

y and ne

gatively

Electroosmotic flow (EOF) is the motion of

Advantages. EOF enables movement of compounds re

gardless


DPJ Based on si

gnificant chan

velocities of the analytes between different pH

narrow range of p

acidic or basic analytes.

Limitations. Electrokinetic dispersion pla

in this technique. Only for ionizable analytes (weak

acids/bases). Analysis of neutral compounds is not possible.

ys an important role

values (BGE solution vs. sample solution with

a suppressor of analyte ionization). Lon

of sample zone is

graduall

from the BGE solution and the anal

ionized in the neutralized zone. The anal

focused at the neutralization boundar

the neutralization of the sample zone.

ytes are picked up and accumulated by

the appropriate pseudostationary phase (e.

micelles) that penetrates the micelle free

sample zone. The length of the analyte zone

after sweep is inversel

retention factor of the analyte (the ratio of the

yte amounts present in micelle and in

anal

solution), Lsweep ~ 1 / k.

y proportional to the

g.,

homogeneous electric field stren

Advantages. Electrokinetic dispersion is minimized by

gth throu

capillary. High concentration efficiency, possibly up to 5000-fold

(according to the affinity analyte vs. pseudophase), or even up

to several million-fold (when the length to the detection point is

very short and very narrow detection window is emplo

in MCE), or even more (with electrokinetic injection). Suitable

for charged as well as neutral analytes. Sample purification

aspect (migrate only analytes interacting with sweeping

pseudophase), useful for complex matrices. Combinable with

nearly every other stacking mechanism.

Limitations. The narrow analyte zone created by sweeping tends

to broaden quickl

(separation efficiency si

capillar

pseudophase.

CFGF Counter-flow gradient focusing (CFGF)

methods can focus the analyte into a

g via simultaneous actin

y and the opposite bulk

solution flow so that the total velocity (the sum

of both velocities) is equal to zero at a unique

point (characteristic for the analyte).

g an

section in this table.

versatile wa

concentrated plu

electrophoretic velocit

y). Application is limited b

Advantages. CFGF approaches are a potentiall

y in which to simultaneousl

separate charged and neutral analytes. See also Pressure flow

Limitations. Specific electrolytes must be used in some of these

techniques. See also Pressure flow section in this table.

gnificantl

y decreases with the len

y availabilit

y of appropriate

y simple and

y concentrate and

gth of

y accordin

g to the diffusion alon

g the capillary

yed, e.

g.,

ghout the whole

SWP Anal

SWP

y titrated b

y the ion

yte will be

yte is

y during

g plug

ges in electrophoretic

Advantages. Selective concentration of the analytes havin

*K*a. Useful for the concentration of weakly

g a


Pressure flow Driving pressure or counter-pressure based

flows are provided simpl

they are controlled electronicall

important tool also in CFGF.

y b

y the pumps and

Advanta

enablin

ges. One of the basic tools in the advanced s

g a controlled transfer of solutes in various se

ystems

gments of

y. This is

the h

yphenated s

ystems (e.

electrophoretic). A counter-flow enables to increase virtually

the effective length of the capillary or channel and, b

increase the separability.

the reproducibility of the anal

instrumentation (pumps and auxiliaries) must be implemented

into the anal

Advanced Sample Preparation 63

Table 3.2. The most important electrophoretic and nonelectrophoretic techniques and tools applicable in electrophoresis.

FESS = field-enhanced sample stacking; NSM = normal stacking mode; LVSS = large volume sample stacking; FESI = field-

ent isotachophoresis; DPJ = dynamic pH junction; SWP = sweeping; EOF = electroosmotic

enhanced sample injection; tITP = transi

flow; CEC = capillary; SPE = solid-phase extraction; LLE = liquid-liquid extraction

ytical s

connections) decrease the separation efficiency.

ystem. Dead volumes (tubin

g and

ysis. An additional

Limitations. Fluctuations of these non selective flows decrease

y that,

g., electrophoretic and non


enhanced sample injection; tITP = transi ent isotachophoresis; DPJ = dynamic pH junction; SWP = sweeping; EOF = electroosmotic

flow; CEC = capillary; SPE = solid-phase extraction; LLE = liquid-liquid extraction

62 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Extraction The analytes are trapped on/in a suitable

solid/liquid-phase according to the affinities.

The distribution constant of the analytes in the

sample vs. extraction phase system determines

the extraction recovery. Separations are based

on chemical principles. The concentration

factor depends on the ratio of sample volume :

preconcentration method. The extraction selectivity can be

easily modified by the type of extractor and a specific or more

universal extraction can then be reached. Limitations. The whole analytical procedure is complex

(extraction requires conditioning, loading/sorption, washing,

labelling, if necessary, elution/desorption). In the single

capillary systems, the entire sample must pass through the

capillary, which can lead to fouling or clogging of the

separation capillary and significant decreasing of

reproducibility of the analyses when particularly problematic

samples (like biological ones) are used. A hyphenation of the

extraction techniques with electrophoresis is difficult in terms

of the instrumentation, however, SPE can be implemented to

Advantages. Microdialysis is a widely accepted sampling and

infusion technique frequently used to sample small molecules

from complex, often biological, matrices. Filtration can be

applied directly in electrophoresis avoiding a sample

collection. Both techniques can concentrate large molecules,

and purify large as well as small molecules. Limitations. Do not concentrate small molecules. In the

microdialysis, the minimum volume required for analysis

often determines the rate at which the dialysate can be

sampled. The whole analytical procedure is complex (dialysis

requires preconcentration of the analyte from dialysate). Single

column filtration systems are relatively simple but less flexible

and versatile. On the other hand, a hyphenation of the

membrane techniques with electrophoresis is more difficult in

terms of the instrumentation.

the electrophoresis easier than LLE. Membrane techniques Separations are based on physical principles,

i.e., size exclusion. In the microdialysis or

filtration, small molecules are able to diffuse

across the membrane while large molecules,

such as proteins and cell fragments, are

excluded. This is the principle of the sample

cleanup. Moreover, large molecules are

preconcentrated on the membrane in this way.

extractor volume. Advantages. An important sample purification (clean-up) and

### **3.2.1 Single column (in-capillary) electrophoretic techniques**

### **3.2.1.1 Field-enhanced sample stacking**

The field-enhanced sample stacking (FESS) is easy to perform in a zone electrophoretic mode (Figure 2.2a) by simply optimizing the sample solution and the separation solution, mainly in their concentrations or electrical conductivities, to constitute different electrical field strengths between the two solutions. To perform preconcentration, the discontinuous zones having different electrical conductivities (*G*) must be constructed along the capillary axis. The analyte from a low-conductivity sample solution zone is concentrated at the boundary of a high-conductivity separation solution zone. The concentration effect is related to the conductivity ratio of these two zones according to Equation 3.1 [Simpson et al., 2008]:

$$\mathcal{C}\_{\text{stacked}} = \mathcal{C}\_{\text{t}\text{\textquotedblleft}\text{\textquotedblright}\textquotedblright}\mathcal{Y} \tag{3.1}$$

done for other stacking techniques with the zone electrophoresis separation step (see the techniques described below). Depending on the charge of chiral selector and polarity of electric field the normal or reversed stacking EKC models can be created that offer different separation selectivities. For example, depending on the charge of micelles and the polarity of the electric field, the normal (Figure 3.3a) or reversed stacking MEKC

Advanced Sample Preparation 65

(ii) Large volume sample stacking (LVSS) requires removing the sample matrix. This can be done with polarity switching where the analysed anion has higher velocity in the opposite direction than the velocity of EOF, or without polarity switching where EOF has to be suppressed as the cations migrate oppositely to EOF. It is possible to separate only cations or anions in one run and more than 100-fold concentration can be achieved.

Figure 3.1. Effect of sample matrix on LVSS. Effect of NaCl on the stacking and enantioseparation of the analytes studied. Analytes 0.01 mg/mL in 20% ACN with [NaCl] in A = 0%, B = 0.1%, C = 0.2% and D = 0.4% w/v. Injection length: 20% capillary volume.

models (Figure 3.3b) can be created.

Reprinted from ref. [Denola et al., 2007].

For schematic diagrams of the LVSS model see Figure 3.4.

where *cstacked* is the concentration of the analyte concentrated by FESS, *cinjected* is the concentration of the analyte in the sample solution injected, is the ratio of the electrophoretic velocities of the ions between the two discontinuous zones (sample zone and BGS, *1* and *2*) having different conductivities (the ratios can be written for velocities, *v1/v2*, intensities of electric field, *E1/E2*, as well as resistivities, *1/<sup>2</sup>*). From this it is obvious that the sample solution should be prepared in a low-conductivity matrix (as in other stacking modes) that is limiting in terms of application, see Figure 3.1. This technique requires suppressing EOF as EOF velocity is also proportional to the field strength and mixing of the two solutions can occur at the boundary, causing broadening of the focused zone. In practice, we often stack in the presence of EOF, albeit at lesser enrichment. In fact, at high pH, stacking occurs for anions at the rear zone boundary as opposed to the front boundary for cations at low pH. The suppression (reduction) of EOF is more important when electrokinetic injection is employed. The FESS approach is useful for charged analytes, while neutral analytes cannot be directly concentrated (with the exception of their charged complexes) [Kim J.B. & Terabe, 2003]. The FESS can be carried out in the CZE or MEKC mode where the stacking action (see below) is the same while the final separation is based on the CZE or MEKC principles [Lin C.H., 2004; Kim J.B. & Terabe, 2003]. In fact, when a chiral separation is required, the final step following stacking procedure must be principally based on the EKC (chiral MEKC, CDEKC, etc.) mechanism, see chapter 2.

Several techniques have been developed by utilizing the FESS for sample preconcentration [Lin C.H. & Kaneta, 2004; Quirino & Terabe, 2000; Chien R.L. & Burgi, 1992].

(i) Normal stacking mode is the simplest mode, it requires a rather low amount of the injected sample, the EOF must be suppressed and ca. 10-fold concentration can be easily achieved. For schematic diagrams of the normal FESS model carried out in CZE or MEKC modes see Figure 3.2 and Figure 3.3a, respectively. The schemes of FESS with CZE or MEKC separation in Figure 3.2 and Figure 3.3 can be easily modified to the chiral EKC regime implementing chiral selector into the system. Then, the final step after stacking is a chiral EKC separation (i.e., FESS-EKC). For example, the scheme of FESS with MEKC separation in Figure 3.3 can be changed from the achiral (with achiral micelles) to the chiral one (with chiral micelles). Such chiral modification can also be schematic

The field-enhanced sample stacking (FESS) is easy to perform in a zone electrophoretic mode (Figure 2.2a) by simply optimizing the sample solution and the separation solution, mainly in their concentrations or electrical conductivities, to constitute different electrical field strengths between the two solutions. To perform preconcentration, the discontinuous zones having different electrical conductivities (*G*) must be constructed along the capillary axis. The analyte from a low-conductivity sample solution zone is concentrated at the boundary of a high-conductivity separation solution zone. The concentration effect is related to the conductivity ratio of these two zones according to Equation 3.1 [Simpson et al., 2008]:

where *cstacked* is the concentration of the analyte concentrated by FESS, *cinjected* is the

*cc*

electrophoretic velocities of the ions between the two discontinuous zones (sample zone and BGS, *1* and *2*) having different conductivities (the ratios can be written for velocities, *v1/v2*,

sample solution should be prepared in a low-conductivity matrix (as in other stacking modes) that is limiting in terms of application, see Figure 3.1. This technique requires suppressing EOF as EOF velocity is also proportional to the field strength and mixing of the two solutions can occur at the boundary, causing broadening of the focused zone. In practice, we often stack in the presence of EOF, albeit at lesser enrichment. In fact, at high pH, stacking occurs for anions at the rear zone boundary as opposed to the front boundary for cations at low pH. The suppression (reduction) of EOF is more important when electrokinetic injection is employed. The FESS approach is useful for charged analytes, while neutral analytes cannot be directly concentrated (with the exception of their charged complexes) [Kim J.B. & Terabe, 2003]. The FESS can be carried out in the CZE or MEKC mode where the stacking action (see below) is the same while the final separation is based on the CZE or MEKC principles [Lin C.H., 2004; Kim J.B. & Terabe, 2003]. In fact, when a chiral separation is required, the final step following stacking procedure must be principally

Several techniques have been developed by utilizing the FESS for sample preconcentration

(i) Normal stacking mode is the simplest mode, it requires a rather low amount of the injected sample, the EOF must be suppressed and ca. 10-fold concentration can be easily achieved. For schematic diagrams of the normal FESS model carried out in CZE or MEKC modes see Figure 3.2 and Figure 3.3a, respectively. The schemes of FESS with CZE or MEKC separation in Figure 3.2 and Figure 3.3 can be easily modified to the chiral EKC regime implementing chiral selector into the system. Then, the final step after stacking is a chiral EKC separation (i.e., FESS-EKC). For example, the scheme of FESS with MEKC separation in Figure 3.3 can be changed from the achiral (with achiral micelles) to the chiral one (with chiral micelles). Such chiral modification can also be

.

> *1/*

*stacked injected c c* 3.1

is the ratio of the

*<sup>2</sup>*). From this it is obvious that the

**3.2.1 Single column (in-capillary) electrophoretic techniques** 

concentration of the analyte in the sample solution injected,

based on the EKC (chiral MEKC, CDEKC, etc.) mechanism, see chapter 2.

[Lin C.H. & Kaneta, 2004; Quirino & Terabe, 2000; Chien R.L. & Burgi, 1992].

intensities of electric field, *E1/E2*, as well as resistivities,

**3.2.1.1 Field-enhanced sample stacking** 

done for other stacking techniques with the zone electrophoresis separation step (see the techniques described below). Depending on the charge of chiral selector and polarity of electric field the normal or reversed stacking EKC models can be created that offer different separation selectivities. For example, depending on the charge of micelles and the polarity of the electric field, the normal (Figure 3.3a) or reversed stacking MEKC models (Figure 3.3b) can be created.

(ii) Large volume sample stacking (LVSS) requires removing the sample matrix. This can be done with polarity switching where the analysed anion has higher velocity in the opposite direction than the velocity of EOF, or without polarity switching where EOF has to be suppressed as the cations migrate oppositely to EOF. It is possible to separate only cations or anions in one run and more than 100-fold concentration can be achieved. For schematic diagrams of the LVSS model see Figure 3.4.

Figure 3.1. Effect of sample matrix on LVSS. Effect of NaCl on the stacking and enantioseparation of the analytes studied. Analytes 0.01 mg/mL in 20% ACN with [NaCl] in A = 0%, B = 0.1%, C = 0.2% and D = 0.4% w/v. Injection length: 20% capillary volume. Reprinted from ref. [Denola et al., 2007].

Figure 3.2. Schematic diagrams of the normal FESS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and a high positive voltage is applied; (B) focusing of the analytes occurs near the boundaries between the sample zone and the BGS because of its mobility changes; (C) stacked analytes migrate and are separated by the CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

(iii) Field-enhanced sample injection (FESI) is based on the injection of a short (usually 2- 3 mm, i.e., ca. 0.5% of the effective capillary length) water plug prior to the electrokinetical injection of the analyte. The EOF has to be reduced. Injection of a larger amount of the sample than in (ii) is possible. The injected amount is biased by the electrophoretic mobility. More than 1000-fold concentration is possible, however, injection reproducibility is influenced by depletion of the analyte in the sample solution. Selective injection is given by the charge of the sample constituents.

Figure 3.3. Schematic diagrams of stacking MEKC models. (a) A normal stacking MEKC model. (A) The sample is dissolved in a low-conductivity buffer, BGS, consisting of SDS to form the micelles; after the background and sample solution are injected, respectively, a positive voltage is applied; (B) the SDS micelles from the inlet enter the sample zone and then permit the analytes to migrate and become stacked; (C) then the SDS-analytes are separated by the MEKC mode. (b) A reversed stacking MEKC model. (A) The sample and BGS are prepared as described in Figure 3.3aA but a negative polarity is applied; (B) the EOF moves toward the inlet, the anionic analytes move toward the outlet and stack at one side of the boundary; (C) the electrophoretic current reaches approximately 95–99% of its original value, the polarity is quickly returned to positive, reversing the EOF; (D) then the SDS-analytes are separated by the MEKC mode. Reprinted from ref. [Lin C.H. & Kaneta,

Advanced Sample Preparation 67

2004].

It should be highlighted that some of the above mentioned electrophoretic on-line preconcentration approaches can be utilized simultaneously also for on-line sample purification. For example, the use of the FESI technique can eliminate potential interfering compounds from the matrix via electrokinetic injection being selective to the sample constituents according to their charge. The selectivity of the electrokinetic injection can be easily influenced by the pH of the sample solution (adjusted by an appropriate buffer if necessary) where the solute of interest has unconditionally to be ionized while the ionization of potential interfering compounds should be suppressed. The FESI can be used for the exhaustive sample injection, e.g., almost all of the ions in a sample can be injected when the volume is small. This is a real advantage to the off-line solvent extraction, evaporation to dryness and redissolving of the analyte in a small amount of dilute buffer.

Figure 3.2. Schematic diagrams of the normal FESS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and a high positive voltage is applied; (B) focusing of the analytes occurs near the boundaries between the sample zone and the BGS because of its mobility changes; (C) stacked analytes migrate and are separated by the CZE mode.

(iii) Field-enhanced sample injection (FESI) is based on the injection of a short (usually 2- 3 mm, i.e., ca. 0.5% of the effective capillary length) water plug prior to the electrokinetical injection of the analyte. The EOF has to be reduced. Injection of a larger amount of the sample than in (ii) is possible. The injected amount is biased by the electrophoretic mobility. More than 1000-fold concentration is possible, however, injection reproducibility is influenced by depletion of the analyte in the sample solution.

It should be highlighted that some of the above mentioned electrophoretic on-line preconcentration approaches can be utilized simultaneously also for on-line sample purification. For example, the use of the FESI technique can eliminate potential interfering compounds from the matrix via electrokinetic injection being selective to the sample constituents according to their charge. The selectivity of the electrokinetic injection can be easily influenced by the pH of the sample solution (adjusted by an appropriate buffer if necessary) where the solute of interest has unconditionally to be ionized while the ionization of potential interfering compounds should be suppressed. The FESI can be used for the exhaustive sample injection, e.g., almost all of the ions in a sample can be injected when the volume is small. This is a real advantage to the off-line solvent extraction, evaporation to dryness and redissolving of the analyte in a small

electrokinetic

Selective injection is given by the charge of the sample constituents.

Reprinted from ref. [Lin C.H. & Kaneta, 2004].

amount of dilute buffer.

Figure 3.3. Schematic diagrams of stacking MEKC models. (a) A normal stacking MEKC model. (A) The sample is dissolved in a low-conductivity buffer, BGS, consisting of SDS to form the micelles; after the background and sample solution are injected, respectively, a positive voltage is applied; (B) the SDS micelles from the inlet enter the sample zone and then permit the analytes to migrate and become stacked; (C) then the SDS-analytes are separated by the MEKC mode. (b) A reversed stacking MEKC model. (A) The sample and BGS are prepared as described in Figure 3.3aA but a negative polarity is applied; (B) the EOF moves toward the inlet, the anionic analytes move toward the outlet and stack at one side of the boundary; (C) the electrophoretic current reaches approximately 95–99% of its original value, the polarity is quickly returned to positive, reversing the EOF; (D) then the SDS-analytes are separated by the MEKC mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

high-conductivity BGS. A (chiral) separation by the zone electrophoresis mode then occurs. This is a simple and attractive approach for high-conductivity samples, which is growing in popularity. Another strategy introducing a sample with a higher ionic strength than the running buffer has been proposed by Landers et al. [Palmer et al., 1999]. For example, using sodium cholate as the pseudostationary phase and simply adding sodium chloride (or other ions) to the sample matrix, a reasonable enhancement in sensitivity has been achieved for a series of corticosteroids. In the first step the micelles, and not the analytes, are stacked at the boundary between the sample and buffer zone. Subsequently, the analytes migrating with the EOF are enriched in the zone with the high micelle concentration as the micelles are negatively charged and migrate in the opposite direction (Figure 3.6). Summarizing, the stacking effect is dependent on the affinity of the analytes to the micelles that were stacked before, at the boundary between the sample (with increased conductivity, e.g., by adding salt) and the buffer zone. This approach is attractive due to its robustness towards other sample constituents. Although these last two procedures have not been used for chiral analysis so far, their potential in this field is apparent. Other strategies for samples with high ionic strengths, also used in the chiral field, are based on appropriate combinations of different stacking techniques as briefly discussed, inter alia, in

Advanced Sample Preparation 69

Figure 3.5. Schematic diagrams of a pH-mediated stacking model. (A) The capillary is conditioned with a high-conductivity BGS, the cationic analytes dissolved in a lowconductivity buffer are electrokinetic injected into the capillary, and then a plug of strong acid is also electrokinetically injected; (B) a positive separation voltage is applied; (C) the strong acid titrates the sample solution to create a neutral zone causing the ions to migrate faster and become stacked; (D) the subsequent separation occurs by the CZE mode.

Reprinted from ref. [Lin C.H. & Kaneta, 2004].

section 3.2.1.5.

Figure 3.4. Schematic diagrams of the LVSS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and then a high negative voltage is applied (EOF is toward the inlet); (B) the anionic analytes move toward the detection end (outlet) and stack at one side of the boundary, whereas the cations and neutral species move and exit the capillary at the injection end (inlet); (C) the electrophoretic current is carefully monitored until it reaches approximately 95–99% of its original value, and the polarity is then quickly returned to positive (EOF is reversed); (D) the following separation occurs by CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

However, the FESS methods suffer generally from poor applicability to real samples. (i) For optimal precision, hydrodynamic injection is preferred since we start with the correct number of ions in the capillary. On the other hand, sample depletion and injection reproducibility could be a problem when making more than one electrokinetic injection from a vial. In a practical sense, there is a balance between the degree of enrichment and the stability of the system. This is a significant problem when performing extreme enrichment from real samples. (ii) Poor applicability to real samples is primarily because the complicated (high-conductivity) matrices increase the conductivity of the sample and reduce the efficiency of stacking. This is typical for urine or blood samples which contain salts as matrix macroconstituents. Therefore, they are usually applied in conjunction with off-line sample pretreatment. A pH-mediated FESS method was introduced by the group of Lunte and is an indirect way of changing a high-conductivity sample into a low-conductivity sample to allow field-enhanced sample stacking [Weiss et al., 2001]. Schematic diagrams are shown in Figure 3.5. In the initial step, the sample is prepared in a high-ionic strength medium and is electrokinetically injected into the capillary. Then, a plug of strong acid is electrophoretically injected and a positive separation voltage is applied. The strong acid titrates the sample solution to create a neutral zone (a high-resistance zone). Thus, a proportionally greater field will develop across the neutral zone, causing the ions to migrate faster. As a result, the analytes are stacked at the boundary between the low-conductivity zone (prepared by on-line titration of the sample solution zone, e.g., by a strong acid plug) and the at to

Figure 3.4. Schematic diagrams of the LVSS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and then a high negative voltage is applied (EOF is toward the inlet); (B) the anionic analytes move toward the detection end (outlet) and stack at one side of the boundary, whereas the cations and neutral species move and exit the capillary at the injection end (inlet); (C) the electrophoretic current is carefully monitored until it reaches approximately 95–99% of its original value, and the polarity is then quickly returned to positive (EOF is reversed); (D) the

following separation occurs by CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

However, the FESS methods suffer generally from poor applicability to real samples. (i) For optimal precision, hydrodynamic injection is preferred since we start with the correct number of ions in the capillary. On the other hand, sample depletion and injection reproducibility could be a problem when making more than one electrokinetic injection from a vial. In a practical sense, there is a balance between the degree of enrichment and the stability of the system. This is a significant problem when performing extreme enrichment from real samples. (ii) Poor applicability to real samples is primarily because the complicated (high-conductivity) matrices increase the conductivity of the sample and reduce the efficiency of stacking. This is typical for urine or blood samples which contain salts as matrix macroconstituents. Therefore, they are usually applied in conjunction with off-line sample pretreatment. A pH-mediated FESS method was introduced by the group of Lunte and is an indirect way of changing a high-conductivity sample into a low-conductivity sample to allow field-enhanced sample stacking [Weiss et al., 2001]. Schematic diagrams are shown in Figure 3.5. In the initial step, the sample is prepared in a high-ionic strength medium and is electrokinetically injected into the capillary. Then, a plug of strong acid is electrophoretically injected and a positive separation voltage is applied. The strong acid titrates the sample solution to create a neutral zone (a high-resistance zone). Thus, a proportionally greater field will develop across the neutral zone, causing the ions to migrate faster. As a result, the analytes are stacked at the boundary between the low-conductivity zone (prepared by on-line titration of the sample solution zone, e.g., by a strong acid plug) and the high-conductivity BGS. A (chiral) separation by the zone electrophoresis mode then occurs. This is a simple and attractive approach for high-conductivity samples, which is growing in popularity. Another strategy introducing a sample with a higher ionic strength than the running buffer has been proposed by Landers et al. [Palmer et al., 1999]. For example, using sodium cholate as the pseudostationary phase and simply adding sodium chloride (or other ions) to the sample matrix, a reasonable enhancement in sensitivity has been achieved for a series of corticosteroids. In the first step the micelles, and not the analytes, are stacked at the boundary between the sample and buffer zone. Subsequently, the analytes migrating with the EOF are enriched in the zone with the high micelle concentration as the micelles are negatively charged and migrate in the opposite direction (Figure 3.6). Summarizing, the stacking effect is dependent on the affinity of the analytes to the micelles that were stacked before, at the boundary between the sample (with increased conductivity, e.g., by adding salt) and the buffer zone. This approach is attractive due to its robustness towards other sample constituents. Although these last two procedures have not been used for chiral analysis so far, their potential in this field is apparent. Other strategies for samples with high ionic strengths, also used in the chiral field, are based on appropriate combinations of different stacking techniques as briefly discussed, inter alia, in section 3.2.1.5.

Figure 3.5. Schematic diagrams of a pH-mediated stacking model. (A) The capillary is conditioned with a high-conductivity BGS, the cationic analytes dissolved in a lowconductivity buffer are electrokinetic injected into the capillary, and then a plug of strong acid is also electrokinetically injected; (B) a positive separation voltage is applied; (C) the strong acid titrates the sample solution to create a neutral zone causing the ions to migrate faster and become stacked; (D) the subsequent separation occurs by the CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004]. migrate

The principle of ITP can be applied to preconcentration in zone electrophoresis techniques (CZE, EKC), which is termed as transient ITP (tITP) [Beckers & Boček, 2000; Schwer et al., 1993]. Schematic diagrams of tITP are shown in Figure 3.7. There are several modifications in tITP to perform sample preconcentration, such as the following configurations (i) a low electrophoretic mobility background solution (e.g., borate) is inside the capillary, a leading electrolyte (e.g., chloride ion) solution is injected as the first plug, the sample solution is injected as the second plug, sample solution zones are preconcentrated by the tITP mechanism and further migrate as zones in zone electrophoresis, (ii) as in (i) but the sample and leading solutions are mixed and then injected as one plug. It is particularly suited to the analysis of trace components in samples with a significant matrix ion in which that matrix ion functions as a leader. This is called 'sample self-stacking' in the literature. (iii) Transient pseudoisotachophoresis is a modification of previous tITP modes, based on the addition of a water miscible organic solvent (e.g., acetonitrile, acetone, methanol, 2-propanol) serving as a pseudoterminating ion [Shihabi, 2002]. Of course, it cannot be a terminating ion in the true sense. The organic solvent serves as a zero mobility low-conductivity zone. This approach is attractive for the analysis of biological fluids because acetonitrile added 2:1 to the sample (making 66% acetonitrile) is used for protein precipitation and it is therefore possible to

Advanced Sample Preparation 71

directly inject the supernatant and achieve a 20-30 fold improvement in sensitivity.

Figure 3.7. Schematic diagrams of a tITP model for cations. (A) The capillary is conditioned with a BGS, the leading electrolyte and sample solution, and terminating electrolyte are then injected in turn - a high positive voltage is also applied; (B) concentration of the analytes occurs between the leading and the terminating ions during tITP migration; (C) the concentrated analyte zones are separated by the CZE mode. Reprinted from ref. [Lin C.H. &

Like FESI, tITP and ITP is adversely affected by changes in sample conductivity. The ITP and tITP techniques are applicable to charged analytes only and simultaneous analysis of

ref.

Kaneta, 2004].

Figure 3.6. Mechanism for stacking in MEKC with a high ionic strength matrix. Reprinted from ref. [Palmer et al., 1999].

### **3.2.1.2 Isotachophoresis and transient isotachophoresis**

Analytes are separated in isotachophoresis (ITP) as adjoining successive zones in the order of decreasing electrophoretic mobilities, migrating between the leading (the highest electrophoretic mobility) and terminating (the lowest electrophoretic mobility) electrolyte solution zones (Figure 2.2b). In the presence of voltage applied, the concentration of each separated zone is automatically adjusted to that determined by the concentration of the ion in the foregoing neighbour zone according to Equation 3.2 [Simpson et al., 2008]:

$$\mathcal{L}\_A = \frac{\mathcal{L}\_L \mu\_A (\mu\_L + \mu\_Q)}{\mu\_L (\mu\_A + \mu\_Q)} \tag{3.2}$$

resulting in the preconcentration of diluted sample zones. In this equation, *L* > *<sup>A</sup>* and *cA*, *cL* are concentrations of analyte ion, *A*, in the adjoining zone to the leading ion, *L*, zone and *A*, *L*, *<sup>Q</sup>* electrophoretic mobility of *A*, that of *L*, and that of the counter ion *Q* (assumed the same ion for *A* and *L*), respectively. ITP is a flexible and powerful method for on-line concentration. It is directly applicable to samples with a significant matrix ion and can also provide very high enrichment factors for low-conductivity samples.

Figure 3.6. Mechanism for stacking in MEKC with a high ionic strength matrix. Reprinted

Analytes are separated in isotachophoresis (ITP) as adjoining successive zones in the order of decreasing electrophoretic mobilities, migrating between the leading (the highest electrophoretic mobility) and terminating (the lowest electrophoretic mobility) electrolyte solution zones (Figure 2.2b). In the presence of voltage applied, the concentration of each separated zone is automatically adjusted to that determined by the concentration of the ion

3.2

*L* > 

*<sup>A</sup>* and *cA*, *cL*

*A*,

 *QAL QLAL*

*<sup>Q</sup>* electrophoretic mobility of *A*, that of *L*, and that of the counter ion *Q* (assumed the same ion for *A* and *L*), respectively. ITP is a flexible and powerful method for on-line concentration. It is directly applicable to samples with a significant matrix ion and can also

in the foregoing neighbour zone according to Equation 3.2 [Simpson et al., 2008]:

*c*

are concentrations of analyte ion, *A*, in the adjoining zone to the leading ion, *L*, zone and

*A*

resulting in the preconcentration of diluted sample zones. In this equation,

*c*

provide very high enrichment factors for low-conductivity samples.

from ref. [Palmer et al., 1999].

*L*, 

**3.2.1.2 Isotachophoresis and transient isotachophoresis** 

The principle of ITP can be applied to preconcentration in zone electrophoresis techniques (CZE, EKC), which is termed as transient ITP (tITP) [Beckers & Boček, 2000; Schwer et al., 1993]. Schematic diagrams of tITP are shown in Figure 3.7. There are several modifications in tITP to perform sample preconcentration, such as the following configurations (i) a low electrophoretic mobility background solution (e.g., borate) is inside the capillary, a leading electrolyte (e.g., chloride ion) solution is injected as the first plug, the sample solution is injected as the second plug, sample solution zones are preconcentrated by the tITP mechanism and further migrate as zones in zone electrophoresis, (ii) as in (i) but the sample and leading solutions are mixed and then injected as one plug. It is particularly suited to the analysis of trace components in samples with a significant matrix ion in which that matrix ion functions as a leader. This is called 'sample self-stacking' in the literature. (iii) Transient pseudoisotachophoresis is a modification of previous tITP modes, based on the addition of a water miscible organic solvent (e.g., acetonitrile, acetone, methanol, 2-propanol) serving as a pseudoterminating ion [Shihabi, 2002]. Of course, it cannot be a terminating ion in the true sense. The organic solvent serves as a zero mobility low-conductivity zone. This approach is attractive for the analysis of biological fluids because acetonitrile added 2:1 to the sample (making 66% acetonitrile) is used for protein precipitation and it is therefore possible to directly inject the supernatant and achieve a 20-30 fold improvement in sensitivity.

Figure 3.7. Schematic diagrams of a tITP model for cations. (A) The capillary is conditioned with a BGS, the leading electrolyte and sample solution, and terminating electrolyte are then injected in turn - a high positive voltage is also applied; (B) concentration of the analytes occurs between the leading and the terminating ions during tITP migration; (C) the concentrated analyte zones are separated by the CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

Like FESI, tITP and ITP is adversely affected by changes in sample conductivity. The ITP and tITP techniques are applicable to charged analytes only and simultaneous analysis of

solution from the cathodic side and the analyte will be ionized in the neutralized zone. The negatively ionized analyte will migrate toward the anode, but if it enters into the acidic sample zone it will be protonated again to neutral and stop the electrophoretic migration. Thus, the weakly acidic analyte can be focused at the neutralization boundary during the on-line neutralization of the sample zone. Two modes of the DPJ can be used for the analyte preconcentration, namely the normal (Figure 3.8a) and reversed (Figure 3.8b) DPJ model, providing conditions for preconcentration of anionic or cationic analytes, respectively. This focusing technique is different from sample stacking since the conductivity of the sample matrix is not of great importance; it can be less than or greater than that of the BGS. The electrokinetic dispersion plays an important role in this technique [Kim J.B. et al., 2003]. The DPJ technique can selectively concentrate analytes having a narrow range of p*K*a and it is useful for the concentration of weakly acidic or basic analytes, as well as zwitterionic analytes. This technique has been used in the chiral field in appropriate combinations with

Advanced Sample Preparation 73

Sweeping (SWP) can be defined as a phenomenon whereby analytes are picked up and accumulated by the appropriate pseudostationary phase (micelles, microemulsions, charged cyclodextrins) that penetrates the sample zone. The homogeneous electric field strength is given by the similar conductivity of the nonmicelle sample zone (provided by the addition of salt if needed) and the running micelle solution. Homogeneous electric field strength is assumed throughout the whole capillary under SWP conditions different from fieldenhanced stacking techniques. The schematic diagrams of SWP preconcentration are shown in Figure 3.9. The length of the analyte zone after sweep, *Lsweep*, is inversely proportional to the retention factor of the analyte, *k* (the ratio of the analyte amounts present in micelle and

> 

3.3

1

*<sup>k</sup> sweep LL inj* <sup>1</sup>

*LsweepLinj*

where *Linj* is the length of the sample solution injected. It is apparent that the analyte having a higher *k* value is more efficiently concentrated. According to the affinity of the analyte to the pseudostationary phase the concentration efficiency can be very high, possibly up to 5000-fold. The narrow analyte zone created by SWP, however, tends to broaden quickly according to the diffusion along the capillary (CE) or channel (MCE). Therefore, when the length to the detection point is very short and a very narrow detection window is employed, very high concentration efficiency, up to several million-fold, can be observed [Kitagawa et al., 2006]. Electrokinetic injection in combination with SWP has made it possible to inject a large volume of samples, and, by that, additionally increase sensitivity [Palmer et al., 2001]. However, commonly used SWP pseudophases do not provide specific interactions with the analyte and many interfering compounds from the sample matrices with affinity to the pseudophase can be swept (enriched) together with the analyte. Therefore, a sample cleanup technique along with the SWP technique can be required to introduce into analytical protocol too, especially when analysing biological samples [Hempel, 2000; Baldacci &

other on-line preconcentration techniques, see section 3.2.1.5.

in solution), according to Equation 3.3. [Simpson et al., 2008]:

**3.2.1.4 Sweeping** 

Thormann, 2006].

oppositely charged analytes is not possible. On the other hand, this limitation can be understood as an advantage in terms of selective removal of neutral or oppositely charged sample matrix constituents.

Figure 3.8. Schematic diagrams of dynamic pH junction models. (a) The normal and (b) reversed dynamic pH junction models. (a) (A) The capillary is filled with a high pH-BGS and a section of sample solution (prepared in a lower-pH buffer); (B) a high positive voltage is applied, resulting in a discontinuous electrolyte zone; (C) the anionic analytes are focused on the boundary of the pH junction; (D) separation of the analytes occurs by the CZE mode. (b) (A) The capillary is filled with BGS (prepared in a higher-pH buffer containing CTAC) and sample solution (prepared in a lower-pH matrix); (B) a negative voltage is applied because of the addition of CTAC the EOF moves toward the outlet; the cationic analytes move toward the inlet and change to neutral at the rear boundary due to the change in pH; (C) separation of the analytes occurs by the CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004]. (B)

The dynamic pH junction (DPJ) technique utilizes significant changes in ionization states of the analytes or electrophoretic velocities between different pH values [Britz-McKibbin & Chen, 2000; Aebersold & Morrison, 1990]. Possible configuration is as follows: the capillary is filled with an alkaline background solution (high concentration, low electrophoretic mobility), a long plug of a weakly acidic analyte dissolved in an acidic matrix (high concentration, high mobility) is injected, a positive voltage is applied at the injection end, the acidic sample zone is gradually titrated by the hydroxide ion in the alkaline background

oppositely charged analytes is not possible. On the other hand, this limitation can be understood as an advantage in terms of selective removal of neutral or oppositely charged

Figure 3.8. Schematic diagrams of dynamic pH junction models. (a) The normal and (b) reversed dynamic pH junction models. (a) (A) The capillary is filled with a high pH-BGS and a section of sample solution (prepared in a lower-pH buffer); (B) a high positive voltage is applied, resulting in a discontinuous electrolyte zone; (C) the anionic analytes are focused on the boundary of the pH junction; (D) separation of the analytes occurs by the CZE mode. (b) (A) The capillary is filled with BGS (prepared in a higher-pH buffer containing CTAC) and sample solution (prepared in a lower-pH matrix); (B) a negative voltage is applied because of the addition of CTAC the EOF moves toward the outlet; the cationic analytes move toward the inlet and change to neutral at the rear boundary due to the change in pH; (C) separation of the

The dynamic pH junction (DPJ) technique utilizes significant changes in ionization states of the analytes or electrophoretic velocities between different pH values [Britz-McKibbin & Chen, 2000; Aebersold & Morrison, 1990]. Possible configuration is as follows: the capillary is filled with an alkaline background solution (high concentration, low electrophoretic mobility), a long plug of a weakly acidic analyte dissolved in an acidic matrix (high concentration, high mobility) is injected, a positive voltage is applied at the injection end, the acidic sample zone is gradually titrated by the hydroxide ion in the alkaline background

analytes occurs by the CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

sample matrix constituents.

**3.2.1.3 Dynamic pH junction** 

solution from the cathodic side and the analyte will be ionized in the neutralized zone. The negatively ionized analyte will migrate toward the anode, but if it enters into the acidic sample zone it will be protonated again to neutral and stop the electrophoretic migration. Thus, the weakly acidic analyte can be focused at the neutralization boundary during the on-line neutralization of the sample zone. Two modes of the DPJ can be used for the analyte preconcentration, namely the normal (Figure 3.8a) and reversed (Figure 3.8b) DPJ model, providing conditions for preconcentration of anionic or cationic analytes, respectively. This focusing technique is different from sample stacking since the conductivity of the sample matrix is not of great importance; it can be less than or greater than that of the BGS. The electrokinetic dispersion plays an important role in this technique [Kim J.B. et al., 2003]. The DPJ technique can selectively concentrate analytes having a narrow range of p*K*a and it is useful for the concentration of weakly acidic or basic analytes, as well as zwitterionic analytes. This technique has been used in the chiral field in appropriate combinations with other on-line preconcentration techniques, see section 3.2.1.5.

### **3.2.1.4 Sweeping**

Sweeping (SWP) can be defined as a phenomenon whereby analytes are picked up and accumulated by the appropriate pseudostationary phase (micelles, microemulsions, charged cyclodextrins) that penetrates the sample zone. The homogeneous electric field strength is given by the similar conductivity of the nonmicelle sample zone (provided by the addition of salt if needed) and the running micelle solution. Homogeneous electric field strength is assumed throughout the whole capillary under SWP conditions different from fieldenhanced stacking techniques. The schematic diagrams of SWP preconcentration are shown in Figure 3.9. The length of the analyte zone after sweep, *Lsweep*, is inversely proportional to the retention factor of the analyte, *k* (the ratio of the analyte amounts present in micelle and in solution), according to Equation 3.3. [Simpson et al., 2008]:

$$L\_{sweep} = L\_{\text{inj}} \left[ \frac{1}{\left(1 + k\right)} \right] \tag{3.3}$$

where *Linj* is the length of the sample solution injected. It is apparent that the analyte having a higher *k* value is more efficiently concentrated. According to the affinity of the analyte to the pseudostationary phase the concentration efficiency can be very high, possibly up to 5000-fold. The narrow analyte zone created by SWP, however, tends to broaden quickly according to the diffusion along the capillary (CE) or channel (MCE). Therefore, when the length to the detection point is very short and a very narrow detection window is employed, very high concentration efficiency, up to several million-fold, can be observed [Kitagawa et al., 2006]. Electrokinetic injection in combination with SWP has made it possible to inject a large volume of samples, and, by that, additionally increase sensitivity [Palmer et al., 2001]. However, commonly used SWP pseudophases do not provide specific interactions with the analyte and many interfering compounds from the sample matrices with affinity to the pseudophase can be swept (enriched) together with the analyte. Therefore, a sample cleanup technique along with the SWP technique can be required to introduce into analytical protocol too, especially when analysing biological samples [Hempel, 2000; Baldacci & Thormann, 2006]. focused with

**3.2.1.5 Single column combinations of sample preparation techniques** 

can be found in the cited references or in ref. [Lin, C.H. & Kaneta, 2004]):

A combination of two of the electrophoretic on-line sample preconcentration techniques described in sections 3.2.1.1-3.2.1.4 can be more efficient in increasing detection sensitivity, preconcentration selectivity and spreading application capabilities (i.e., analysis of a wider range of analytes, differing by their charge, polarity, etc., in one experiment), according to particular

Advanced Sample Preparation 75

Most of these combinations have been used only in achiral analyses so far, however, their potential in the chiral field is apparent. As it is believed that they will appear in the chiral field in the near future, it is useful to list them (corresponding schemes of these techniques

(i) FESI + tITP (i.e., electrokinetic supercharging, EKS): 3000-fold concentration, analysis of ionic analytes in diluted samples [Fang L. et al., 2006]. Compared with conventional electrokinetic injection, the enhancement factors can be greatly improved, e.g., to be 250-

(ii) pH-mediated FESS + DPJ: only for ionisable analytes [Chou Y.W. et al., 2008]. This technique has been developed to enhance analyte focusing for CE for the analysis of physiological samples. The process results in ultra-narrow peak widths and no dilution of the sample to lower ionic strength is necessary. In comparison with normal base stacking and electrokinetic injection, mass loading capacity can be increased with this technique without degradation in peak shape and resolution is dramatically improved. (iii) tITP + DPJ: 50-fold increase in sensitivity, nM LODs of cationic metabolites [Hou J. et al., 2007]. The authors depict three major transitions experimentally observed in the process: (a) initial tryptophan (Trp) focusing at the back end of the sample-BGE boundary, (b) partial focusing of Trp with residual peak fronting and (c) complete focusing of Trp within the original sample plug. The authors demonstrated that CE could serve as an effective preconcentrator, desalter and separator prior to ESI-MS, while providing additional qualitative information for unambiguous identification among isobaric and isomeric metabolites. The proposed strategy is particularly relevant for characterizing yet unknown biologically relevant metabolites that are not readily

(iv) FESI + SWP: up to million-fold increase in sensitivity [Rudaz et al., 2005; Servais et al., 2006]. This technique has two modes. (a) The cation selective exhaustive injection (CSEI) + SWP. The CSEI-SWP-MEKC method provides for a more sensitive detection than sweeping and is sufficiently flexible to offer the potential for achieving an increase in the detection limit of more than 100 000-fold, for positively chargeable analytes [Rudaz et al., 2005]. (b) Alternatively, anion selective exhaustive injection (ASEI)-SWP-MEKC, using a cationic surfactant, offers under optimized conditions an approximately 1000 to 6000-fold improvement in LODs of negatively chargeable analytes [Servais et al., 2006]. Applications of the CSEI-SWP-MEKC method: (methamphetamine, ketamine, morphine and codeine in hair, LODs of 50–200 pg/mg hair) [Huang Y.S. et al., 2003], (amphetamine, methamphetamine and hydroxymethamphetamine in urine, LODs of 15–20 ppm) [Theurillat & Thormann, 2008], (amphetamine, methamphetamine and methylenedioxymethamphetamine, 6–8 ppt LODs, several 1000-fold improvement in detection sensitivity compared with typical

demands. It can also overcome some limitations of these methods when used separately.

**based on electrophoretic principles** 

fold [Huang, L. et al., 2011].

synthesized or commercially available.

injection) [Tsimachidis et al., 2008; Theurillat et al., 2007].

Figure 3.9. Schematic diagrams of a reversed sweeping MEKC model. (A) The BGS consists of a surfactant (for example, SDS, a negatively charged surfactant) and electrolytes to form a micelle buffer, but the samples are dissolved in a nonmicelle buffer; (B) after the injection of the BGS and the sample solution, a negative polarity is applied to power the CE separation; (C) the cations and anions move toward the inlet and outlet , respectively, and anionic SDS micelles enter the capillary sweeping the analytes; (D) the analytes are completely swept by SDS, the subsequent separation occurs by the MEKC mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004]. of nM

SWP was originally developed for the on-line concentration of neutral analytes for MEKC separation [Quirino & Terabe, 1998]. The SWP approach is suitable also for charged analytes, regardless of the charge of the analyte and the direction of EOF. Strong Coulomb interactions between oppositely charged analytes and the pseudophase can be reflected in a higher k value and, subsequently, more efficient preconcentration [Quirino & Terabe, 1999; Quirino et al., 2000]. SWP is a perfect combination for nearly every other stacking mechanism. Its complementarity so far has really only been used for sensitivity enhancement, but it could also be used to selectively enrich specific analytes. This will become increasingly important as more integrated methods are developed for the analysis of more complex samples with less off-line sample pretreatment (clean-up).

Figure 3.9. Schematic diagrams of a reversed sweeping MEKC model. (A) The BGS consists of a surfactant (for example, SDS, a negatively charged surfactant) and electrolytes to form a micelle buffer, but the samples are dissolved in a nonmicelle buffer; (B) after the injection of the BGS and the sample solution, a negative polarity is applied to power the CE separation; (C) the cations and anions move toward the inlet and outlet , respectively, and anionic SDS micelles enter the capillary sweeping the analytes; (D) the analytes are completely swept by SDS, the subsequent separation occurs by the MEKC mode. Reprinted from ref. [Lin C.H. &

SWP was originally developed for the on-line concentration of neutral analytes for MEKC separation [Quirino & Terabe, 1998]. The SWP approach is suitable also for charged analytes, regardless of the charge of the analyte and the direction of EOF. Strong Coulomb interactions between oppositely charged analytes and the pseudophase can be reflected in a higher k value and, subsequently, more efficient preconcentration [Quirino & Terabe, 1999; Quirino et al., 2000]. SWP is a perfect combination for nearly every other stacking mechanism. Its complementarity so far has really only been used for sensitivity enhancement, but it could also be used to selectively enrich specific analytes. This will become increasingly important as more integrated methods are developed for the analysis

of more complex samples with less off-line sample pretreatment (clean-up).

Kaneta, 2004].

### **3.2.1.5 Single column combinations of sample preparation techniques based on electrophoretic principles**

A combination of two of the electrophoretic on-line sample preconcentration techniques described in sections 3.2.1.1-3.2.1.4 can be more efficient in increasing detection sensitivity, preconcentration selectivity and spreading application capabilities (i.e., analysis of a wider range of analytes, differing by their charge, polarity, etc., in one experiment), according to particular demands. It can also overcome some limitations of these methods when used separately.

Most of these combinations have been used only in achiral analyses so far, however, their potential in the chiral field is apparent. As it is believed that they will appear in the chiral field in the near future, it is useful to list them (corresponding schemes of these techniques can be found in the cited references or in ref. [Lin, C.H. & Kaneta, 2004]):

(i) FESI + tITP (i.e., electrokinetic supercharging, EKS): 3000-fold concentration, analysis of ionic analytes in diluted samples [Fang L. et al., 2006]. Compared with conventional electrokinetic injection, the enhancement factors can be greatly improved, e.g., to be 250 fold [Huang, L. et al., 2011].

(ii) pH-mediated FESS + DPJ: only for ionisable analytes [Chou Y.W. et al., 2008]. This technique has been developed to enhance analyte focusing for CE for the analysis of physiological samples. The process results in ultra-narrow peak widths and no dilution of the sample to lower ionic strength is necessary. In comparison with normal base stacking and electrokinetic injection, mass loading capacity can be increased with this technique without degradation in peak shape and resolution is dramatically improved.

(iii) tITP + DPJ: 50-fold increase in sensitivity, nM LODs of cationic metabolites [Hou J. et al., 2007]. The authors depict three major transitions experimentally observed in the process: (a) initial tryptophan (Trp) focusing at the back end of the sample-BGE boundary, (b) partial focusing of Trp with residual peak fronting and (c) complete focusing of Trp within the original sample plug. The authors demonstrated that CE could serve as an effective preconcentrator, desalter and separator prior to ESI-MS, while providing additional qualitative information for unambiguous identification among isobaric and isomeric metabolites. The proposed strategy is particularly relevant for characterizing yet unknown biologically relevant metabolites that are not readily synthesized or commercially available.

(iv) FESI + SWP: up to million-fold increase in sensitivity [Rudaz et al., 2005; Servais et al., 2006]. This technique has two modes. (a) The cation selective exhaustive injection (CSEI) + SWP. The CSEI-SWP-MEKC method provides for a more sensitive detection than sweeping and is sufficiently flexible to offer the potential for achieving an increase in the detection limit of more than 100 000-fold, for positively chargeable analytes [Rudaz et al., 2005]. (b) Alternatively, anion selective exhaustive injection (ASEI)-SWP-MEKC, using a cationic surfactant, offers under optimized conditions an approximately 1000 to 6000-fold improvement in LODs of negatively chargeable analytes [Servais et al., 2006]. Applications of the CSEI-SWP-MEKC method: (methamphetamine, ketamine, morphine and codeine in hair, LODs of 50–200 pg/mg hair) [Huang Y.S. et al., 2003], (amphetamine, methamphetamine and hydroxymethamphetamine in urine, LODs of 15–20 ppm) [Theurillat & Thormann, 2008], (amphetamine, methamphetamine and methylenedioxymethamphetamine, 6–8 ppt LODs, several 1000-fold improvement in detection sensitivity compared with typical injection) [Tsimachidis et al., 2008; Theurillat et al., 2007].

(i) SWP + DPJ: suitable for a mixture of neutral and weakly acidic/basic analytes [Britz-McKibbin et al., 2002], for illustrative scheme see Figure 3.10. This method is very effective for overcoming the often poor band-narrowing efficiency of conventional SWP (using anionic micelles) and the DPJ for hydrophilic and neutral analytes, respectively, even if the migration time needed for separation is much longer than that of a conventional DPJ or SWP. This method was applied for plasma and urine samples. A picomolar detectability can be achieved by CE-LIF detection without the need for

Advanced Sample Preparation 77

(ii) LVSS (a combination of field-enhanced stacking and pH-mediated stacking) + SWP: LODs up to 10-10 M with LIF detection, applied for animal tissues [Kirschner et al., 2007],

(iii) FESI + tITP: applied for the simultaneous on-line preconcentration and enantioseparation of drugs in urine samples. The detection limits in ng/mL levels can be

can

Figure 3.11. Schematic diagram of the stacking/sweeping: (i) hydrodynamic injection of large volume (1/3 of the capillary) of the CBI–amino acids in water at pH 6.0; (ii) migration toward the pH junction at the outlet side of the injection plug of the anionic CBI–amino acids; (iii) pumping water out of the capillary and movement of the stacked analyte band toward the inlet by the EOF; (iv) sweeping of the HS--CD through the stacked band of

laborious off-line preconcentration and clean-up [Britz-McKibbin et al., 2003].

for illustrative scheme see Figure 3.11.

easily obtained [Huang, L. et al., 2011].

analyte. Adapted from ref. [Kirschner et al., 2007].

(v) Electrocapture. Horáková et al. [Horáková et al., 2007] presented a novel approach which they called 'electrocapture'. This technique makes possible the determination of nanomolar concentrations of weak acidic analytes in CE. The method consists of longrunning electrokinetic sample injection and stacking (electrokinetic immobilization) of the analytes at a boundary of two electrolytes with different pH values (pH 9.5 and 2.5), and consequent mobilization of the stacked uncharged analytes in a micelle system (containing SDS micelles). The micelle system can provide for further concentrating the analytes by sweeping. An approximate 4600-fold increase of the sample concentration (in comparison with the standard CZE) can be achieved during the preconcentration step.

Some of the techniques based on combined electrophoretic principles in a single column arrangement have also been used for chiral analyses, or, at least, a chiral separation environment has been utilized in these techniques. These are the following methods:

Figure 3.10. Schematic diagrams of the DPJ-SWP model. (A) The micelle (such as SDS) BGS and the sample solution (a nonmicelle buffer) are injected into the capillary, respectively; (B) when the injection is complete, a positive polarity is applied (if a negatively charged SDS surfactant is used) to power the CE separation; (C) the neutral analytes are converted to anions and are swept by the SDS micelles; (D) separation occurs by the MEKC mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

Some of the techniques based on combined electrophoretic principles in a single column arrangement have also been used for chiral analyses, or, at least, a chiral separation

Figure 3.10. Schematic diagrams of the DPJ-SWP model. (A) The micelle (such as SDS) BGS and the sample solution (a nonmicelle buffer) are injected into the capillary, respectively; (B) when the injection is complete, a positive polarity is applied (if a negatively charged SDS surfactant is used) to power the CE separation; (C) the neutral analytes are converted to anions and are swept by the SDS micelles; (D) separation occurs by the MEKC mode.

Reprinted from ref. [Lin C.H. & Kaneta, 2004].

with the standard CZE) can be achieved during the preconcentration step.

environment has been utilized in these techniques. These are the following methods:

(v) Electrocapture. Horáková et al. [Horáková et al., 2007] presented a novel approach which they called 'electrocapture'. This technique makes possible the determination of nanomolar concentrations of weak acidic analytes in CE. The method consists of longrunning electrokinetic sample injection and stacking (electrokinetic immobilization) of the analytes at a boundary of two electrolytes with different pH values (pH 9.5 and 2.5), and consequent mobilization of the stacked uncharged analytes in a micelle system (containing SDS micelles). The micelle system can provide for further concentrating the analytes by sweeping. An approximate 4600-fold increase of the sample concentration (in comparison (i) SWP + DPJ: suitable for a mixture of neutral and weakly acidic/basic analytes [Britz-McKibbin et al., 2002], for illustrative scheme see Figure 3.10. This method is very effective for overcoming the often poor band-narrowing efficiency of conventional SWP (using anionic micelles) and the DPJ for hydrophilic and neutral analytes, respectively, even if the migration time needed for separation is much longer than that of a conventional DPJ or SWP. This method was applied for plasma and urine samples. A picomolar detectability can be achieved by CE-LIF detection without the need for laborious off-line preconcentration and clean-up [Britz-McKibbin et al., 2003].

(ii) LVSS (a combination of field-enhanced stacking and pH-mediated stacking) + SWP: LODs up to 10-10 M with LIF detection, applied for animal tissues [Kirschner et al., 2007], for illustrative scheme see Figure 3.11.

(iii) FESI + tITP: applied for the simultaneous on-line preconcentration and enantioseparation of drugs in urine samples. The detection limits in ng/mL levels can be easily obtained [Huang, L. et al., 2011]. in

Figure 3.11. Schematic diagram of the stacking/sweeping: (i) hydrodynamic injection of large volume (1/3 of the capillary) of the CBI–amino acids in water at pH 6.0; (ii) migration toward the pH junction at the outlet side of the injection plug of the anionic CBI–amino acids; (iii) pumping water out of the capillary and movement of the stacked analyte band toward the inlet by the EOF; (iv) sweeping of the HS--CD through the stacked band of analyte. Adapted from ref. [Kirschner et al., 2007].

In-capillary sample preconcentration with chemical derivatization (SPCD) is included as an innovative strategy [Ptolemy et al., 2005, 2006; Ptolemy & Britz-McKibbin, 2006]. The general principles of SPCD–CE for single-step enantioselective analysis of submicromolar levels of analytes are: (i) multiple hydrodynamic injection sequence (appropriate arrangement of sample and derivatization reagent(s) zones); (ii) on-line sample preconcentration (electrokinetic focusing); (iii) in-capillary chemical labelling by zone passing of derivatization reagent(s); (iv) chiral separation of diastereomeric analyte adducts formed, see illustrative schemes in Figure 3.12a and corresponding electropherograms in

Advanced Sample Preparation 79

**3.2.1.7 Single column combination of electrophoretic migration with counter-flow**  Counter-flow gradient focusing (CFGF) approaches [Shackman & Ross, 2007] are a potentially simple and versatile way in which to simultaneously concentrate and separate charged and neutral analytes. These types of techniques have the potential to concentrate each sample component in a uniquely different place in the separation space (similarly to the IEF where each component is concentrated at its pI region, see Figure 2.2c). Over the last decade a number of new approaches that can focus analytes at positions according to their electrophoretic mobility (electric field gradient focusing, temperature gradient focusing) and interaction with a pseudophase (micellar affinity gradient focusing) have been developed. It is worthwhile noting that counter-flow gradient focusing methods are exceptionally flexible: they can be used as an analytical method in itself or as an electrophoretic equivalent to solid-phase extraction (SPE), whereby analytes can be selectively captured and/or released

Figure 3.13. TGF method. (a) Schematic illustration of the TGF apparatus. A linear temperature gradient is formed along the capillary in the 2-mm space between the copper blocks regulated at temperatures T1 and T2. (b) Schematic of chiral TGF separations. T1, 13°C (left side in image); T2, 40°C; +1000 V/cm; BGS,10 mM -CD in 1 M Tris–borate (pH 8.3). The D-enantiomer peak is to the left in the figure; the L-enantiomer is to the right.

Figure 3.12b.

in a well-defined and controlled manner.

Reprinted from ref. [Balss et al., 2004].

**3.2.1.6 Single column combination of sample preparation techniques based on electrophoretic principles with chemical reaction** 

Figure 3.12. SPCD-CE method. (a) Proposed mechanism and kinetics of analyte electrokinetic focusing with in-capillary derivatization of amino acid enantiomers by SPCD-CE using OPA/NAC based on time-resolved electrophoretic experiments depicted in Figure 3.12b. Note the rapid analyte focusing with labelling of the long sample plug via tITP with subsequent zone passing of reagents, as well as the distinct time-dependent electrokinetic focusing of labelled amino acid-adducts at later stages by the moving pH boundary. (b) Series of electropherograms showing the distinct time-dependent processes of electrokinetic focusing and in-capillary OPA/NAC derivatization by SPCD-CE. Electropherograms were monitored with UV absorbance: (i) 214 nm and (ii) 340 nm. Arrows note time-delayed analyte electrokinetic focusing mediated by a dynamic pH junction. All samples contained 20 M D-Ala and D-Glu in 40mM phosphate, pH 6.0. A long sample plug (6.1 cm, using a sample injection of 100 s) was placed at different positions from the capillary window using a low pressure rinse (0.5 psi or 3.5 kPa) in order to change the effective capillary length (Ld) from (a) 0 cm, (b) 11.4 cm, (c) 22.8 cm and (d) 30.5 cm. Other conditions: 140mM borate buffer, pH 9.5; voltage 25 kV; capillary length 67 cm; internal diameter; 50m; UV 340 nm. Analyte peak number corresponds amino acid-isoindole adducts, where 2a: D-Ala, 3a: D-Glu. Reprinted from ref. [Ptolemy & Britz-McKibbin, 2006].

Figure 3.12. SPCD-CE method. (a) Proposed mechanism and kinetics of analyte electrokinetic focusing with in-capillary derivatization of amino acid enantiomers by SPCD-CE using OPA/NAC based on time-resolved electrophoretic experiments depicted in Figure 3.12b. Note the rapid analyte focusing with labelling of the long sample plug via tITP with subsequent zone passing of reagents, as well as the distinct time-dependent electrokinetic focusing of labelled amino acid-adducts at later stages by the moving pH boundary. (b) Series of electropherograms showing the distinct time-dependent processes of electrokinetic focusing and in-capillary OPA/NAC derivatization by SPCD-CE. Electropherograms were monitored with UV absorbance: (i) 214 nm and (ii) 340 nm. Arrows note time-delayed analyte electrokinetic focusing mediated by a dynamic pH junction. All samples contained 20 M D-Ala and D-Glu in 40mM phosphate, pH 6.0. A long sample plug (6.1 cm, using a sample injection of 100 s) was placed at different positions from the capillary window using a low pressure rinse (0.5 psi or 3.5 kPa) in order to change the effective capillary length (Ld) from (a) 0 cm, (b) 11.4 cm, (c) 22.8 cm and (d) 30.5 cm. Other conditions: 140mM borate buffer, pH 9.5; voltage 25 kV; capillary length 67 cm; internal diameter; 50m; UV 340 nm. Analyte peak number corresponds amino acid-isoindole adducts, where 2a: D-Ala, 3a: D-

Glu. Reprinted from ref. [Ptolemy & Britz-McKibbin, 2006].

**3.2.1.6 Single column combination of sample preparation techniques** 

**based on electrophoretic principles with chemical reaction** 

In-capillary sample preconcentration with chemical derivatization (SPCD) is included as an innovative strategy [Ptolemy et al., 2005, 2006; Ptolemy & Britz-McKibbin, 2006]. The general principles of SPCD–CE for single-step enantioselective analysis of submicromolar levels of analytes are: (i) multiple hydrodynamic injection sequence (appropriate arrangement of sample and derivatization reagent(s) zones); (ii) on-line sample preconcentration (electrokinetic focusing); (iii) in-capillary chemical labelling by zone passing of derivatization reagent(s); (iv) chiral separation of diastereomeric analyte adducts formed, see illustrative schemes in Figure 3.12a and corresponding electropherograms in Figure 3.12b.

### **3.2.1.7 Single column combination of electrophoretic migration with counter-flow**

Counter-flow gradient focusing (CFGF) approaches [Shackman & Ross, 2007] are a potentially simple and versatile way in which to simultaneously concentrate and separate charged and neutral analytes. These types of techniques have the potential to concentrate each sample component in a uniquely different place in the separation space (similarly to the IEF where each component is concentrated at its pI region, see Figure 2.2c). Over the last decade a number of new approaches that can focus analytes at positions according to their electrophoretic mobility (electric field gradient focusing, temperature gradient focusing) and interaction with a pseudophase (micellar affinity gradient focusing) have been developed. It is worthwhile noting that counter-flow gradient focusing methods are exceptionally flexible: they can be used as an analytical method in itself or as an electrophoretic equivalent to solid-phase extraction (SPE), whereby analytes can be selectively captured and/or released in a well-defined and controlled manner.

Figure 3.13. TGF method. (a) Schematic illustration of the TGF apparatus. A linear temperature gradient is formed along the capillary in the 2-mm space between the copper blocks regulated at temperatures T1 and T2. (b) Schematic of chiral TGF separations. T1, 13°C (left side in image); T2, 40°C; +1000 V/cm; BGS,10 mM -CD in 1 M Tris–borate (pH 8.3). The D-enantiomer peak is to the left in the figure; the L-enantiomer is to the right. Reprinted from ref. [Balss et al., 2004].

compounds (up to 99% or even more interfering compounds can be isolated [Danková et al., 1999]) into the second CE stage for the final separation and detection. It should also be mentioned that the use of tubes with larger internal diameter in CE analysis (300-800m, typical in hydrodynamically closed separation systems) is favourable for their enhanced sample load capacities (30l sample injection volumes are then common) linked with lower LODs [Kaniansky et al., 1997]. Using an ITP-CZE method, it is possible to analyse directly ultratrace charged analytes (lower ng/mL regions even with conventional detectors, e.g., UV absorbance detector) in complex ionic matrices (e.g., biological fluids) with minimum sample handling (e.g., only dilution) and to improve the LOD commonly more than 100-fold (depending on ionic matrix composition/concentration) in comparison with single column

Advanced Sample Preparation 81

Figure 3.14. ITP-EKC technique in the capillary format and column coupling configuration of the separation units for the direct analysis of samples with the unpretreated complex matrices. The instrumental schemes (left and middle) and the photo of the corresponding commercial equipment, capillary electrophoresis analyser EA-102 (Villa-Labeco, Spišská Nová Ves, Slovakia), (right). On-line sample preparation: removing matrices X, preconcentration of enantiomers Y, Z in the first ITP stage (column C1). Final separation: enantioseparation of Y and Z in the second EKC stage (column C2). C1 – ITP column, C2 – EKC column, B – bifurcation block for coupling C1 and C2, D – positions of detectors. The

instrumental schemes are adapted from ref. [Tekeľ & Mikuš, 2005].

CE.

The method termed temperature gradient focusing (TGF) [Ross & Locascio, 2002; Balss, 2004; Danger & Ross, 2008; Kitagawa & Otsuka, 2011], applied also in the chiral field, relied upon the use of a buffer with a temperature-dependent ionic strength (such as tris-borate) so that application of a temperature gradient would result in an electrophoretic velocity gradient. For the scheme of the TGF apparatus and separation mechanism see Figure 3.13. For real-time chiral TGF separation see an application example in section 3.5. The advantages of the TGF method included easier implementation than electric-field gradient focusing (EFGF), as well as the ability to focus wider classes of analytes, see review [Shackman & Ross, 2007]. The maximum reported concentration enhancement was 10 000 fold in 100 min. A disadvantage of TGF can be the limited variety of buffers having the necessary temperature-dependent ionic strength.

### **3.2.2 Column-coupled (hyphenated) electrophoretic techniques**

The theory and analytical potentialities of the column coupled electrophoretic techniques carried out in the capillary, as well as channel (microchip), formats and applied in the field of advanced pharmaceutical and biomedical analysis are comprehensively presented in the latest monograph chapter of the authors [Mikuš & Maráková, 2011]. On the other hand, in the present book/section the theory and potentialities of the column-coupled electrophoresis with chiral aspect are given.

The CE performed in a hydrodynamically closed separation system (hydrodynamic flow is eliminated by semipermeable membranes at the ends of the separation compartment) can be easily implemented into advanced CE systems, e.g., into those operating with coupled columns [Kaniansky & Marák, 1990; Kaniansky et al., 1993, 1994a, 1994b]. In addition to the single column (in-capillary) sample preconcentration and purification approaches (section 3.2.1), an on-line column combination of two CE methods can effectively solve problems of sample preparation and final analysis in one run in a well-defined way, i.e., producing high reproducibility of analyses. The Kaniansky group has been carrying out detailed research on the basic aspects, instrumentation and utilization of on-line coupled CE techniques for many years with interesting results. The proposed and commercially available CE-CE systems have a modular composition that provides a high flexibility in arranging particular modules in the separation unit, creating desirable CE-CE combinations (e.g., ITP-ITP, ITP-EKC, EKC-EKC, small or large volume injection) capable of solving a wide range of advanced analytical problems. 2002; of C1 EKC column, B – bifurcation block for coupling C1 and C2, D – positions of detectors. The

One pioneering work [Kaniansky & Marák, 1990] demonstrates the analytical potential of on-line coupling of ITP with CZE in the trace analysis of multicomponent ionic mixtures, see the instrumental scheme in Figure 3.14 for a general point of view. The ITP stage serves for the sample preparation and CZE stage for the final separation and detection of the analyte, and the following benefits can be recognized: (i) the ITP technique is useful for the separation of only cations or only anions in one run and this can be considered as an incolumn ITP sample clean-up. (ii) ITP provides preconcentration of the sample constituents along with the analyte (see 3.2.1.2). (iii) In the column coupling separation system the postcolumn ITP sample clean-up (removing of undesired zones migrating in ITP) is performed via a proper switching of the direction of the driving current to the counter-electrodes. This enables transfer of an ITP zone of the concentrated analyte with only minimum interfering

The method termed temperature gradient focusing (TGF) [Ross & Locascio, 2002; Balss, 2004; Danger & Ross, 2008; Kitagawa & Otsuka, 2011], applied also in the chiral field, relied upon the use of a buffer with a temperature-dependent ionic strength (such as tris-borate) so that application of a temperature gradient would result in an electrophoretic velocity gradient. For the scheme of the TGF apparatus and separation mechanism see Figure 3.13. For real-time chiral TGF separation see an application example in section 3.5. The advantages of the TGF method included easier implementation than electric-field gradient focusing (EFGF), as well as the ability to focus wider classes of analytes, see review [Shackman & Ross, 2007]. The maximum reported concentration enhancement was 10 000 fold in 100 min. A disadvantage of TGF can be the limited variety of buffers having the

The theory and analytical potentialities of the column coupled electrophoretic techniques carried out in the capillary, as well as channel (microchip), formats and applied in the field of advanced pharmaceutical and biomedical analysis are comprehensively presented in the latest monograph chapter of the authors [Mikuš & Maráková, 2011]. On the other hand, in the present book/section the theory and potentialities of the column-coupled

The CE performed in a hydrodynamically closed separation system (hydrodynamic flow is eliminated by semipermeable membranes at the ends of the separation compartment) can be easily implemented into advanced CE systems, e.g., into those operating with coupled columns [Kaniansky & Marák, 1990; Kaniansky et al., 1993, 1994a, 1994b]. In addition to the single column (in-capillary) sample preconcentration and purification approaches (section 3.2.1), an on-line column combination of two CE methods can effectively solve problems of sample preparation and final analysis in one run in a well-defined way, i.e., producing high reproducibility of analyses. The Kaniansky group has been carrying out detailed research on the basic aspects, instrumentation and utilization of on-line coupled CE techniques for many years with interesting results. The proposed and commercially available CE-CE systems have a modular composition that provides a high flexibility in arranging particular modules in the separation unit, creating desirable CE-CE combinations (e.g., ITP-ITP, ITP-EKC, EKC-EKC, small or large volume injection) capable of solving a wide range of advanced

One pioneering work [Kaniansky & Marák, 1990] demonstrates the analytical potential of on-line coupling of ITP with CZE in the trace analysis of multicomponent ionic mixtures, see the instrumental scheme in Figure 3.14 for a general point of view. The ITP stage serves for the sample preparation and CZE stage for the final separation and detection of the analyte, and the following benefits can be recognized: (i) the ITP technique is useful for the separation of only cations or only anions in one run and this can be considered as an incolumn ITP sample clean-up. (ii) ITP provides preconcentration of the sample constituents along with the analyte (see 3.2.1.2). (iii) In the column coupling separation system the postcolumn ITP sample clean-up (removing of undesired zones migrating in ITP) is performed via a proper switching of the direction of the driving current to the counter-electrodes. This enables transfer of an ITP zone of the concentrated analyte with only minimum interfering

necessary temperature-dependent ionic strength.

electrophoresis with chiral aspect are given.

analytical problems.

**3.2.2 Column-coupled (hyphenated) electrophoretic techniques** 

compounds (up to 99% or even more interfering compounds can be isolated [Danková et al., 1999]) into the second CE stage for the final separation and detection. It should also be mentioned that the use of tubes with larger internal diameter in CE analysis (300-800m, typical in hydrodynamically closed separation systems) is favourable for their enhanced sample load capacities (30l sample injection volumes are then common) linked with lower LODs [Kaniansky et al., 1997]. Using an ITP-CZE method, it is possible to analyse directly ultratrace charged analytes (lower ng/mL regions even with conventional detectors, e.g., UV absorbance detector) in complex ionic matrices (e.g., biological fluids) with minimum sample handling (e.g., only dilution) and to improve the LOD commonly more than 100-fold (depending on ionic matrix composition/concentration) in comparison with single column CE.

Figure 3.14. ITP-EKC technique in the capillary format and column coupling configuration of the separation units for the direct analysis of samples with the unpretreated complex matrices. The instrumental schemes (left and middle) and the photo of the corresponding commercial equipment, capillary electrophoresis analyser EA-102 (Villa-Labeco, Spišská Nová Ves, Slovakia), (right). On-line sample preparation: removing matrices X, preconcentration of enantiomers Y, Z in the first ITP stage (column C1). Final separation: enantioseparation of Y and Z in the second EKC stage (column C2). C1 – ITP column, C2 – EKC column, B – bifurcation block for coupling C1 and C2, D – positions of detectors. The instrumental schemes are adapted from ref. [Tekeľ & Mikuš, 2005]. EKC column, B – bifurcation block for coupling C1 and C2, D – positions of detectors. The

molecules can influence these mechanisms through competitive complexing equilibria. For example, a partial filling of the separation capillary with chiral electrolyte can be employed to avoid the elution of the enriched solutes during flushing of the sorbent with chiral

Advanced Sample Preparation 83

A microcolumn liquid chromatography (MLC) can be used in an on-line arrangement with the CE for sample purification and concentration allowing the injection of microlitre volumes into the electrophoresis capillary [Bushey & Jorgenson, 1990; Pálmarsdóttir et al., 1995]. For the instrumental scheme of the MLC-CE see Figure 3.16. The combined system has a much greater resolving power and peak capacity than either of the two systems used independently of each other. The selectivity and sensitivity gain of combining MLC with CE for determination of low concentrations of chiral drugs in biosamples is exemplified in section 3.5. However, the MLC-CE coupling is technically much more difficult than the CE-CE because it has to be accompanied by collection, evaporation and reconstitution of

Figure 3.16. Experimental set-up of MLC coupled on line with CE. (1) Pump; (2) flow processor; (3) loop; (4) valve; (5) analytical column; (6) p.-dumper interface; (7) Tee adapter.

Extraction techniques now play a major role in sample preparation in CE. These techniques can be used not only for reconstitution of the sample from small volumes, but also for sample clean-up in complex matrices and desalting for very saline samples that would interfere with the electrophoretic process. Considerable progress has been made towards the coupling of solid-phase extraction (SPE) with subsequent electrophoresis, while coupling of liquid-phase extraction (LLE) with electrophoresis is less used. The review by Breadmore et al. [Breadmore et al., 2009] gives attention to on-line or in-line extraction methods that have

electrolyte [Petersson et al., 1999].

fraction isolated by MLC.

**3.3.1 Chromatographic techniques** 

Reprinted from ref. [Pálmarsdóttir et al., 1995].

**3.3.2 Extraction techniques** 

been used for electrophoresis.

Figure 3.15. Schematic layout and dimensions of the microchip with on-line coupled separation channels. Reservoirs are labelled as: (A) run buffer; (B) sample; (C) sample waste; (D) buffer waste; (E) run buffer containing a chiral selector; and (F) buffer waste containing the chiral selector. The channel depth was 35 m and width at a half-depth was 60 m. The dots represent the detection points, which are located at 32 and 38 mm from the first and second injection crosses, respectively. Reprinted from ref. [Cho et al., 2004].

The CE-CE methodology is easily adaptable to chiral analysis. ITP-EKC is the most popular combination for the ultrasensitive determinations of chiral analytes present in complex matrices, see the schematic separation of electrophoretic zones in Figure 3.14 and several application examples in section 3.5. Neutral, as well as charged, chiral selectors are usually implemented in the EKC stage of the ITP-EKC combination [Danková et al., 1999; Fanali et al., 2000; Mikuš et al., 2006a, 2008a, 2008b; Marák et al., 2007]. Nevertheless, an ITP-ITP combination is also applicable for chiral analyses as the implementation of both neutral and charged chiral selectors in the ITP is possible [Ölvecká et al., 2001, Mikuš et al., 2006b; Kubačák et al., 2006a, 2006b, 2007].

On-line combinations of the electrophoretic techniques performed on chip (coupled channels), see Figure 3.15, offer additional advantages of microscale analysis as they are described in the introduction part of chapter 3 and section 2.2.3 (and references given therein) and also provide good possibilities for speed chiral analyses of drugs in minute amounts of sample with the model [Ölvecká et al., 2001], as well as complex matrices [Cho et al., 2004], as illustrated by an application example in section 3.5.

### **3.3 On-line sample preparation techniques based on nonelectrophoretic principles**

The on-line sample preparation can be carried out advantageously also combining CE with other than electrophoretic techniques. Most of these approaches are based on extraction or chromatographic principles, but also other techniques, such as membrane filtration or microdialysis (separations based on physical principles), can be used. Electrophoresis and nonelectrophoretic on-line sample preparation techniques can be properly combined to achieve a desired effect. Lately several such approaches have been introduced, as illustrated by application examples in section 3.5.

On the other hand, attention must be paid when on-line sample preparation based on sorption-desorption or distribution mechanism is combined with a chiral system, as chiral

Figure 3.15. Schematic layout and dimensions of the microchip with on-line coupled separation channels. Reservoirs are labelled as: (A) run buffer; (B) sample; (C) sample waste; (D) buffer waste; (E) run buffer containing a chiral selector; and (F) buffer waste containing the chiral selector. The channel depth was 35 m and width at a half-depth was 60 m. The dots represent the detection points, which are located at 32 and 38 mm from the first and

The CE-CE methodology is easily adaptable to chiral analysis. ITP-EKC is the most popular combination for the ultrasensitive determinations of chiral analytes present in complex matrices, see the schematic separation of electrophoretic zones in Figure 3.14 and several application examples in section 3.5. Neutral, as well as charged, chiral selectors are usually implemented in the EKC stage of the ITP-EKC combination [Danková et al., 1999; Fanali et al., 2000; Mikuš et al., 2006a, 2008a, 2008b; Marák et al., 2007]. Nevertheless, an ITP-ITP combination is also applicable for chiral analyses as the implementation of both neutral and charged chiral selectors in the ITP is possible [Ölvecká et al., 2001, Mikuš et al., 2006b;

On-line combinations of the electrophoretic techniques performed on chip (coupled channels), see Figure 3.15, offer additional advantages of microscale analysis as they are described in the introduction part of chapter 3 and section 2.2.3 (and references given therein) and also provide good possibilities for speed chiral analyses of drugs in minute amounts of sample with the model [Ölvecká et al., 2001], as well as complex matrices [Cho

**3.3 On-line sample preparation techniques based on nonelectrophoretic principles**  The on-line sample preparation can be carried out advantageously also combining CE with other than electrophoretic techniques. Most of these approaches are based on extraction or chromatographic principles, but also other techniques, such as membrane filtration or microdialysis (separations based on physical principles), can be used. Electrophoresis and nonelectrophoretic on-line sample preparation techniques can be properly combined to achieve a desired effect. Lately several such approaches have been introduced, as illustrated

On the other hand, attention must be paid when on-line sample preparation based on sorption-desorption or distribution mechanism is combined with a chiral system, as chiral

second injection crosses, respectively. Reprinted from ref. [Cho et al., 2004].

et al., 2004], as illustrated by an application example in section 3.5.

Kubačák et al., 2006a, 2006b, 2007].

by application examples in section 3.5.

molecules can influence these mechanisms through competitive complexing equilibria. For example, a partial filling of the separation capillary with chiral electrolyte can be employed to avoid the elution of the enriched solutes during flushing of the sorbent with chiral electrolyte [Petersson et al., 1999].

### **3.3.1 Chromatographic techniques**

A microcolumn liquid chromatography (MLC) can be used in an on-line arrangement with the CE for sample purification and concentration allowing the injection of microlitre volumes into the electrophoresis capillary [Bushey & Jorgenson, 1990; Pálmarsdóttir et al., 1995]. For the instrumental scheme of the MLC-CE see Figure 3.16. The combined system has a much greater resolving power and peak capacity than either of the two systems used independently of each other. The selectivity and sensitivity gain of combining MLC with CE for determination of low concentrations of chiral drugs in biosamples is exemplified in section 3.5. However, the MLC-CE coupling is technically much more difficult than the CE-CE because it has to be accompanied by collection, evaporation and reconstitution of fraction isolated by MLC.

Figure 3.16. Experimental set-up of MLC coupled on line with CE. (1) Pump; (2) flow processor; (3) loop; (4) valve; (5) analytical column; (6) p.-dumper interface; (7) Tee adapter. Reprinted from ref. [Pálmarsdóttir et al., 1995].

#### **3.3.2 Extraction techniques**

Extraction techniques now play a major role in sample preparation in CE. These techniques can be used not only for reconstitution of the sample from small volumes, but also for sample clean-up in complex matrices and desalting for very saline samples that would interfere with the electrophoretic process. Considerable progress has been made towards the coupling of solid-phase extraction (SPE) with subsequent electrophoresis, while coupling of liquid-phase extraction (LLE) with electrophoresis is less used. The review by Breadmore et al. [Breadmore et al., 2009] gives attention to on-line or in-line extraction methods that have been used for electrophoresis.

### **3.3.2.1 Solid-phase extraction / microextraction**

Solid-phase extraction (SPE) is the most attractive way of coupling extraction with CE and, especially, MCE. This is in particular because it can provide significant improvements in sensitivity without the use of electrokinetic injection [Puig et al., 2007a, 2008; Bertoncini & Hennion, 2004]. Depending on the nature of the adsorbent chemistry, this can be specific for certain analytes, such as through the use of a biopolymeric phase (see a generalized mechanism in Figure 3.17), or more generic for the extraction of a range or classes of compounds, such as a C18 reverse phase material, ion-exchanger resins, etc. These solid phases can be present in various formats, such as particles, parallel open channels and monoliths.

Figure 3.18. Miniaturised on-line SPE for enhancement of concentration sensitivity in CE. Cross-section of (A) the extractor and (B) the enrichment capillary where *L*<sup>t</sup> (28–58 cm) is the enrichment capillary total length, *L*d (21.2–51.2 cm) is the length to the detector, *Li* (5.4 cm) is the length of the inlet capillary and *le* (1–3 mm) is the extractor length. (C) Sample enrichment procedure for terbutaline dissolved in water. Arrows indicate flow directions. The post-sorption washing with water is optional, as the electrolyte filling, in which nonretained solutes are flushed out of the capillary, usually is enough for rather clean samples.

Advanced Sample Preparation 85

Figure 3.19. Schematic diagram of the three types of interfaces for on-line SPE–CE coupling: (a) vial interface; (b) valve interface; (c) T-split interface. Reproduced from refs. (a) [Stroink

In order to overcome this issue, on-line methods may be used, although care must be taken to ensure that no efficiency is lost in the transferral (e.g., dead volume must be minimized). Nowadays, the most used on-line SPE interfaces are the vial-type, the valve-type and the T-

on-line T-

et al., 2003], (b) [Tempels et al., 2007] and (c) [Puig et al., 2007b].

Reprinted from ref. [Petersson et al., 1999].

Figure 3.17. Diagrammatic representation of the 3-D biospecific extraction employing biopolymer coupled to CE technique. (A) Analyte percolation and capture phase. (B) Washing of non-retained compounds. (C) Acid elution of the analytes and separation by CE. V, S, N, C = chiral analytes. Adapted from ref. [Phillips, 1998].

In-line systems are created inserting solid-phase column into capillary (see Figure 3.18a, b) and they are attractive thanks to their low cost and easy construction. The whole analytical procedure includes conditioning, loading/sorption, washing, (labelling, if necessary), filling (by electrolyte), elution/desorption, separation and detection, see an example in Figure 3.18c. One of the main limitations of performing in-line SPE is that the entire sample, washing and elution solvents must pass through the capillary, which can lead to fouling of the separation capillary, particularly when problematic samples are used.

Solid-phase extraction (SPE) is the most attractive way of coupling extraction with CE and, especially, MCE. This is in particular because it can provide significant improvements in sensitivity without the use of electrokinetic injection [Puig et al., 2007a, 2008; Bertoncini & Hennion, 2004]. Depending on the nature of the adsorbent chemistry, this can be specific for certain analytes, such as through the use of a biopolymeric phase (see a generalized mechanism in Figure 3.17), or more generic for the extraction of a range or classes of compounds, such as a C18 reverse phase material, ion-exchanger resins, etc. These solid phases can be present in various formats, such as particles, parallel open channels and

Figure 3.17. Diagrammatic representation of the 3-D biospecific extraction employing biopolymer coupled to CE technique. (A) Analyte percolation and capture phase. (B) Washing of non-retained compounds. (C) Acid elution of the analytes and separation by CE.

In-line systems are created inserting solid-phase column into capillary (see Figure 3.18a, b) and they are attractive thanks to their low cost and easy construction. The whole analytical procedure includes conditioning, loading/sorption, washing, (labelling, if necessary), filling (by electrolyte), elution/desorption, separation and detection, see an example in Figure 3.18c. One of the main limitations of performing in-line SPE is that the entire sample, washing and elution solvents must pass through the capillary, which can lead to fouling of

V, S, N, C = chiral analytes. Adapted from ref. [Phillips, 1998].

the separation capillary, particularly when problematic samples are used.

**3.3.2.1 Solid-phase extraction / microextraction** 

monoliths.

Figure 3.18. Miniaturised on-line SPE for enhancement of concentration sensitivity in CE. Cross-section of (A) the extractor and (B) the enrichment capillary where *L*<sup>t</sup> (28–58 cm) is the enrichment capillary total length, *L*d (21.2–51.2 cm) is the length to the detector, *Li* (5.4 cm) is the length of the inlet capillary and *le* (1–3 mm) is the extractor length. (C) Sample enrichment procedure for terbutaline dissolved in water. Arrows indicate flow directions. The post-sorption washing with water is optional, as the electrolyte filling, in which nonretained solutes are flushed out of the capillary, usually is enough for rather clean samples. Reprinted from ref. [Petersson et al., 1999].

Figure 3.19. Schematic diagram of the three types of interfaces for on-line SPE–CE coupling: (a) vial interface; (b) valve interface; (c) T-split interface. Reproduced from refs. (a) [Stroink et al., 2003], (b) [Tempels et al., 2007] and (c) [Puig et al., 2007b].

In order to overcome this issue, on-line methods may be used, although care must be taken to ensure that no efficiency is lost in the transferral (e.g., dead volume must be minimized). Nowadays, the most used on-line SPE interfaces are the vial-type, the valve-type and the T-

Figure 3.20. Interface for SPME–CE–MS coupling. Reproduced from Santos et al. [Santos et

Advanced Sample Preparation 87

The miniaturization of liquid-phase (or liquid-liquid) extraction (LLE) has benefits in minimizing organic solvent consumption and sample amount requirements. Moreover, it simplifies and (partially) automates the extraction process. However, there are a number of technical issues that must be overcome for the development of an on-line integrated system. Recent progress covering the whole field of liquid-phase microextraction can be found in reviews on the subject by Bjergaard and Rasmussen [Bjergaard & Rasmussen, 2008], Lee et al. [Lee et al., 2008] and Xu et al. [Xu L. et al.; 2007]. The authors summarize miniaturized and highly flexible formats for LLE combinable with separation techniques, including CE, and they also gives views on environmental and bioanalytical applications of this coupled

Integration of LLE with CE is based on an on-line back extraction system with FESI [Fang H.F. et al., 2006a]. In this approach the weak bases are first extracted into an organic solvent following a conventional off-line LLE protocol. The organic solvent containing the analytes is then placed in the sample vial and a small amount of water is placed on top. The analytes distribute between the organic phase (donor solution) and aqueous phase (acceptor solution), where they are partially protonated. Electrokinetic injection of the charged analytes depletes the analytes from the aqueous layer, disrupting the equilibrium, and more analytes are transferred from the organic phase into the water plug. Upon entering the capillary, analytes stack by normal FESI principles. Using this approach, a several thousandfold increase in sensitivity can be obtained accompanied with sample purification. Other modifications of this approach are based on the use of a Teflon micromembrane [Almeda et al., 2007] or propylene hollow fibre [Nozal et al., 2007] filled with an acceptor solution and placed between the capillary and sample vial, see a scheme of LLE-CE equipment in Figure 3.21. For a detailed illustration, a schematic description of the construction of the liquid-

several

al., 2007].

technique.

**3.3.2.2 Liquid-phase extraction** 

phase microextraction unit can be seen in Figure 3.22.

split-type [Bonneil & Waldron, 2000; Tempels et al., 2006; Puig et al., 2007b; Jiménez & de Castro, 2008]. Figure 3.19 shows a schematic representation of these interfaces. On-line SPE interface ensures that during the extraction waste solvents from the wash step are redirected, providing a cleaner extract for analysis. However, one of the major limitations of this approach is that because of the dead volume of the system, the entire eluate is not injected for separation, hence, some of the sensitivity gain is lost. Zhang and Wu [Zhang L.H. & Wu X. Z., 2007] presented a novel and conceptually simple approach to overcome this by creating a small hole in the capillary just after the SPE phase. The hole in the capillary allowed sample and wash solutions to be redirected to waste away from the separation capillary, while ensuring that the entire volume of solution used to elute the analytes was used for electrophoretic separation. While the integration level is impressive and the improvement in the LOD is sufficient (more than 10 000-fold is not unusual), practical analyses can be limited by the loading times in some cases (injection time can even be several hours). With respect to shortening analysis time, microchips offer a more attractive way of integrating SPE with electrophoresis, see examples in the review paper by Breadmore et al. [Breadmore et al., 2009]. However, unlike capillary format (SPE-CE), online coupled SPE-MCE has not been applied in chiral analysis so far.

Solid-phase microextraction (SPME) is an increasingly used technique because it is simple, can be used to extract analytes from very small samples and provides a rapid extraction and transfer to the analytical instrument. Moreover, it can be easily combined with other extraction and/or analytical procedures, improving to a large extent the sensitivity and selectivity of the whole method [Pawliszyn, 1997; Lord & Pawliszyn, 2000; Ouyang & Pawliszyn, 2006*;* Saito & Jinno, 2003]. As an example, an interface for SPME–CE–MS coupling is given in Figure 3.20. The on-line coupling of microextraction with the chiral CE has been described in the literature. For example, a direct chiral analysis of primary amine drugs in human urine by single drop microextraction in-line coupled to CE was demonstrated by Choi et al. [Choi K et al., 2009]. Examples on sensitive chiral analyses by means of microextraction-CE are stated in section 3.5. However, such coupling has not been widely used in practice because of its inherent drawbacks regarding the low injection volumes typically required in the CE (which are crucial to obtaining a good separation efficiency) and also because the different sizes of the separation capillaries usually used for CE and the SPME fibres [Liu Z. & Pawliszyn, 2006].

A general problem with the SPE/SPME-CE is the poisoning of the concentrator by matrix components and also their adsorption on the capillary wall. The use of coated capillaries, like poly(vinyl alcohol) (PVA), can decrease problems involved with protein adsorption. However, PVA capillaries are not available in the dimensions often required. Washing with sodium hydroxide is excluded due to incompatibility with the silica-based sorbent. Alternatives, then, are the use of polymer-based sorbents [Knudsen & Beattie, 1997] or a detergent such as SDS [Lloyd & Wätzig, 1995].

split-type [Bonneil & Waldron, 2000; Tempels et al., 2006; Puig et al., 2007b; Jiménez & de Castro, 2008]. Figure 3.19 shows a schematic representation of these interfaces. On-line SPE interface ensures that during the extraction waste solvents from the wash step are redirected, providing a cleaner extract for analysis. However, one of the major limitations of this approach is that because of the dead volume of the system, the entire eluate is not injected for separation, hence, some of the sensitivity gain is lost. Zhang and Wu [Zhang L.H. & Wu X. Z., 2007] presented a novel and conceptually simple approach to overcome this by creating a small hole in the capillary just after the SPE phase. The hole in the capillary allowed sample and wash solutions to be redirected to waste away from the separation capillary, while ensuring that the entire volume of solution used to elute the analytes was used for electrophoretic separation. While the integration level is impressive and the improvement in the LOD is sufficient (more than 10 000-fold is not unusual), practical analyses can be limited by the loading times in some cases (injection time can even be several hours). With respect to shortening analysis time, microchips offer a more attractive way of integrating SPE with electrophoresis, see examples in the review paper by Breadmore et al. [Breadmore et al., 2009]. However, unlike capillary format (SPE-CE), on-

The

Solid-phase microextraction (SPME) is an increasingly used technique because it is simple, can be used to extract analytes from very small samples and provides a rapid extraction and transfer to the analytical instrument. Moreover, it can be easily combined with other extraction and/or analytical procedures, improving to a large extent the sensitivity and selectivity of the whole method [Pawliszyn, 1997; Lord & Pawliszyn, 2000; Ouyang & Pawliszyn, 2006*;* Saito & Jinno, 2003]. As an example, an interface for SPME–CE–MS coupling is given in Figure 3.20. The on-line coupling of microextraction with the chiral CE has been described in the literature. For example, a direct chiral analysis of primary amine drugs in human urine by single drop microextraction in-line coupled to CE was demonstrated by Choi et al. [Choi K et al., 2009]. Examples on sensitive chiral analyses by means of microextraction-CE are stated in section 3.5. However, such coupling has not been widely used in practice because of its inherent drawbacks regarding the low injection volumes typically required in the CE (which are crucial to obtaining a good separation efficiency) and also because the different sizes of the separation capillaries usually used for

A general problem with the SPE/SPME-CE is the poisoning of the concentrator by matrix components and also their adsorption on the capillary wall. The use of coated capillaries, like poly(vinyl alcohol) (PVA), can decrease problems involved with protein adsorption. However, PVA capillaries are not available in the dimensions often required. Washing with sodium hydroxide is excluded due to incompatibility with the silica-based sorbent. Alternatives, then, are the use of polymer-based sorbents [Knudsen & Beattie, 1997] or a

line coupled SPE-MCE has not been applied in chiral analysis so far.

CE and the SPME fibres [Liu Z. & Pawliszyn, 2006].

detergent such as SDS [Lloyd & Wätzig, 1995].

Figure 3.20. Interface for SPME–CE–MS coupling. Reproduced from Santos et al. [Santos et al., 2007].

### **3.3.2.2 Liquid-phase extraction**

The miniaturization of liquid-phase (or liquid-liquid) extraction (LLE) has benefits in minimizing organic solvent consumption and sample amount requirements. Moreover, it simplifies and (partially) automates the extraction process. However, there are a number of technical issues that must be overcome for the development of an on-line integrated system. Recent progress covering the whole field of liquid-phase microextraction can be found in reviews on the subject by Bjergaard and Rasmussen [Bjergaard & Rasmussen, 2008], Lee et al. [Lee et al., 2008] and Xu et al. [Xu L. et al.; 2007]. The authors summarize miniaturized and highly flexible formats for LLE combinable with separation techniques, including CE, and they also gives views on environmental and bioanalytical applications of this coupled technique.

Integration of LLE with CE is based on an on-line back extraction system with FESI [Fang H.F. et al., 2006a]. In this approach the weak bases are first extracted into an organic solvent following a conventional off-line LLE protocol. The organic solvent containing the analytes is then placed in the sample vial and a small amount of water is placed on top. The analytes distribute between the organic phase (donor solution) and aqueous phase (acceptor solution), where they are partially protonated. Electrokinetic injection of the charged analytes depletes the analytes from the aqueous layer, disrupting the equilibrium, and more analytes are transferred from the organic phase into the water plug. Upon entering the capillary, analytes stack by normal FESI principles. Using this approach, a several thousandfold increase in sensitivity can be obtained accompanied with sample purification. Other modifications of this approach are based on the use of a Teflon micromembrane [Almeda et al., 2007] or propylene hollow fibre [Nozal et al., 2007] filled with an acceptor solution and placed between the capillary and sample vial, see a scheme of LLE-CE equipment in Figure 3.21. For a detailed illustration, a schematic description of the construction of the liquidphase microextraction unit can be seen in Figure 3.22.

**3.3.3 Membrane filtration, microdialysis** 

Analytes can also be concentrated by inducing a velocity change due to their size by physically restricting their movement, so called concentration by physically induced changes in velocity. This has traditionally been most easily performed with large molecules, such as proteins and DNA [Yu C.J. et al., 2008]. Implementation of nanoporous media (nafion membrane, anionic hydrogel plug, etc.) in microchips (MCE is dominant in this field) has led to a number of interesting developments where the concentration of much smaller molecules is possible [de Jong et al., 2006; Holtzel & Tallarek, 2007; Dhopeshwarkar et al., 2008; Long et al., 2006]. Sensitivity enhancements of 4–6 orders of magnitude make this method powerful for sensitivity enhancement. A small piece of membrane (e.g., with 10 nm pores) can be integrated into a microchip also for the isolation of small molecules from crude samples. The potential of this approach was demonstrated with the analysis of biomarkers in blood without any off-line protein removal [Long et al., 2006]. A scheme of membrane – preconcentration/purification device implemented on-line into the CE is given as an example in Figure 3.23, where the detail of membrane insertion can be clearly seen.

Advanced Sample Preparation 89

Figure 3.23. Membrane – preconcentration device with styrene-divinyl-benzene membrane

A microdialysis is frequently used to sample small molecules from complex, often biological, matrices [Adell & Artigas, 1998; Robinson & Justice, 1991; Chaurasia, 1999]. For example, some amino acid neurotransmitters are heterogeneously distributed in the brain and colocalized with *N*-methyl-D-aspartate (NMDA) receptors, suggesting a role in neurotransmission. In this analytical field, initial tissue assays for D-serine and D-aspartate biomarkers were based on a somewhat labour-intensive clean-up procedure followed by, e.g., a 70 min HPLC separation and LIF detection [Hashimoto et al., 1992, 1995]. On the other hand, in microdialysis, small molecules are able to diffuse across the dialysis membrane into the probe, while large molecules, such as proteins and cell fragments, are excluded. The clean-up provided by the microdialysis can allow analysing for neurotransmitters in tissue (e.g., brain) homogenates directly [Thompson, J.E. et al., 1999]. In the microdialysis, the minimum volume required for analysis often determines the rate at which the dialysate can be sampled. On-line microdialysis-CE-LIF assays (for the instrumental scheme see Figure 3.24) eliminate fraction collection. This elimination of fraction collection, combined with the high mass sensitivity of LIF or electrochemical detectors, makes sampling rates on the order of seconds possible [Thompson, J.E. et al., 1999; Hogan et al., 1994; Zhou S.Y. et al., 1995, 1999; Lada & Kennedy, 1995, 1997; Lada et al., 1998]. On-line microdialysis-CE assays for neurotransmitters to date have been most

to concentrate samples on-line in CE. Reprinted from ref. [Barroso & de Jong, 1998].

Figure 3.21. In-line liquid-phase microextraction – capillary electrophoresis (LLE-CE) arrangement for the determination of nonsteroidal antiinflammatory drugs in urine. Reprinted from ref. [Nozal et al., 2007].

Figure 3.22. (A) Description of the construction of the liquid-phase microextraction unit. (1) Capillaries lining up and insertion in the hollow fibre; (2) protection of the hollow fibre with a Teflon tube and burning of the free part for capillary-hollow fibre connexion; (3) system with one connexion; (4) performance of the second connexion and (5) microextraction unit integrated into the capillary. (B) Comparison of connexions performed with epoxy glue and by burning the hollow fibre. Reprinted from ref. [Nozal et al., 2007].

Figure 3.21. In-line liquid-phase microextraction – capillary electrophoresis (LLE-CE) arrangement for the determination of nonsteroidal antiinflammatory drugs in urine.

Figure 3.22. (A) Description of the construction of the liquid-phase microextraction unit. (1) Capillaries lining up and insertion in the hollow fibre; (2) protection of the hollow fibre with a Teflon tube and burning of the free part for capillary-hollow fibre connexion; (3) system with one connexion; (4) performance of the second connexion and (5) microextraction unit integrated into the capillary. (B) Comparison of connexions performed with epoxy glue and

by burning the hollow fibre. Reprinted from ref. [Nozal et al., 2007].

Reprinted from ref. [Nozal et al., 2007].

### **3.3.3 Membrane filtration, microdialysis**

Analytes can also be concentrated by inducing a velocity change due to their size by physically restricting their movement, so called concentration by physically induced changes in velocity. This has traditionally been most easily performed with large molecules, such as proteins and DNA [Yu C.J. et al., 2008]. Implementation of nanoporous media (nafion membrane, anionic hydrogel plug, etc.) in microchips (MCE is dominant in this field) has led to a number of interesting developments where the concentration of much smaller molecules is possible [de Jong et al., 2006; Holtzel & Tallarek, 2007; Dhopeshwarkar et al., 2008; Long et al., 2006]. Sensitivity enhancements of 4–6 orders of magnitude make this method powerful for sensitivity enhancement. A small piece of membrane (e.g., with 10 nm pores) can be integrated into a microchip also for the isolation of small molecules from crude samples. The potential of this approach was demonstrated with the analysis of biomarkers in blood without any off-line protein removal [Long et al., 2006]. A scheme of membrane – preconcentration/purification device implemented on-line into the CE is given as an example in Figure 3.23, where the detail of membrane insertion can be clearly seen.

Figure 3.23. Membrane – preconcentration device with styrene-divinyl-benzene membrane to concentrate samples on-line in CE. Reprinted from ref. [Barroso & de Jong, 1998].

A microdialysis is frequently used to sample small molecules from complex, often biological, matrices [Adell & Artigas, 1998; Robinson & Justice, 1991; Chaurasia, 1999]. For example, some amino acid neurotransmitters are heterogeneously distributed in the brain and colocalized with *N*-methyl-D-aspartate (NMDA) receptors, suggesting a role in neurotransmission. In this analytical field, initial tissue assays for D-serine and D-aspartate biomarkers were based on a somewhat labour-intensive clean-up procedure followed by, e.g., a 70 min HPLC separation and LIF detection [Hashimoto et al., 1992, 1995]. On the other hand, in microdialysis, small molecules are able to diffuse across the dialysis membrane into the probe, while large molecules, such as proteins and cell fragments, are excluded. The clean-up provided by the microdialysis can allow analysing for neurotransmitters in tissue (e.g., brain) homogenates directly [Thompson, J.E. et al., 1999]. In the microdialysis, the minimum volume required for analysis often determines the rate at which the dialysate can be sampled. On-line microdialysis-CE-LIF assays (for the instrumental scheme see Figure 3.24) eliminate fraction collection. This elimination of fraction collection, combined with the high mass sensitivity of LIF or electrochemical detectors, makes sampling rates on the order of seconds possible [Thompson, J.E. et al., 1999; Hogan et al., 1994; Zhou S.Y. et al., 1995, 1999; Lada & Kennedy, 1995, 1997; Lada et al., 1998]. On-line microdialysis-CE assays for neurotransmitters to date have been most

The great potential with the hybrid on-line sample preparation techniques lies in their complementarity that enables the accumulation of positive effects and/or overcoming the weak points of the individual sample preparation techniques (discussed in sections 3.2 and 3.3). For example, the extraction used as the first step of the sample preparation can simplify the sample matrix (e.g., removing major interfering ionic constituents from the sample matrix) that is essential for some of the stacking modes (e.g., FESS requires a low-

Advanced Sample Preparation 91

A combination of on-line sample preparation techniques based on electrophoretic and nonelectrophoretic principles can be performed in a single column or column coupling arrangement. Another interesting possibility with how to implement various sample preparation procedures on-line is a combination of the flow injection (FI) with electrophoresis. The combination of FI with electrophoresis using capillaries and chips is reviewed by Lü et al. [Lü W.J. et al., 2009]. Here, the basic principles, instrumental developments (including newly designed interfaces for FI-CE) and applications of FI-CE system from 2006 to 2008 are reviewed. The technique of combined flow injection CE (FI-CE) integrates the essential favourable merits of FI and CE. It utilizes the various excellent on-line sample pretreatments and preconcentration (such as cloud point extraction, SPE, ion-exchange, DPJ and head-column FESS technique, analyte derivatization) of FI, which has the advantages of high-speed, accuracy, precision and avoiding manual handling of sample and reagents. Therefore, the coupling of FI-CE is an attractive technique; it can significantly expand the application of CE and has achieved many publications since its first appearance. The significant potential of the FI-CE method in the automatization of sample derivatization and chiral separation was demonstrated by Mardones et al. [Mardones et al.,

Figure 3.25. FI manifold used for the derivatization of the carnitine enantiomers and their

The sample preparation techniques on-line combined with the CE, described in sections 3.2-3.4 (see for the theory and schemes), have been applied in many models, as well as real situations. In a lesser extent, given by (i) the higher complexity of experimental arrangement with chiral additive(s), as well as (ii) the natural proportion between currently solved chiral and achiral analytical problems, these techniques have been on-line combined with chiral CE systems.

on-line introduction into the CE system. Reprinted from ref. [Mardones et al., 1999].

conductivity sample) used as the second step.

1999], and the proposed FI-CE scheme is shown in Figure 3.25.

**3.4 Flow injection** 

**3.5 Applications** 

successful for easily resolved analytes such as glutamate and aspartate [Thompson, J.E. et al., 1999; Zhou S.Y. et al., 1995; Lada et al., 1997, 1998; Lada & Kennedy, 1996]. Efficiency and peak capacity of high-speed CE separations are often not high enough to resolve complex mixtures. Recently, improvements in injection technique and detection limits have improved separation efficiency, e.g., allowing singly charged amine derivatives, such as amino-*n*butyric acid (GABA), to be analysed [Bowser & Kennedy, 2001]. Recently, on-line microdialysis-CE has been adapted to chiral determinations of neurotransmitters in multicomponent amino acid mixtures, as well as biological matrices [O'Brien et al., 2003], as shown in section 3.5.

Figure 3.24. Diagram of the microdialysis-CE-LIF instrument. The whole automated procedure consists of following steps/modules performed on-line: (i) Microdialysis, (ii) derivatization, (iii) Flow-gated injection interface, (iv) High-speed CE with LIF detection. Reprinted from ref. [O'Brien et al., 2003].

### **3.3.4 Combination of electrophoretic stacking with nonelectrophoretic techniques**

The electrophoretic stacking and nonelectrophoretic on-line sample preparation principles can be properly combined with each other to achieve the desired effect. Lately several such hybrid on-line sample preparation techniques have been introduced into the CE that offer excellent solutions, especially, for the sample clean-up (often accomplished by nonelectrophoretic principles) and analyte preconcentration (often accomplished by electrophoretic principles) in one experiment. Some of them, namely (i) extraction + stacking [Fang H.F. et al., 2006a, 2006b], (ii) dialysis + stacking [Hadwiger et al., 1996], (iii) chromatography + stacking [Pálmarsdóttir & Edholm, 1995; Pálmarsdóttir et al., 1996, 1997], were successfully applied also in chiral analyses of biologically active compounds in biological samples as presented in section 3.5.

successful for easily resolved analytes such as glutamate and aspartate [Thompson, J.E. et al., 1999; Zhou S.Y. et al., 1995; Lada et al., 1997, 1998; Lada & Kennedy, 1996]. Efficiency and peak capacity of high-speed CE separations are often not high enough to resolve complex mixtures. Recently, improvements in injection technique and detection limits have improved separation efficiency, e.g., allowing singly charged amine derivatives, such as amino-*n*butyric acid (GABA), to be analysed [Bowser & Kennedy, 2001]. Recently, on-line microdialysis-CE has been adapted to chiral determinations of neurotransmitters in multicomponent amino acid mixtures, as well as biological matrices [O'Brien et al., 2003], as

Figure 3.24. Diagram of the microdialysis-CE-LIF instrument. The whole automated procedure consists of following steps/modules performed on-line: (i) Microdialysis, (ii) derivatization, (iii) Flow-gated injection interface, (iv) High-speed CE with LIF detection.

**3.3.4 Combination of electrophoretic stacking with nonelectrophoretic techniques**  The electrophoretic stacking and nonelectrophoretic on-line sample preparation principles can be properly combined with each other to achieve the desired effect. Lately several such hybrid on-line sample preparation techniques have been introduced into the CE that offer excellent solutions, especially, for the sample clean-up (often accomplished by nonelectrophoretic principles) and analyte preconcentration (often accomplished by electrophoretic principles) in one experiment. Some of them, namely (i) extraction + stacking [Fang H.F. et al., 2006a, 2006b], (ii) dialysis + stacking [Hadwiger et al., 1996], (iii) chromatography + stacking [Pálmarsdóttir & Edholm, 1995; Pálmarsdóttir et al., 1996, 1997], were successfully applied also in chiral analyses of biologically active compounds in

shown in section 3.5.

Reprinted from ref. [O'Brien et al., 2003].

biological samples as presented in section 3.5.

The great potential with the hybrid on-line sample preparation techniques lies in their complementarity that enables the accumulation of positive effects and/or overcoming the weak points of the individual sample preparation techniques (discussed in sections 3.2 and 3.3). For example, the extraction used as the first step of the sample preparation can simplify the sample matrix (e.g., removing major interfering ionic constituents from the sample matrix) that is essential for some of the stacking modes (e.g., FESS requires a lowconductivity sample) used as the second step.

### **3.4 Flow injection**

A combination of on-line sample preparation techniques based on electrophoretic and nonelectrophoretic principles can be performed in a single column or column coupling arrangement. Another interesting possibility with how to implement various sample preparation procedures on-line is a combination of the flow injection (FI) with electrophoresis. The combination of FI with electrophoresis using capillaries and chips is reviewed by Lü et al. [Lü W.J. et al., 2009]. Here, the basic principles, instrumental developments (including newly designed interfaces for FI-CE) and applications of FI-CE system from 2006 to 2008 are reviewed. The technique of combined flow injection CE (FI-CE) integrates the essential favourable merits of FI and CE. It utilizes the various excellent on-line sample pretreatments and preconcentration (such as cloud point extraction, SPE, ion-exchange, DPJ and head-column FESS technique, analyte derivatization) of FI, which has the advantages of high-speed, accuracy, precision and avoiding manual handling of sample and reagents. Therefore, the coupling of FI-CE is an attractive technique; it can significantly expand the application of CE and has achieved many publications since its first appearance. The significant potential of the FI-CE method in the automatization of sample derivatization and chiral separation was demonstrated by Mardones et al. [Mardones et al., 1999], and the proposed FI-CE scheme is shown in Figure 3.25.

Figure 3.25. FI manifold used for the derivatization of the carnitine enantiomers and their on-line introduction into the CE system. Reprinted from ref. [Mardones et al., 1999].

### **3.5 Applications**

The sample preparation techniques on-line combined with the CE, described in sections 3.2-3.4 (see for the theory and schemes), have been applied in many models, as well as real situations. In a lesser extent, given by (i) the higher complexity of experimental arrangement with chiral additive(s), as well as (ii) the natural proportion between currently solved chiral and achiral analytical problems, these techniques have been on-line combined with chiral CE systems.

The increase of sensitivity by applying a FESS procedure was necessary for the analysis of amphetamine and its metabolic products (3,4-ethylenedioxymethamphetamine, MDMA, 3- 4-methylenedioxyamphetamine, MDA, 3,4-methalenedioxyethylamphetamine, MDE) in hair samples [Tagliaro et al., 1998]. The FESS-CE-DAD method allowed the chiral determination of the metabolites at concentrations occurring in real samples from ecstasy users, with the possibility of recording UV spectra of the peaks. The analytical precision was characterized by relative standard deviation values <0.8 % (≤0.15% with internal standardization) for migration times intra-day and <2.0% (≤0.54% with internal standardization) day-to-day; linearity, in the range 0.156-40 g/mL, and accuracy were also satisfactory. Even when evaluated at the lowest concentration of the standard curve, 0.156 pg/mL (with a signal-to-noise ratio of 5 for MDMA and 3 for amphetamine), the betweendays area reproducibility was acceptable, ranging from 7.93% RSD for MDMA, to 16.07% RSD for amphetamine. The intra-day RSDs of migration times were always <0.8%, while the between-days RSDs were <2.0%; for relative migration times, RSDs were ≤0.15% and ≤0.54%, respectively. Absolute peak area reproducibility was still acceptable at 20 pg/mL (RSD was about 5% intra-day and ≤8% inter-days), but at 0.2 pg/mL the variability was relevant (RSDs between 9 and 12%). The area normalization on the basis of an IS (Dmethamphetamine) was helpful, but for MDMA, MDA and MDE, RSDs remained around

Advanced Sample Preparation 93

FESI as an on-line sample stacking method was employed in order to increase the detection sensitivity for six -blockers, namely (±) carteolol, (±) atenolol, (±) sotalol, (±) metoprolol, (±) esmolol and (±) propranolol, in human serum samples [Huang L. et al., 2008]. The final serum sample solution had to be diluted before injection since the ion concentration in the sample solution strongly influenced the signal enhancement. When the human serum sample was not diluted, the signal enhancement was not great because of the matrix effect. For validation of the FESI-CE-UV method, sensitivity (Table 3.1), linearity and precision were evaluated. The study showed that the repeatabilities of migration times (intra-day RSD 5.5-6.1%, inter-day RSD 4.4-5.2%) and peak heights (intra-day RSD 1.5-12.9%, inter-day RSD 3.6-5.2%) for the enantiomers of -blockers were satisfactory. Recoveries (calculated using

method for

The FESI-CE-UV method was used to determine the concentration of sulindac (SU) and its two active metabolites, sulindac sulfide (SI) and sulindac sulfone (SO), in human plasma [Chen Y.L. et al., 2006]. During method validation, calibration plots were linear (*r* > 0.994) over a range of 0.3–30.0 M for SU and SO, and 0.5–30.0 M for SI. During intra- and interday analysis, relative standard deviations (RSD) and relative errors (RE) were all less than 16%. Compared to the peak area ratio of the standard analytes, the absolute recoveries for SU, SO and SI were about 82, 81 and 60%, respectively. This method was feasible for the investigation of a pharmacokinetic profile of SU in plasma after oral administration of one

The LVSS-CE-DAD method was applied for the sensitivity enhancement of four basic racemic drugs (methoxamine, metaproterenol, terbutaline and carvedilol) in serum samples [Denola et al., 2007]. Accuracy ranges were 96.4–103.4% for the hydrodynamic injection and 102.3–115.5% for the electrokinetic injection. Better recovery values were achieved with

the 5 mg/mL racemic standard solutions) ranged from 82.1 to 98.2%.

SU tablet (Clinoril, 200 mg/tab) to a female volunteer.

10%.

Chiral CE determinations of biologically active compounds in various real matrices, e.g., environmental, food, beverage and mainly biological (clinical, forensic), as well as pharmaceutical samples, employing an on-line sample preparation, are listed in Table 3.1.

Figure 3.26. Analysis of plasma extracts containing 50 ng/mL of drug enantiomers and internal standard by CE with optimized FESI injection. Capillary column, 57 cm x 50 m) i.d.; applied voltage, +20 kV; temperature 25°C; buffer, formic acid-ammonia (pH 4, ionic strength 50 mM) and 3.5 mM HP--CD. FESI, hydrodynamic injection of water plug (5 s) then electrokinetic injection (+10 kV, 20 s) of racemic drug dissolved in a water-methanol (10/90 v/v) and 80 M H3PO4 mixture. SPE pretreatment of plasma sample was carried out before FESI-CE. Reprinted from ref. [Grard et al., 2002].

In this Table we tried to emphasize briefly the most important features of the methods and purpose of sample preparation, i.e., sample clean-up, analyte preconcentration, analyte derivatization. Here, the selectivity and sensitivity gain of combining on-line sample preparation procedures with chiral CE for the determination of low concentrations of drugs in complex matrices was clearly exemplified.

From among those examples, selected applications in biological and model (presented here for a given sample preparation method only in the case when no bioapplication is available) samples are described in the text of this section in detail, emphasizing the practical aspects of the proposed methods via their performance parameters (precision, recovery, etc.). In this way, the usefulness of the various techniques in routine analysis can be clearly ascertained.

*Field-enhanced sample stacking.* The FESI-CE-UV method has been developed for the quantification of cationic enantiomers of the new adrenoreceptor antagonists in plasma samples, for the illustrative electropherogram see Figure 3.26 [Grard et al., S., 2002]. An excellent accuracy on retention times and peak efficiencies was found with relative standard deviations (RSDs) being generally less than 1% and 2%, respectively. The FESI method also provided good reproducibility of ratio between the corrected areas of enantiomer and of the internal standard, since the RSD never exceeded 3%. These experimental results attest to the reliability of the FESI method in analysing chiral drugs by CE.

Chiral CE determinations of biologically active compounds in various real matrices, e.g., environmental, food, beverage and mainly biological (clinical, forensic), as well as pharmaceutical samples, employing an on-line sample preparation, are listed in Table 3.1.

Figure 3.26. Analysis of plasma extracts containing 50 ng/mL of drug enantiomers and internal standard by CE with optimized FESI injection. Capillary column, 57 cm x 50 m) i.d.; applied voltage, +20 kV; temperature 25°C; buffer, formic acid-ammonia (pH 4, ionic strength 50 mM) and 3.5 mM HP--CD. FESI, hydrodynamic injection of water plug (5 s) then electrokinetic injection (+10 kV, 20 s) of racemic drug dissolved in a water-methanol (10/90 v/v) and 80 M H3PO4 mixture. SPE pretreatment of plasma sample was carried out

In this Table we tried to emphasize briefly the most important features of the methods and purpose of sample preparation, i.e., sample clean-up, analyte preconcentration, analyte derivatization. Here, the selectivity and sensitivity gain of combining on-line sample preparation procedures with chiral CE for the determination of low concentrations of drugs

From among those examples, selected applications in biological and model (presented here for a given sample preparation method only in the case when no bioapplication is available) samples are described in the text of this section in detail, emphasizing the practical aspects of the proposed methods via their performance parameters (precision, recovery, etc.). In this way, the usefulness of the various techniques in routine analysis can be clearly ascertained.

*Field-enhanced sample stacking.* The FESI-CE-UV method has been developed for the quantification of cationic enantiomers of the new adrenoreceptor antagonists in plasma samples, for the illustrative electropherogram see Figure 3.26 [Grard et al., S., 2002]. An excellent accuracy on retention times and peak efficiencies was found with relative standard deviations (RSDs) being generally less than 1% and 2%, respectively. The FESI method also provided good reproducibility of ratio between the corrected areas of enantiomer and of the internal standard, since the RSD never exceeded 3%. These experimental results attest to the

before FESI-CE. Reprinted from ref. [Grard et al., 2002].

reliability of the FESI method in analysing chiral drugs by CE.

in complex matrices was clearly exemplified.

The increase of sensitivity by applying a FESS procedure was necessary for the analysis of amphetamine and its metabolic products (3,4-ethylenedioxymethamphetamine, MDMA, 3- 4-methylenedioxyamphetamine, MDA, 3,4-methalenedioxyethylamphetamine, MDE) in hair samples [Tagliaro et al., 1998]. The FESS-CE-DAD method allowed the chiral determination of the metabolites at concentrations occurring in real samples from ecstasy users, with the possibility of recording UV spectra of the peaks. The analytical precision was characterized by relative standard deviation values <0.8 % (≤0.15% with internal standardization) for migration times intra-day and <2.0% (≤0.54% with internal standardization) day-to-day; linearity, in the range 0.156-40 g/mL, and accuracy were also satisfactory. Even when evaluated at the lowest concentration of the standard curve, 0.156 pg/mL (with a signal-to-noise ratio of 5 for MDMA and 3 for amphetamine), the betweendays area reproducibility was acceptable, ranging from 7.93% RSD for MDMA, to 16.07% RSD for amphetamine. The intra-day RSDs of migration times were always <0.8%, while the between-days RSDs were <2.0%; for relative migration times, RSDs were ≤0.15% and ≤0.54%, respectively. Absolute peak area reproducibility was still acceptable at 20 pg/mL (RSD was about 5% intra-day and ≤8% inter-days), but at 0.2 pg/mL the variability was relevant (RSDs between 9 and 12%). The area normalization on the basis of an IS (Dmethamphetamine) was helpful, but for MDMA, MDA and MDE, RSDs remained around 10%.

FESI as an on-line sample stacking method was employed in order to increase the detection sensitivity for six -blockers, namely (±) carteolol, (±) atenolol, (±) sotalol, (±) metoprolol, (±) esmolol and (±) propranolol, in human serum samples [Huang L. et al., 2008]. The final serum sample solution had to be diluted before injection since the ion concentration in the sample solution strongly influenced the signal enhancement. When the human serum sample was not diluted, the signal enhancement was not great because of the matrix effect. For validation of the FESI-CE-UV method, sensitivity (Table 3.1), linearity and precision were evaluated. The study showed that the repeatabilities of migration times (intra-day RSD 5.5-6.1%, inter-day RSD 4.4-5.2%) and peak heights (intra-day RSD 1.5-12.9%, inter-day RSD 3.6-5.2%) for the enantiomers of -blockers were satisfactory. Recoveries (calculated using the 5 mg/mL racemic standard solutions) ranged from 82.1 to 98.2%. of

The FESI-CE-UV method was used to determine the concentration of sulindac (SU) and its two active metabolites, sulindac sulfide (SI) and sulindac sulfone (SO), in human plasma [Chen Y.L. et al., 2006]. During method validation, calibration plots were linear (*r* > 0.994) over a range of 0.3–30.0 M for SU and SO, and 0.5–30.0 M for SI. During intra- and interday analysis, relative standard deviations (RSD) and relative errors (RE) were all less than 16%. Compared to the peak area ratio of the standard analytes, the absolute recoveries for SU, SO and SI were about 82, 81 and 60%, respectively. This method was feasible for the investigation of a pharmacokinetic profile of SU in plasma after oral administration of one SU tablet (Clinoril, 200 mg/tab) to a female volunteer.

The LVSS-CE-DAD method was applied for the sensitivity enhancement of four basic racemic drugs (methoxamine, metaproterenol, terbutaline and carvedilol) in serum samples [Denola et al., 2007]. Accuracy ranges were 96.4–103.4% for the hydrodynamic injection and 102.3–115.5% for the electrokinetic injection. Better recovery values were achieved with

was equal or less than 5.6% (for seven out of nine values), which is considered as acceptable for the application. The accuracy at 1.9% of the enantiomeric impurity for three different BGEs where the composition was varied (±1%) was in the range 91.3–99.7% and the precision was 1.7–8.7% (n = 2). Thus, the method is robust and small differences in the BGE composition should not have any major influence in the determination of the 1R,2S-ephedrine impurity.

Advanced Sample Preparation 95

Figure 3.28. Peak sharpening of S-timolol by tITP. BGE: 100mM (+)-ketopinic acid and 40mM KOH in methanol:ethanol (3:2, v/v). 30 kV, *L*det 23 cm. Sample: 2mM *S*-timolol and 0.05mM *R*-timolol (2.4%) dissolved in methanol. (A) Normal injection pressure injection at 35 mbar over 5 s, (B) tITP, leading electrolyte: 100mM sodium acetate in methanol (25 mbar over 1 s). Terminating electrolyte: 200mM triethanolamine in methanol (anodic vial). (C) tITP at LOD (0.2% *R-*timolol). Conditions as in (B). The arrow in the electropherograms (A– C) points out the position of the *R*-timolol peak. Reprinted from ref. [Hedeland et al., 2007]. *Combined stacking techniques.* A combination of LVSS and SWP, where LVSS involves a subcombination of field-amplified stacking and pH-mediated stacking, provided a very high sensitivity for the anionic cyanobenz[*f*]isoindole (CBI)-amino acids [Kirschner et al., 2007]. Average and standard deviation in peak area ratios (CBI-amino acid/ internal standard) for nine injections of 2.4 M CBI-amino acid were as follows: CBI-L-Glu 0.461±0.070, CBI-D-Ser 0.400±0.085, CBI-L-Asp 0.358±0.036. A procedure for the LVSS-SWP-CE-LIF determination of CBI-D-Ser was applied for squirrel brain samples. This preconcentration technique was also applied to multicomponent mixtures of CBI amino acid

derivatives without loss of resolution, see electropherogram in Figure 3.29.

hydrodynamic injection wherein values ranged from 62.8–74.5% as compared to that of the electrokinetic injection in which recovery values were all lower (31.1–69.4%). The deviations in the resolution values were higher for the electrokinetic (RSD 3.6–6.0%) than for the hydrodynamic injection (RSD <1.9%). However, the repeatability of migration times was better when control samples were loaded electrokinetically (RSD 0.6–0.8%) rather than hydrodynamically (RSD 2.1–2.6%). Migration times do not vary significantly from those obtained with the control samples. Good repeatability in terms of migration times and enantioresolution was evident in the very low RSD values (migration time RSD ≤0.5; enantioresolution RSD ≤5.5) in both types of injections.

Figure 3.27. SWP-MEKC electropherograms obtained from a 30 psi x s injection of four pooled human liver microsomes incubations (A) without lorazepam (blank) and (B) with 50 M lorazepam. Two insets show the comparison of blank subtracted, standardized spectra of lorazepam metabolites M1 and M2 (broken lines), and lorazepam (full line). Analytical conditions: detection was effected at 200 nm and, for solute identification purpose, the fast scanning mode (range: 195-320 nm, scanning interval: 5 nm) was employed, temperature 25°C, applied voltage 20kV (current about 28 A). Separation electrolyte: 6 mM Na2B4O7, 10 mM Na2HPO4, and 75 mM SDS, pH 9.1 (before addition of SDS). Reprinted from ref. [Baldacci & Thormann, 2006].

*Transient isotachophoresis.* tITP was used for the preconcentration of timolol and ephedrine in standard solutions and dosage forms [Hedeland et al., 2007]. tITP served for peak sharpening of S-timolol and therefore, the tITP-CE-DAD determination of the enantiomeric impurity (Rtimolol) was possible with sufficient enantioresolution, as illustrated in Figure 3.28. For ephedrine, the tITP-CE-DAD method was validated. The intermediate precision for the quality control (QC) samples was in the interval 4.2–5.6% and the precision for each set of samples

hydrodynamic injection wherein values ranged from 62.8–74.5% as compared to that of the electrokinetic injection in which recovery values were all lower (31.1–69.4%). The deviations in the resolution values were higher for the electrokinetic (RSD 3.6–6.0%) than for the hydrodynamic injection (RSD <1.9%). However, the repeatability of migration times was better when control samples were loaded electrokinetically (RSD 0.6–0.8%) rather than hydrodynamically (RSD 2.1–2.6%). Migration times do not vary significantly from those obtained with the control samples. Good repeatability in terms of migration times and enantioresolution was evident in the very low RSD values (migration time RSD ≤0.5;

Figure 3.27. SWP-MEKC electropherograms obtained from a 30 psi x s injection of four pooled human liver microsomes incubations (A) without lorazepam (blank) and (B) with 50 M lorazepam. Two insets show the comparison of blank subtracted, standardized spectra of lorazepam metabolites M1 and M2 (broken lines), and lorazepam (full line). Analytical conditions: detection was effected at 200 nm and, for solute identification purpose, the fast scanning mode (range: 195-320 nm, scanning interval: 5 nm) was employed, temperature 25°C, applied voltage 20kV (current about 28 A). Separation electrolyte: 6 mM Na2B4O7, 10 mM Na2HPO4, and 75 mM SDS, pH 9.1 (before addition of SDS). Reprinted from ref.

*Transient isotachophoresis.* tITP was used for the preconcentration of timolol and ephedrine in standard solutions and dosage forms [Hedeland et al., 2007]. tITP served for peak sharpening of S-timolol and therefore, the tITP-CE-DAD determination of the enantiomeric impurity (Rtimolol) was possible with sufficient enantioresolution, as illustrated in Figure 3.28. For ephedrine, the tITP-CE-DAD method was validated. The intermediate precision for the quality control (QC) samples was in the interval 4.2–5.6% and the precision for each set of samples

enantioresolution RSD ≤5.5) in both types of injections.

[Baldacci & Thormann, 2006].

was equal or less than 5.6% (for seven out of nine values), which is considered as acceptable for the application. The accuracy at 1.9% of the enantiomeric impurity for three different BGEs where the composition was varied (±1%) was in the range 91.3–99.7% and the precision was 1.7–8.7% (n = 2). Thus, the method is robust and small differences in the BGE composition should not have any major influence in the determination of the 1R,2S-ephedrine impurity.

Figure 3.28. Peak sharpening of S-timolol by tITP. BGE: 100mM (+)-ketopinic acid and 40mM KOH in methanol:ethanol (3:2, v/v). 30 kV, *L*det 23 cm. Sample: 2mM *S*-timolol and 0.05mM *R*-timolol (2.4%) dissolved in methanol. (A) Normal injection pressure injection at 35 mbar over 5 s, (B) tITP, leading electrolyte: 100mM sodium acetate in methanol (25 mbar over 1 s). Terminating electrolyte: 200mM triethanolamine in methanol (anodic vial). (C) tITP at LOD (0.2% *R-*timolol). Conditions as in (B). The arrow in the electropherograms (A– C) points out the position of the *R*-timolol peak. Reprinted from ref. [Hedeland et al., 2007].

*Combined stacking techniques.* A combination of LVSS and SWP, where LVSS involves a subcombination of field-amplified stacking and pH-mediated stacking, provided a very high sensitivity for the anionic cyanobenz[*f*]isoindole (CBI)-amino acids [Kirschner et al., 2007]. Average and standard deviation in peak area ratios (CBI-amino acid/ internal standard) for nine injections of 2.4 M CBI-amino acid were as follows: CBI-L-Glu 0.461±0.070, CBI-D-Ser 0.400±0.085, CBI-L-Asp 0.358±0.036. A procedure for the LVSS-SWP-CE-LIF determination of CBI-D-Ser was applied for squirrel brain samples. This preconcentration technique was also applied to multicomponent mixtures of CBI amino acid derivatives without loss of resolution, see electropherogram in Figure 3.29.

whereas precision in CE is often limited by short hydrodynamic injections. Anyway, flavin analysis by DPJ-SWP-CE-LIF offers a simple, yet sensitive way to analyse trace levels of

Advanced Sample Preparation 97

Transient isotachophoresis with field-enhanced sample injection, using -CD as the chiral selector and tetrabutylammonium hydroxide (TBAOH) as the additive, was applied for online preconcentration and enantioseparation of three beta-agonists, namely, cimaterol, clenbuterol and terbutaline. Under the optimum conditions, the detection limits (defined as S/N = 3) of this method were found to be 1 ng/mL for all three pairs of beta-agonists enantiomers. Compared with conventional electrokinetic injection, the enhancement factors were greatly improved to be 250-fold. The proposed method has been applied for the

*Stacking techniques with derivatization.* An on-line sample preconcentration approach coupled with in-capillary derivatization (SPCD-CE) has recently been applied, among others, to the (indirect) chiral separation of amino acids, whereby a special application represents the detection of D-amino acids as biomarkers in connection with bacterial growth, see Figure 3.30 [Ptolemy et al., 2006; Ptolemy & Britz-McKibbin, 2006]. In comparison with conventional CE, SPCD-CE provided a 100-fold improvement in concentration sensitivity, reduced sample handling and shorter analysis time. The reproducibility (n = 5) of the integrated SPCD-CE-UV technique was acceptable with average coefficients of variation of approximately 7.4 and 1.2% for quantitation (peak height) and apparent migration time,

flavin metabolites from complex biological samples.

analysis of human urine samples [Huang, L. et al., 2011].

respectively [Ptolemy et al., 2006].

Figure 3.29. Electropherogram showing the potential of stacking/sweeping-EKC combination to the enantioseparation of a complex sample of CBI–amino acids (0.5 M each). Electrophoretic conditions: fused-silica capillary, 70 cm total length (45 cm detector length) and 25 m i.d.; separation buffer, 25 mM phosphate buffer (pH 2.0) containing 2% HS--CD; applied voltage, 230 kV; hydrodynamic injection, 380 mbar for 180 s. LIF detection with lexc at 420 nm. Peak identification: 1, CBI-D-Arg; 2, CBI-L-Arg; 3, CBI-D-His; 4, CBI-L-His; 5, CBI-Gly; 6, CBI-L-Tyr; 7, CBI-L-Glu; 8, CBI-D-Ser; 9, CBI-L-Ser; 10, CBI-L-Glu; 11, CBI-D-Glu. Adapted from ref. [Kirschner et al., 2007].

Sensitive CE methods are required for emerging areas of biochemical research such as the metabolome. In ref. [Britz-McKibbin et al., 2003], DPJ-SWP-CE-LIF was applied as a robust single method to analyse trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein and biological fluids. For the separation of flavins a chiral selector mediated CE separation system was needed. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of *Bacillus subtilis*. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration (e.g., SPE). The method was validated in terms of reproducibility, sensitivity (Table 3.1), linearity and specificity. The linearity of the method within a 100-fold concentration range was excellent as reflected by the correlation coefficient (*R*2) of 0.999 for all three flavin calibration curves. However, significant nonlinearity was observed to occur at concentrations above 0.1 μM. Because of the lack of native fluorescent species in most biological matrices, the method is considered to be extremely specific with few chemical interferences. Reproducibility of the CE method was assessed by analysis of five replicate injections of formic acid cell extract (malate) sample performed on two consecutive days. Inter-day coefficients of variance (CV, n=10) for migration time and peak area of flavin coenzymes were determined to be 0.68 and 3.8%, respectively. The low reproducibility of this technique may be attributed in part to the long injection time (60 s) used for analysis,

Figure 3.29. Electropherogram showing the potential of stacking/sweeping-EKC combination to the enantioseparation of a complex sample of CBI–amino acids (0.5 M each). Electrophoretic conditions: fused-silica capillary, 70 cm total length (45 cm detector length) and 25 m i.d.; separation buffer, 25 mM phosphate buffer (pH 2.0) containing 2% HS--CD; applied voltage, 230 kV; hydrodynamic injection, 380 mbar for 180 s. LIF detection with lexc at 420 nm. Peak identification: 1, CBI-D-Arg; 2, CBI-L-Arg; 3, CBI-D-His; 4, CBI-L-His; 5, CBI-Gly; 6, CBI-L-Tyr; 7, CBI-L-Glu; 8, CBI-D-Ser; 9, CBI-L-Ser; 10, CBI-L-Glu; 11,

Sensitive CE methods are required for emerging areas of biochemical research such as the metabolome. In ref. [Britz-McKibbin et al., 2003], DPJ-SWP-CE-LIF was applied as a robust single method to analyse trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein and biological fluids. For the separation of flavins a chiral selector mediated CE separation system was needed. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of *Bacillus subtilis*. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration (e.g., SPE). The method was validated in terms of reproducibility, sensitivity (Table 3.1), linearity and specificity. The linearity of the method within a 100-fold concentration range was excellent as reflected by the correlation coefficient (*R*2) of 0.999 for all three flavin calibration curves. However, significant nonlinearity was observed to occur at concentrations above 0.1 μM. Because of the lack of native fluorescent species in most biological matrices, the method is considered to be extremely specific with few chemical interferences. Reproducibility of the CE method was assessed by analysis of five replicate injections of formic acid cell extract (malate) sample performed on two consecutive days. Inter-day coefficients of variance (CV, n=10) for migration time and peak area of flavin coenzymes were determined to be 0.68 and 3.8%, respectively. The low reproducibility of this technique may be attributed in part to the long injection time (60 s) used for analysis,

CBI-D-Glu. Adapted from ref. [Kirschner et al., 2007].

whereas precision in CE is often limited by short hydrodynamic injections. Anyway, flavin analysis by DPJ-SWP-CE-LIF offers a simple, yet sensitive way to analyse trace levels of flavin metabolites from complex biological samples.

Transient isotachophoresis with field-enhanced sample injection, using -CD as the chiral selector and tetrabutylammonium hydroxide (TBAOH) as the additive, was applied for online preconcentration and enantioseparation of three beta-agonists, namely, cimaterol, clenbuterol and terbutaline. Under the optimum conditions, the detection limits (defined as S/N = 3) of this method were found to be 1 ng/mL for all three pairs of beta-agonists enantiomers. Compared with conventional electrokinetic injection, the enhancement factors were greatly improved to be 250-fold. The proposed method has been applied for the analysis of human urine samples [Huang, L. et al., 2011]. 

*Stacking techniques with derivatization.* An on-line sample preconcentration approach coupled with in-capillary derivatization (SPCD-CE) has recently been applied, among others, to the (indirect) chiral separation of amino acids, whereby a special application represents the detection of D-amino acids as biomarkers in connection with bacterial growth, see Figure 3.30 [Ptolemy et al., 2006; Ptolemy & Britz-McKibbin, 2006]. In comparison with conventional CE, SPCD-CE provided a 100-fold improvement in concentration sensitivity, reduced sample handling and shorter analysis time. The reproducibility (n = 5) of the integrated SPCD-CE-UV technique was acceptable with average coefficients of variation of approximately 7.4 and 1.2% for quantitation (peak height) and apparent migration time, respectively [Ptolemy et al., 2006].

Figure 3.31. Example of TGF focusing and separation. Total image length is 1.9 mm. Realtime chiral TGF of DNS-D,L-Glutamic acid. Working conditions as in Figure 3.13. The Denantiomer peak is to the left in the figure; the L-enantiomer is to the right. Reprinted from

Advanced Sample Preparation 99

*Column-coupled electrophoretic techniques.* An ITP-EKC-UV method was successfully applied for the determination of trace enantiomers of various drugs and their biodegradation products (pheniramine and its metabolites, dioxopromethazine, dimethindene, amlodipine) in model complex ionic matrices and clinical samples [Mikuš et al., 2006, 2008a, 2008b; Marák et al., 2007], see an example in Figure 3.32. RSD values of migration times were lower than 2.0%. The concentrations of the analytes in tested samples, corresponding to the quantitation limits, were determined with acceptable precisions (RSD values ranged in interval 3.95–4.54%, n = 5) and accuracies (relative errors ranged in interval 5.84–6.22%, n = 5) under the stated conditions. The mean relative errors (REs) indicated by the recovery tests, characterizing accuracy of the chiral method for pheniramine, dimethindene and

The ITP-EKC-DAD method was shown to be a powerful tool in enantioselective pharmacokinetic studies of the -blocker drug, amlodipine, in clinical urine samples [Mikuš et al., 2008a] and the enantioselective metabolic study of cationic H1-antihistamine, pheniramine, present in clinical urine samples [Marák et al., 2007]. Performance parameters of the ITP-EKC-UV/DAD methods were sufficient for the routine enantioselective biomedical analyses of unpretreated biological samples. Besides all the benefits of the ITP-EKC combination, as mentioned above, speed spectral evaluation of separated zones enabled preliminary characterization of their homogeneity (presence of mixed zones) and preliminary indication of structurally (spectrally) similar compounds (potential metabolites of the drugs) in unknown electrophoretic peaks. To complete the evaluation of the performance parameters, the selectivity of the ITP-EKC separation method can be examined

dioxopromethazine, were 4.5, 4.8 and 3.6%, respectively [Mikuš et al., 2006a].

by the spectral evaluation of purity of analyte zones.

ref. [Balss et al., 2004].

Figure 3.30. Direct enantioselective amino acid flux analyses in the extra-cellular medium of *E. coli* by SPCD-CE. Electropherograms represent the extra-cellular media with 5% seeding volume incubated for (a) 0 h (control), (b) 3 h and (c) 5 h. Other conditions: sample solutions contained 25 and 50M of the D and L-amino acids, respectively, prepared in 40mM phosphate, pH 6.0 using a sample injection of 100 s. Conditions: 140mM borate buffer, pH 9.5; voltage 25 kV; capillary length 67 cm; internal diameter; 50m; UV 340 nm. Analyte peak number corresponds amino acid-isoindole adducts, where 1a: D-Ser, 1b: L-Ser, 2a: D-Ala, 2b: L-Ala, 3a: D-Glu, 3b: L-Glu, 4a: D-Asp, 4b: L-Asp, 5: taurine (6 M, internal standard), 6: Gly and 7a,b: L-Lys side-product. The direction of arrow indicates a net release (↑) or uptake (↓) of amino acid has been observed in the extra-cellular medium during bacterial growth. Note the rapid uptake of L-Ser and L-Asp and the steady-state enantioselective release of L-Ala. There was no detection of net efflux of D-Ala and D-Glu from *E. coli* into extra-cellular matrix during bacterial growth. Reprinted from ref. [Ptolemy & Britz-McKibbin, 2006].

*Counter-flow gradient focusing.* TGF has been shown to be effective for a 1200-fold concentration enrichment in 30 min for the baseline-separated enantiomers of glutamic acid, as illustrated in Figure 3.31 [Balss et al., 2004]. No validation data are available for this method.

Figure 3.30. Direct enantioselective amino acid flux analyses in the extra-cellular medium of *E. coli* by SPCD-CE. Electropherograms represent the extra-cellular media with 5% seeding volume incubated for (a) 0 h (control), (b) 3 h and (c) 5 h. Other conditions: sample solutions contained 25 and 50M of the D and L-amino acids, respectively, prepared in 40mM phosphate, pH 6.0 using a sample injection of 100 s. Conditions: 140mM borate buffer, pH 9.5; voltage 25 kV; capillary length 67 cm; internal diameter; 50m; UV 340 nm. Analyte peak number corresponds amino acid-isoindole adducts, where 1a: D-Ser, 1b: L-Ser, 2a: D-Ala, 2b: L-Ala, 3a: D-Glu, 3b: L-Glu, 4a: D-Asp, 4b: L-Asp, 5: taurine (6 M, internal standard), 6: Gly and 7a,b: L-Lys side-product. The direction of arrow indicates a net release (↑) or uptake (↓) of amino acid has been observed in the extra-cellular medium during bacterial growth. Note the rapid uptake of L-Ser and L-Asp and the steady-state enantioselective release of L-Ala. There was no detection of net efflux of D-Ala and D-Glu from *E. coli* into extra-cellular matrix during bacterial growth. Reprinted from ref. [Ptolemy

*Counter-flow gradient focusing.* TGF has been shown to be effective for a 1200-fold concentration enrichment in 30 min for the baseline-separated enantiomers of glutamic acid, as illustrated in Figure 3.31 [Balss et al., 2004]. No validation data are available for this

& Britz-McKibbin, 2006].

method.

Figure 3.31. Example of TGF focusing and separation. Total image length is 1.9 mm. Realtime chiral TGF of DNS-D,L-Glutamic acid. Working conditions as in Figure 3.13. The Denantiomer peak is to the left in the figure; the L-enantiomer is to the right. Reprinted from ref. [Balss et al., 2004].

*Column-coupled electrophoretic techniques.* An ITP-EKC-UV method was successfully applied for the determination of trace enantiomers of various drugs and their biodegradation products (pheniramine and its metabolites, dioxopromethazine, dimethindene, amlodipine) in model complex ionic matrices and clinical samples [Mikuš et al., 2006, 2008a, 2008b; Marák et al., 2007], see an example in Figure 3.32. RSD values of migration times were lower than 2.0%. The concentrations of the analytes in tested samples, corresponding to the quantitation limits, were determined with acceptable precisions (RSD values ranged in interval 3.95–4.54%, n = 5) and accuracies (relative errors ranged in interval 5.84–6.22%, n = 5) under the stated conditions. The mean relative errors (REs) indicated by the recovery tests, characterizing accuracy of the chiral method for pheniramine, dimethindene and dioxopromethazine, were 4.5, 4.8 and 3.6%, respectively [Mikuš et al., 2006a]. in the

The ITP-EKC-DAD method was shown to be a powerful tool in enantioselective pharmacokinetic studies of the -blocker drug, amlodipine, in clinical urine samples [Mikuš et al., 2008a] and the enantioselective metabolic study of cationic H1-antihistamine, pheniramine, present in clinical urine samples [Marák et al., 2007]. Performance parameters of the ITP-EKC-UV/DAD methods were sufficient for the routine enantioselective biomedical analyses of unpretreated biological samples. Besides all the benefits of the ITP-EKC combination, as mentioned above, speed spectral evaluation of separated zones enabled preliminary characterization of their homogeneity (presence of mixed zones) and preliminary indication of structurally (spectrally) similar compounds (potential metabolites of the drugs) in unknown electrophoretic peaks. To complete the evaluation of the performance parameters, the selectivity of the ITP-EKC separation method can be examined by the spectral evaluation of purity of analyte zones.

Figure 3.33. Chiral separation of gemifloxacin dissolved in a urinary solution with microchip electrophoresis. Peaks: (1) K+; (2) Na+; and (3) gemifloxacin racemate. Peaks for gemifloxacin enantiomers are denoted by 'g'. (a) The removal of metal ions was performed in the first separation channel. Run buffer: (50mM Bis–Tris + 10 M quinine)/Citric acid of pH 4.0. (b) The chiral separation was performed in the second separation channel using a run buffer of 50mM Bis–Tris/Citric acid containing 50 M 18C6H4 (pH 4.0). A urinary solution was fivefold diluted using 50mM Bis–Tris/Citric acid (pH 4.0). Gemifloxacin was 100 M in five-fold diluted urinary solution. Gemifloxacin was injected into the second separation channel by floating reservoir D, with reservoir F grounded, for 15 s right after the analyte passed the first detection point to ensure that all gemifloxacin was introduced into the second channel. For the analysis in the second channel, the applied voltage was 3.0 kV at reservoir E with reservoir F grounded and all other reservoirs floating. For the instrumental scheme, see Figure 3.15. Light source: He–Cd laser (325 nm); indirect laser-induced fluorescence detection at 405 nm using a photomultiplier tube. Analytes were detected at 32 and 38mm from the first and second injection crosses, respectively. Reprinted from ref. [Cho S.I. et al.,

Advanced Sample Preparation 101

A channel-coupled microchip electrophoresis device was designed to clean-up alkaline metal ions (interfering with chiral selector) from a sample matrix for the chiral analysis of gemifloxacin in urine, see Figure 3.33 [Cho S.I. et al., 2004]. The total analysis time of directly injected urine samples was less than 4 min with micromolar amounts of chiral selector (50

chiral

M CWE). No validation data are available for this method.

2004].

Figure 3.32. ITP-EKC method in column coupling configuration of the separation units for the direct analysis of unpretreated complex matrices sample; electrophoretic traces. Determination of pheniramine enantiomers in model urine sample demonstrates the effectivity of on-line sample preparation (removing matrices, preconcentration of enantiomers) in the first ITP stage (upper trace) and countercurrent separation mechanism (enantioseparation) in the second EKC stage (lower trace) of the ITP-EKC method. The separations were carried out using 10 mM sodium acetate - acetic acid, pH 4.75 as a leading electrolyte (ITP), 5 mM -aminocaproic acid - acetic acid, pH 4.5 as a terminating electrolyte (ITP), and 25 mM -aminocaproic acid - acetic acid, pH 4.5 as a carrier electrolyte (EKC). 0.1% (w/v) methyl-hydroxyethylcellulose served as an EOF suppressor in leading and carrier electrolytes. Carboxyethyl--CD (5 mg/mL) was used as a chiral selector in carrier electrolyte. Concentration of pheniramine was 7.10-8 M in the injected sample (a 10 times diluted spiked urine). The detection wavelength in EKC stage was 261 nm. The driving currents in the ITP and EKC stages were 200 A and 80 A, respectively. LE – leading cation; TE – terminating cation; PHM1, PHM2– migration positions of the first and second pheniramine enantiomer, respectively, MMC – major matrix constituents, SMC – semiminor matrix constituents. Reprinted from ref. [Mikuš et al., 2008b].

Figure 3.32. ITP-EKC method in column coupling configuration of the separation units for the direct analysis of unpretreated complex matrices sample; electrophoretic traces. Determination of pheniramine enantiomers in model urine sample demonstrates the effectivity of on-line sample preparation (removing matrices, preconcentration of enantiomers) in the first ITP stage (upper trace) and countercurrent separation mechanism (enantioseparation) in the second EKC stage (lower trace) of the ITP-EKC method. The separations were carried out using 10 mM sodium acetate - acetic acid, pH 4.75 as a leading electrolyte (ITP), 5 mM -aminocaproic acid - acetic acid, pH 4.5 as a terminating electrolyte (ITP), and 25 mM -aminocaproic acid - acetic acid, pH 4.5 as a carrier electrolyte (EKC). 0.1% (w/v) methyl-hydroxyethylcellulose served as an EOF suppressor in leading and carrier electrolytes. Carboxyethyl--CD (5 mg/mL) was used as a chiral selector in carrier electrolyte. Concentration of pheniramine was 7.10-8 M in the injected sample (a 10 times diluted spiked urine). The detection wavelength in EKC stage was 261 nm. The driving currents in the ITP and EKC stages were 200 A and 80 A, respectively. LE – leading cation; TE – terminating cation; PHM1, PHM2– migration positions of the first and second pheniramine enantiomer, respectively, MMC – major matrix constituents, SMC – semiminor

matrix constituents. Reprinted from ref. [Mikuš et al., 2008b].

Figure 3.33. Chiral separation of gemifloxacin dissolved in a urinary solution with microchip electrophoresis. Peaks: (1) K+; (2) Na+; and (3) gemifloxacin racemate. Peaks for gemifloxacin enantiomers are denoted by 'g'. (a) The removal of metal ions was performed in the first separation channel. Run buffer: (50mM Bis–Tris + 10 M quinine)/Citric acid of pH 4.0. (b) The chiral separation was performed in the second separation channel using a run buffer of 50mM Bis–Tris/Citric acid containing 50 M 18C6H4 (pH 4.0). A urinary solution was fivefold diluted using 50mM Bis–Tris/Citric acid (pH 4.0). Gemifloxacin was 100 M in five-fold diluted urinary solution. Gemifloxacin was injected into the second separation channel by floating reservoir D, with reservoir F grounded, for 15 s right after the analyte passed the first detection point to ensure that all gemifloxacin was introduced into the second channel. For the analysis in the second channel, the applied voltage was 3.0 kV at reservoir E with reservoir F grounded and all other reservoirs floating. For the instrumental scheme, see Figure 3.15. Light source: He–Cd laser (325 nm); indirect laser-induced fluorescence detection at 405 nm using a photomultiplier tube. Analytes were detected at 32 and 38mm from the first and second injection crosses, respectively. Reprinted from ref. [Cho S.I. et al., 2004].

A channel-coupled microchip electrophoresis device was designed to clean-up alkaline metal ions (interfering with chiral selector) from a sample matrix for the chiral analysis of gemifloxacin in urine, see Figure 3.33 [Cho S.I. et al., 2004]. The total analysis time of directly injected urine samples was less than 4 min with micromolar amounts of chiral selector (50 M CWE). No validation data are available for this method.

Figure 3.35. Miniaturized on-line SPE for enhancement of concentration sensitivity in CE. (a) Terbutaline enantiomer separation with on-line enrichment. A resolution of 1.6 and a separation efficiency of 300 000 plates were obtained. Enrichment capillary: *Lt* 58.0 cm, *Ld* 51.2 cm, *Li* 5.5 cm, *le* 2.5 mm, wash: water x 1.6 min x 140 kPa, wetting: methanol x 2.4 min x 140 kPa, conditioning: water x 2.4 min x 140 kPa, injection: 100 nM *rac*-terbutaline in water x 1.0 min x 140 kPa, wash/ filling: 40 mM potassium phosphate (pH 6.4) x 0.1 min x 140 kPa, 15 mM dimethyl--CD in 40 mM potassium phosphate (pH 6.4) x 0.7 min x 140 kPa, desorption: acetonitrile x 40 s x 3.4 kPa followed by 15 mM dimethyl--CD in 40 mM potassium phosphate (pH 6.4) x 4.0 min x 3.4 kPa, voltage: 14 kV, detection wavelength: 200 nm, temperature: 25°C. (b) Direct injection of terbutaline in plasma with on-line enrichment. The enrichment capillary had not been subjected to plasma samples before this run. Enrichment capillary: *L*<sup>t</sup> 58.0 cm, *L*<sup>d</sup> 51.2 cm, *L*<sup>i</sup> 5.4 cm, *l*<sup>e</sup> 1.25 mm, wash: water x 1.4 min x 140 kPa, 200 mM SDS in 100 mM sodium borate (pH 9.0) x 3.5 min x 140 kPa, wetting: methanol x 7.0 min x 140 kPa, conditioning: water x 2.1 min x 140 kPa, injection: 2 mM terbutaline in water–bovine plasma (3:1) x 0.1 min x 140 kPa, wash: water x 1.4 min x 140 kPa, filling: 40 mM potassium phosphate (pH 6.4) x 0.8 min x 140 kPa, desorption: acetonitrile x 30 s x 3.4 kPa followed by 40 mM potassium phosphate (pH 6.4) x 3.0 min x 3.4 kPa, voltage: 20 kV, detection wavelength: 200 nm, temperature: 25°C. Adapted from ref.

Advanced Sample Preparation 103

The separation efficiency was 300 000 plates. The relative average deviation from the mean was 17% for the enrichment capillaries. Within each enrichment capillary the relative average deviation from the mean was below 2% (n=6). Fouling of the capillary wall with plasma protein during the analysis was prevented by an SDS washing step. However, partial clogging of the enrichment capillary was observed after repeated plasma injections.

Electro membrane extraction as a new microextraction method was applied for the extraction of amlodipine (AML) enantiomers from biological samples [Nojavan & Fakhari, 2010]. During the extraction time of 15 min AML enantiomers migrated from a 3 mL sample solution through a supported liquid membrane into a 20 l acceptor solution presented inside the lumen of the hollow fibre. The driving force of the extraction was 200 V potential

[Petersson et al., 1999].

Figure 3.34. Isotachopherogram from the separations of racemic mixture of tryptophan enantiomers on the CC chip. (a) Single-column separation in the first channel using C1 to monitor the situation. The separation was carried out in the electrolyte system L1 with 8.5 A driving current. The injected sample contained the racemate at a 6x10-4 M concentration. (b) Separation in the tandem-coupled separation channels using C2 to monitor the separation. The separation was carried out in the electrolyte system L1 placed in both separation channels. The driving current was 8.5 A during the run in the first channel and it was reduced to 5 A during the separation in the second channel. The injected sample contained the racemate at a 1.5x10-3 M concentration. R, increasing resistance; L1 leading anion (propionate), T, termination anion (-aminocaproate). Reprinted from ref. [Ölvecká et al., 2001].

The use of a poly(methylmethacrylate) chip, provided with a pair of on-line hyphenated separation channels and on-column conductivity detectors, to isotachophoresis (ITP) separations of optical isomers was investigated by Ölvecká et al. [Ölvecká et al., 2001]. Single-column ITP, ITP in the tandem-coupled columns, and concentration-cascade ITP in the tandem-coupled columns were employed in this investigation using tryptophan enantiomers as model analytes, see Figure 3.34. Although providing a high production rate (about 2 pmol of a pure tryptophan enantiomer separated per second), single-column ITP was found suitable only to the analysis of samples containing the enantiomers at close concentrations. The best results in the respect of the resolution were achieved by using a concentration-cascade of the leading anions in the tandem-coupled separation channels.

*Extraction.* A miniaturized SPE (1-3 mm capillary of 200 m internal diameter packed with C18 alkyl-diol silica and capped by glass-fibre filters for the sorbent retaining) coupled online with CE significantly enhanced the concentration sensitivity for terbutaline [Petersson et al., 1999]. Experimental arrangement of the extractor and speed (10 min) sample enrichment procedure for the terbutaline enantioselective analysis are illustrated in Figure 3.18. The chiral application of the SPE-CE-UV method for model sample, as well as achiral application of the same method for plasma sample, is shown in Figure 3.35 [Petersson et al., 1999].

Figure 3.34. Isotachopherogram from the separations of racemic mixture of tryptophan enantiomers on the CC chip. (a) Single-column separation in the first channel using C1 to monitor the situation. The separation was carried out in the electrolyte system L1 with 8.5 A driving current. The injected sample contained the racemate at a 6x10-4 M concentration. (b) Separation in the tandem-coupled separation channels using C2 to monitor the separation. The separation was carried out in the electrolyte system L1 placed in both separation channels. The driving current was 8.5 A during the run in the first channel and it was reduced to 5 A during the separation in the second channel. The injected sample contained the racemate at a 1.5x10-3 M concentration. R, increasing resistance; L1 leading anion (propionate), T, termination anion (-aminocaproate). Reprinted from ref. [Ölvecká et al.,

The use of a poly(methylmethacrylate) chip, provided with a pair of on-line hyphenated separation channels and on-column conductivity detectors, to isotachophoresis (ITP) separations of optical isomers was investigated by Ölvecká et al. [Ölvecká et al., 2001]. Single-column ITP, ITP in the tandem-coupled columns, and concentration-cascade ITP in the tandem-coupled columns were employed in this investigation using tryptophan enantiomers as model analytes, see Figure 3.34. Although providing a high production rate (about 2 pmol of a pure tryptophan enantiomer separated per second), single-column ITP was found suitable only to the analysis of samples containing the enantiomers at close concentrations. The best results in the respect of the resolution were achieved by using a concentration-cascade of the leading anions in the tandem-coupled separation channels.

*Extraction.* A miniaturized SPE (1-3 mm capillary of 200 m internal diameter packed with C18 alkyl-diol silica and capped by glass-fibre filters for the sorbent retaining) coupled online with CE significantly enhanced the concentration sensitivity for terbutaline [Petersson et al., 1999]. Experimental arrangement of the extractor and speed (10 min) sample enrichment procedure for the terbutaline enantioselective analysis are illustrated in Figure 3.18. The chiral application of the SPE-CE-UV method for model sample, as well as achiral application of the same method for plasma sample, is shown in Figure 3.35 [Petersson et al.,

2001].

1999].

Figure 3.35. Miniaturized on-line SPE for enhancement of concentration sensitivity in CE. (a) Terbutaline enantiomer separation with on-line enrichment. A resolution of 1.6 and a separation efficiency of 300 000 plates were obtained. Enrichment capillary: *Lt* 58.0 cm, *Ld* 51.2 cm, *Li* 5.5 cm, *le* 2.5 mm, wash: water x 1.6 min x 140 kPa, wetting: methanol x 2.4 min x 140 kPa, conditioning: water x 2.4 min x 140 kPa, injection: 100 nM *rac*-terbutaline in water x 1.0 min x 140 kPa, wash/ filling: 40 mM potassium phosphate (pH 6.4) x 0.1 min x 140 kPa, 15 mM dimethyl--CD in 40 mM potassium phosphate (pH 6.4) x 0.7 min x 140 kPa, desorption: acetonitrile x 40 s x 3.4 kPa followed by 15 mM dimethyl--CD in 40 mM potassium phosphate (pH 6.4) x 4.0 min x 3.4 kPa, voltage: 14 kV, detection wavelength: 200 nm, temperature: 25°C. (b) Direct injection of terbutaline in plasma with on-line enrichment. The enrichment capillary had not been subjected to plasma samples before this run. Enrichment capillary: *L*<sup>t</sup> 58.0 cm, *L*<sup>d</sup> 51.2 cm, *L*<sup>i</sup> 5.4 cm, *l*<sup>e</sup> 1.25 mm, wash: water x 1.4 min x 140 kPa, 200 mM SDS in 100 mM sodium borate (pH 9.0) x 3.5 min x 140 kPa, wetting: methanol x 7.0 min x 140 kPa, conditioning: water x 2.1 min x 140 kPa, injection: 2 mM terbutaline in water–bovine plasma (3:1) x 0.1 min x 140 kPa, wash: water x 1.4 min x 140 kPa, filling: 40 mM potassium phosphate (pH 6.4) x 0.8 min x 140 kPa, desorption: acetonitrile x 30 s x 3.4 kPa followed by 40 mM potassium phosphate (pH 6.4) x 3.0 min x 3.4 kPa, voltage: 20 kV, detection wavelength: 200 nm, temperature: 25°C. Adapted from ref. [Petersson et al., 1999]. the applied

The separation efficiency was 300 000 plates. The relative average deviation from the mean was 17% for the enrichment capillaries. Within each enrichment capillary the relative average deviation from the mean was below 2% (n=6). Fouling of the capillary wall with plasma protein during the analysis was prevented by an SDS washing step. However, partial clogging of the enrichment capillary was observed after repeated plasma injections.

Electro membrane extraction as a new microextraction method was applied for the extraction of amlodipine (AML) enantiomers from biological samples [Nojavan & Fakhari, 2010]. During the extraction time of 15 min AML enantiomers migrated from a 3 mL sample solution through a supported liquid membrane into a 20 l acceptor solution presented inside the lumen of the hollow fibre. The driving force of the extraction was 200 V potential

*Extraction + stacking.* A sufficient selectivity and sensitivity enhancement of two orders of magnitude was achieved for ephedrine derivatives {(1R,2S)-ephedrine, (1R,2R) pseudoephedrine, (1S,2S)-pseudoephedrine} in human urine samples using an on-line combination of headspace SPME with FESI, see ref. [Fang H.F. et al., 2006b]. Electropherogram from the SPME-FESI-EKC method applied on spiked urine samples is shown in Figure 3.36. The intra-assay relative standard deviations were between 4.38 and 7.76% (concentration of analytes: 0.3 μg/mL; n = 5). Recovery was in range 88.7-97.5%. The performance parameters indicate a good potential of the SPME-FESI-CE-DAD method for

Advanced Sample Preparation 105

The back LLE integrated with FESI and supported by centrifuge microextraction (CME) was applied for a significant increase in sensitivity for (1*R*, 2*R*)-pseudoephedrine, (1*R*, 2*S*) ephedrine, (1*S*, 2*S*)-pseudoephedrine and (*S*)-(+)-methamphetamine in spiked serum [Fang H.F. et al., 2006a]. In this method, the CME effectively combined removal of macromolecular contaminants and other interfering components, desalting and preconcentration into one single step. Performance parameters of the CME-LLE-FESI-CE-DAD method indicate good potential of the proposed method for its routine use. The relative standard deviations (RSD, n=6) of all of the analytes were between 8.7-17.7% on the basis of peak areas. Utilizing (-) pseudoephedrine as an internal standard, RSD results showed significant improvement (5.3- 8.9%). For a 0.1 g/mL concentration of the ephedrine derivates, recoveries were in the range of 97-114%. Relative recoveries, defined as a ratio of CE peak areas of two different sets of spiked urine extracts, were calculated to evaluate the effect of the matrix. The relative recoveries for all target drugs were from 90-112%. This means that the matrix had little effect

Figure 3.37. On-line microdialysis-CE-LIF analysis of the supernatant over an intact larval tiger salamander retina. A single salamander retina was incubated for 3 h in 50 L Amphibian Ringers solution. D-Serine (5.2±2.1 M, standard error, *n* = 4) was detected in the supernatant. Separation buffer, 50 mM borate, 20 mM HP--CD, pH 10.5. Separation

its routine use.

on the method, particularly on the CME step.

distance, 8 cm. Reprinted from ref. [O'Brien et al., 2003].

with the negative electrode in the acceptor solution and the positive electrode in the sample solution. 2-Nitro-phenyl octylether was used as the supported liquid membrane. Using 10 mM HCl as background electrolyte in the sample and acceptor solution, enrichment up to 124 times was achieved. Then the extract was analysed using the CD modified CE method for separation of AML enantiomers. The best results were achieved using a phosphate running buffer (100 mM, pH 2.0) containing 5 mM hydroxypropyl alpha CD. The range of quantitation for both enantiomers was 10-500 ng/mL. Intra- and inter-day RSDs (n = 6) were less than 14%. The limits of quantitation and detection for both enantiomers were 10 and 3 ng/mL respectively. Finally this procedure was applied to determine the concentration of AML enantiomers in plasma and urine samples.

Figure 3.36. Separation of spiked urine sample (a) with directly injected (concentration of each analyte: 5.00 g/mL); (b) after SPME-FESI (concentration of each analyte: 0.25g/mL) followed by CE separation. The optimized SPME conditions: temperature of 90°C, time of 30 min, 2.00 g sodium hydroxide and 0.50 g sodium chloride for headspace extraction; 20% acetonitrile aqueous phase solvent with desorption time of 20 min for desorption. CE conditions: the run buffer contained 17.5mM -CD and 150mM phosphate (pH 2.5) in the running voltage 25 kV at 20°C. Sample injection: 7 kV×10 s. (1R,2S)-ephedrine ((−)-E), (1R,2R)-pseudoephedrine ((−)-PE) and (1S,2S)-pseuodephedrine ((+)-PE). Reprinted from ref. [Fang H.F. et al., 2006b].

with the negative electrode in the acceptor solution and the positive electrode in the sample solution. 2-Nitro-phenyl octylether was used as the supported liquid membrane. Using 10 mM HCl as background electrolyte in the sample and acceptor solution, enrichment up to 124 times was achieved. Then the extract was analysed using the CD modified CE method for separation of AML enantiomers. The best results were achieved using a phosphate running buffer (100 mM, pH 2.0) containing 5 mM hydroxypropyl alpha CD. The range of quantitation for both enantiomers was 10-500 ng/mL. Intra- and inter-day RSDs (n = 6) were less than 14%. The limits of quantitation and detection for both enantiomers were 10 and 3 ng/mL respectively. Finally this procedure was applied to determine the

Figure 3.36. Separation of spiked urine sample (a) with directly injected (concentration of each analyte: 5.00 g/mL); (b) after SPME-FESI (concentration of each analyte: 0.25g/mL) followed by CE separation. The optimized SPME conditions: temperature of 90°C, time of 30 min, 2.00 g sodium hydroxide and 0.50 g sodium chloride for headspace extraction; 20% acetonitrile aqueous phase solvent with desorption time of 20 min for desorption. CE conditions: the run buffer contained 17.5mM -CD and 150mM phosphate (pH 2.5) in the running voltage 25 kV at 20°C. Sample injection: 7 kV×10 s. (1R,2S)-ephedrine ((−)-E), (1R,2R)-pseudoephedrine ((−)-PE) and (1S,2S)-pseuodephedrine ((+)-PE). Reprinted from

ref. [Fang H.F. et al., 2006b].

concentration of AML enantiomers in plasma and urine samples.

*Extraction + stacking.* A sufficient selectivity and sensitivity enhancement of two orders of magnitude was achieved for ephedrine derivatives {(1R,2S)-ephedrine, (1R,2R) pseudoephedrine, (1S,2S)-pseudoephedrine} in human urine samples using an on-line combination of headspace SPME with FESI, see ref. [Fang H.F. et al., 2006b]. Electropherogram from the SPME-FESI-EKC method applied on spiked urine samples is shown in Figure 3.36. The intra-assay relative standard deviations were between 4.38 and 7.76% (concentration of analytes: 0.3 μg/mL; n = 5). Recovery was in range 88.7-97.5%. The performance parameters indicate a good potential of the SPME-FESI-CE-DAD method for its routine use.

The back LLE integrated with FESI and supported by centrifuge microextraction (CME) was applied for a significant increase in sensitivity for (1*R*, 2*R*)-pseudoephedrine, (1*R*, 2*S*) ephedrine, (1*S*, 2*S*)-pseudoephedrine and (*S*)-(+)-methamphetamine in spiked serum [Fang H.F. et al., 2006a]. In this method, the CME effectively combined removal of macromolecular contaminants and other interfering components, desalting and preconcentration into one single step. Performance parameters of the CME-LLE-FESI-CE-DAD method indicate good potential of the proposed method for its routine use. The relative standard deviations (RSD, n=6) of all of the analytes were between 8.7-17.7% on the basis of peak areas. Utilizing (-) pseudoephedrine as an internal standard, RSD results showed significant improvement (5.3- 8.9%). For a 0.1 g/mL concentration of the ephedrine derivates, recoveries were in the range of 97-114%. Relative recoveries, defined as a ratio of CE peak areas of two different sets of spiked urine extracts, were calculated to evaluate the effect of the matrix. The relative recoveries for all target drugs were from 90-112%. This means that the matrix had little effect on the method, particularly on the CME step.

Figure 3.37. On-line microdialysis-CE-LIF analysis of the supernatant over an intact larval tiger salamander retina. A single salamander retina was incubated for 3 h in 50 L Amphibian Ringers solution. D-Serine (5.2±2.1 M, standard error, *n* = 4) was detected in the supernatant. Separation buffer, 50 mM borate, 20 mM HP--CD, pH 10.5. Separation distance, 8 cm. Reprinted from ref. [O'Brien et al., 2003].

*Dialysis.* A microdialysis coupled on-line with CE has been used for the rapid determination of Asp enantiomers in tissue samples from rats [Thompson, J.E. et al., 1999]. Filtration and deproteination was carried out inserting a microdialysis probe into a homogenized tissue sample. The variability that was observed by the microdialysis-CE method was due to variations in the sample as the same sample analysed repeatedly gave a RSD of 2.6%. A similar set-up for the on-line microdialysis-CE was used for the enantioselective analysis of Ser (separated from other primary amines commonly found in biological samples) in tissue homogenates [O'Brien et al., 2003], see electropherogram in Figure 3.37. Recovery was in the range of 71.5-86.8%.

*Dialysis + stacking.* An EKC analysis method designed for use with microdialysis sampling and electrophoretic sample stacking has been developed for the determination of isoproterenol enantiomers [Hadwiger et al., 1996]. The half-life of isoproterenol is less than l0 min, therefore, a 1 min sampling frequency, with a dialysis perfusion flow-rate of 500 nL/min, was needed to sufficiently define the pharmacokinetic curve. The optimized chiral CE method was applied to the analysis of intravenous microdialysis samples collected following administration of racemic isoproterenol. A typical electropherogram of a microdialysis sample collected over the first 5 min after dosing is shown in Figure 3.38. The enantiomers of isoproterenol are resolved from each other and from all endogenous compounds. Unfortunately, the detection limits were not sufficient to follow the concentration of isoproterenol for long enough to establish pharmacokinetic parameters. However, no off-line sample preconcentration was possible because of very low sample volumes for the analysis. Using an on-column concentration technique (*pH-mediated peak stacking*, i.e., injecting a plug of acidic solution directly after the sample) essential for analysing highly ionic sample, i.e., the microdialysis perfusate of plasma, the pharmacokinetic data/curve for the enantioselective elimination of isoproterenol could be obtained. A five-fold increase in sensitivity was achieved inserting an in-capillary stacking preconcentration of microdialyzed plasma samples, as shown in Figure 3.39. Using a catecholamine-based internal standard with similar electrochemistry (important for the electrochemical detection, EC) to isoproterenol, the precision of analysis increased from 3.2% RSD to 1.4%. This example illustrated a practical situation where the on-line sample preparation essentially replaced an off-line procedure. i.e.,

Figure 3.38. CE-UV electropherograms of isoproterenol obtained with CE and CEmicrodialysis method. (A) Standard containing 100M of (-)- and (+)-isoproterenol (ISP) bitartrate dissolved in Ringers-8.0 mM Na2EDTA-97 M NaHSO3; (B) Microdialysate

Advanced Sample Preparation 107

Figure 3.39. CE-EC electropherograms of standard solutions using normal electrokinetic injection (A) and acid stacked electrokinetic injection (B). Peaks: I=DHBA; 2=5NMHT; 3=(-)-

iso -proterenol (ISP); 4=(+)-ISP. Reprinted from ref. [Hadwiger et al., 1996].

acquired from a rat 5 min after dosing. Reprinted from ref. [Hadwiger et al., 1996]

*Dialysis.* A microdialysis coupled on-line with CE has been used for the rapid determination of Asp enantiomers in tissue samples from rats [Thompson, J.E. et al., 1999]. Filtration and deproteination was carried out inserting a microdialysis probe into a homogenized tissue sample. The variability that was observed by the microdialysis-CE method was due to variations in the sample as the same sample analysed repeatedly gave a RSD of 2.6%. A similar set-up for the on-line microdialysis-CE was used for the enantioselective analysis of Ser (separated from other primary amines commonly found in biological samples) in tissue homogenates [O'Brien et al., 2003], see electropherogram in Figure 3.37. Recovery was

*Dialysis + stacking.* An EKC analysis method designed for use with microdialysis sampling and electrophoretic sample stacking has been developed for the determination of isoproterenol enantiomers [Hadwiger et al., 1996]. The half-life of isoproterenol is less than l0 min, therefore, a 1 min sampling frequency, with a dialysis perfusion flow-rate of 500 nL/min, was needed to sufficiently define the pharmacokinetic curve. The optimized chiral CE method was applied to the analysis of intravenous microdialysis samples collected following administration of racemic isoproterenol. A typical electropherogram of a microdialysis sample collected over the first 5 min after dosing is shown in Figure 3.38. The enantiomers of isoproterenol are resolved from each other and from all endogenous compounds. Unfortunately, the detection limits were not sufficient to follow the concentration of isoproterenol for long enough to establish pharmacokinetic parameters. However, no off-line sample preconcentration was possible because of very low sample volumes for the analysis. Using an on-column concentration technique (*pH-mediated peak stacking*, i.e., injecting a plug of acidic solution directly after the sample) essential for analysing highly ionic sample, i.e., the microdialysis perfusate of plasma, the pharmacokinetic data/curve for the enantioselective elimination of isoproterenol could be obtained. A five-fold increase in sensitivity was achieved inserting an in-capillary stacking preconcentration of microdialyzed plasma samples, as shown in Figure 3.39. Using a catecholamine-based internal standard with similar electrochemistry (important for the electrochemical detection, EC) to isoproterenol, the precision of analysis increased from 3.2% RSD to 1.4%. This example illustrated a practical situation where the on-line sample

in the range of 71.5-86.8%.

preparation essentially replaced an off-line procedure.

Figure 3.38. CE-UV electropherograms of isoproterenol obtained with CE and CEmicrodialysis method. (A) Standard containing 100M of (-)- and (+)-isoproterenol (ISP) bitartrate dissolved in Ringers-8.0 mM Na2EDTA-97 M NaHSO3; (B) Microdialysate acquired from a rat 5 min after dosing. Reprinted from ref. [Hadwiger et al., 1996]

Figure 3.39. CE-EC electropherograms of standard solutions using normal electrokinetic injection (A) and acid stacked electrokinetic injection (B). Peaks: I=DHBA; 2=5NMHT; 3=(-) iso -proterenol (ISP); 4=(+)-ISP. Reprinted from ref. [Hadwiger et al., 1996].

Figure 3.41. FI-CE derivatization and separation of carnitine. Electrophoretic separation of a synthetic sample containing a D-:L-carnitine ratio of 1:100, obtained with automatized FI-CE system based on FMOC derivatization and 2,6 dimethyl--CD as chiral selector. Reprinted

Advanced Sample Preparation 109

from ref. [Mardones et al., 1999].

Figure 3.40. Electropherogram obtained after using the double stacking procedure followed by CE enantiomer separation. 3 L of 400 nM *rac-terbutaline* (200 nM of each enantiomer) was injected. Arrows indicate the different events. (1) Stacking step one begins; (2) stacking peak of positive species; (3) voltage off, backpressure on; (4) zone of 5 mM phosphate buffer pH 7.5; (5) back-pressure off; (6) voltage on, stacking step two at the inlet end of the capillary; (7) back-pressure on; (8) back-pressure off, final separation step begins. The plasma samples were pretreated by on-line MLC before double stacking and CE enantioseparation. Reprinted from ref. [Pálmarsdóttir & Edholm, 1995].

*Chromatography + stacking.* The supported liquid membrane technique coupled on-line with CE through a MLC interface and additionally combined with a doublestacking preconcentration has been applied to a sensitive enantioselective determination of bambuterol in human plasma [Pálmarsdóttir et al., 1996, 1997]. No validation data are available for this method.

Plasma samples were pretreated and the concentration sensitivity increased by on-line MLC before double stacking and CE enantioseparation of terbutaline, see the electropherogram in Figure 3.40 [Pálmarsdóttir & Edholm, 1995]. Microlitre volumes of the cleaned sample from the MLC were concentrated directly in the electrophoresis capillary without significant loss of separation performance. The whole procedure was performed with a high degree of precision. Reproducibility of the double stacking procedure at different concentrations was as follows: RSD of migration times was 0.5-1.5%, peak areas 0.7-2.4% and peak heights 1.4- 3.9% for enantiomer 1, and RSD of migration times 0.6-1.0%, peak areas 0.9-3.5%, peak heights 2.8-3.3 % for enantiomer 2. RSD of resolution was in the range of 1.1-1.9%. Other enantiomers of chiral drugs, namely bambuterol, brompheniramine, propranolol, ephedrine, were also separated using the same procedure.

*Flow injection.* An automatized system with on-line FI derivatization coupled to chiral CE has been developed for the enantiomeric separation of carnitine [Mardones et al., 1999]. This method allowed the determination of D-carnitine in a large excess of L-carnitine (1:100, D:L) in synthetic samples, see Figure 3.41. The reproducibility of the migration time was about 2.3%.

Figure 3.40. Electropherogram obtained after using the double stacking procedure followed by CE enantiomer separation. 3 L of 400 nM *rac-terbutaline* (200 nM of each enantiomer) was injected. Arrows indicate the different events. (1) Stacking step one begins; (2) stacking peak of positive species; (3) voltage off, backpressure on; (4) zone of 5 mM phosphate buffer pH 7.5; (5) back-pressure off; (6) voltage on, stacking step two at the inlet end of the capillary; (7) back-pressure on; (8) back-pressure off, final separation step begins. The plasma samples were pretreated by on-line MLC before double stacking and CE

*Chromatography + stacking.* The supported liquid membrane technique coupled on-line with CE through a MLC interface and additionally combined with a doublestacking preconcentration has been applied to a sensitive enantioselective determination of bambuterol in human plasma [Pálmarsdóttir et al., 1996, 1997]. No validation data are

Plasma samples were pretreated and the concentration sensitivity increased by on-line MLC before double stacking and CE enantioseparation of terbutaline, see the electropherogram in Figure 3.40 [Pálmarsdóttir & Edholm, 1995]. Microlitre volumes of the cleaned sample from the MLC were concentrated directly in the electrophoresis capillary without significant loss of separation performance. The whole procedure was performed with a high degree of precision. Reproducibility of the double stacking procedure at different concentrations was as follows: RSD of migration times was 0.5-1.5%, peak areas 0.7-2.4% and peak heights 1.4- 3.9% for enantiomer 1, and RSD of migration times 0.6-1.0%, peak areas 0.9-3.5%, peak heights 2.8-3.3 % for enantiomer 2. RSD of resolution was in the range of 1.1-1.9%. Other enantiomers of chiral drugs, namely bambuterol, brompheniramine, propranolol,

*Flow injection.* An automatized system with on-line FI derivatization coupled to chiral CE has been developed for the enantiomeric separation of carnitine [Mardones et al., 1999]. This method allowed the determination of D-carnitine in a large excess of L-carnitine (1:100, D:L) in synthetic samples, see Figure 3.41. The reproducibility of the migration time was about

enantioseparation. Reprinted from ref. [Pálmarsdóttir & Edholm, 1995].

ephedrine, were also separated using the same procedure.

available for this method.

2.3%.

Figure 3.41. FI-CE derivatization and separation of carnitine. Electrophoretic separation of a synthetic sample containing a D-:L-carnitine ratio of 1:100, obtained with automatized FI-CE system based on FMOC derivatization and 2,6 dimethyl--CD as chiral selector. Reprinted from ref. [Mardones et al., 1999].

**4** 

**Advanced Combinations of** 

**Detection and Electrophoresis** 

In the past two decades, the on-line coupling of separation techniques and appropriate (e.g., spectral, electrochemic) detection, also referred to as hyphenation, has become an important topic in analytical chemistry [Ewing et al., 1989]. In most instances hyphenation is used to obtain more information on the identity of the sample constituents, to improve analyte detectability or to enhance the speed of analysis. Therefore, in the following sections on-line detection approaches will be discussed. A list of the detection techniques hyphenated with

Detection in CE is a significant challenge as a result of the small dimensions of the capillary. Poor concentration sensitivity of CE is due to the limited sample volume that can be introduced into the capillary (typically nanolitre volumes in 25-75 m internal diameter capillaries) and the low optical path length when the most available and used UV-VIS absorbance detection is employed. This is even worse when combining photometric absorbance detection with the MCE, resulting in a preference for other detection techniques for the microfluidic devices (e.g., fluorescence, electrochemical) [Belder, 2006]. A review of the major on-line CE detection modalities with their advantages and limitations is presented by Swinney and Bornhop [Swinney & Bornhop, 2000]. Hempel [Hempel, 2000] aimed at ascertaining strategies for how to improve the sensitivity in CE for the analysis of drugs in biological fluids, discussing sensitive detection modes such as laser-induced fluorescence detection (LIF) and mass spectrometry (MS). Hernández et al. [Hernández et al., 2008, 2010] give a general view of the different strategies used in recent years (2005-2007) to enhance the detection sensitivity in chiral analysis by CE. Molecular fluorescence, phosphorescence and chemiluminescence spectrometry as very sensitive and selective detection techniques are presented by Powe et al. [Powe et al. 2010]*.* Alternatively to the sensitive detection techniques (advanced detection), an advanced CE (i.e., CE with on-line sample preconcentration) or, at best, a combination of both advanced approaches, has been proposed for a significant enhancement of the sensitivity in chiral CE analyses, as

Analyte identification aspects in CE and CEC separation techniques are discussed by Kok et al. [Kok et al., 1998]. Attention is paid to the spectrometric techniques covering UV-VIS absorption, fluorescence line-narrowing spectroscopy, Raman spectroscopy, nuclear magnetic resonance (NMR) and MS. The most important/applied detection techniques, regarding an on-line structural identification in chiral CE analyses, are comprehensively

CE and several important characteristics of these techniques are given in Table 4.1.

comprehensively illustrated in chapters 3 and 4 of this monograph.

presented in chapter 4 of this monograph.

**4.1 Detection in electrophoresis - introduction** 

### **Advanced Combinations of Detection and Electrophoresis**

### **4.1 Detection in electrophoresis - introduction**

In the past two decades, the on-line coupling of separation techniques and appropriate (e.g., spectral, electrochemic) detection, also referred to as hyphenation, has become an important topic in analytical chemistry [Ewing et al., 1989]. In most instances hyphenation is used to obtain more information on the identity of the sample constituents, to improve analyte detectability or to enhance the speed of analysis. Therefore, in the following sections on-line detection approaches will be discussed. A list of the detection techniques hyphenated with CE and several important characteristics of these techniques are given in Table 4.1.

Detection in CE is a significant challenge as a result of the small dimensions of the capillary. Poor concentration sensitivity of CE is due to the limited sample volume that can be introduced into the capillary (typically nanolitre volumes in 25-75 m internal diameter capillaries) and the low optical path length when the most available and used UV-VIS absorbance detection is employed. This is even worse when combining photometric absorbance detection with the MCE, resulting in a preference for other detection techniques for the microfluidic devices (e.g., fluorescence, electrochemical) [Belder, 2006]. A review of the major on-line CE detection modalities with their advantages and limitations is presented by Swinney and Bornhop [Swinney & Bornhop, 2000]. Hempel [Hempel, 2000] aimed at ascertaining strategies for how to improve the sensitivity in CE for the analysis of drugs in biological fluids, discussing sensitive detection modes such as laser-induced fluorescence detection (LIF) and mass spectrometry (MS). Hernández et al. [Hernández et al., 2008, 2010] give a general view of the different strategies used in recent years (2005-2007) to enhance the detection sensitivity in chiral analysis by CE. Molecular fluorescence, phosphorescence and chemiluminescence spectrometry as very sensitive and selective detection techniques are presented by Powe et al. [Powe et al. 2010]*.* Alternatively to the sensitive detection techniques (advanced detection), an advanced CE (i.e., CE with on-line sample preconcentration) or, at best, a combination of both advanced approaches, has been proposed for a significant enhancement of the sensitivity in chiral CE analyses, as comprehensively illustrated in chapters 3 and 4 of this monograph.

Analyte identification aspects in CE and CEC separation techniques are discussed by Kok et al. [Kok et al., 1998]. Attention is paid to the spectrometric techniques covering UV-VIS absorption, fluorescence line-narrowing spectroscopy, Raman spectroscopy, nuclear magnetic resonance (NMR) and MS. The most important/applied detection techniques, regarding an on-line structural identification in chiral CE analyses, are comprehensively presented in chapter 4 of this monograph.

upon the analyte being analysed). These LODs are clearly insufficient to solve many analytical problems, especially to analyse ultratrace analytes in complex matrices, being variable in qualitative and quantitative composition. Therefore, various strategies based on increasing absorbance signal (A=dc) have been proposed to overcome the poor concentration sensitivity of UV-VIS-CE, such as (i) the selection of lower-UV wavelengths, 190–205 nm [Swinney & Bornhop, 2000], (ii) indirect absorbance detection for analytes with low values of molar absorptivity [Yeung, 1989], for the principle see Figure 4.1, (iii) increasing the pathlength d [Kaniansky et al., 1997; Grant & Steuer, 1990; Tsueda et al., 1990; Taylor & Yeung, 1991; Poppe, 1980; Mainka & Bachmann, 1997; Kaltenbach et al., 1997, Anaheim, CA 1997; Aiken et al., 1991] and (iv) derivatization of analytes [Mikuš & Kaniansky, 2000; Hu T. et al., 1995]. However, the strategies (i-iv) have several limitations and specific features that have to be carefully considered before being employed. These are (i) a relatively poor signal-to-noise ratio, chiral selector can also significantly absorb, (ii) use of strongly absorbing chromophores, inserted chromofores can influence complex equilibrium of analyte with chiral selector, (iii) use of capillaries with higher internal diameter, e.g., 300-800 m (temperature dissipation and, by that, separation efficiency decrease with internal diameter), or properly designed detection window, e.g., bubble cells (see Figure 4.2a and a photo detail in Figure 4.3), Z-shaped cells (see Figure 4.2b), multireflection nanolitre scale cell (often accompanied by larger probe volumes and reduction in separation efficiency and resolution) and (iv) additional sample preparation step is needed, electrophoretic mobilities, as well as the complexing properties of derivatives, can differ from those of their native forms. Nevertheless, the detection sensitivity with UV-VIS absorbance detectors usually cannot be increased more than several tenths times, even if applying any of the presented approaches, so that sample preparation is required. On the other hand, an appropriate use of the on-line sample preparation procedures (as given in chapter 3) enables an application of UV-VIS absorbance detectors, even in ultrasensitive

Advanced Combinations of Detection and Electrophoresis 113

bioanalyses, as illustrated on several examples (in section 4.6. and Table 3.1).

Figure 4.1. Schematic illustration of indirect absorption detection. The separation electrolyte additive provides a large background signal at the detector. Displacement occurs in the analyte zone and lower absorbance intensity is observed. White dots represent absorbing species, blue dots represent non-absorbing species. Reprinted from ref. [Yeung, 1989].

When performing chiral analysis based on the implementation of chiral selector into the separation system, potential interferences of the chiral selector with the detection technique have to be considered and avoided, in order not to deteriorate the detection signal. Therefore, great attention must be paid to the selection of the type of chiral selector and to studying the compatibility of the chiral separation system with the linked detection technique in each particular case. Thus, chiral selectors with suppressed absorption properties must be used in absorbance detection, chiral selectors with suppressed electrochemical activity in electrochemical detection, etc. Another possibility in avoiding detection interferences caused by a chiral selector is to utilize specific electromigration effects (countercurrent migration of charged chiral selector, manipulation of the selector velocity by EOF, see chapter 2) [Iio et al., 2005; Yang L. et al., 1997; Lu W.Z. et al., 1996; Fanali et al., 1998a; Schulte et al., 1998; Lu W., 1998] or techniques (the partial filling technique, a zone of the capillary where the enantiomeric separation takes place is filled with BGE containing the chiral selector while the zone close to the detector only contains buffer without chiral selector) [Rudaz et al., 2005; Schappler et al., 2006]. These approaches are used to prevent entering the selector into the detection space. In analogy, the compatibility of chiral selectors with on-line sample preparation techniques also has to be considered in advanced CE systems (those with on-line sample preparation), as discussed in chapter 3. Therefore, the most complex situation is given when considering chiral selector (or, generally, selector) vs. on-line sample preparation vs. on-line detection. This hyphenation requires sophisticated solutions based on a deep knowledge of the particular fields, which are presented in the chapters 2-4 of this monograph.

The most important and/or the most frequently used on- and end-column detection techniques and corresponding hyphenations suitable for (advanced) CE are critically described in the following sections, also giving examples of their utilization in chiral bioanalyses of drugs (section 4.6. and Tables 2.1 and 3.1). Other, more rarely used detection techniques for CE (radioisotope detection, nuclear magnetic resonance, photothermal refraction, refractive index detection, circular dichroism, Raman-based detection, electrochemiluminescence, etc.), with their description and utilization, can be found in review papers given above. Those rare detection techniques have been mostly applied in achiral analyses, while their chiral applications available in literature are given in Table 2.1.

### **4.2 Photometric absorbance detection**

### **4.2.1 Single wavelength UV-VIS absorbance detection**

UV–VIS absorbance detectors working at single wavelength are the most commonly used on-column detectors for microseparations, particularly for CE, due to their interesting features, such as commercial availability, simplicity, versatility, relatively low cost, and frequently used as universal detection techniques because many organics can be detected at 195–210 nm. UV-VIS absorbance detectors produce LODs in CE corresponding to a few femtomoles of analyte (at subpicomole levels), i.e., high mass sensitivity. However, due to the need for small volumes employed in CE to avoid peak broadening that decrease the efficiency of the separation (pl to nL volumes), such sensitivity is in the micromolar range, i.e., appears modest when expressed in terms of concentration (mass detection limits are in the range of 10-13-10-15 mol while concentration LODs ranging from 10-5 to 10-7 M depending

When performing chiral analysis based on the implementation of chiral selector into the separation system, potential interferences of the chiral selector with the detection technique have to be considered and avoided, in order not to deteriorate the detection signal. Therefore, great attention must be paid to the selection of the type of chiral selector and to studying the compatibility of the chiral separation system with the linked detection technique in each particular case. Thus, chiral selectors with suppressed absorption properties must be used in absorbance detection, chiral selectors with suppressed electrochemical activity in electrochemical detection, etc. Another possibility in avoiding detection interferences caused by a chiral selector is to utilize specific electromigration effects (countercurrent migration of charged chiral selector, manipulation of the selector velocity by EOF, see chapter 2) [Iio et al., 2005; Yang L. et al., 1997; Lu W.Z. et al., 1996; Fanali et al., 1998a; Schulte et al., 1998; Lu W., 1998] or techniques (the partial filling technique, a zone of the capillary where the enantiomeric separation takes place is filled with BGE containing the chiral selector while the zone close to the detector only contains buffer without chiral selector) [Rudaz et al., 2005; Schappler et al., 2006]. These approaches are used to prevent entering the selector into the detection space. In analogy, the compatibility of chiral selectors with on-line sample preparation techniques also has to be considered in advanced CE systems (those with on-line sample preparation), as discussed in chapter 3. Therefore, the most complex situation is given when considering chiral selector (or, generally, selector) vs. on-line sample preparation vs. on-line detection. This hyphenation requires sophisticated solutions based on a deep knowledge of the particular

The most important and/or the most frequently used on- and end-column detection techniques and corresponding hyphenations suitable for (advanced) CE are critically described in the following sections, also giving examples of their utilization in chiral bioanalyses of drugs (section 4.6. and Tables 2.1 and 3.1). Other, more rarely used detection techniques for CE (radioisotope detection, nuclear magnetic resonance, photothermal refraction, refractive index detection, circular dichroism, Raman-based detection, electrochemiluminescence, etc.), with their description and utilization, can be found in review papers given above. Those rare detection techniques have been mostly applied in achiral analyses, while their chiral applications available in literature are given in Table 2.1.

UV–VIS absorbance detectors working at single wavelength are the most commonly used on-column detectors for microseparations, particularly for CE, due to their interesting features, such as commercial availability, simplicity, versatility, relatively low cost, and frequently used as universal detection techniques because many organics can be detected at 195–210 nm. UV-VIS absorbance detectors produce LODs in CE corresponding to a few femtomoles of analyte (at subpicomole levels), i.e., high mass sensitivity. However, due to the need for small volumes employed in CE to avoid peak broadening that decrease the efficiency of the separation (pl to nL volumes), such sensitivity is in the micromolar range, i.e., appears modest when expressed in terms of concentration (mass detection limits are in the range of 10-13-10-15 mol while concentration LODs ranging from 10-5 to 10-7 M depending

fields, which are presented in the chapters 2-4 of this monograph.

**4.2 Photometric absorbance detection** 

**4.2.1 Single wavelength UV-VIS absorbance detection** 

upon the analyte being analysed). These LODs are clearly insufficient to solve many analytical problems, especially to analyse ultratrace analytes in complex matrices, being variable in qualitative and quantitative composition. Therefore, various strategies based on increasing absorbance signal (A=dc) have been proposed to overcome the poor concentration sensitivity of UV-VIS-CE, such as (i) the selection of lower-UV wavelengths, 190–205 nm [Swinney & Bornhop, 2000], (ii) indirect absorbance detection for analytes with low values of molar absorptivity [Yeung, 1989], for the principle see Figure 4.1, (iii) increasing the pathlength d [Kaniansky et al., 1997; Grant & Steuer, 1990; Tsueda et al., 1990; Taylor & Yeung, 1991; Poppe, 1980; Mainka & Bachmann, 1997; Kaltenbach et al., 1997, Anaheim, CA 1997; Aiken et al., 1991] and (iv) derivatization of analytes [Mikuš & Kaniansky, 2000; Hu T. et al., 1995]. However, the strategies (i-iv) have several limitations and specific features that have to be carefully considered before being employed. These are (i) a relatively poor signal-to-noise ratio, chiral selector can also significantly absorb, (ii) use of strongly absorbing chromophores, inserted chromofores can influence complex equilibrium of analyte with chiral selector, (iii) use of capillaries with higher internal diameter, e.g., 300-800 m (temperature dissipation and, by that, separation efficiency decrease with internal diameter), or properly designed detection window, e.g., bubble cells (see Figure 4.2a and a photo detail in Figure 4.3), Z-shaped cells (see Figure 4.2b), multireflection nanolitre scale cell (often accompanied by larger probe volumes and reduction in separation efficiency and resolution) and (iv) additional sample preparation step is needed, electrophoretic mobilities, as well as the complexing properties of derivatives, can differ from those of their native forms. Nevertheless, the detection sensitivity with UV-VIS absorbance detectors usually cannot be increased more than several tenths times, even if applying any of the presented approaches, so that sample preparation is required. On the other hand, an appropriate use of the on-line sample preparation procedures (as given in chapter 3) enables an application of UV-VIS absorbance detectors, even in ultrasensitive bioanalyses, as illustrated on several examples (in section 4.6. and Table 3.1).

Figure 4.1. Schematic illustration of indirect absorption detection. The separation electrolyte additive provides a large background signal at the detector. Displacement occurs in the analyte zone and lower absorbance intensity is observed. White dots represent absorbing species, blue dots represent non-absorbing species. Reprinted from ref. [Yeung, 1989].


Table 4.1. Detection techniques in CEa 

a adapted from refs. [Heiger, 2000; Swinney & Bornhop, 2000] b assume 10 nL injection volume Figure 4.2. Techniques for increasing the pathlength of the capillary: (a) a bubble cell and (b)

Advanced Combinations of Detection and Electrophoresis 115

A diode array detector (DAD) serves as a typical multi-wavelength on-column CE detector providing UV-VIS spectra in the required interval of wavelengths [Heiger D.N. et al., 1994; Beck et al., 1993], for the instrumental scheme of the CE-DAD see Figure 4.3. Many organic compounds have characteristic spectra in the UV which can be used to help identify the

Figure 4.3. Schematic overview of the CE diode array detector (DAD) optics. Reprinted from

a z-cell (additional tubing). Reprinted from ref. [Skoog et al., 2007].

**4.2.2 Multi-wavelength (spectral) UV-VIS absorbance detection** 

substance passing through the sensor cell.

ref. [Agilent Technologies, 2009].

Method Mass detection limit (moles) Concentration detection limit (M)b Advantages/ disadvantages UV-VIS absorption 10-13 – 10-15 10-5 – 10-7 ● Universal

Fluorescence 10-15 – 10-17 10-7 – 10-9 ● Sensitive

Laser-induced

fluorescence 10-18 – 10-20 10-10 – 10-16

Amperometry 10-18 – 10-19 10-10 – 10-11

Conductivity 10-15 – 10-16 10-7 – 10-8

Indirect UV,

1-2 orders less than direct

method –

fluorescence,

amperometry

LODs of other detection techniques [M]:

derivatization, 10-5, native)

Table 4.1. Detection techniques in CEa

a adapted from refs. [Heiger, 2000; Swinney & Bornhop, 2000]

b assume 10 nL injection volume

Phototermal refraction (10-7-10-8), Potentiometric (10-7-10-8), Refractive index (10-5-10-6, capillary, 10-5, chip-scale), Raman (10-3-10-6,

preconcentration needed), Radiofrequency, NMR (10-3), Radioisotope (10-10), Laser-induced capillary vibration (10-8, chemical

● Diode array offers spectral information

● Usually requires sample derivatization

● Extremely sensitive

● Usually requires sample derivatization

● Post-column LIF is 3-6 orders more

sensitive than direct on-column LIF

● Sensitive

analyses

● Selective but useful only for electroactive

● Requires special electronics and capillary

modification

● Universal

● Requires special electronics and capillary

● Interface between CE and MS complicated

● Universal

● Lower sensitivity than direct methods

modification

Mass spectrometry 10-16 – 10-17 10-8 – 10-9 ● Sensitive and offers structural information

● Expensive

Figure 4.2. Techniques for increasing the pathlength of the capillary: (a) a bubble cell and (b) a z-cell (additional tubing). Reprinted from ref. [Skoog et al., 2007].

### **4.2.2 Multi-wavelength (spectral) UV-VIS absorbance detection**

A diode array detector (DAD) serves as a typical multi-wavelength on-column CE detector providing UV-VIS spectra in the required interval of wavelengths [Heiger D.N. et al., 1994; Beck et al., 1993], for the instrumental scheme of the CE-DAD see Figure 4.3. Many organic compounds have characteristic spectra in the UV which can be used to help identify the substance passing through the sensor cell.

Figure 4.3. Schematic overview of the CE diode array detector (DAD) optics. Reprinted from ref. [Agilent Technologies, 2009].

the same possibility to be combined with CE (e.g., on-line combination via optical fibres) when compared to the single wavelength UV-VIS absorbance detectors. Therefore, CE-DAD was proposed as an advantageous alternative to conventional CE-UV with the single wavelength detection and, at the same time, a pragmatic solution to the expensive CE-MS or CE-NMR, for many pharmaceutical and biomedical applications including those with the enantioselective aspects [Beck et al., 1993], see also examples in section 4.6 and Table 3.1.

Advanced Combinations of Detection and Electrophoresis 117

Fluorescence detection is a more sensitive and selective alternative to UV-VIS absorbance detection, and it is usually carried out in the on-column arrangement. For a schematic layout of on-column fluorescence detection see Figure 4.5. Mass detection limits are in the range of 10-15-10-17 mol, while concentration LODs range from 10-7 to 10-9 M depending upon the

Figure 4.5. A schematic layout of on-column fluorescence detection. Adapted from ref.

Generally, there is a considerably reduced number of compounds exhibiting natural fluorescence in comparison with UV-VIS absorbing compounds and therefore, this detection mode is very often linked with analyte derivatization. However, the complicated chemistry and time-consuming procedures associated with derivatization make fluorescence detection {conventional, as well as LIF (see below)} less attractive than universal detection methods. Another possibility to perform fluorescence detection is its indirect regime (universal). The concept of indirect fluorescence measurements is essentially the same as indirect absorbance measurements, except the background (buffer) fluoresces instead of absorbing, see Figure 4.1. Unfortunately, indirect fluorescence (likewise UV absorbance and other indirect methods) has 10-100 times less sensitivity than direct methods. Poor LODs for indirect fluorescence measurements are the result of the noisy fluorescence background. Therefore, it

is not surprising that the indirect fluorescence mode has rare use in CE.

**4.3 Fluorescence detection** 

[Amankwa et al., 1992].

**4.3.1 Conventional fluorescence detection** 

analyte's fluorofore [Ewing et al., 1989].

Figure 4.4. Processed UV-VIS spectra of amlodipine (AML) in different matrices. (a) AML present in demineralized water, serving as a reference spectrum, (b) AML present in 10 times diluted model urine, at a 500 ng/mL concentration level of the drug in the samples. Separating and other working conditions of the ITP-EKC-DAD method modified with (2 hydroxypropyl)--cyclodextrin (HP--CD), serving as the chiral selector, were the same for (a) and (b). AML1, AML2 – migration positions of the first migrating enantiomer of amlodipine (red spectrum), the second migrating enantiomer of amlodipine (green spectrum), respectively. Reprinted from ref. [Mikuš et al., 2008a].

Although UV-VIS spectra are not as informative as those of NMR, MS or infra-red spectrometry, they still can serve for a preliminary confirmation of peak/analyte identity and validation of peak purity [Strašík et al., 2003]. The similarity of spectral profiles can be used for the indication of structurally related compounds separated from each other while differences in spectra can indicate impurity in analyte peak. As an example, the similar profiles of the UV-VIS absorption spectra of the amlodipine enantiomers recorded in the model water and urine samples, indicating an acceptable purity of the separated electrophoretic peaks of the amlodipine enantiomers in the complex matrices (urine), are shown in Figure 4.4. With respect to spectral analysis, DAD also has significant advantages over rapid scanning detectors such as the signal-to-noise ratio of spectral data being independent of the number of wavelengths acquired, the bandwidth of individual wavelengths not being predetermined and on-line spectra being available at all times [Heiger D., 2000]. CE-DAD provides comparable sensitivity and linear detection range, and the same possibility to be combined with CE (e.g., on-line combination via optical fibres) when compared to the single wavelength UV-VIS absorbance detectors. Therefore, CE-DAD was proposed as an advantageous alternative to conventional CE-UV with the single wavelength detection and, at the same time, a pragmatic solution to the expensive CE-MS or CE-NMR, for many pharmaceutical and biomedical applications including those with the enantioselective aspects [Beck et al., 1993], see also examples in section 4.6 and Table 3.1.

### **4.3 Fluorescence detection**

116 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Figure 4.4. Processed UV-VIS spectra of amlodipine (AML) in different matrices. (a) AML present in demineralized water, serving as a reference spectrum, (b) AML present in 10 times diluted model urine, at a 500 ng/mL concentration level of the drug in the samples. Separating and other working conditions of the ITP-EKC-DAD method modified with (2 hydroxypropyl)--cyclodextrin (HP--CD), serving as the chiral selector, were the same for (a) and (b). AML1, AML2 – migration positions of the first migrating enantiomer of amlodipine (red spectrum), the second migrating enantiomer of amlodipine (green

Although UV-VIS spectra are not as informative as those of NMR, MS or infra-red spectrometry, they still can serve for a preliminary confirmation of peak/analyte identity and validation of peak purity [Strašík et al., 2003]. The similarity of spectral profiles can be used for the indication of structurally related compounds separated from each other while differences in spectra can indicate impurity in analyte peak. As an example, the similar profiles of the UV-VIS absorption spectra of the amlodipine enantiomers recorded in the model water and urine samples, indicating an acceptable purity of the separated electrophoretic peaks of the amlodipine enantiomers in the complex matrices (urine), are shown in Figure 4.4. With respect to spectral analysis, DAD also has significant advantages over rapid scanning detectors such as the signal-to-noise ratio of spectral data being independent of the number of wavelengths acquired, the bandwidth of individual wavelengths not being predetermined and on-line spectra being available at all times [Heiger D., 2000]. CE-DAD provides comparable sensitivity and linear detection range, and

spectrum), respectively. Reprinted from ref. [Mikuš et al., 2008a].

### **4.3.1 Conventional fluorescence detection**

Fluorescence detection is a more sensitive and selective alternative to UV-VIS absorbance detection, and it is usually carried out in the on-column arrangement. For a schematic layout of on-column fluorescence detection see Figure 4.5. Mass detection limits are in the range of 10-15-10-17 mol, while concentration LODs range from 10-7 to 10-9 M depending upon the analyte's fluorofore [Ewing et al., 1989].

Figure 4.5. A schematic layout of on-column fluorescence detection. Adapted from ref. [Amankwa et al., 1992].

Generally, there is a considerably reduced number of compounds exhibiting natural fluorescence in comparison with UV-VIS absorbing compounds and therefore, this detection mode is very often linked with analyte derivatization. However, the complicated chemistry and time-consuming procedures associated with derivatization make fluorescence detection {conventional, as well as LIF (see below)} less attractive than universal detection methods. Another possibility to perform fluorescence detection is its indirect regime (universal). The concept of indirect fluorescence measurements is essentially the same as indirect absorbance measurements, except the background (buffer) fluoresces instead of absorbing, see Figure 4.1. Unfortunately, indirect fluorescence (likewise UV absorbance and other indirect methods) has 10-100 times less sensitivity than direct methods. Poor LODs for indirect fluorescence measurements are the result of the noisy fluorescence background. Therefore, it is not surprising that the indirect fluorescence mode has rare use in CE.

The application examples shown in section 4.6. and Tables 2.1 and 3.1 indicate that CE-LIF is a powerful tool, due to its excellent sensitivity, for the analysis of samples ranging from tissue extracts to single cells. However, one challenge for the chiral CE analysis of complex biological samples is the accurate peak identification in complex electropherograms, as often matching a migration time between an analyte and the corresponding standard may be insufficient to confirm the peak's identity. Therefore, sample preparation procedures, and, especially, their on-line modes (chapter 3), should be used to eliminate matrix interferents and, by that, simplifying the confirmation of the peak's identity, see e.g., [Cho et al., 2004] and Figure 3.33. Another interesting possibility to confirm the peak's identity reliably is based on the confirmation of analyte signal identity via a combination of single-step immunoprecipitation (specific elimination of analyte signal) and CE-LIF analysis [Miao et

Advanced Combinations of Detection and Electrophoresis 119

Figure 4.7. D-Asp antibody allows confirmation of analyte peak assignment in the CE-LIF analysis of complex biological samples such as nerve tissue (a) or single cell (b). Chemical information output of CE-LIF analysis is significantly enhanced by use of the antibody for signal identification. (a) Electropherograms of an anterior tentacular nerve sample before (A) and after incubation (B) with D-Asp antibody and L-Asp conjugate. (b) Electropherograms of an MCC neuron sample before (A) and after incubation (B) with D-Asp antibody and L-Asp conjugate. Increased intensity of the L-Asp signal and disappearance or decrease in the amplitude of the D-Asp peak are noticeable in (B). Internal

standard (I.S.), L-cysteic acid. Adapted from ref. [Miao et al., 2006].

al., 2006], see Figure 4.7.

### **4.3.2 Laser-induced fluorescence detection**

Laser-induced fluorescence (LIF), based on a fluorescence signal produced by the molecule after its illumination by a laser beam, is the most sensitive, extremely selective, small volume detection method, suitable for an on-line arrangement with the CE. It is also one of the most favoured detection techniques for microfluidic devices such as MCE. The first oncolumn LIF detection system was developed by Zare et al. [Gassmann et al., 1985]. For the scheme of the CE-LIF see Figure 4.5. The concentration LODs of the best CE/laser-based systems are in interval 10-14-10-16 M with mass detection limits 10-18-10-20 mol (individual molecules can be detected) [Ewing et al., 1989]. A variability (sensitivity, selectivity) of LIF detection is given by employing different lasers {the common argon laser (458 and 488 nm), He-Cd laser (440 nm), blue diode laser (420 nm), frequency-doubled Ar-ion laser (257 nm), frequency-doubled Kr-ion laser (284 nm)} [Hernández et al., 2008, 2010; Nie et al., 1993; Chang & Yeung, 1995; Craig D.B. et al., 1998]. To overcome problems associated with oncolumn detection, such as broad-band luminescence background and poor S/N ratios, Rayleigh and Raman light scattering from the capillary, several techniques based on the spatial or spectral separation of the fluorescence signal from the high background signal have been proposed (the best results have been reached with a sheath flow cuvette incorporated at the end of the capillary, performing an end-column LIF) [Cheng Y.F. & Dovichi, 1988; Chen D.Y. & Dovichi, 1996; Craig D.B. et al., 1998; Xue & Yeung, 1995]. For example, the schematic diagram of sheath-flow CE-LIF system with multichannel photomultiplier tube detection is shown in Figure 4.6. background

Figure 4.6. Schematic diagram of sheath-flow CE-LIF system with multichannel photomultiplier tube (PMT) detection. The beam from a HeAg laser is used for excitation of the analyte as it elutes from the capillary. Fluorescence is collected orthogonally to the excitation optics and transmitted or reflected by one or both of two custom dichroic beamsplitters. The spectrally distributed fluorescence is detected by one of three PMTs corresponding to wavelength ranges 250–310 nm (PMT "Blue"), 310–400 nm (PMT "Green"), and 400+ nm (PMT "Red"). Reprinted from ref. [Lapainis et al., 2007]. 

Laser-induced fluorescence (LIF), based on a fluorescence signal produced by the molecule after its illumination by a laser beam, is the most sensitive, extremely selective, small volume detection method, suitable for an on-line arrangement with the CE. It is also one of the most favoured detection techniques for microfluidic devices such as MCE. The first oncolumn LIF detection system was developed by Zare et al. [Gassmann et al., 1985]. For the scheme of the CE-LIF see Figure 4.5. The concentration LODs of the best CE/laser-based systems are in interval 10-14-10-16 M with mass detection limits 10-18-10-20 mol (individual molecules can be detected) [Ewing et al., 1989]. A variability (sensitivity, selectivity) of LIF detection is given by employing different lasers {the common argon laser (458 and 488 nm), He-Cd laser (440 nm), blue diode laser (420 nm), frequency-doubled Ar-ion laser (257 nm), frequency-doubled Kr-ion laser (284 nm)} [Hernández et al., 2008, 2010; Nie et al., 1993; Chang & Yeung, 1995; Craig D.B. et al., 1998]. To overcome problems associated with oncolumn detection, such as broad-band luminescence background and poor S/N ratios, Rayleigh and Raman light scattering from the capillary, several techniques based on the spatial or spectral separation of the fluorescence signal from the high background signal have been proposed (the best results have been reached with a sheath flow cuvette incorporated at the end of the capillary, performing an end-column LIF) [Cheng Y.F. & Dovichi, 1988; Chen D.Y. & Dovichi, 1996; Craig D.B. et al., 1998; Xue & Yeung, 1995]. For example, the schematic diagram of sheath-flow CE-LIF system with multichannel

Figure 4.6. Schematic diagram of sheath-flow CE-LIF system with multichannel photomultiplier tube (PMT) detection. The beam from a HeAg laser is used for excitation of the analyte as it elutes from the capillary. Fluorescence is collected orthogonally to the excitation optics and transmitted or reflected by one or both of two custom dichroic beamsplitters. The spectrally distributed fluorescence is detected by one of three PMTs corresponding to wavelength ranges 250–310 nm (PMT "Blue"), 310–400 nm (PMT

"Green"), and 400+ nm (PMT "Red"). Reprinted from ref. [Lapainis et al., 2007].

**4.3.2 Laser-induced fluorescence detection** 

photomultiplier tube detection is shown in Figure 4.6.

The application examples shown in section 4.6. and Tables 2.1 and 3.1 indicate that CE-LIF is a powerful tool, due to its excellent sensitivity, for the analysis of samples ranging from tissue extracts to single cells. However, one challenge for the chiral CE analysis of complex biological samples is the accurate peak identification in complex electropherograms, as often matching a migration time between an analyte and the corresponding standard may be insufficient to confirm the peak's identity. Therefore, sample preparation procedures, and, especially, their on-line modes (chapter 3), should be used to eliminate matrix interferents and, by that, simplifying the confirmation of the peak's identity, see e.g., [Cho et al., 2004] and Figure 3.33. Another interesting possibility to confirm the peak's identity reliably is based on the confirmation of analyte signal identity via a combination of single-step immunoprecipitation (specific elimination of analyte signal) and CE-LIF analysis [Miao et al., 2006], see Figure 4.7.

Figure 4.7. D-Asp antibody allows confirmation of analyte peak assignment in the CE-LIF analysis of complex biological samples such as nerve tissue (a) or single cell (b). Chemical information output of CE-LIF analysis is significantly enhanced by use of the antibody for signal identification. (a) Electropherograms of an anterior tentacular nerve sample before (A) and after incubation (B) with D-Asp antibody and L-Asp conjugate. (b) Electropherograms of an MCC neuron sample before (A) and after incubation (B) with D-Asp antibody and L-Asp conjugate. Increased intensity of the L-Asp signal and disappearance or decrease in the amplitude of the D-Asp peak are noticeable in (B). Internal standard (I.S.), L-cysteic acid. Adapted from ref. [Miao et al., 2006].

Amperometric detection is based on electron transfer to or from the analyte of interest at an electrode surface that is under the influence on an applied DC voltage. The result of electron transfer is a redox reaction at the electrode that produces a current that is directly related to the analyte concentration. Therefore, chiral selectors giving a redox reaction at the electrode, comparable with the redox reaction of the analyte, cannot be employed for chiral

Advanced Combinations of Detection and Electrophoresis 121

Figure 4.8. Schematic diagram of the capillary electrophoresis system with end-column amperometric detection: R, reference electrode; W, working electrode; C, counter electrode.

Pulsed amperometric detection (including the electrode cleaning step), an advanced detection mode, can reduce the major problem of this detection, linked with strong adsorption of the intermediate reaction products of the analyte to the electrode surface (reducing activity, i.e., electron transfer of the electrode and interfering with detection) [Ewing et al., 1994]. In this way, reproducibility of measurements, as well as sensitivity, can

The conventional amperometric mode, usually used in the end-column configuration, is selective (restricted only for electroactive analytes) and can be tuned to the analyte of interest [O'Shea et al., 1993; Lu W.Z. & Cassidy, 1993]. On the other hand, the square-wave mode can be successfully applied for detection of typical nonelectroactive organic compounds, where currents associated with the non-Faradaic interfacial process due to analyte adsorption at a Pt microelectrode were used to provide the analytical signal [Gerhardt et al., 2000]. The detection limits obtained in such measurements are at least one order of magnitude lower than the ones that could be obtained by UV absorption. Moreover, such an amperometric detection mode is more universal and provides a wider

Compared to the conductimetric mode, amperometric detection is much more sensitive, mass detection limits are in the range of 10-18-10-19 mol corresponding to 10-10-10-11 M concentration LODs [Ewing et al., 1989]. Thus, amperometric detection represents a good alternative to LIF for ultrasensitive CE determinations of another specific

Reprinted from ref. [His et al., 1997].

application range.

be considerably improved [Trojanowicz, 2009].

separations with this type of detection and a modified chiral system has to be used.

### **4.4 Electrochemical detection**

Electrochemical detection for CE can be divided into three main categories: conductimetric (based on conductivity changes monitored by sensors during electrophoretic migration process) [Landers, 1997; Everaerts et al., 1979; Gebauer et al., 1997], potentiometric (based on classical ion-selective microelectrodes) [Haber et al., 1990, 1991] and amperometric (based on electrode vs. analyte redox reaction that produces a current, directly related to the analyte concentration) [Ewing et al., 1994]. Electrochemical detection provides an alternative solution for non-absorbing or non-fluorescing analytes. This detection approach offers both universal and selective modes, and provides sensitive measurements in an end-column, as well as an on-column, arrangement in the CE and MCE format [de Silva, 2003].

The most difficult problem to solve in applying electrochemical detection in CE is the need to instrumentally eliminate the effect of the strong electric field used in electrophoretic separation from the conditions of electrochemical detections. The main drawback of electrochemical detectors in their implementation with CE is that it can be extremely difficult and tedious since sensor preparation can involve complicated fabrication and handling procedures, and accurate spatial alignment of the electrodes within the capillary [Swinney & Bornhop, 2000]. Moreover, limited sensor lifetime and required recalibration, strong adsorption of the intermediate reaction products of the analyte to the electrode surface reducing its activity/response (this can be overcome by the electronic cleaning of the electrode) [Haber et al., 1991; Ewing et al., 1994] are other limiting factors that should be carefully considered when electrochemical detectors are applied. These limitations can be pronounced, especially, when complex biological samples are analysed, where sample pretreatment (especially purification) is strongly recommended for reliable and reproducible detection.

Nevertheless, recent years have provided numerous new examples of applying flowthrough electrochemical detectors in chemical analysis. The review by Trojanowicz [Trojanowicz, 2009], based on about 250 original research papers cited from the current analytical literature, presents their application in flow analysis and capillary electrophoretic methods. Potentiometric detection with ion-selective electrodes predominates in flow analysis carried out mostly in a flow injection system, while amperometric and conductivity detections are most commonly employed in capillary electrophoresis and they have been applied also in the chiral field. For application examples of electrochemical detection in chiral CE, see section 4.6. and Tables 2.1 and 3.1.

### **4.4.1. Amperometric detection**

As with other electrochemical detection methods in CE, amperometric detection is also carried out with the use of microelectrodes because of the need to adjust their size to the diameter of capillaries. There are several most commonly reported constructions of various types used for amperometric detection cells in CE, as well as various types of working electrodes resulting in interesting application possibilities and very low detection limits obtainable, as reviewed by Trojanowicz [Trojanowicz, 2009]. Schemes of the amperometric detector coupled with the hydrodynamically open (conventional) and hydrodynamically closed (unconventional) CE systems are shown in Figure 4.8 and Figure 4.9, respectively. of

Electrochemical detection for CE can be divided into three main categories: conductimetric (based on conductivity changes monitored by sensors during electrophoretic migration process) [Landers, 1997; Everaerts et al., 1979; Gebauer et al., 1997], potentiometric (based on classical ion-selective microelectrodes) [Haber et al., 1990, 1991] and amperometric (based on electrode vs. analyte redox reaction that produces a current, directly related to the analyte concentration) [Ewing et al., 1994]. Electrochemical detection provides an alternative solution for non-absorbing or non-fluorescing analytes. This detection approach offers both universal and selective modes, and provides sensitive measurements in an end-column, as

The most difficult problem to solve in applying electrochemical detection in CE is the need to instrumentally eliminate the effect of the strong electric field used in electrophoretic separation from the conditions of electrochemical detections. The main drawback of electrochemical detectors in their implementation with CE is that it can be extremely difficult and tedious since sensor preparation can involve complicated fabrication and handling procedures, and accurate spatial alignment of the electrodes within the capillary [Swinney & Bornhop, 2000]. Moreover, limited sensor lifetime and required recalibration, strong adsorption of the intermediate reaction products of the analyte to the electrode surface reducing its activity/response (this can be overcome by the electronic cleaning of the electrode) [Haber et al., 1991; Ewing et al., 1994] are other limiting factors that should be carefully considered when electrochemical detectors are applied. These limitations can be pronounced, especially, when complex biological samples are analysed, where sample pretreatment (especially purification) is strongly recommended for reliable and

Nevertheless, recent years have provided numerous new examples of applying flowthrough electrochemical detectors in chemical analysis. The review by Trojanowicz [Trojanowicz, 2009], based on about 250 original research papers cited from the current analytical literature, presents their application in flow analysis and capillary electrophoretic methods. Potentiometric detection with ion-selective electrodes predominates in flow analysis carried out mostly in a flow injection system, while amperometric and conductivity detections are most commonly employed in capillary electrophoresis and they have been applied also in the chiral field. For application examples of electrochemical detection in

As with other electrochemical detection methods in CE, amperometric detection is also carried out with the use of microelectrodes because of the need to adjust their size to the diameter of capillaries. There are several most commonly reported constructions of various types used for amperometric detection cells in CE, as well as various types of working electrodes resulting in interesting application possibilities and very low detection limits obtainable, as reviewed by Trojanowicz [Trojanowicz, 2009]. Schemes of the amperometric detector coupled with the hydrodynamically open (conventional) and hydrodynamically closed (unconventional) CE systems are shown in Figure 4.8 and Figure 4.9, respectively.

well as an on-column, arrangement in the CE and MCE format [de Silva, 2003].

**4.4 Electrochemical detection** 

reproducible detection.

chiral CE, see section 4.6. and Tables 2.1 and 3.1.

**4.4.1. Amperometric detection** 

Amperometric detection is based on electron transfer to or from the analyte of interest at an electrode surface that is under the influence on an applied DC voltage. The result of electron transfer is a redox reaction at the electrode that produces a current that is directly related to the analyte concentration. Therefore, chiral selectors giving a redox reaction at the electrode, comparable with the redox reaction of the analyte, cannot be employed for chiral separations with this type of detection and a modified chiral system has to be used.

Figure 4.8. Schematic diagram of the capillary electrophoresis system with end-column amperometric detection: R, reference electrode; W, working electrode; C, counter electrode. Reprinted from ref. [His et al., 1997]. electrode;

Pulsed amperometric detection (including the electrode cleaning step), an advanced detection mode, can reduce the major problem of this detection, linked with strong adsorption of the intermediate reaction products of the analyte to the electrode surface (reducing activity, i.e., electron transfer of the electrode and interfering with detection) [Ewing et al., 1994]. In this way, reproducibility of measurements, as well as sensitivity, can be considerably improved [Trojanowicz, 2009].

The conventional amperometric mode, usually used in the end-column configuration, is selective (restricted only for electroactive analytes) and can be tuned to the analyte of interest [O'Shea et al., 1993; Lu W.Z. & Cassidy, 1993]. On the other hand, the square-wave mode can be successfully applied for detection of typical nonelectroactive organic compounds, where currents associated with the non-Faradaic interfacial process due to analyte adsorption at a Pt microelectrode were used to provide the analytical signal [Gerhardt et al., 2000]. The detection limits obtained in such measurements are at least one order of magnitude lower than the ones that could be obtained by UV absorption. Moreover, such an amperometric detection mode is more universal and provides a wider application range.

Compared to the conductimetric mode, amperometric detection is much more sensitive, mass detection limits are in the range of 10-18-10-19 mol corresponding to 10-10-10-11 M concentration LODs [Ewing et al., 1989]. Thus, amperometric detection represents a good alternative to LIF for ultrasensitive CE determinations of another specific

practical importance of this detection is justified by the fact that commercially available detectors are on the market only for this electrochemical detection for CE [Trojanowicz, 2009].

Advanced Combinations of Detection and Electrophoresis 123

Figure 4.10. General schemes of the arrangements of the detection electrodes in contact conductivity detection cells for CZE. (a) An in-column placement of the detection electrodes exposed to direct contact with the separated constituents; (b) a post-column placement of the detection electrodes exposed to direct contact with the separated constituents; (c) an oncolumn placement of the detection electrodes with an electrolyte solution mediated contact with the detection compartment of the cell. CD, measuring circuitry of the detector.

In general, conductivity detectors consist of two sensors (electrodes), in direct or indirect contact with the electrolyte solution, across which an electrical potential has been applied. General schemes of the arrangements of the detection electrodes in contact conductivity detection cells for CZE are shown in Figure 4.10. Conductimetric mode is attractive by providing universal detection in an on-line arrangement and it is the most frequently used electrochemical detection for CE. Therefore, it can be the first choice when analysing nonabsorbing/fluorescing organic ions avoiding the derivatization step in the analytical protocol. However, sensitivity of the contact conductivity detection mode is moderate, mass detection limits are usually in the range of 10-14-10-15 mol corresponding to 10-6-10-7 M concentration LODs [Landers, 1997]. This is not sufficient for the majority of clinical and

Improvement of detectability can be additionally achieved to the sub-ppb level by applying on-capillary preconcentration by electrostacking [Haber et al., 1998]. Another method of lowering the value of the detection limit in CE determinations with conductivity detection, following wide use in ion-chromatography, is to apply on-line suppressing of conductivity of BGE by the ion-exchange process [Avdalovic et al., 1993]. For example, the suppressor for determination of anions can be made of an ion-exchange sulfonated Teflon capillary tubing (1 cm long) placed between the separation capillary and the detector electrode in the wall-jet arrangement. This method can improve the detectability of about two orders of magnitude

biomedical analytical problems when performed by conventional CE.

compared to indirect detection of non-UV-absorbing ions.

Reprinted from ref. [Bodor et al., 2001].

group of analytes. For application examples of amperometric detection in chiral CE, see section 4.6. and Tables 2.1 and 3.1.

Figure 4.9. (a) Schematic diagram of the hydrodynamically closed CE separation unit with a post-column amperometric detector. IB = injection block for a microsyringe sample injection; C= capillary tube; CD =on-column conductivity detection cell; "T"=elution T-piece; =bifurcation point; AD= amperometric detection cell; RB-1 = refilling block for the capillary tube (C); C-E=counter-electrode compartment; M = Cellophane membranes; LE = inlet for the leading electrolyte solution; P-in, P-out = inlet and outlet for the elution solution, respectively; RB-2 = refilling block for the terminating compartment (TC) and the injection block (IB); TE = inlet for the terminating electrolyte solution; W = waste; S = position for the injection of the sample; V1-V4 = valves. (b) Detail of the amperometric detection cell attached to the T-piece. l=T-piece; 2=channel to the counter electrode; 3= connection for the column; 4=elution channel (detection capillary) of 4 mm × 0.2 mm i.d.; 5=PTFE spacer (0.05 mm); 6=O-ring for a leak-proof connection; 7= amperometric cell; 8=working electrode; 9=connecting cable to the electronics of the detector; 10 = epoxy body of the electrode; 11 =connecting channel for the reference electrode. Reprinted from ref. [Kaniansky et al., 1995]. conductivity

#### **4.4.2 Conductivity detection**

Conductivity detection, due to separation ions, charged particles or microorganisms in CE, has a special importance for capillary electrophoresis. The charged species are detected in the solution of electrolyte, which makes use of this detection process difficult. Nevertheless, the

group of analytes. For application examples of amperometric detection in chiral CE, see

Figure 4.9. (a) Schematic diagram of the hydrodynamically closed CE separation unit with a post-column amperometric detector. IB = injection block for a microsyringe sample injection; C= capillary tube; CD =on-column conductivity detection cell; "T"=elution T-piece; =bifurcation point; AD= amperometric detection cell; RB-1 = refilling block for the capillary tube (C); C-E=counter-electrode compartment; M = Cellophane membranes; LE = inlet for the leading electrolyte solution; P-in, P-out = inlet and outlet for the elution solution, respectively; RB-2 = refilling block for the terminating compartment (TC) and the injection block (IB); TE = inlet for the terminating electrolyte solution; W = waste; S = position for the injection of the sample; V1-V4 = valves. (b) Detail of the amperometric detection cell attached to the T-piece. l=T-piece; 2=channel to the counter electrode; 3= connection for the column; 4=elution channel (detection capillary) of 4 mm × 0.2 mm i.d.; 5=PTFE spacer (0.05 mm); 6=O-ring for a leak-proof connection; 7= amperometric cell; 8=working electrode; 9=connecting cable to the electronics of the detector; 10 = epoxy body of the electrode; 11 =connecting channel for the reference electrode. Reprinted from ref. [Kaniansky et al., 1995].

Conductivity detection, due to separation ions, charged particles or microorganisms in CE, has a special importance for capillary electrophoresis. The charged species are detected in the solution of electrolyte, which makes use of this detection process difficult. Nevertheless, the

section 4.6. and Tables 2.1 and 3.1.

**4.4.2 Conductivity detection** 

practical importance of this detection is justified by the fact that commercially available detectors are on the market only for this electrochemical detection for CE [Trojanowicz, 2009].

Figure 4.10. General schemes of the arrangements of the detection electrodes in contact conductivity detection cells for CZE. (a) An in-column placement of the detection electrodes exposed to direct contact with the separated constituents; (b) a post-column placement of the detection electrodes exposed to direct contact with the separated constituents; (c) an oncolumn placement of the detection electrodes with an electrolyte solution mediated contact with the detection compartment of the cell. CD, measuring circuitry of the detector. Reprinted from ref. [Bodor et al., 2001].

In general, conductivity detectors consist of two sensors (electrodes), in direct or indirect contact with the electrolyte solution, across which an electrical potential has been applied. General schemes of the arrangements of the detection electrodes in contact conductivity detection cells for CZE are shown in Figure 4.10. Conductimetric mode is attractive by providing universal detection in an on-line arrangement and it is the most frequently used electrochemical detection for CE. Therefore, it can be the first choice when analysing nonabsorbing/fluorescing organic ions avoiding the derivatization step in the analytical protocol. However, sensitivity of the contact conductivity detection mode is moderate, mass detection limits are usually in the range of 10-14-10-15 mol corresponding to 10-6-10-7 M concentration LODs [Landers, 1997]. This is not sufficient for the majority of clinical and biomedical analytical problems when performed by conventional CE. be

Improvement of detectability can be additionally achieved to the sub-ppb level by applying on-capillary preconcentration by electrostacking [Haber et al., 1998]. Another method of lowering the value of the detection limit in CE determinations with conductivity detection, following wide use in ion-chromatography, is to apply on-line suppressing of conductivity of BGE by the ion-exchange process [Avdalovic et al., 1993]. For example, the suppressor for determination of anions can be made of an ion-exchange sulfonated Teflon capillary tubing (1 cm long) placed between the separation capillary and the detector electrode in the wall-jet arrangement. This method can improve the detectability of about two orders of magnitude compared to indirect detection of non-UV-absorbing ions.

Chiral selectors, as well as sample matrix constituents, can influence contact conductivity detection. To reduce a decrease in detection sensitivity and reproducibility caused by a modification of the electrode surface in an ionic environment, various approaches have been proposed. A contact conductivity detection cell for CZE with an electrolyte solution mediated contact of the separated constituents with the detection electrodes (ESMC cell) was proposed to eliminate detection disturbances due to electrode reactions and adsorption of the separated constituents when these are coming into direct contact with the detection electrodes, see Figure 4.12 [Bodor et al., 2001]. Despite precautions in the construction of sensing and measuring parts of the conductivity detector, the detection in the ITP runs can be accompanied by enhanced noise of the detection signal and some detection disturbances when injecting common real samples, i.e., complex ionic samples (Figure 4.13a). Here, an electrochemical cleaning of the detection electrodes as be recommended for some conventional conductivity detection cells [Kaniansky, 1981; Kaniansky et al., 1983] provided a simple and effective solution also in microchip format [Masár et al., 2001]. Here, a pair of 100-V pulses of reversed polarities is applied between the electrodes of a particular sensor along with a stream of the leading electrolyte solution. An overall effect of this cleaning

Advanced Combinations of Detection and Electrophoresis 125

procedure is clearly illustrated by isotachopherograms in Figure 4.13a and b.

Figure 4.12. A schematic drawing of the conductivity detection cell with an electrolyte solution mediated contact of the separated constituents with the detection electrodes. (a) A connection of the cell to the separation capillary; (b) holes with screw plugs for filling the electrode channels (d) with the detection electrodes (c); (e) the detection compartment; (f) a connection of the cell to the counter-electrode compartment. A body of the cell was made of

A contactless conductivity detector (instrumental scheme see in Figure 4.14), although a bit less sensitive than contact conductivity detector, is an advantageous alternative for the analyses of complex samples as it does not suffer from the contamination of the conductivity sensors by compounds from the electrolyte or sample (adsorption, electrochemical reactions) and, hence, the high reproducibility of detection signal can be maintained [Mikkers et al., 1979; Gebauer et al., 1997; de Silva et al., 2002; Kaniansky et al., 1999]. This detection approach was successfully applied also in ITP-EKC analyses of enantiomers in multicomponent ionic matrices, see refs. [Mikuš & Maráková, 2009, 2010] and references

poly(methyl methacrylate). Reprinted from ref. [Bodor et al., 2001]

cited therein.

Figure 4.11. Graphical illustration of the principle of the electronic cutting of the zone of interest in the ITP stage of the ITP-CZE combination. L = leading ion, T = terminating ion, X = matrix compound(s), Y, Z = analytes, R = resistance. Reprinted from ref. [Tekeľ & Mikuš, 2005].

Jumps in voltage (conductivity) between neighbouring zones result in permanently sharp boundaries between zones and are very convenient for conductivity detection. Although extremely convenient to the detection of the ITP zones (the preferred detection in this field), the conductivity detection technique has a limited applicability in the CZE separations of enantiomers. It can be caused by the measurements of small conductivity changes due to the analyte zones on a relatively high-conductivity background of the carrier electrolyte. Therefore, when monitoring the enantioseparations by the conductivity detection, mobility difference of the analyte vs. chiral selector must be carefully considered.

On the other hand, one interesting possibility of the sensitive analyses of enantiomers in multicomponent ionic samples with the aid of conductivity detection is its implementation into column-coupled CE-CE systems, see examples in ref. [Gebauer et al., 2009, 2011; Mikuš & Maráková, 2009, 2010]. Conductivity detectors in ITP produce a staircase signal, resistance (or conductivity) vs. migration time, in which the relative high of step is constant for a given analyte under given separation conditions (qualitative parameter independent on analysis time). Such an ITP profile can be beneficial in the preliminary mapping of the composition of complex (e.g., biological) samples. This also gives a good possibility of an accurately "cut" (by electronic switching) desired zone (analyte) in the preseparation stage (ITP) of ITP-EKC combination and transferring it into the analytical stage (EKC) without potential interfering compounds from the matrices, avoiding any analytical capillary overloading by the major matrix constituents [Danková et al., 1999], see Figure 4.11. In this way, chiral analytes in multicomponent matrices (e.g., biological) can be monitored and on-line preseparated/pretreated in an achiral environment before a chiral CZE (i.e., EKC) step [Mikuš et al., 2006a, 2008a, 2008b; Marák et al., 2007, 2008; Mikuš, 2010]. Anyway, the conductivity detection can be advantageously used also for the detection of enantiomers in a chiral environment as it is well documented in the literature [Mikuš et al., 2006b; Kubačák et al., 2006a, 2006b, 2007].

Figure 4.11. Graphical illustration of the principle of the electronic cutting of the zone of interest in the ITP stage of the ITP-CZE combination. L = leading ion, T = terminating ion, X = matrix

Jumps in voltage (conductivity) between neighbouring zones result in permanently sharp boundaries between zones and are very convenient for conductivity detection. Although extremely convenient to the detection of the ITP zones (the preferred detection in this field), the conductivity detection technique has a limited applicability in the CZE separations of enantiomers. It can be caused by the measurements of small conductivity changes due to the analyte zones on a relatively high-conductivity background of the carrier electrolyte. Therefore, when monitoring the enantioseparations by the conductivity detection, mobility

On the other hand, one interesting possibility of the sensitive analyses of enantiomers in multicomponent ionic samples with the aid of conductivity detection is its implementation into column-coupled CE-CE systems, see examples in ref. [Gebauer et al., 2009, 2011; Mikuš & Maráková, 2009, 2010]. Conductivity detectors in ITP produce a staircase signal, resistance (or conductivity) vs. migration time, in which the relative high of step is constant for a given analyte under given separation conditions (qualitative parameter independent on analysis time). Such an ITP profile can be beneficial in the preliminary mapping of the composition of complex (e.g., biological) samples. This also gives a good possibility of an accurately "cut" (by electronic switching) desired zone (analyte) in the preseparation stage (ITP) of ITP-EKC combination and transferring it into the analytical stage (EKC) without potential interfering compounds from the matrices, avoiding any analytical capillary overloading by the major matrix constituents [Danková et al., 1999], see Figure 4.11. In this way, chiral analytes in multicomponent matrices (e.g., biological) can be monitored and on-line preseparated/pretreated in an achiral environment before a chiral CZE (i.e., EKC) step [Mikuš et al., 2006a, 2008a, 2008b; Marák et al., 2007, 2008; Mikuš, 2010]. Anyway, the conductivity detection can be advantageously used also for the detection of enantiomers in a chiral environment as it is well documented in the literature [Mikuš et al., 2006b; Kubačák et

compound(s), Y, Z = analytes, R = resistance. Reprinted from ref. [Tekeľ & Mikuš, 2005].

difference of the analyte vs. chiral selector must be carefully considered.

al., 2006a, 2006b, 2007].

Chiral selectors, as well as sample matrix constituents, can influence contact conductivity detection. To reduce a decrease in detection sensitivity and reproducibility caused by a modification of the electrode surface in an ionic environment, various approaches have been proposed. A contact conductivity detection cell for CZE with an electrolyte solution mediated contact of the separated constituents with the detection electrodes (ESMC cell) was proposed to eliminate detection disturbances due to electrode reactions and adsorption of the separated constituents when these are coming into direct contact with the detection electrodes, see Figure 4.12 [Bodor et al., 2001]. Despite precautions in the construction of sensing and measuring parts of the conductivity detector, the detection in the ITP runs can be accompanied by enhanced noise of the detection signal and some detection disturbances when injecting common real samples, i.e., complex ionic samples (Figure 4.13a). Here, an electrochemical cleaning of the detection electrodes as be recommended for some conventional conductivity detection cells [Kaniansky, 1981; Kaniansky et al., 1983] provided a simple and effective solution also in microchip format [Masár et al., 2001]. Here, a pair of 100-V pulses of reversed polarities is applied between the electrodes of a particular sensor along with a stream of the leading electrolyte solution. An overall effect of this cleaning procedure is clearly illustrated by isotachopherograms in Figure 4.13a and b.

Figure 4.12. A schematic drawing of the conductivity detection cell with an electrolyte solution mediated contact of the separated constituents with the detection electrodes. (a) A connection of the cell to the separation capillary; (b) holes with screw plugs for filling the electrode channels (d) with the detection electrodes (c); (e) the detection compartment; (f) a connection of the cell to the counter-electrode compartment. A body of the cell was made of poly(methyl methacrylate). Reprinted from ref. [Bodor et al., 2001]

A contactless conductivity detector (instrumental scheme see in Figure 4.14), although a bit less sensitive than contact conductivity detector, is an advantageous alternative for the analyses of complex samples as it does not suffer from the contamination of the conductivity sensors by compounds from the electrolyte or sample (adsorption, electrochemical reactions) and, hence, the high reproducibility of detection signal can be maintained [Mikkers et al., 1979; Gebauer et al., 1997; de Silva et al., 2002; Kaniansky et al., 1999]. This detection approach was successfully applied also in ITP-EKC analyses of enantiomers in multicomponent ionic matrices, see refs. [Mikuš & Maráková, 2009, 2010] and references cited therein.

fragmentation of analysed substances. In this way, the on-line CE–MS technique has been established as a powerful separation and identification analytical tool. Moreover, this endcolumn hyphenation may enhance the sensitivity of certain compounds depending on their characteristics such as proton affinity, absorptivity, etc. In the last 10 years there have been many significant developments in CE-MS instrumentation and applications that have made

Advanced Combinations of Detection and Electrophoresis 127

Figure 4.15. Generic illustration of a CE-MS instrumental arrangement. HV= High voltage.

Several recent review papers have been devoted to a combination of CE-MS, applied to the structural identification of analytes and trace analytes. Surveys on developments and applications of CE-MS in the years of 2003-2004 and 2005-2006 are given by Schmitt and Englmann [Schmitt-Kopplin & Englmann, 2005] and Gaspar et al. [Gaspar et al., 2008], respectively. The reader will rapidly find the mostly used CE-MS combinations, as well as applications classified as forensic, environmental, bioanalytical, pharmaceutical and metabolomics. Ramautar et al. [Ramautar et al., 2009] pay attention to the utilization of CE-MS in metabolomics, considering papers published between 2000 and 2008. The authors presented CE-MS to be a powerful technique for the profiling of polar metabolites in biological samples. The combination of CE with MS was chosen as a review topic also in conjunction with chiral separations in aqueous and non-aqueous media [Cai & Henion, 1995; Shamsi, 2002; Scriba, 2007]. Methodological aspects of CE–MS for quantitation were summarized and evaluated by Ohnesorge et al. [Ohnesorge et al., 2005]. Pantučková et al. [Pantučková et al., 2009] reviewed electrolyte systems for on-line CE-MS with respect to detection requirements and separation possibilities. Smyth and Rodriguez [Smyth & Rodriguez, 2007] summarized recent studies of the electro spray ionization (ESI) behaviour of selected drugs and their application in CE-MS and HPLC-MS. For application examples

The electrolyte systems in CE must be carefully selected regarding all the requirements of the on-line coupled MS analyser where terms such as the analyte ionization and evaporation play the key role (see section 4.5.2 CE-MS interfaces). It is obvious that both electrophoresis BGE and the solvent must support the ionization process and evaporation of droplets (i.e.,

CE-MS a competitive tool.

Reprinted from ref. [Cai & Henion, 1995].

of chiral CE-MS, see section 4.6 and Table 3.1.

**4.5.1 CE stage in CE-MS** 

Figure 4.13. Impacts of cleaning of contaminated surfaces of the detection electrodes on the responses of the conductivity sensors in the ITP separations on the CC chips. Isotachopherograms from the separations of a three-component test mixture of anions as obtained by a particular sensor (a) before and (b) after electrochemical cleaning of its electrodes. LE=Leading anion; 1=succinate; 2=acetate; 3=benzoate; TE=terminating anion. The concentrations of the analytes in the test sample were 300 M. The driving current was 10 A. Reprinted from ref. [Masár et al., 2001].

Figure 4.14. Block scheme of a high-frequency contactless conductivity detector used in this work. 1, high-frequency generator (consisting of a 4-MHz oscillator, an amplifier and inverter, phasing elements and output voltage amplifiers); 2, four-electrode (capacitive) detection cell; 3. receiver (consisting of a pre-amplifier, a narrow-band amplifier, a selective filter, a highfrequency rectifier, a d.c. amplifier and a low band-pass filter); 4, a data acquisition unit; E –E , detection electrodes. Generated from refs. [Gaš et al., 1980; Vacík et al., 1985].

#### **4.5 Mass spectrometry**

An on-line combination of CE and MS (representative scheme of CE-MS is in Figure 4.15) is a highly effective technique that separates analytes according to differences in their electrophoretic mobilities and then provides information on molecular masses and/or

Figure 4.13. Impacts of cleaning of contaminated surfaces of the detection electrodes on the responses of the conductivity sensors in the ITP separations on the CC chips. Isotachopherograms from the separations of a three-component test mixture of anions as obtained by a particular sensor (a) before and (b) after electrochemical cleaning of its electrodes. LE=Leading anion; 1=succinate; 2=acetate; 3=benzoate; TE=terminating anion. The concentrations of the analytes in the test sample were 300 M. The driving current was

Figure 4.14. Block scheme of a high-frequency contactless conductivity detector used in this work. 1, high-frequency generator (consisting of a 4-MHz oscillator, an amplifier and inverter, phasing elements and output voltage amplifiers); 2, four-electrode (capacitive) detection cell; 3. receiver (consisting of a pre-amplifier, a narrow-band amplifier, a selective filter, a highfrequency rectifier, a d.c. amplifier and a low band-pass filter); 4, a data acquisition unit; E –E ,

An on-line combination of CE and MS (representative scheme of CE-MS is in Figure 4.15) is a highly effective technique that separates analytes according to differences in their electrophoretic mobilities and then provides information on molecular masses and/or

detection electrodes. Generated from refs. [Gaš et al., 1980; Vacík et al., 1985].

10 A. Reprinted from ref. [Masár et al., 2001].

**4.5 Mass spectrometry** 

fragmentation of analysed substances. In this way, the on-line CE–MS technique has been established as a powerful separation and identification analytical tool. Moreover, this endcolumn hyphenation may enhance the sensitivity of certain compounds depending on their characteristics such as proton affinity, absorptivity, etc. In the last 10 years there have been many significant developments in CE-MS instrumentation and applications that have made CE-MS a competitive tool.

Figure 4.15. Generic illustration of a CE-MS instrumental arrangement. HV= High voltage. Reprinted from ref. [Cai & Henion, 1995].

Several recent review papers have been devoted to a combination of CE-MS, applied to the structural identification of analytes and trace analytes. Surveys on developments and applications of CE-MS in the years of 2003-2004 and 2005-2006 are given by Schmitt and Englmann [Schmitt-Kopplin & Englmann, 2005] and Gaspar et al. [Gaspar et al., 2008], respectively. The reader will rapidly find the mostly used CE-MS combinations, as well as applications classified as forensic, environmental, bioanalytical, pharmaceutical and metabolomics. Ramautar et al. [Ramautar et al., 2009] pay attention to the utilization of CE-MS in metabolomics, considering papers published between 2000 and 2008. The authors presented CE-MS to be a powerful technique for the profiling of polar metabolites in biological samples. The combination of CE with MS was chosen as a review topic also in conjunction with chiral separations in aqueous and non-aqueous media [Cai & Henion, 1995; Shamsi, 2002; Scriba, 2007]. Methodological aspects of CE–MS for quantitation were summarized and evaluated by Ohnesorge et al. [Ohnesorge et al., 2005]. Pantučková et al. [Pantučková et al., 2009] reviewed electrolyte systems for on-line CE-MS with respect to detection requirements and separation possibilities. Smyth and Rodriguez [Smyth & Rodriguez, 2007] summarized recent studies of the electro spray ionization (ESI) behaviour of selected drugs and their application in CE-MS and HPLC-MS. For application examples of chiral CE-MS, see section 4.6 and Table 3.1.

### **4.5.1 CE stage in CE-MS**

The electrolyte systems in CE must be carefully selected regarding all the requirements of the on-line coupled MS analyser where terms such as the analyte ionization and evaporation play the key role (see section 4.5.2 CE-MS interfaces). It is obvious that both electrophoresis BGE and the solvent must support the ionization process and evaporation of droplets (i.e.,

Figure 4.16. Operational regions in CZE recommended for use in practice for on-line CE– MS. The concentration of NH4Ac usually ranges from 0.01 to 0.3M and the concentration of NH4Fo from 0.02 to 0.9M. The pH of the buffer in the acidic range is adjusted with either HAc (0.01–1.2 M) or HFo (0.02–2 M) according to the co-ion used. Alkaline pH is usually adjusted by adding ammonia solution or alkylamine. Separation in alkaline pH is sometimes done in NH4HCO3 or (NH4)2CO3 buffers in concentrations between 0.01 and 0.1

Advanced Combinations of Detection and Electrophoresis 129

LE *L ueff,L* a) pH*L* TE *T ueff,T* a) pH*<sup>T</sup>*,*adj*

NH4+ 56.3 8.80 10mM

CO32- -47.8 9.19 10mM EACA

Fo- -38.5 4.06 10mM MES +

Fortunately, a CE methodology improvement for advanced CE-MS hyphenation can be performed in several ways. A number of on-line sample preparation techniques have been introduced to improve the concentration sensitivity of CE, as given in chapter 3. One of the approaches for on-line analyte concentration for CE hyphenated with MS is the chromatographic method. This concept has been incorporated into CE-ESI-MS applications [Tomlinson et al., 1994]. The pre-column was made of a small bed of HPLC packing attached

a) Effective mobility (*ueff*) of leading (*L*) or terminating (*T*) ion, in 10-9m2V-1 s-1.

Table 4.2. Separation properties of examples of ITP systems

Reprinted from ref. [Pantučková et al., 2009].

NH4+ 76.2 5.06 10mM HAc H+ 14.7 3.25

imidazol + 20mM Asp

> + 20mM NH4OH

10mM NH4OH b)

Imid+ 7.8 7.91

MES- -11.2 5.92


EAC A-

M. Reprinted from ref. [Pantučková et al., 2009].

 *Cationic ITP*  1 10mM NH4Ac + 5mM HAc

2 10mM NH4OH + 20mM Asp

4 10mM NH4Fo + 5mM HFo

b) pH adjusted.

 *Anionic ITP*  3 10mM (NH4)2CO3

volatile and low ionic strength buffers, such as acetic acid, formic acid and their ammonium salts, buffers with carbonate as a volatile anion, and alkylamines as volatile cations), otherwise, the MS signal suppression due to salt deposits on the source and decreased response due to ion-pairing with the analyte could occur (e.g., citrate, phosphate, phthalate, morpholine, Tris buffers). Operational regions in CZE recommended for use in practice for on-line CE–MS are shown in Figure 4.16. The limited choice of electrolytes that are suitable for online CE–MS brings considerable limitations not only to the CE separation itself, but also to the potential sample stacking [Pantučková et al., 2009]. For example, separation properties of typical suitable ITP systems are given in Table 4.2. Due to the high volatility and low surface tension of organic solvents like methanol or acetonitrile, the use of nonaqueous media in NACE-ESI-MS may enhance ionization, resulting in improved detection limits compared to separation in aqueous buffer systems [Servais et al., 2006].

Although numerous publications have appeared on CE-MS, this technique is still developing to be widely accepted for routine use. The major limitation of CE is the limited sample volumes that can be analysed without compromising separation efficiency. Consequently, the concentration detection limit for CE can be several orders of magnitude higher than that of chromatographic methods. Using the currently available instrumentation, CE-MS detection limits for some applications can be too high, making it unlikely to be used in routine analysis. For example, a study was conducted comparing the performance of CE-MS with that of microbore LC-MS in the determination of endogenous amounts of leucine-enkephalin and methionine-enkephalin in equine cerebrospinal fluid using identical sample clean-up and enrichment procedures [Muck & Henion, 1989]. CE-MS was found to be limited in its concentration detection capacity owing to its much smaller injection volume. Another drawback with CE-MS is that migration times tend to fluctuate with a change of temperature in the environment. Although some manufacturers have incorporated a temperature controlling system into their CE instruments, these devices cannot be effectively utilized in CE-MS applications because a large portion of the CE capillary is extended between the CE instrument and the mass spectrometer. For applications such as regulatory work or those involving unknown components in a mixture, the use of a suitable internal standard would be necessary [Pleasance et al., 1992; Henion et al., 1994]. The chemical condition of the CE capillary inner walls also plays an important role in CE separation. The reproducibility and ruggedness of CE-MS are not currently as good as those of LC-MS. Like LC-MS, the use of non-volatile buffers in CE-MS is generally avoided. Compromises are often made in choosing appropriate operational conditions for CE-MS. Sensitivity limitations, as well as ion source plugging problems, created by the use of nonvolatile buffers have impeded the direct transfer of CE separation conditions to on-line CE-MS. Non-volatile additives, such as cyclodextrins, are widely used in the separation of closely related analytes including optical isomers [Ward, 1994; Novotny et al., 1994], however, the concentrations of these additives are often limited by practical restrictions [Varghese & Cole, 1993]. The MS sensitivity may deteriorate as the bulk flow of surfactant enters the mass spectrometer source region.

volatile and low ionic strength buffers, such as acetic acid, formic acid and their ammonium salts, buffers with carbonate as a volatile anion, and alkylamines as volatile cations), otherwise, the MS signal suppression due to salt deposits on the source and decreased response due to ion-pairing with the analyte could occur (e.g., citrate, phosphate, phthalate, morpholine, Tris buffers). Operational regions in CZE recommended for use in practice for on-line CE–MS are shown in Figure 4.16. The limited choice of electrolytes that are suitable for online CE–MS brings considerable limitations not only to the CE separation itself, but also to the potential sample stacking [Pantučková et al., 2009]. For example, separation properties of typical suitable ITP systems are given in Table 4.2. Due to the high volatility and low surface tension of organic solvents like methanol or acetonitrile, the use of nonaqueous media in NACE-ESI-MS may enhance ionization, resulting in improved detection

Although numerous publications have appeared on CE-MS, this technique is still developing to be widely accepted for routine use. The major limitation of CE is the limited sample volumes that can be analysed without compromising separation efficiency. Consequently, the concentration detection limit for CE can be several orders of magnitude higher than that of chromatographic methods. Using the currently available instrumentation, CE-MS detection limits for some applications can be too high, making it unlikely to be used in routine analysis. For example, a study was conducted comparing the performance of CE-MS with that of microbore LC-MS in the determination of endogenous amounts of leucine-enkephalin and methionine-enkephalin in equine cerebrospinal fluid using identical sample clean-up and enrichment procedures [Muck & Henion, 1989]. CE-MS was found to be limited in its concentration detection capacity owing to its much smaller injection volume. Another drawback with CE-MS is that migration times tend to fluctuate with a change of temperature in the environment. Although some manufacturers have incorporated a temperature controlling system into their CE instruments, these devices cannot be effectively utilized in CE-MS applications because a large portion of the CE capillary is extended between the CE instrument and the mass spectrometer. For applications such as regulatory work or those involving unknown components in a mixture, the use of a suitable internal standard would be necessary [Pleasance et al., 1992; Henion et al., 1994]. The chemical condition of the CE capillary inner walls also plays an important role in CE separation. The reproducibility and ruggedness of CE-MS are not currently as good as those of LC-MS. Like LC-MS, the use of non-volatile buffers in CE-MS is generally avoided. Compromises are often made in choosing appropriate operational conditions for CE-MS. Sensitivity limitations, as well as ion source plugging problems, created by the use of nonvolatile buffers have impeded the direct transfer of CE separation conditions to on-line CE-MS. Non-volatile additives, such as cyclodextrins, are widely used in the separation of closely related analytes including optical isomers [Ward, 1994; Novotny et al., 1994], however, the concentrations of these additives are often limited by practical restrictions [Varghese & Cole, 1993]. The MS sensitivity may deteriorate as the bulk flow of surfactant

limits compared to separation in aqueous buffer systems [Servais et al., 2006].

enters the mass spectrometer source region.

Figure 4.16. Operational regions in CZE recommended for use in practice for on-line CE– MS. The concentration of NH4Ac usually ranges from 0.01 to 0.3M and the concentration of NH4Fo from 0.02 to 0.9M. The pH of the buffer in the acidic range is adjusted with either HAc (0.01–1.2 M) or HFo (0.02–2 M) according to the co-ion used. Alkaline pH is usually adjusted by adding ammonia solution or alkylamine. Separation in alkaline pH is sometimes done in NH4HCO3 or (NH4)2CO3 buffers in concentrations between 0.01 and 0.1 M. Reprinted from ref. [Pantučková et al., 2009].


Table 4.2. Separation properties of examples of ITP systems

a) Effective mobility (*ueff*) of leading (*L*) or terminating (*T*) ion, in 10-9m2V-1 s-1.

b) pH adjusted.

Reprinted from ref. [Pantučková et al., 2009].

Fortunately, a CE methodology improvement for advanced CE-MS hyphenation can be performed in several ways. A number of on-line sample preparation techniques have been introduced to improve the concentration sensitivity of CE, as given in chapter 3. One of the approaches for on-line analyte concentration for CE hyphenated with MS is the chromatographic method. This concept has been incorporated into CE-ESI-MS applications [Tomlinson et al., 1994]. The pre-column was made of a small bed of HPLC packing attached to the inlet of the CE capillary. It serves as an on-line device for sample clean-up such as desalting and preconcentration for subsequent analysis by CE-ESI-MS. The capability of the ITP-CZE-ESI-MS system was demonstrated for the trace analysis of a complex matrix such as calf urine extracts. Several research groups have evaluated the ITP-CZE-ESI-MS approach [Kelly et al., 1994; Severs & Games, 1994; Moseley, 1994]. On-line ITP preconcentration has lowered the concentration detection limits of CE-MS by two orders of magnitude [Kelly et al., 1994; Moseley, 1994*.*]. Such an improved CE system provided much better separation efficiency than conventional LC systems. The two dimensional ITP approach based on a large volume sample injection (30 l injection volume) and large volume ITP preseparation (800 m i.d.) was applied for a very efficient sample preconcentration and analyte injection into a hyphenated MS stage [Tomáš et al., 2010]. The experimental arrangement of this ITP-ITP approach is illustrated in Figure 4.17. Although it has not been used for any enantiomeric separation so far, the ITP-CE approach is very promising for advanced hyphenation with the MS detection, overcoming many of the practical limitations of the CE-MS combination.

The use of small-ID capillaries was found to have improved the absolute sensitivity of CE-ESI-MS [Wahl et al., 1992, 1993]. This improvement in sensitivity is due to the high ionization efficiency associated with the very low bulk flow-rate from the small-ID capillary into the mass spectrometer. Since the ESI ion current is nearly independent of flowrate, ESI-MS will operate as a mass-sensitive detection system when the ESI current is limited by the flow-rate of the charged species in the solution to the ESI source [Wahl et al., 1994]. For example, a 25- to 50-fold increase in absolute sensitivity can be obtained by reducing the i.d. of CE capillary from 100 to 10 m [Wahl et al., 1993]. Another instrumental development is the use of coated capillaries. Coated capillaries are often used in the analysis of biological samples to minimize band broadening and peak tailing due to adsorption of analytes onto the capillary walls [Kohr & Engelhardt, 1993]. Moreover, the elimination of sample adsorption (mainly proteins) and EOF considerably increases reproducibility of the measurements. Several types of coated capillaries have been used in CE-MS applications such as aminopropyl silylated fused-silica capillaries [Goodlett et al., 1994; Wahl et al., 1993], capillaries coated with polyacrylamide [Thompson, T.J. et al., 1993], different hydrophilic derivatized capillaries [Cole et al., 1994] and non-covalent coated CE capillary with an overall positive charge [Thibault et al., 1991]. The use of coated capillaries to eliminate the electroosmotic flow may also be useful in minimizing the flow of non-volatile additives into the mass spectrometer [Kirby et al., 1994]. This may be of particular importance in applications such as chiral separations by CE-MS.

Figure 4.17. Scheme of the hydrodynamically closed isotachophoretic system. X, valves; HVPS, high voltage power supply; LE, TE, leading and terminating electrolytes and electrode reservoirs, respectively; C1, C2, preseparation and separation capillaries; CD, conductivity detectors; S, high voltage switch. Reprinted from ref. [Tomáš et al., 2010].

Advanced Combinations of Detection and Electrophoresis 131

The transfer of analytes from the liquid phase of CE to the gas-phase for MS is facilitated via interfaces, where practically all solvent and BGE are selectively removed, and the MS inlet is reached by analytes in the gas-phase with only some rests of the BGE. Several ionization methods have been used for the hyphenation of CE with MS. These include electro spray ionization (ESI), ion spray or pneumatically assisted electro spray ionization, and continuous-flow fast atom bombardment [Cai & Henion, 1995]. These are the on-line interfaces applied for the small, as well as large, molecules. Schematic diagrams of these interfaces are illustrated and described in the next subsections. On the other hand, desorption MS interfaces are preferred for the large non-volatile molecules like proteins,

**4.5.2 CE-MS interfaces** 

One solution to improving the sensitivity of CE-MS is the development of alternative types of mass spectrometers which offer the potential for greater sensitivity, as discussed in section 4.5.3.

to the inlet of the CE capillary. It serves as an on-line device for sample clean-up such as desalting and preconcentration for subsequent analysis by CE-ESI-MS. The capability of the ITP-CZE-ESI-MS system was demonstrated for the trace analysis of a complex matrix such as calf urine extracts. Several research groups have evaluated the ITP-CZE-ESI-MS approach [Kelly et al., 1994; Severs & Games, 1994; Moseley, 1994]. On-line ITP preconcentration has lowered the concentration detection limits of CE-MS by two orders of magnitude [Kelly et al., 1994; Moseley, 1994*.*]. Such an improved CE system provided much better separation efficiency than conventional LC systems. The two dimensional ITP approach based on a large volume sample injection (30 l injection volume) and large volume ITP preseparation (800 m i.d.) was applied for a very efficient sample preconcentration and analyte injection into a hyphenated MS stage [Tomáš et al., 2010]. The experimental arrangement of this ITP-ITP approach is illustrated in Figure 4.17. Although it has not been used for any enantiomeric separation so far, the ITP-CE approach is very promising for advanced hyphenation with the MS detection, overcoming many of the practical limitations of the CE-

The use of small-ID capillaries was found to have improved the absolute sensitivity of CE-ESI-MS [Wahl et al., 1992, 1993]. This improvement in sensitivity is due to the high ionization efficiency associated with the very low bulk flow-rate from the small-ID capillary into the mass spectrometer. Since the ESI ion current is nearly independent of flowrate, ESI-MS will operate as a mass-sensitive detection system when the ESI current is limited by the flow-rate of the charged species in the solution to the ESI source [Wahl et al., 1994]. For example, a 25- to 50-fold increase in absolute sensitivity can be obtained by reducing the i.d. of CE capillary from 100 to 10 m [Wahl et al., 1993]. Another instrumental development is the use of coated capillaries. Coated capillaries are often used in the analysis of biological samples to minimize band broadening and peak tailing due to adsorption of analytes onto the capillary walls [Kohr & Engelhardt, 1993]. Moreover, the elimination of sample adsorption (mainly proteins) and EOF considerably increases reproducibility of the measurements. Several types of coated capillaries have been used in CE-MS applications such as aminopropyl silylated fused-silica capillaries [Goodlett et al., 1994; Wahl et al., 1993], capillaries coated with polyacrylamide [Thompson, T.J. et al., 1993], different hydrophilic derivatized capillaries [Cole et al., 1994] and non-covalent coated CE capillary with an overall positive charge [Thibault et al., 1991]. The use of coated capillaries to eliminate the electroosmotic flow may also be useful in minimizing the flow of non-volatile additives into the mass spectrometer [Kirby et al., 1994]. This may be of particular importance in

One solution to improving the sensitivity of CE-MS is the development of alternative types of mass spectrometers which offer the potential for greater sensitivity, as discussed in

MS combination.

section 4.5.3.

applications such as chiral separations by CE-MS.

Figure 4.17. Scheme of the hydrodynamically closed isotachophoretic system. X, valves; HVPS, high voltage power supply; LE, TE, leading and terminating electrolytes and electrode reservoirs, respectively; C1, C2, preseparation and separation capillaries; CD, conductivity detectors; S, high voltage switch. Reprinted from ref. [Tomáš et al., 2010].

### **4.5.2 CE-MS interfaces**

The transfer of analytes from the liquid phase of CE to the gas-phase for MS is facilitated via interfaces, where practically all solvent and BGE are selectively removed, and the MS inlet is reached by analytes in the gas-phase with only some rests of the BGE. Several ionization methods have been used for the hyphenation of CE with MS. These include electro spray ionization (ESI), ion spray or pneumatically assisted electro spray ionization, and continuous-flow fast atom bombardment [Cai & Henion, 1995]. These are the on-line interfaces applied for the small, as well as large, molecules. Schematic diagrams of these interfaces are illustrated and described in the next subsections. On the other hand, desorption MS interfaces are preferred for the large non-volatile molecules like proteins,

**4.5.2.1 Ion spray interface** 

schematic diagram of CE-ISP-MS interfaces see Figure 4.19.

Ion spray (ISP) is closely related to the commonly used electro spray ionization interface (ESI), the difference being the application of a nebulizing gas which permits stable electro spray operation at flow-rates up to 1 mL/min [Hopfgartner et al., 1993], whereas pure electro spray has been restricted to flow-rate below 10 l/min [Whitehouse et al., 1985]. ISP provides a mild ionization via an ion evaporation process which results in primarily molecular mass information in the form of singly and multiply charged ions. For the

Advanced Combinations of Detection and Electrophoresis 133

Figure 4.19. Schematic diagram of CE-ISP-MS interfaces. (A) Liquid junction and (B) coaxial

The continuous-flow fast atom bombardment (CF-FAB) technique can dramatically extend the capability of MS for the determination of fragile and polar compounds [Minard et al., 1988; Moseley et al., 1989a, 1989b]. For the schematic diagram of CE-CF-FAB-MS interfaces see Figure 4.20. There are several attractive features of CE-MS using the ESI interface in comparison with CF-FAB interface. Since larger-ID capillaries can be used for the ESI interface, the loading capacity should be greater than those using the coaxial sheath-flow CF-FAB interface where only small-ID capillaries (ca. 10 m) can be used due to the

sheath-flow configurations. Reprinted from ref. [Cai & Henion, 1995].

**4.5.2.2 Continuous-flow fast atom bombardment interface** 

DNA etc. The coupling of CE to desorption MS can usually be off-line, although advanced (on-line) hyphenations have also recently appeared. The off-line coupling of CE with matrixassisted laser resorption ionization and 252Cf plasma desorption MS using fraction collection are reviewed by Cai and Henion [Cai & Henion, 1995]. In the recent review by Huck et al. [Huck et al., 2006] the most important techniques developed to hyphenate CE to matrixassisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) are summarized. The principles of the different interfaces and ways to solve the hyphenation problem are explained and discussed in detail. Very recently, modified matrix-assisted ionization techniques have been adapted also for the analysis of low molecular weight compounds like drugs and their metabolites [Wang et al., 2011]. Therefore, this interface is principally applicable also in chiral CE-MS and a special analyte treatment is briefly mentioned here. A schematic diagram of the mechanism of MALDI is shown in Figure 4.18.

Figure 4.18. A schematic diagram of the mechanism of MALDI. The mechanism of MALDI is believed to consist of three basic steps: (i) *Formation of a 'Solid Solution'*: this is essential for the matrix to be in access thus leading to the analyte molecules being completely isolated from each other. This eases the formation of the homogenous 'solid solution' required to produce a stable desorption of the analyte. (ii) *Matrix Excitation*: the laser beam is focused onto the surface consist of the matrix-analyte solid solution. The chromaphore of the matrix couples with the laser frequency causing rapid vibrational excitation, bringing about localised disintegration of the solid solution. The clusters ejected from the surface consist of analyte molecules surrounded by matrix and salt ions. The matrix molecules evaporate away from the clusters to leave the free analyte in the gas-phase. (iii) *Analyte Ionisation*: the photo-excited matrix molecules are stabilized through proton transfer to the analyte. Cation attachment to the analyte is also encouraged during this process. In this way the characteristic [M+X]+ (X= H, Na, K etc.) analyte ions are formed. These ionisation reactions take place in the desorbed matrix-analyte cloud just above the surface. The ions are then extracted into the mass spectrometer for analysis. Reprinted from ref. [http://www.chm.bris.ac.uk/ms/theory/maldi-ionisation.html].

### **4.5.2.1 Ion spray interface**

132 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

DNA etc. The coupling of CE to desorption MS can usually be off-line, although advanced (on-line) hyphenations have also recently appeared. The off-line coupling of CE with matrixassisted laser resorption ionization and 252Cf plasma desorption MS using fraction collection are reviewed by Cai and Henion [Cai & Henion, 1995]. In the recent review by Huck et al. [Huck et al., 2006] the most important techniques developed to hyphenate CE to matrixassisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) are summarized. The principles of the different interfaces and ways to solve the hyphenation problem are explained and discussed in detail. Very recently, modified matrix-assisted ionization techniques have been adapted also for the analysis of low molecular weight compounds like drugs and their metabolites [Wang et al., 2011]. Therefore, this interface is principally applicable also in chiral CE-MS and a special analyte treatment is briefly mentioned here. A schematic diagram of the mechanism of MALDI is shown in Figure 4.18.

Figure 4.18. A schematic diagram of the mechanism of MALDI. The mechanism of MALDI is believed to consist of three basic steps: (i) *Formation of a 'Solid Solution'*: this is essential for the matrix to be in access thus leading to the analyte molecules being completely isolated from each other. This eases the formation of the homogenous 'solid solution' required to produce a stable desorption of the analyte. (ii) *Matrix Excitation*: the laser beam is focused onto the surface consist of the matrix-analyte solid solution. The chromaphore of the matrix couples with the laser frequency causing rapid vibrational excitation, bringing about localised disintegration of the solid solution. The clusters ejected from the surface consist of analyte molecules surrounded by matrix and salt ions. The matrix molecules evaporate away from the clusters to leave the free analyte in the gas-phase. (iii) *Analyte Ionisation*: the photo-excited matrix molecules are stabilized through proton transfer to the analyte. Cation attachment to the analyte is also encouraged during this process. In this way the characteristic [M+X]+ (X= H, Na, K etc.) analyte ions are formed. These ionisation reactions take place in the desorbed matrix-analyte cloud just above the surface. The ions are then extracted into the mass spectrometer for analysis. Reprinted from ref.

[http://www.chm.bris.ac.uk/ms/theory/maldi-ionisation.html].

Ion spray (ISP) is closely related to the commonly used electro spray ionization interface (ESI), the difference being the application of a nebulizing gas which permits stable electro spray operation at flow-rates up to 1 mL/min [Hopfgartner et al., 1993], whereas pure electro spray has been restricted to flow-rate below 10 l/min [Whitehouse et al., 1985]. ISP provides a mild ionization via an ion evaporation process which results in primarily molecular mass information in the form of singly and multiply charged ions. For the schematic diagram of CE-ISP-MS interfaces see Figure 4.19. 10

Figure 4.19. Schematic diagram of CE-ISP-MS interfaces. (A) Liquid junction and (B) coaxial sheath-flow configurations. Reprinted from ref. [Cai & Henion, 1995].

### **4.5.2.2 Continuous-flow fast atom bombardment interface**

The continuous-flow fast atom bombardment (CF-FAB) technique can dramatically extend the capability of MS for the determination of fragile and polar compounds [Minard et al., 1988; Moseley et al., 1989a, 1989b]. For the schematic diagram of CE-CF-FAB-MS interfaces see Figure 4.20. There are several attractive features of CE-MS using the ESI interface in comparison with CF-FAB interface. Since larger-ID capillaries can be used for the ESI interface, the loading capacity should be greater than those using the coaxial sheath-flow CF-FAB interface where only small-ID capillaries (ca. 10 m) can be used due to the

Figure 4.21. A schematic of an ESI source. The analyte is introduced to the source in solution as the eluent flow from the CE analyser. Flow-rates are typically of the order of 1µl min-1. The analyte solution flow passes through the electro spray needle that has a high potential difference (with respect to the counter electrode) applied to it (typically in the range from 2.5 to 4 kV). This forces the spraying of charged droplets from the needle with a surface charge of the same polarity to the charge on the needle. The droplets are repelled from the needle towards the source sampling cone on the counter electrode (shown in blue). As the droplets traverse the space between the needle tip and the cone and solvent evaporation occurs. This is circled on the Figure 4.21 and enlarged in Figure 4.22. Reprinted from ref.

Advanced Combinations of Detection and Electrophoresis 135

Nearly all commercially available CE–MS interfaces are nowadays based upon electro spray ionization (ESI) [von Brocke et al., 2001; Schmitt-Kopplin & Frommberger, 2003]. For the schemes of ESI interfaces see Figure 4.23. Here, various configurations of ESI have been proposed in order to eliminate dead volume at the capillary terminus (Figure 4.23A) and to produce and maintain a stable electro spray signal during a CE-ESI-MS experiment (Figure 4.23B,C). Gale and Smith [Gale & Smith, 1993] compared the performance of the two ESI interfaces (i.e., sheath and sheathless) using pressure infusion. Their study showed several advantages of the sheathless version, including great sensitivity, low required flow-rates and long-term stability. In another study [Wahl et al., 1994], the performance of a goldcoated sheathless interface was compared with that of a sheath-flow interface regarding their dependence on a buffer system and concentration, as well as capillary i.d. The sheathless interface turned out to offer better analyte detectability. It was found that the sheathless configuration also eliminated the possible interferences from the sheath solvents

[http://www.chm.bris.ac.uk/ms/theory].

such as charge state distribution shift [Kriger et al., 1994].

pressure drop at the source region which leads to hydrodynamic flow within the CE capillary distorting the plug profile of electroosmotic flow and causing band broadening. The ESI interface also gives reduced background noise and higher sensitivity [Deterding et al., 1991; Edmonds et al., 1991]. band

Figure 4.20. Schematic diagram of CE-CF-FAB-MS interfaces. (A) Liquid junction and (B) coaxial sheath-flow configurations. Reprinted from ref. [Cai & Henion, 1995].

### **4.5.2.3 Electro spray ionization interface**

The ESI source is currently the preferred interface for CE-MS, as it can produce ions directly from liquids at atmospheric pressure and with high sensitivity and selectivity for a wide range of analytes. A schematic of an ESI source is in Figure 4.21. ESI is based on electrostatic effects on solution starting with the nebulization of a sample into electrically charged droplets and finishing with the formation of the gas-phase ions. A schematic of the mechanism of ion formation in ESI is in Figure 4.22.

pressure drop at the source region which leads to hydrodynamic flow within the CE capillary distorting the plug profile of electroosmotic flow and causing band broadening. The ESI interface also gives reduced background noise and higher sensitivity [Deterding et

band

Figure 4.20. Schematic diagram of CE-CF-FAB-MS interfaces. (A) Liquid junction and (B)

The ESI source is currently the preferred interface for CE-MS, as it can produce ions directly from liquids at atmospheric pressure and with high sensitivity and selectivity for a wide range of analytes. A schematic of an ESI source is in Figure 4.21. ESI is based on electrostatic effects on solution starting with the nebulization of a sample into electrically charged droplets and finishing with the formation of the gas-phase ions. A schematic of the

coaxial sheath-flow configurations. Reprinted from ref. [Cai & Henion, 1995].

**4.5.2.3 Electro spray ionization interface** 

mechanism of ion formation in ESI is in Figure 4.22.

al., 1991; Edmonds et al., 1991].

Figure 4.21. A schematic of an ESI source. The analyte is introduced to the source in solution as the eluent flow from the CE analyser. Flow-rates are typically of the order of 1µl min-1. The analyte solution flow passes through the electro spray needle that has a high potential difference (with respect to the counter electrode) applied to it (typically in the range from 2.5 to 4 kV). This forces the spraying of charged droplets from the needle with a surface charge of the same polarity to the charge on the needle. The droplets are repelled from the needle towards the source sampling cone on the counter electrode (shown in blue). As the droplets traverse the space between the needle tip and the cone and solvent evaporation occurs. This is circled on the Figure 4.21 and enlarged in Figure 4.22. Reprinted from ref. [http://www.chm.bris.ac.uk/ms/theory].

Nearly all commercially available CE–MS interfaces are nowadays based upon electro spray ionization (ESI) [von Brocke et al., 2001; Schmitt-Kopplin & Frommberger, 2003]. For the schemes of ESI interfaces see Figure 4.23. Here, various configurations of ESI have been proposed in order to eliminate dead volume at the capillary terminus (Figure 4.23A) and to produce and maintain a stable electro spray signal during a CE-ESI-MS experiment (Figure 4.23B,C). Gale and Smith [Gale & Smith, 1993] compared the performance of the two ESI interfaces (i.e., sheath and sheathless) using pressure infusion. Their study showed several advantages of the sheathless version, including great sensitivity, low required flow-rates and long-term stability. In another study [Wahl et al., 1994], the performance of a goldcoated sheathless interface was compared with that of a sheath-flow interface regarding their dependence on a buffer system and concentration, as well as capillary i.d. The sheathless interface turned out to offer better analyte detectability. It was found that the sheathless configuration also eliminated the possible interferences from the sheath solvents such as charge state distribution shift [Kriger et al., 1994].

**4.5.3 Mass spectrometry detection approaches** 

[www.noble.org/PlantBio/Sumner/metabolomics.html].

reduced sensitivity in FT-ICR MS [Baidoo et al., 2008].

So far, the triple quadrupole (TQ) and ion trap (IT) have been the most commonly used mass analysers in CE-MS for the analysis of low-molecular-weight compounds (including drugs and their metabolites) in biological samples [Soga, 2007]. A schematic of the basic setup of a TQ mass analyser is shown in Figure 4.24 while that of IT is in Figure 4.25. These MS instruments provide (i) high sensitivity (mainly TQ) with (ii) the capability to obtain structural information on unknown compounds (mainly IT). However, a disadvantage of these mass analysers, especially with respect to fast and highly efficient CE separations, is the relatively slow scanning process, therefore, they are not able to obtain sufficient data points across a very narrow CE peak to accurately define it [Soga et al., 2003]. In addition, the mass resolution of these instruments is limited providing lower selectivity for distinguishing analysed compounds. In general, TQ and IT mass analysers are more suited for targeted metabolomic studies by CE-MS [Ramautar et al., 2006; Monton & Soga, 2007].

Advanced Combinations of Detection and Electrophoresis 137

Figure 4.24. Conceptual view of tandem mass spectrometry with a tandem-in-space triple quadrupole mass analyser. The first mass analyser (Q1) selects the precursor ion of interest by allowing it only to pass while discriminating against all others. The precursor ion is then fragmented, usually by energetic collisions, in the second quadrupole (q2) that is operated in transmissive mode allowing all fragment ions to be collimated and passed into the third quadrupole (Q3). Q3 performs mass analysis on the product ions that compose the tandem mass spectra and are rationalized to a structure. Reprinted from ref.

The limitations of TQ- and IT-MS can be overcome by the time-of-flight (TOF) mass analysers where ions are accelerated by an electric field [Bajad & Shulaev, 2007; Lacorte & Fernandez-Albaz, 2006]. The working principle of a linear time-of-flight mass spectrometer is illustrated in Figure 4.26. The high scan speed (e.g., 10 spectra/s), high mass accuracy and high mass resolution of TOF-MS makes this method very suitable for non-targeted metabolomics studies [Soga et al., 2003]. Despite the increased resolution of TOF analysers, potential interferences from solvent ions, adducts and compounds with the same nominal mass as the metabolites in the biological sample can still disturb the analysis. Therefore, efficient separations prior to MS analysis are of key importance. These problems could also be circumvented by using Fourier transform ion cyclotron resonance (FT-ICR) MS, which has a mass resolution of >500 000 and provides very accurate mass measurements with subppm errors. However, accurate mass measurements with FT-ICR MS are relatively slow which can compromise the analysis of narrow CE peaks, and faster analysis may also lead to

is resolution

Figure 4.22. A schematic of the mechanism of ion formation in ESI. As the solvent evaporation occurs, the droplet shrinks until it reaches the point that the surface tension can no longer sustain the charge (the Rayleigh limit) at which point a "Coulombic explosion" occurs and the droplet is ripped apart. This produces smaller droplets that can repeat the process as well as naked charged analyte molecules. These charged analyte molecules (they are not strictly ions) can be singly or multiply charged. This is a very soft method of ionisation as very little residual energy is retained by the analyte upon ionisation. This is why ESI-MS is such an important technique in biological studies where the analyst often requires that non-covalent molecule-ligand (e.g., protein) interactions are representatively transferred into the gas-phase. By this technique very little (usually no) fragmentation is produced. For structural elucidation studies, this leads to the requirement for tandem mass spectrometry where the analyte molecules can be fragmented. Reprinted from ref. [http://www.chm.bris.ac.uk/ms/theory].

Figure 4.23. Schematic diagram of CE-ESI-MS interfaces. (A) Coaxial sheath-flow configuration, (B) sheathless interface with tapered and conductive coated CE capillary and (C) sheathless interface using a gold wire electrode. Reprinted from ref. [Cai & Henion, 1995].

Figure 4.22. A schematic of the mechanism of ion formation in ESI. As the solvent evaporation occurs, the droplet shrinks until it reaches the point that the surface tension can no longer sustain the charge (the Rayleigh limit) at which point a "Coulombic explosion" occurs and the droplet is ripped apart. This produces smaller droplets that can repeat the process as well as naked charged analyte molecules. These charged analyte molecules (they are not strictly ions) can be singly or multiply charged. This is a very soft method of ionisation as very little residual energy is retained by the analyte upon ionisation. This is why ESI-MS is such an important technique in biological studies where the analyst often requires that non-covalent molecule-ligand (e.g., protein) interactions are representatively transferred into the gas-phase. By this technique very little (usually no) fragmentation is produced. For structural elucidation studies, this leads to the requirement for tandem mass spectrometry where the analyte molecules can be fragmented. Reprinted from ref. [http://www.chm.bris.ac.uk/ms/theory].

Figure 4.23. Schematic diagram of CE-ESI-MS interfaces. (A) Coaxial sheath-flow configuration, (B) sheathless interface with tapered and conductive coated CE capillary and (C) sheathless interface using a gold wire electrode. Reprinted from ref. [Cai & Henion, 1995].

### **4.5.3 Mass spectrometry detection approaches**

So far, the triple quadrupole (TQ) and ion trap (IT) have been the most commonly used mass analysers in CE-MS for the analysis of low-molecular-weight compounds (including drugs and their metabolites) in biological samples [Soga, 2007]. A schematic of the basic setup of a TQ mass analyser is shown in Figure 4.24 while that of IT is in Figure 4.25. These MS instruments provide (i) high sensitivity (mainly TQ) with (ii) the capability to obtain structural information on unknown compounds (mainly IT). However, a disadvantage of these mass analysers, especially with respect to fast and highly efficient CE separations, is the relatively slow scanning process, therefore, they are not able to obtain sufficient data points across a very narrow CE peak to accurately define it [Soga et al., 2003]. In addition, the mass resolution of these instruments is limited providing lower selectivity for distinguishing analysed compounds. In general, TQ and IT mass analysers are more suited for targeted metabolomic studies by CE-MS [Ramautar et al., 2006; Monton & Soga, 2007]. 

Figure 4.24. Conceptual view of tandem mass spectrometry with a tandem-in-space triple quadrupole mass analyser. The first mass analyser (Q1) selects the precursor ion of interest by allowing it only to pass while discriminating against all others. The precursor ion is then fragmented, usually by energetic collisions, in the second quadrupole (q2) that is operated in transmissive mode allowing all fragment ions to be collimated and passed into the third quadrupole (Q3). Q3 performs mass analysis on the product ions that compose the tandem mass spectra and are rationalized to a structure. Reprinted from ref. [www.noble.org/PlantBio/Sumner/metabolomics.html].

The limitations of TQ- and IT-MS can be overcome by the time-of-flight (TOF) mass analysers where ions are accelerated by an electric field [Bajad & Shulaev, 2007; Lacorte & Fernandez-Albaz, 2006]. The working principle of a linear time-of-flight mass spectrometer is illustrated in Figure 4.26. The high scan speed (e.g., 10 spectra/s), high mass accuracy and high mass resolution of TOF-MS makes this method very suitable for non-targeted metabolomics studies [Soga et al., 2003]. Despite the increased resolution of TOF analysers, potential interferences from solvent ions, adducts and compounds with the same nominal mass as the metabolites in the biological sample can still disturb the analysis. Therefore, efficient separations prior to MS analysis are of key importance. These problems could also be circumvented by using Fourier transform ion cyclotron resonance (FT-ICR) MS, which has a mass resolution of >500 000 and provides very accurate mass measurements with subppm errors. However, accurate mass measurements with FT-ICR MS are relatively slow which can compromise the analysis of narrow CE peaks, and faster analysis may also lead to reduced sensitivity in FT-ICR MS [Baidoo et al., 2008].

**4.5.4 Chiral CE-ESI-MS** 

into ESI-MS.

**4.6 Applications** 

The key factor in manipulating enantioselectivity is the usage of suitable chiral selectors (see section 2.3). They are generally non-volatile molecules, whose introduction into the MS system should be avoided because they damage electro spray efficiency, and increase background noise, decreasing the sensitivity of detection [Shamsi, 2002]. It can be generalized that buffer components that interact strongly with the sample degrade the sensitivity of MS detection. For example, amphiphilic molecules give rise to intense signals in both positive and negative ion ESI, presenting a major barrier for MEKC-MS applications. The main target in such cases is to circumvent these difficulties. Therefore, an important aspect to be considered when a chiral analysis is going to be performed by CE-MS is the type of (i) chiral selector and (ii) separation mode, minimizing introduction of chiral selector

Advanced Combinations of Detection and Electrophoresis 139

As for the selection of the proper type of chiral selector, micelles vs. micelle polymers can serve as a model example. Assuming that the enantioselective capability is comparable, micelle polymers provide several advantages over conventional micelles for MEKC-ESI-MS hyphenation (see section 2.3.8.) resulting in less background noise and higher obtainable

As for the selection of proper separation mode minimizing the introduction of chiral selector into ESI-MS, the first possibility is immobilization of chiral selectors (CEC mode) which does not affect MS sensitivity (for CEC see section 2.2.2). The second possibility is based on the movement of the selector in the opposite direction toward the detector that can be provided by the selector's charge or by EOF in CE [Iio et al., 2005; Yang et al., 1997; Lu W. Z. et al., 1996; Fanali et al., 1998a; Schulte et al., 1998; Lu W. & Cole, 1998]. For advanced separation mechanisms see section 2.2.1.2. For example, EOF manipulation was used for micelles [Yang L. et al., 1997] and the countercurrent approach was used for antibiotics [Fanali et al., 1998a] and CD derivatives [Iio et al., 2005; Schulte et al., 1998] as chiral selectors. The partial filling of the capillary with the separation media containing the chiral selector in combination with its countercurrent migration can enhance the overall effect [Rudaz et al., 2005; Schappler et al., 2006]. Then, the concentration LODs obtainable by EKC-ESI-MS can be 10-8-10-9 M

*Absorbance detection.* Mikuš et al. [Mikuš et al., 2006a] demonstrated that advanced CE systems based on (i) on-line coupled columns (ITP-EKC), enabling efficient on-line sample preparation (preconcentration of analytes, elimination of matrix constituents) and providing enhanced sample loadability (30 l sample injection volume), and (ii) countercurrent migration of chiral selector (carboxyethyl--CD), allowed the use of a conventional UV detector (240 or 265 nm wavelengths were applied) for a highly efficient (~8x104 theoretical plates), selective (baseline enantioresolution and complete resolution from matrix compounds) and ultra-trace (LODs were in the concentration range of 1.1-4.8 ng/mL) direct determination of enantiomers of various drugs (H1-antihistamines like pheniramine, dimethindene, dioxopromethazine) in the multicomponent ionic matrices (urine samples). The method was successfully applied for enantioselective metabolic study of pheniramine in the externally unpretreated urine samples

corresponding with mass detection limits of 10-16-10-17 mol [Ewing et al., 1989].

S/N [Akbay et al., 2005; Hou et al., 2006; Rizvi et al., 2007].

showing its analytical potential in clinical research.

Figure 4.25. A schematic (cutaway view) of a quadrupole ion trap mass analyser. The ions, produced in the source of the instrument, enter into the trap through the inlet and are trapped through the action of the three hyperbolic electrodes: the ring electrode and the entrance and exit endcap electrodes. Various voltages are applied to these electrodes which results in the formation of a cavity in which ions are trapped. The ring electrode RF potential, an a.c. potential of constant frequency but variable amplitude, produces a 3D quadrupolar potential field within the trap. This traps the ions in a stable oscillating trajectory. The exact motion of the ions is dependent on the voltages applied and their individual mass-to-charge (*m/z*) ratios. For detection of the ions, the potentials are altered to destabilize the ion motions resulting in ejection of the ions through the exit endcap. The ions are usually ejected in order of increasing *m/z* by a gradual change in the potentials. This 'stream' of ions is focused onto the detector of the instrument to produce the mass spectrum. The very nature of trapping and ejection makes a quadrupolar ion trap especially suited to performing MSn experiments in structural elucidation studies. It is possible to selectively isolate a particular *m/z* in the trap by ejecting all the other ions from the trap. Fragmentation of this isolated precursor ion can then be induced by collision-induced dissociation (CID) experiments. The isolation and fragmentation steps can be repeated a number of times and this is only limited by the trapping efficiency of the instrument. Reprinted from ref. [www.chm.bris.ac.uk/ms/theory/qit-massspec.html]. of the

Figure 4.26. The working principle of a linear time-of-flight mass spectrometer. Time-offlight mass spectrometers identify molecules by measuring the time that sample molecules, all starting with the same kinetic energy, require to fly a known distance. Reprinted from ref. [www.kore.co.uk/tutorial.html].

### **4.5.4 Chiral CE-ESI-MS**

138 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Figure 4.25. A schematic (cutaway view) of a quadrupole ion trap mass analyser. The ions, produced in the source of the instrument, enter into the trap through the inlet and are trapped through the action of the three hyperbolic electrodes: the ring electrode and the entrance and exit endcap electrodes. Various voltages are applied to these electrodes which results in the formation of a cavity in which ions are trapped. The ring electrode RF potential, an a.c. potential of constant frequency but variable amplitude, produces a 3D quadrupolar potential field within the trap. This traps the ions in a stable oscillating trajectory. The exact motion of the ions is dependent on the voltages applied and their individual mass-to-charge (*m/z*) ratios. For detection of the ions, the potentials are altered to destabilize the ion motions resulting in ejection of the ions through the exit endcap. The ions are usually ejected in order of increasing *m/z* by a gradual change in the potentials. This 'stream' of ions is focused onto the detector of the instrument to produce the mass spectrum. The very nature of trapping and ejection makes a quadrupolar ion trap especially suited to performing MSn experiments in structural elucidation studies. It is possible to selectively isolate a particular *m/z* in the trap by ejecting all the other ions from the trap. Fragmentation of this isolated precursor ion can then be induced by collision-induced dissociation (CID) experiments. The isolation and fragmentation steps can be repeated a number of times and this is only limited by the trapping efficiency of the instrument. Reprinted from ref.

Figure 4.26. The working principle of a linear time-of-flight mass spectrometer. Time-offlight mass spectrometers identify molecules by measuring the time that sample molecules, all starting with the same kinetic energy, require to fly a known distance. Reprinted from

[www.chm.bris.ac.uk/ms/theory/qit-massspec.html].

motion of

ref. [www.kore.co.uk/tutorial.html].

The key factor in manipulating enantioselectivity is the usage of suitable chiral selectors (see section 2.3). They are generally non-volatile molecules, whose introduction into the MS system should be avoided because they damage electro spray efficiency, and increase background noise, decreasing the sensitivity of detection [Shamsi, 2002]. It can be generalized that buffer components that interact strongly with the sample degrade the sensitivity of MS detection. For example, amphiphilic molecules give rise to intense signals in both positive and negative ion ESI, presenting a major barrier for MEKC-MS applications. The main target in such cases is to circumvent these difficulties. Therefore, an important aspect to be considered when a chiral analysis is going to be performed by CE-MS is the type of (i) chiral selector and (ii) separation mode, minimizing introduction of chiral selector into ESI-MS.

As for the selection of the proper type of chiral selector, micelles vs. micelle polymers can serve as a model example. Assuming that the enantioselective capability is comparable, micelle polymers provide several advantages over conventional micelles for MEKC-ESI-MS hyphenation (see section 2.3.8.) resulting in less background noise and higher obtainable S/N [Akbay et al., 2005; Hou et al., 2006; Rizvi et al., 2007].

As for the selection of proper separation mode minimizing the introduction of chiral selector into ESI-MS, the first possibility is immobilization of chiral selectors (CEC mode) which does not affect MS sensitivity (for CEC see section 2.2.2). The second possibility is based on the movement of the selector in the opposite direction toward the detector that can be provided by the selector's charge or by EOF in CE [Iio et al., 2005; Yang et al., 1997; Lu W. Z. et al., 1996; Fanali et al., 1998a; Schulte et al., 1998; Lu W. & Cole, 1998]. For advanced separation mechanisms see section 2.2.1.2. For example, EOF manipulation was used for micelles [Yang L. et al., 1997] and the countercurrent approach was used for antibiotics [Fanali et al., 1998a] and CD derivatives [Iio et al., 2005; Schulte et al., 1998] as chiral selectors. The partial filling of the capillary with the separation media containing the chiral selector in combination with its countercurrent migration can enhance the overall effect [Rudaz et al., 2005; Schappler et al., 2006]. Then, the concentration LODs obtainable by EKC-ESI-MS can be 10-8-10-9 M corresponding with mass detection limits of 10-16-10-17 mol [Ewing et al., 1989].

### **4.6 Applications**

*Absorbance detection.* Mikuš et al. [Mikuš et al., 2006a] demonstrated that advanced CE systems based on (i) on-line coupled columns (ITP-EKC), enabling efficient on-line sample preparation (preconcentration of analytes, elimination of matrix constituents) and providing enhanced sample loadability (30 l sample injection volume), and (ii) countercurrent migration of chiral selector (carboxyethyl--CD), allowed the use of a conventional UV detector (240 or 265 nm wavelengths were applied) for a highly efficient (~8x104 theoretical plates), selective (baseline enantioresolution and complete resolution from matrix compounds) and ultra-trace (LODs were in the concentration range of 1.1-4.8 ng/mL) direct determination of enantiomers of various drugs (H1-antihistamines like pheniramine, dimethindene, dioxopromethazine) in the multicomponent ionic matrices (urine samples). The method was successfully applied for enantioselective metabolic study of pheniramine in the externally unpretreated urine samples showing its analytical potential in clinical research.

A very complex solution in applied biomedical analytical research was demonstrated by the hyphenation of the ITP-EKC (on-line sample preparation plus countercurrent chiral selector migration) with the spectral detector such as diode array detector (DAD). A DAD spectral characterization/identification in combination with the ITP-EKC separation was successfully applied for the enantioselective determination of drugs and their potential metabolites in unpretreated biological samples that were utilized in enantioselective pharmacokinetic and metabolic studies. Spectral monitoring of the real urine samples, taken after oral administration of 20 mg of amlodipine, carried out over a 54 hour time span, revealed the time vs. concentration profiles typical for the elimination of amlodipine enantiomers in the human body [Mikuš et al., 2008a]. These pharmacokinetic dependences clearly indicated the maxima of excluded amlodipine enantiomers and revealed the differences in elimination between the two enantiomers (enantioselective elimination). High reliability of the results was based on the fact that each point in the pharmacokinetic curves was characterized through spectra and only zones/peaks with acceptable purity (spectral homogeneity) and that matched to the reference spectra were further considered for the pharmacokinetic curve. In another work [Marák et al., 2007], ITP-EKC-DAD enabled potential pheniramine metabolites present at lower ng/mL concentration levels in real urine samples (taken after oral administration of pheniramine tablets to a female volunteer) to be distinguished, see Figure 4.27. Calculated match factors (reference vs. real spectra) gave a high statistical probability of peak identity of pheniramine enantiomers, as well as their potential metabolites. Two unknown peaks migrating close to pheniramine enantiomers peaks (Figure 4.27b) were supposed to be N-desmethyl pheniramine enantiomers, taking into account the former findings concerning the pheniramine metabolism and excretion, as well as migration times of the native and metabolized compounds. The DAD spectral analysis confirmed the high separation selectivity (i.e., producing of spectrally homogeneous analytes zones) of the ITP-EKC method with possible direct injection of unpretreated urine samples. Thus, the ITP-EKC-DAD method illustrates the benefits in (i) chiral field {countercurrent (enantio)resolution enhancement}, (ii) sample preparation (online preconcentration and purification) and (iii) detection (sensitivity enhancement and preliminary identification aspect) in analysis of a chiral drug and its metabolites in complex

Advanced Combinations of Detection and Electrophoresis 141

*Fluorescence detection.* LIF was used for the chiral determination of many biologically active compounds [Hernández et al., 2008, 2010]. For example, CBI derivatized baclofen was determined in human plasma with LOD 5x10-8 M using the CD-EKC-LIF method (highly sulfated--CD as chiral selector, excitation wavelength was 442 nm, emission wavelength

The main application of LIF in CE has been the analysis of amino acid enantiomers, including also biological samples [Quan et al., 2005; Zhao S.L. et al., 2005a, 2005b; Kirschner et al., 2007; Miao et al., 2005, 2006; Sheeley et al., 2005]. In all cases, the methods involved precapillary chiral derivatization of the amino acids. EKC-LIF (2-hydroxypropyl--CD and 2-hydroxypropyl--CD as chiral selectors, excitation wavelength was 457.9 nm) was used for the chiral separation and detection of Ser, primary endogenous amino acid that binds to the Gly site of *N*-methyl-D-Asp receptor involved in a variety of physiological functions and disorders. The sensitive detection of this amino acid is essential, because it is at low

matrices.

was 500 nm) [Kavran-Belin et al., 2005].

Figure 4.27. ITP-EKC-DAD method for the direct sensitive determination of enantiomers in a sample of unpretreated complex matrices with spectral characterization of electrophoretic zones. 3D traces were obtained combining electrophoretic (EKC) and spectral (DAD) data where the spectra were scanned in the interval of wavelengths 200-400 nm. (a) 3D trace illustrating the whole EKC enantioseparation of pheniramine and its metabolites in the on-line pretreated clinical urine sample (spectra of matrix constituents, well separated from the analytes, are pronounced), (b) detail on the 3D spectra showing the migration positions of pheniramine enantiomers (E1 and E2) and their structurally related metabolites (M1 and M2). The spectrum of the little unknown peak marked with the asterisk differed from the pheniramine spectrum significantly and, therefore, it was not considered as a pheniramine biodegradation product. The urine sample was taken 8.5 hours after the administration of one dose of Fervex (containing 25 mg of racemic pheniramine) to a female volunteer and it was diluted 10-fold before the injection. The separations were carried out using 10 mM sodium acetate - acetic acid, pH 4.75 as a leading electrolyte (ITP), 5 mM -aminocaproic acid - acetic acid, pH 4.5 as a terminating electrolyte (ITP) and 25 mM -aminocaproic acid - acetic acid, pH 4.5 as a carrier electrolyte (EKC). 0.1% (w/v) methyl-hydroxyethylcellulose served as an EOF suppressor in leading and carrier electrolytes. Carboxyethyl--CD (5 mg/mL) was used as a chiral selector in carrier electrolyte. Reprinted from ref. [Marák et al., 2007].

Figure 4.27. ITP-EKC-DAD method for the direct sensitive determination of enantiomers in a sample of unpretreated complex matrices with spectral characterization of electrophoretic zones. 3D traces were obtained combining electrophoretic (EKC) and spectral (DAD) data where the spectra were scanned in the interval of wavelengths 200-400 nm. (a) 3D trace illustrating the whole EKC enantioseparation of pheniramine and its metabolites in the on-line pretreated clinical urine sample (spectra of matrix constituents, well separated from the analytes, are pronounced), (b) detail on the 3D spectra showing the migration positions of pheniramine enantiomers (E1 and E2) and their structurally related metabolites (M1 and M2). The spectrum of the little unknown peak marked with the asterisk differed from the pheniramine spectrum significantly and, therefore, it was not considered as a pheniramine biodegradation product. The urine sample was taken 8.5 hours after the administration of one dose of Fervex (containing 25 mg of racemic pheniramine) to a female volunteer and it was diluted 10-fold before the injection. The separations were carried out using 10 mM sodium acetate - acetic acid, pH 4.75 as a leading electrolyte (ITP), 5 mM -aminocaproic acid - acetic acid, pH 4.5 as a terminating electrolyte (ITP) and 25 mM -aminocaproic acid - acetic acid, pH 4.5 as a carrier electrolyte (EKC). 0.1% (w/v) methyl-hydroxyethylcellulose served as an EOF suppressor in leading and carrier electrolytes. Carboxyethyl--CD (5 mg/mL) was used as a

chiral selector in carrier electrolyte. Reprinted from ref. [Marák et al., 2007].

A very complex solution in applied biomedical analytical research was demonstrated by the hyphenation of the ITP-EKC (on-line sample preparation plus countercurrent chiral selector migration) with the spectral detector such as diode array detector (DAD). A DAD spectral characterization/identification in combination with the ITP-EKC separation was successfully applied for the enantioselective determination of drugs and their potential metabolites in unpretreated biological samples that were utilized in enantioselective pharmacokinetic and metabolic studies. Spectral monitoring of the real urine samples, taken after oral administration of 20 mg of amlodipine, carried out over a 54 hour time span, revealed the time vs. concentration profiles typical for the elimination of amlodipine enantiomers in the human body [Mikuš et al., 2008a]. These pharmacokinetic dependences clearly indicated the maxima of excluded amlodipine enantiomers and revealed the differences in elimination between the two enantiomers (enantioselective elimination). High reliability of the results was based on the fact that each point in the pharmacokinetic curves was characterized through spectra and only zones/peaks with acceptable purity (spectral homogeneity) and that matched to the reference spectra were further considered for the pharmacokinetic curve. In another work [Marák et al., 2007], ITP-EKC-DAD enabled potential pheniramine metabolites present at lower ng/mL concentration levels in real urine samples (taken after oral administration of pheniramine tablets to a female volunteer) to be distinguished, see Figure 4.27. Calculated match factors (reference vs. real spectra) gave a high statistical probability of peak identity of pheniramine enantiomers, as well as their potential metabolites. Two unknown peaks migrating close to pheniramine enantiomers peaks (Figure 4.27b) were supposed to be N-desmethyl pheniramine enantiomers, taking into account the former findings concerning the pheniramine metabolism and excretion, as well as migration times of the native and metabolized compounds. The DAD spectral analysis confirmed the high separation selectivity (i.e., producing of spectrally homogeneous analytes zones) of the ITP-EKC method with possible direct injection of unpretreated urine samples. Thus, the ITP-EKC-DAD method illustrates the benefits in (i) chiral field {countercurrent (enantio)resolution enhancement}, (ii) sample preparation (online preconcentration and purification) and (iii) detection (sensitivity enhancement and preliminary identification aspect) in analysis of a chiral drug and its metabolites in complex matrices.

*Fluorescence detection.* LIF was used for the chiral determination of many biologically active compounds [Hernández et al., 2008, 2010]. For example, CBI derivatized baclofen was determined in human plasma with LOD 5x10-8 M using the CD-EKC-LIF method (highly sulfated--CD as chiral selector, excitation wavelength was 442 nm, emission wavelength was 500 nm) [Kavran-Belin et al., 2005].

The main application of LIF in CE has been the analysis of amino acid enantiomers, including also biological samples [Quan et al., 2005; Zhao S.L. et al., 2005a, 2005b; Kirschner et al., 2007; Miao et al., 2005, 2006; Sheeley et al., 2005]. In all cases, the methods involved precapillary chiral derivatization of the amino acids. EKC-LIF (2-hydroxypropyl--CD and 2-hydroxypropyl--CD as chiral selectors, excitation wavelength was 457.9 nm) was used for the chiral separation and detection of Ser, primary endogenous amino acid that binds to the Gly site of *N*-methyl-D-Asp receptor involved in a variety of physiological functions and disorders. The sensitive detection of this amino acid is essential, because it is at low

this pH, a charged cyclodextrin derivative had to be used for chiral resolution. It was observed that the detection sensitivity is also affected by the type and concentration of chiral and achiral additives applied for achieving resolution. As mentioned by the authors, electrochemical detection has the advantage that derivatization can be omitted. On the other hand, the freedom of buffer choice is limited as conditions have to be arrived which satisfy the requirements for both separation and detection. Another important aspect is that

Advanced Combinations of Detection and Electrophoresis 143

In another paper, the detection limits using UV absorbance detector were found to be too high to determine the concentration of isoproterenol (with a speed elimination pharmacokinetics) in intravenous microdialysis samples for a sufficient time following administration to establish the pharmacokinetics. The LODs of this catecholamine were decreased three orders of magnitude (to 3 ng/mL) by using an amperometric detector. Moreover, further sensitivity enhancement (to 0.6 ng/mL) was achieved by inserting incapillary stacking preconcentration of microdialyzed plasma samples. This provided conditions suitable for obtaining pharmacokinetic curves of isoproterenol enantiomers in

Figure 4.29. Typical CE-electrochemical detection (CE-EC) electropherograms from a pharmacokinetic experiment using on-line sample pretreatment combining dialysis and stacking. (A) Blank sample acquired prior to administering an ISP dose; (B) microdialysate acquired 6 min after dosing and (C) microdialysate acquired 54 min after dosing. Peaks: 1=3,4-Dihydroxybenzylamine(DHBA); 2=5NMHT; 3=(-)-isoproterenol (ISP); 4=(+)-ISP.

While conductivity detection is regarded as rather intensive in zone electrophoresis, it can be very attractive in isotachophoresis due to the on-line focusing effect during analyses.

electrochemical detection lacks universality.

plasma, see Figure 4.29 [Hadviger et al., 1996].

Reprinted from ref. [Hadwiger et al., 1996].

concentration in biological samples. D-Ser (NBD-F or CBI derivatives) was detected in rat brain achieving LODs of about 10-7 M [Quan et al., 2005; Zhao S.L. et al., 2005a]. The EKC-LIF method (a highly sulfated--CD as chiral selector, excitation wavelength was 420 nm) for CBI-Ser was improved (and applied also for other amino acids like aspartate and Glu) by on-line preconcentration procedures. Subsequently a very high sensitivity (LODs up to 10-10 M) was achieved in squirrel brain samples [Kirschner et al., 2007]. Derivatized amino acids, such as CBI-Ser and NDA-Asp, were also enantioseparated and detected in invertebrate tissue (mollusc neurons) [Quan et al., 2005; Miao et al., 2005; Zhao S.L. et al., 2005b] where LODs ranged from ~10-7-10-10 M using EKC-LIF and excitation wavelength 457.9 nm. An effectivity of the dual chiral selector system and LIF detection for the enantioseparation and identification of CBI-D/L-serine enantiomers in real biological matrices is shown in Figure 4.28 [Zhao S.L. et al., 2005b]. In this way, for the first time, peaks corresponding to L-serine and D-serine were well identified and sensitively detected (LOD was 3x10-8 M) in *Aplysia*  ganglian (a sea mollusc widely used as a neuronal model) homogenates. Miao et al. [Miao et al., 2005] demonstrated an approach for the quantitative investigation of the biochemical composition of subcellular regions of single neurons. Such investigations were possible thanks to the excellent LOD (5x10-10 M) of NDA-D-Asp and powerful separation system (CD-MEKC).

Figure 4.28. Separation and detection of CBI-Ser enantiomers in biological matrices. Separation conditions: 100mM borate (pH 9.5), 30mM -CD and 60mM sodium deoxycholate (SDC). Electropherogram obtained from analysing an *Aplysia* pedal ganglion homogenate. Capillary was 50 m i.d.×50 cm effective length. Voltage applied was 15 kV. Reprinted from ref. [Zhao S.L. et al., 2005b]. 

*Electrochemical detection.* Amperometric detection was introduced by Schwarz and Hauser in chiral MCE for the separation of basic drugs and biomarkers [Schwarz & Hauser, 2001, 2003]. Plate numbers of up to 20 000, chiral resolutions of 2.5 and detection limits of the order of 10-7 *M* were achieved. All separations were completed in less than 3 min. Detection was carried out with a two electrode amperomatric detector, eliminating the need for individual counter and reference electrodes. Because the electrochemical oxidation of catecholamines is pH-dependent, higher detection sensitivities could be obtained at higher pH values. Direct electrochemical oxidation of ephedrine and pseudoephedrine was achieved at a gold electrode at a high pH of 12.6. As the basic compounds are uncharged at

concentration in biological samples. D-Ser (NBD-F or CBI derivatives) was detected in rat brain achieving LODs of about 10-7 M [Quan et al., 2005; Zhao S.L. et al., 2005a]. The EKC-LIF method (a highly sulfated--CD as chiral selector, excitation wavelength was 420 nm) for CBI-Ser was improved (and applied also for other amino acids like aspartate and Glu) by on-line preconcentration procedures. Subsequently a very high sensitivity (LODs up to 10-10 M) was achieved in squirrel brain samples [Kirschner et al., 2007]. Derivatized amino acids, such as CBI-Ser and NDA-Asp, were also enantioseparated and detected in invertebrate tissue (mollusc neurons) [Quan et al., 2005; Miao et al., 2005; Zhao S.L. et al., 2005b] where LODs ranged from ~10-7-10-10 M using EKC-LIF and excitation wavelength 457.9 nm. An effectivity of the dual chiral selector system and LIF detection for the enantioseparation and identification of CBI-D/L-serine enantiomers in real biological matrices is shown in Figure 4.28 [Zhao S.L. et al., 2005b]. In this way, for the first time, peaks corresponding to L-serine and D-serine were well identified and sensitively detected (LOD was 3x10-8 M) in *Aplysia*  ganglian (a sea mollusc widely used as a neuronal model) homogenates. Miao et al. [Miao et al., 2005] demonstrated an approach for the quantitative investigation of the biochemical composition of subcellular regions of single neurons. Such investigations were possible thanks to the excellent LOD (5x10-10 M) of NDA-D-Asp and powerful separation system

Figure 4.28. Separation and detection of CBI-Ser enantiomers in biological matrices. Separation conditions: 100mM borate (pH 9.5), 30mM -CD and 60mM sodium deoxycholate (SDC). Electropherogram obtained from analysing an *Aplysia* pedal ganglion homogenate. Capillary was 50 m i.d.×50 cm effective length. Voltage applied was 15 kV.

*Electrochemical detection.* Amperometric detection was introduced by Schwarz and Hauser in chiral MCE for the separation of basic drugs and biomarkers [Schwarz & Hauser, 2001, 2003]. Plate numbers of up to 20 000, chiral resolutions of 2.5 and detection limits of the order of 10-7 *M* were achieved. All separations were completed in less than 3 min. Detection was carried out with a two electrode amperomatric detector, eliminating the need for individual counter and reference electrodes. Because the electrochemical oxidation of catecholamines is pH-dependent, higher detection sensitivities could be obtained at higher pH values. Direct electrochemical oxidation of ephedrine and pseudoephedrine was achieved at a gold electrode at a high pH of 12.6. As the basic compounds are uncharged at

(CD-MEKC).

Reprinted from ref. [Zhao S.L. et al., 2005b].

this pH, a charged cyclodextrin derivative had to be used for chiral resolution. It was observed that the detection sensitivity is also affected by the type and concentration of chiral and achiral additives applied for achieving resolution. As mentioned by the authors, electrochemical detection has the advantage that derivatization can be omitted. On the other hand, the freedom of buffer choice is limited as conditions have to be arrived which satisfy the requirements for both separation and detection. Another important aspect is that electrochemical detection lacks universality.

In another paper, the detection limits using UV absorbance detector were found to be too high to determine the concentration of isoproterenol (with a speed elimination pharmacokinetics) in intravenous microdialysis samples for a sufficient time following administration to establish the pharmacokinetics. The LODs of this catecholamine were decreased three orders of magnitude (to 3 ng/mL) by using an amperometric detector. Moreover, further sensitivity enhancement (to 0.6 ng/mL) was achieved by inserting incapillary stacking preconcentration of microdialyzed plasma samples. This provided conditions suitable for obtaining pharmacokinetic curves of isoproterenol enantiomers in plasma, see Figure 4.29 [Hadviger et al., 1996].

Figure 4.29. Typical CE-electrochemical detection (CE-EC) electropherograms from a pharmacokinetic experiment using on-line sample pretreatment combining dialysis and stacking. (A) Blank sample acquired prior to administering an ISP dose; (B) microdialysate acquired 6 min after dosing and (C) microdialysate acquired 54 min after dosing. Peaks: 1=3,4-Dihydroxybenzylamine(DHBA); 2=5NMHT; 3=(-)-isoproterenol (ISP); 4=(+)-ISP. Reprinted from ref. [Hadwiger et al., 1996].

While conductivity detection is regarded as rather intensive in zone electrophoresis, it can be very attractive in isotachophoresis due to the on-line focusing effect during analyses.

*Mass spectrometry.* Rudaz et al. [Rudaz et al., 2005; Schappler et al., 2006] used the EKC-MS with countercurrent migration of the negatively charged highly sulfated--CD (ammonium formate electrolyte) and the partial filling technique to achieve the enantioseparation of amphetamine derivatives. The use of this strategy with a low concentration of the chiral selector allowed the rapid stereoselective separation of seven amphetamine derivatives in spiked plasma samples in analysis times of less than 6 min and concentration LODs ranging from ~7x10-7-3x10-6 M [Rudaz et al., 2005]. An enhancing of the detection sensitivity was accomplished with an electrokinetic injection accompanied by the introduction of an appropriate buffer plug length between the zone containing the chiral selector and the analyte injection (to overcome drawbacks linked with electrokinetic injection in the presence of a charged chiral selector) [Schappler et al., 2006], see Figure 4.30. When such complex matrices are analysed, MS signal suppression or enhancement effects are generally not reproducible and can compromise results using CE–ESI-MS. Therefore, the matrix effect was investigated with a commercially available coaxial sheath liquid ESI interface used as the post-capillary infusion system. It was found that with PP, signal suppression was observed while for LLE, no relevant matrix effect occurred in all experiments. Concentration LODs were improved by up to 4x10-9 M of each enantiomer present in plasma samples after LLE

Advanced Combinations of Detection and Electrophoresis 145

Anionic CD, heptakis(2,6-diacethyl-6-sulfato)--CD, migrating from the detector to the inlet of the capillary, was used for the simultaneous chiral separation of drugs [Iio et al., 2005]. This selective EKC-ESI-MS method (formic acid electrolyte) was applied to the analysis of methamphetamine, 3,4-methylenedioxyamphetamine and amphetamine in clinical human urine samples pretreated by LLE. Sufficient sensitivity was obtained when concentration

Figure 4.31. Typical electropherogram of salbutamol enantiomers at a concentration close to the LOQ obtained using SPE–NACE–ESI-MS (EIC at 240.4 *m/z*) using a BGE of 10mM ammonium formate and 15mM HDAS--CD in methanol acidified with 0.75M formic acid.

and detected by a single quadrupole MS as a detection device.

LODs ranged from ~6x10-8-10-7 M.

Reprinted from ref. [Servais et al., 2006].

Mikus et al. used conductivity detection with a chiral ITP for the enantioseparation of various H1-antihistaminic drugs in dosage forms [Mikuš et al., 2006b; Kubačák et al., 2006a, 2006b, 2007]. Chiral isotachophoresis in microfluidic devices with on-column conductivity detection was successfully demonstrated by Olvecka et al. [Ölvecká et al., 2001] for the chiral separation of tryptophan enantiomers, however, only in model samples. For this purpose they used a coupled separation channel configuration. In other works, contact, as well as contactless, conductivity detection was successfully used for preliminary scanning of sample profile in the ITP stage of the ITP-EKC method when analysing various drugs present in biological samples. Thus, amlodipine, pheniramine (plus its metabolites), dioxopromethazine, and dimethindene could be easily transferred into the EKC stage for the final enantioseparation without undesired matrix constituents present in urine samples [Mikuš et al., 2006a, 2008a, 2008b; Marák et al., 2007]. The methods were applied in enantioselective metabolic [Mikuš et al., 2006a, 2008b; Marák et al., 2007] and pharmacokinetic [Mikuš et al., 2008a] studies of the drugs, see e.g., Figure 4.27.

Figure 4.30. Chiral analysis of amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxyamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA), tramadol (TMD) and methadone (MTD) amphetamine enantiomers at 0.5 ppb in plasma after LLE with EK injection. Experimental conditions: FS capillary of 75 cm and 650mm (ID); 25 kV; EK injection at 10 kV 610 s; 50% capillary filled with HS--CD 0.15% in BGE 2% capillary filled with BGE post-plug; on-line CE– ESI/MS in SIM mode and extracted ion current (XIC). Reprinted from ref. [Schappler et al., 2006].

Mikus et al. used conductivity detection with a chiral ITP for the enantioseparation of various H1-antihistaminic drugs in dosage forms [Mikuš et al., 2006b; Kubačák et al., 2006a, 2006b, 2007]. Chiral isotachophoresis in microfluidic devices with on-column conductivity detection was successfully demonstrated by Olvecka et al. [Ölvecká et al., 2001] for the chiral separation of tryptophan enantiomers, however, only in model samples. For this purpose they used a coupled separation channel configuration. In other works, contact, as well as contactless, conductivity detection was successfully used for preliminary scanning of sample profile in the ITP stage of the ITP-EKC method when analysing various drugs present in biological samples. Thus, amlodipine, pheniramine (plus its metabolites), dioxopromethazine, and dimethindene could be easily transferred into the EKC stage for the final enantioseparation without undesired matrix constituents present in urine samples [Mikuš et al., 2006a, 2008a, 2008b; Marák et al., 2007]. The methods were applied in enantioselective metabolic [Mikuš et al., 2006a, 2008b; Marák et al., 2007] and

pharmacokinetic [Mikuš et al., 2008a] studies of the drugs, see e.g., Figure 4.27.

Figure 4.30. Chiral analysis of amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxyamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA), tramadol (TMD) and methadone (MTD) amphetamine enantiomers at 0.5 ppb in plasma after LLE with EK injection. Experimental conditions: FS capillary of 75 cm and 650mm (ID); 25 kV; EK injection at 10 kV 610 s; 50% capillary filled with HS--CD 0.15% in BGE 2% capillary filled with BGE post-plug; on-line CE– ESI/MS in SIM mode and extracted ion current (XIC). Reprinted from ref. [Schappler et al., 2006].

*Mass spectrometry.* Rudaz et al. [Rudaz et al., 2005; Schappler et al., 2006] used the EKC-MS with countercurrent migration of the negatively charged highly sulfated--CD (ammonium formate electrolyte) and the partial filling technique to achieve the enantioseparation of amphetamine derivatives. The use of this strategy with a low concentration of the chiral selector allowed the rapid stereoselective separation of seven amphetamine derivatives in spiked plasma samples in analysis times of less than 6 min and concentration LODs ranging from ~7x10-7-3x10-6 M [Rudaz et al., 2005]. An enhancing of the detection sensitivity was accomplished with an electrokinetic injection accompanied by the introduction of an appropriate buffer plug length between the zone containing the chiral selector and the analyte injection (to overcome drawbacks linked with electrokinetic injection in the presence of a charged chiral selector) [Schappler et al., 2006], see Figure 4.30. When such complex matrices are analysed, MS signal suppression or enhancement effects are generally not reproducible and can compromise results using CE–ESI-MS. Therefore, the matrix effect was investigated with a commercially available coaxial sheath liquid ESI interface used as the post-capillary infusion system. It was found that with PP, signal suppression was observed while for LLE, no relevant matrix effect occurred in all experiments. Concentration LODs were improved by up to 4x10-9 M of each enantiomer present in plasma samples after LLE and detected by a single quadrupole MS as a detection device. overcome

Anionic CD, heptakis(2,6-diacethyl-6-sulfato)--CD, migrating from the detector to the inlet of the capillary, was used for the simultaneous chiral separation of drugs [Iio et al., 2005]. This selective EKC-ESI-MS method (formic acid electrolyte) was applied to the analysis of methamphetamine, 3,4-methylenedioxyamphetamine and amphetamine in clinical human urine samples pretreated by LLE. Sufficient sensitivity was obtained when concentration LODs ranged from ~6x10-8-10-7 M.

Figure 4.31. Typical electropherogram of salbutamol enantiomers at a concentration close to the LOQ obtained using SPE–NACE–ESI-MS (EIC at 240.4 *m/z*) using a BGE of 10mM ammonium formate and 15mM HDAS--CD in methanol acidified with 0.75M formic acid. Reprinted from ref. [Servais et al., 2006].

**5** 

**Conclusion** 

This book clearly illustrates that advanced chiral CE can show real advantages over chromatographic methods in the field of chiral analysis of trace biologically active compounds present in complex matrices. Advanced chiral CE, including (i) sophisticated chiral separation mechanisms and CE formats, (ii) on-line sample preparation and (iii) hyphenation with the conventional, as well as current, top detection systems, enables automatization and miniaturization of the analytical method with the possibility of solving

CE offers tremendous flexibility for enantiomeric separations of new drugs, metabolites, biomarkers, etc., because of a wide variety of available chiral additives easily applicable into CE systems with the cooperation of proper electrophoretic effects (e.g., countercurrent migration of chiral selector vs. analyte). In this field the CD derivatives {especially various (selectively) sulphated CDs} dominate, but progress can also be seen in micelle systems (e.g., micelle polymers). Among a huge amount of other chiral selectors, as presented in this book, their success will be determined not only by their enantiorecognition capability, but also

Advanced CE techniques based on effective on-line sample pretreatment (preconcentration, purification, derivatization) offer enhanced sensitivity and selectivity along with minimization of sample manipulation, simplifying the overall analytical procedure and allowing automatization and miniaturization of such systems. Although the analytical potential of the most effective and frequently used on-line sample preparation techniques based on electrophoretic principles (stacking techniques, CE-CE couplings), nonelectrophoretic principles (microextraction, microdialysis, flow injection, etc.) and their combinations is very significant, which can be demonstrated by their microscale implementations (MCE) and obtainable preconcentration factors 102-106 allowing performance of extremely fast and sensitive bioanalyses (pharmacokinetic studies, analysis of single cell contents, etc.), their utilization is still relatively rare and traditional off-line sample preparation procedures dominate. It is believed that future demands on effectivity

Advanced CE methods based on hyphenated detection techniques enabling ultrasensitive quantification and/or providing structural information tend to increase their applicability in (chiral) bioanalyses. Thus, a single molecules analysis (LIF) and structural characterization of unknown biodegradation products and markers (MS) can be accomplished. However, these detection modes are still accompanied, besides their high cost, by many limitations, such as the need for analyte derivatization (LIF), restrictions in applicable buffers and chiral selectors (MS) and the need for further development. Probably, these are possible reasons

many real tasks based on chiral actions in biological systems.

their compatibility with the detection system employed.

and microscale analysis will stimulate their use.

Micelle polymers are a promising class of chiral selectors for MS, which combine powerful chiral recognition in MEKC and high sensitivity of MS. A sensitive MEKC-MS method using poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate (triethylamine + ammonium acetate + acetonitrile buffer) was developed for enantioselective analysis of pseudoephedrine in human urine [Rizvi et al., 2007]. The concentration LOD obtained was ~2x10-6 M. LOD at pH 2.0 was 16 times lower than that obtained at pH 8.0, because the ionization efficiency of the MS, in the positive ionization mode, is higher when acidic solutions are used and the sulfated polymeric surfactant was effective also at low pH because of its permanent charge.

The on-line coupling of NACE and ESI-MS was used for the determination of low levels of the enantiomers of a basic chiral drug (salbutamol) in biological samples (human urine) pretreated by SPE [Servais et al., 2006]. The selected BGE (methanol acidified with formic acid containing heptakis(2,3-di-*O*-acetyl-6-*O-*sulfo)--CD offered good possibilities to be directly applied for MS coupling since the BGE contained volatile solvents and the nonvolatile CD migrated toward the capillary inlet (countercurrent technique) away from the MS detector. After optimization of several parameters, such as sheath liquid composition and flow-rate, nebulizing gas pressure, CE counterpressure and position of the CE capillary outlet, LOQs of 18 and 20 ng/mL were obtained for the salbutamol enantiomers. The RSD at a concentration of 30 ng/mL was below 7% for both enantiomers and *R*-values were 0.9988 and 0.9966. This paper therefore proposed an easy to use and relatively sensitive NACE– ESI-MS method to determine enantiomers of a basic chiral drug in biological fluids preceded by SPE as sample clean-up. Figure 4.31 presents a typical electropherogram of the salbutamol enantiomers at a concentration close to the LOQ using SPE–NACE–ESI-MS. It is noted that, compared with the LOQ of the NACE-UV method (i.e., 375 ng/mL for an injection time of 15 s), the use of an MS detector (with an injection time of 30 s) gives rise to a significant increase in sensitivity.

## **Conclusion**

This book clearly illustrates that advanced chiral CE can show real advantages over chromatographic methods in the field of chiral analysis of trace biologically active compounds present in complex matrices. Advanced chiral CE, including (i) sophisticated chiral separation mechanisms and CE formats, (ii) on-line sample preparation and (iii) hyphenation with the conventional, as well as current, top detection systems, enables automatization and miniaturization of the analytical method with the possibility of solving many real tasks based on chiral actions in biological systems.

146 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Micelle polymers are a promising class of chiral selectors for MS, which combine powerful chiral recognition in MEKC and high sensitivity of MS. A sensitive MEKC-MS method using poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate (triethylamine + ammonium acetate + acetonitrile buffer) was developed for enantioselective analysis of pseudoephedrine in human urine [Rizvi et al., 2007]. The concentration LOD obtained was ~2x10-6 M. LOD at pH 2.0 was 16 times lower than that obtained at pH 8.0, because the ionization efficiency of the MS, in the positive ionization mode, is higher when acidic solutions are used and the sulfated polymeric surfactant was effective also at low pH because of its permanent charge.

The on-line coupling of NACE and ESI-MS was used for the determination of low levels of the enantiomers of a basic chiral drug (salbutamol) in biological samples (human urine) pretreated by SPE [Servais et al., 2006]. The selected BGE (methanol acidified with formic acid containing heptakis(2,3-di-*O*-acetyl-6-*O-*sulfo)--CD offered good possibilities to be directly applied for MS coupling since the BGE contained volatile solvents and the nonvolatile CD migrated toward the capillary inlet (countercurrent technique) away from the MS detector. After optimization of several parameters, such as sheath liquid composition and flow-rate, nebulizing gas pressure, CE counterpressure and position of the CE capillary outlet, LOQs of 18 and 20 ng/mL were obtained for the salbutamol enantiomers. The RSD at a concentration of 30 ng/mL was below 7% for both enantiomers and *R*-values were 0.9988 and 0.9966. This paper therefore proposed an easy to use and relatively sensitive NACE– ESI-MS method to determine enantiomers of a basic chiral drug in biological fluids preceded by SPE as sample clean-up. Figure 4.31 presents a typical electropherogram of the salbutamol enantiomers at a concentration close to the LOQ using SPE–NACE–ESI-MS. It is noted that, compared with the LOQ of the NACE-UV method (i.e., 375 ng/mL for an injection time of 15 s), the use of an MS detector (with an injection time of 30 s) gives rise to a

significant increase in sensitivity.

CE offers tremendous flexibility for enantiomeric separations of new drugs, metabolites, biomarkers, etc., because of a wide variety of available chiral additives easily applicable into CE systems with the cooperation of proper electrophoretic effects (e.g., countercurrent migration of chiral selector vs. analyte). In this field the CD derivatives {especially various (selectively) sulphated CDs} dominate, but progress can also be seen in micelle systems (e.g., micelle polymers). Among a huge amount of other chiral selectors, as presented in this book, their success will be determined not only by their enantiorecognition capability, but also their compatibility with the detection system employed.

Advanced CE techniques based on effective on-line sample pretreatment (preconcentration, purification, derivatization) offer enhanced sensitivity and selectivity along with minimization of sample manipulation, simplifying the overall analytical procedure and allowing automatization and miniaturization of such systems. Although the analytical potential of the most effective and frequently used on-line sample preparation techniques based on electrophoretic principles (stacking techniques, CE-CE couplings), nonelectrophoretic principles (microextraction, microdialysis, flow injection, etc.) and their combinations is very significant, which can be demonstrated by their microscale implementations (MCE) and obtainable preconcentration factors 102-106 allowing performance of extremely fast and sensitive bioanalyses (pharmacokinetic studies, analysis of single cell contents, etc.), their utilization is still relatively rare and traditional off-line sample preparation procedures dominate. It is believed that future demands on effectivity and microscale analysis will stimulate their use.

Advanced CE methods based on hyphenated detection techniques enabling ultrasensitive quantification and/or providing structural information tend to increase their applicability in (chiral) bioanalyses. Thus, a single molecules analysis (LIF) and structural characterization of unknown biodegradation products and markers (MS) can be accomplished. However, these detection modes are still accompanied, besides their high cost, by many limitations, such as the need for analyte derivatization (LIF), restrictions in applicable buffers and chiral selectors (MS) and the need for further development. Probably, these are possible reasons

**6** 

**Acknowledgements** 

This work was supported by the publication fund of the Faculty of Pharmacy Comenius University, the grant from the Slovak Grant Agency for Science under the project VEGA No. 1/0664/12 and the grant from the Cultural and Educational Grant Agency of the Ministry of Education under the project KEGA No. 031UK-4/2012. Much research work by the author, included in this book, was supported by the Excellence Center of Pharmacy (ECP) at the Faculty of Pharmacy Comenius University in Bratislava. The author also would like to give his great thanks to the reviewers, excellent chemists and analysts: Prof. Dr. Ladislav Novotný, Assoc. Prof. Dr. Jozef Polonský, Prof. Dr. Emil Havránek and Prof. Dr. Milan Remko, for reviewing this scientific monograph, their valuable statements and suggestions. Last but not least, the author thanks the deputy head of Book Publishing Department and publishing process manager of InTech, Mr. Davor Vidic, for providing the editorial and

stylistic revision, and his excellent assistance during the whole publication process.

for the significant domination of universal detection modes like UV-VIS absorbance photometric detection or, to a lesser extent, electrochemical detection, accompanied essentially by a sample preparation in (enantioselective) bioanalytical applications. Although the sensitivity of these later mentioned detection techniques is not sufficient for the majority of bioanalytical problems, they still have great potential to create brand new advanced methods, e.g., utilizing some of the on-line sample preparation techniques presented herein.

It is concluded that the potential, effectivity and performance parameters of CE (and also MCE and CEC) for the enantioselective drug bioanalyses can improve with an appropriate combination of progressive enantioseparation, sample preparation and detection approaches, as it was comprehensively demonstrated in this book. Indeed, new advanced combinations could lead to the development of other interesting fully automatized microscale analytical procedures suitable for reference, as well as routine, use in (chiral) biomedical research. It is apparent that their importance will continually increase with new advanced analytical tasks.

### **Acknowledgements**

This work was supported by the publication fund of the Faculty of Pharmacy Comenius University, the grant from the Slovak Grant Agency for Science under the project VEGA No. 1/0664/12 and the grant from the Cultural and Educational Grant Agency of the Ministry of Education under the project KEGA No. 031UK-4/2012. Much research work by the author, included in this book, was supported by the Excellence Center of Pharmacy (ECP) at the Faculty of Pharmacy Comenius University in Bratislava. The author also would like to give his great thanks to the reviewers, excellent chemists and analysts: Prof. Dr. Ladislav Novotný, Assoc. Prof. Dr. Jozef Polonský, Prof. Dr. Emil Havránek and Prof. Dr. Milan Remko, for reviewing this scientific monograph, their valuable statements and suggestions. Last but not least, the author thanks the deputy head of Book Publishing Department and publishing process manager of InTech, Mr. Davor Vidic, for providing the editorial and stylistic revision, and his excellent assistance during the whole publication process.

148 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

for the significant domination of universal detection modes like UV-VIS absorbance photometric detection or, to a lesser extent, electrochemical detection, accompanied essentially by a sample preparation in (enantioselective) bioanalytical applications. Although the sensitivity of these later mentioned detection techniques is not sufficient for the majority of bioanalytical problems, they still have great potential to create brand new advanced methods, e.g., utilizing some of the on-line sample preparation techniques

It is concluded that the potential, effectivity and performance parameters of CE (and also MCE and CEC) for the enantioselective drug bioanalyses can improve with an appropriate combination of progressive enantioseparation, sample preparation and detection approaches, as it was comprehensively demonstrated in this book. Indeed, new advanced combinations could lead to the development of other interesting fully automatized microscale analytical procedures suitable for reference, as well as routine, use in (chiral) biomedical research. It is apparent that their importance will continually increase with new

presented herein.

advanced analytical tasks.

**7** 

**References** 

[1] Adell, A. & Artigas, F. (1998). *In Vivo Neuromethods*; Boulton, A.A.; Baker, G.B.; Bateson

[2] Aebersold, R. & Morrison, H.D. (1990). Analysis of dilute peptide samples by capillary zone electrophoresis. *Journal of Chromatography A*, Vol. 516, No. 1, pp. 79-88. [3] Afshar, M. & Thormann, W. (2006). Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites

[4] Akbay, C.; Rizvi, S.A.A. & Shamsi, S.A. (2005). Simultaneous enantioseparation and tandem UV-MS detection of eight β-blockers in micellar electrokinetic chromatography using a chiral molecular micelle. *Analytical Chemistry,* Vol.77, No.6,

[5] Almeda, S.; Nozal, L.; Arce, L. & Valcárcel, M. (2007). Direct determination of chlorophenols present in liquid samples by using a supported liquid membrane coupled in-line with capillary electrophoresis equipment. *Analytica Chimica Acta*, Vol.

[6] Altria, K.D.; Mahuzier, P.-E. & Clark, B.J. (2003). Background and operating parameters in microemulsion electrokinetic chromatography. *Electrophoresis,* Vol.24, No.3, pp. 315-324. [7] Altria, K.; Marsh, A. & Sanger-van de Griend, C. (2006). Capillary electrophoresis for the analysis of small-molecule pharmaceuticals. *Electrophoresis*, Vol.27, No.12, pp. 2263-2282. [8] Amankwa, L.N.; Albin, M. & Kuhr, W.G. (1992). Fluorescence detection in capillary

[9] Andersen, S.; Halvorsen, T.G.; Pedersen-Bjergaard, S.; Rasmussen, K.E.; Tanum, L. & Refsum, H. (2003). Stereospecific determination of citalopram and desmethylcitalopram by capillary electrophoresis and liquid-phase microextraction.

[10] Armstrong, D.W.; Rundlett, K. & Reid III, G.L. (1994a). Use of a macrocyclic antibiotic, rifamycin B, and indirect detection for the resolution of racemic amino alcohols by CE.

[11] Armstrong, D.W.; Rundlett, K.L. & Chen, J.R. (1994b). Evaluation of the macrocyclic antibiotic vancomycin as a chiral selector for capillary electrophoresis. *Chirality,* Vol.6,

[12] Asami, T. & Imura, H. (2006). Absolute determination method for trace quantities of enantiomer of glufosinate by gamma-cyclodextrin modified capillary zone electrophoresis combined with solid-phase extraction and on-capillary concentration.

[13] Aturki, Z.; Scotti, V.; D'Orazio, G.; Rocco, A.; Raggi, M.A. & Fanali, S. (2007). Enantioselective separation of the novel antidepressant mirtazapine and its main

metabolites by CEC. *Electrophoresis,* Vol.28, No.15, pp. 2717-2725.

electrophoresis. *Trends in Analytical Chemistry*, Vol.11, No.3, pp. 114-120.

*Journal of Pharmaceutical and Biomedical Analysis,* Vol.33, No.2, pp. 263-273.

*Analytical Chemistry,* Vol.66, No.10, pp. 1690-1695.

*Analytical Science*, Vol. 22, No.12, pp. 1489-1493.

A.N. (Eds.) Chapter 1, Humana Press: Totowa, NJ.

pp. 1672-1683.

587, No. 1, pp. 97-103.

No.6, pp. 496-509.

in biological samples. *Electrophoresis.* Vol. 27, No.8, pp.1517-1525.

### **References**


[30] Blanco, M. & Valverde, I. (2003). Choice of chiral selector for enantioseparation by capillary electrophoresis. *TrAC - Trends in Analytical Chemistry,* Vol.22, No.7-8, pp. 428-

References 153

[32] Bodor, R.; Kaniansky, D. & Masár, M. (2001). Conductivity detection cell for capillary zone electrophoresis with a solution mediated contact of the separated constituents with the detection electrodes. *Journal of Chromatography A*, Vol. 916, No.1-2, pp. 31–40. [33] Bonato, P.S. (2003). Recent advances in the determination of enantiomeric drugs and their metabolites in biological fluids by capillary electrophoresis-mediated

[34] Bonneil, E. & Waldron, K.C. (2000). On-line system for peptide mapping by capillary electrophoresis at sub-micromolar concentrations. *Talanta*, Vol. 53, No., 3, pp. 678-699. [35] Bortocan, R. & Bonato, P.S. (2004). Enantioselective analysis of primaquine and its metabolite carboxyprimaquine by capillary electrophoresis. *Electrophoresis,* Vol.25,

[36] Bowser, M.T. & Kennedy, R.T. (2001). In vivo monitoring of amine neurotransmitters using microdialysis with on-line capillary electrophoresis. *Electrophoresis*, Vol.22,

[37] Breadmore, M.C. (2007). Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips.

[38] Breadmore, M.C.; Thabano, J.R.E.; Dawod, M.; Kazarian, A.A.; Quirino, J.P. & Guijt, R.M. (2009). Recent advances in enhancing the sensitivity of electrophoresis and in capillaries and microchips (2006-2008). *Electrophoresis*, Vol.30, No.1, pp. 230-248. [39] Britz-McKibbin, P. & Chen, D.D.Y. (2000). Selective focusing of catecholamines and weakly acidic compounds by capillary electrophoresis using a dynamic pH junction.

[40] Britz-McKibbin, P.; Otsuka, K. & Terabe, S. (2002). On-line focusing of flavin derivatives using dynamic pH junction-sweeping capillary electrophoresis with laserinduced fluorescence detection. *Analytical Chemistry,* Vol.74, No.15, pp. 3736-3743. [41] Britz-McKibbin, P. & Terabe, S. (2003). On-line preconcentration strategies for trace analysis of metabolites by capillary electrophoresis. *Journal of Chromatography A,*

[42] Britz-McKibbin, P.; Markuszewski, M.J.; Iyanagi, T.; Matsuda, K.; Nishioka, T. & Terabe, S. (2003). Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection.

(2003).

*A,*

*of* 

[43] Bushey, M.M. & Jorgenson, J.W. (1990). Automated instrumentation for comprehensive 2-dimensional high-performance liquid-chromatography capillary

[44] Cai, J. & Henion, J. (1995). Capillary electrophoresis-mass spectrometry. *Journal of* 

[45] Camilleri, P. (1991). Biomedical applications of chiral liquid chromatography.

zone electrophoresis. *Analytical Chemistry*, Vol.62, No. 10, pp. 978-984.

[31] Boček, P. (1987). *Analytical capillary isotachophoresis,* Academia, Praha, pp.133.

microanalysis. *Electrophoresis*, Vol.24, No.22-23, pp. 4078-4094.

439.

No.16, pp. 2848-2853.

No.17, pp. 3668-3676.

Vol.1000, No.1-2, pp. 917-934.

*Electrophoresis,* Vol.28, No.1-2, pp. 254-281.

*Analytical Chemistry*, Vol.72, No. 6, pp. 1242-1252.

*Analytical Biochemistry*, Vol.313, No.1, pp. 89-96.

Vol.313, No.1, &

*Chromatography A,* Vol.703, No.1-2, pp. 667-692

P.

*Biomedical Chromatography,* Vol.5, No.3, pp. 128-132.


[14] Avdalovic, N.; Pohl, C.A.; Rocklin, R.D. & Stillian, J.R. (1993). Determination of cations and anions by capillary electrophoresis combined with suppressed conductivity

[15] Awadallah, B; Smidt, P.C & Wahl, M.A. (2003). Quantitation of the enantiomers of ofloxacin by capillary electrophoresis in the parts per billion concentration range for in vitro drug absorption studies. *Journal of Chromatography A,* Vol.988, No.1, pp. 135-143. [16] Baidoo, E.E.K.; Benke, P.I.; Neusüss, C.; Pelzing, M.; Kruppa, G.;Leary, J.A. & Keasling, J.D. (2008). Capillary electrophoresis-fourier transforms ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stackingtransient isotachophoretic method. *Analytical Chemistry,* Vol.80, No.9, pp. 3112-3122. [17] Bajad, S. & Shulaev, V. (2007). Highly-parallel metabolomics approaches using LC-MS2 for pharmaceutical and environmental analysis. *TrAC - Trends in Analytical* 

[18] Baldacci, A. & Thormann, W. (2006). Analysis of lorazepam and its 3O-glucuronide in human urine by capillary electrophoresis: Evidence for the formation of two distinct diastereoisomeric glucuronides. *Journal of Separation Science*, Vol.29, No.1, pp. 153-163. [19] Balss, K.M.; Vreeland, W.N.; Phinney, K.W. & Ross, D. (2004). Simultaneous concentration and separation of enantiomers with chiral temperature gradient

[20] Barroso, M.B. & de Jong, A.P. (1998). A new design for large, dilute sample loading in capillary electrophoresis. *Journal of Capillary electrophoresis,* Vol.5, No. 1-2, pp. 1-7 [21] Bartos, D. & Gorog, S. (2009). Recent Advances in the Impurity Profiling of Drugs.

[22] Beck, W.; Van Hoek, R. & Engelhardt, H. (1993). Application of a diode-array detector

[23] Beckers, J.L. & Boček, P. (2000). Sample stacking in capillary zone electrophoresis: Principles, advantages and limitations. *Electrophoresis*, Vol.21, No. 14, pp. 2747-2767. [24] Behn, F.; Michels, S.; Laer, S. & Blaschke, G. (2001). Separation of carvedilol enantiomers in very small volumes of human plasma by capillary electrophoresis with laser-induced fluorescence. *Journal of Chromatography B,* Vol.755, No.1-2, pp. 111-117. [25] Belder, D.; Kohler, F.; Ludwig, M.; Tolba, K. & Piehl, N. (2006). Coating of powderblasted channels for high-performance microchip electrophoresis. *Electrophoresis,*

[26] Belder, D. (2006). *Chiral Analysis*, Busch, K.W.; M.A. Busch (Eds.), Chapter 9: Chiral separations in microfluidic devices, pp. 277-295, Elsevier B.V., Oxford, UK. [27] Bertoncini, N.D. & Hennion, M.C. (2004). Immunoaffinity solid-phase extraction for pharmaceutical and biomedical trace-analysis—coupling with HPLC and CE perspectives. *Journal of Pharmaceutical and Biomedical Analysis,* Vol.34, No.4, pp.717–736. [28] Bjergaard, S.P. & Rasmussen, K.E. (2008). Liquid-phase microextraction with porous hollow fibers, a miniaturized and highly flexible format for liquid-liquid extraction.

[29] Bjørnsdottir, I.; Hansen, S.H. & Terabe, S. (1996). Chiral separation in non-aqueous media by capillary electrophoresis using the ion-pair principle. *Journal of* 

in capillary electrophoresis. *Electrophoresis,* Vol.14, No.5-6, pp. 540-546.

detection. *Analytical Chemistry,* Vol.65, No.10, pp. 1470-1475.

focusing. *Analytical Chemistry,* Vol.76, No.24, pp. 7243-7249.

*Current Pharmaceutical Analysis,* Vol.4, No.4, pp. 215-230.

*Journal of Chromatography A*, Vol. 1184, No.1-2, pp. 132-142.

*Chromatography A,* Vol.745, No.1-2, pp. 37-44.

*Chemistry,* Vol.26, No.6, pp. 625-636.

Vol.27, No.16, pp. 3277-3283


[61] Chen, Y.M., Chen, C.F. & Xi, F. (1998). Chiral Dendrimers With Axial Chirality.

References 155

[62] Chen, Y.L.; Jong, Y.J. & Wu, S.M. (2006). Capillary electrophoresis combining fieldamplified sample stacking and electroosmotic flow suppressant for analysis of sulindac and its two metabolites in plasma. *Journal of Chromatography A*, Vol.1119,

[63] Chen, Y.; Guo, Z.; Wang, X. & Qiu, C. (2008). Sample preparation. *Journal of* 

[64] Chen, J.; Du, Y.; Zhu, F. & Chen, B. (2010). Evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary

[68] Chiang, M.T.; Chang, S.Y. & Whang, C.W. (2001). Multiple voltage-gradient gel

[69] Chien, R.L. & Burgi, D.S. (1992). On-column sample concentration using field

[70] Cho, S.I.; Shim, J.; Kim, M.S.; Kim, Y.K. & Chung, D.S. (2004). On-line sample cleanup and chiral separation of gemifloxacin in a urinary solution using chiral crown ether as a chiral selector in microchip electrophoresis. *Journal of Chromatography A*, Vol.1055,

[71] Choi, K.; Kim, J.; Jang, Y.O. & Chung, D.S. (2009). Direct chiral analysis of primary amine drugs in human urine by single drop microextraction in-line coupled to CE.

[72] Chou, Y.W.; Huang, W.S.; Ko, C.C. & Chen, S.H. (2008). Enantioseparation of cetirizine by sulfated-β-cyclodextrin-mediated capillary electrophoresis. *Journal of Separation* 

[73] Christodoulou, E.A. (2010). An Overview of HPLC Methods for the Enantiomer Separation of Active Pharmaceutical Ingredients in Bulk and Drug Formulations,

for

[74] Cole, R.B.; Varghese, J. & McCormick, R.M. (1994). Capillary Electrophoresis Electrospray Mass Spectrometry Using a Novel Hydrophilic Derivarized Capillary for Protein Analysis. *42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago,* 

[75] Craig, D.B.; Arriaga, E.; Wong, J.C.Y.; Lu, H. & Dovichi, N.J. (1998). Life and Death of a

[76] Craig, S.L.; Zhong, M. & Brauman, J.I. (1998). Nonstatistical reactivity in a vibrationally excited S(N)2 intermediate [1]. *Journal of the American Chemical Society,*

Single Enzyme Molecule. *Analytical Chemistry,* Vol.70, No.1, pp. 39A-43A.

electrophoresis. *Journal of Chromatography A,* Vol.1217, No.45, pp. 7158-7163. [65] Cheng, Y.F. & Dovichi, N.J. (1988). Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence. *Science,* Vol.242, No.4878, pp. 562-564. [66] Cheng, J. & Kang, J. (2006). An on-column derivatization method for the determination of the enantiomeric excess of chiral primary amines via indirect enantioseparation by micellar electrokinetic chromatography. *Electrophoresis,* Vol.27, No.4, pp. 865-871. [67] Cherkaoui, S.; Rudaz, S.; Varesio, E. & Veuthey, J.L. (2001). On-line capillary electrophoresis-electrospray mass spectrometry for the stereoselective analysis of

drugs and metabolites. *Electrophoresis,* Vol.22, No.15, pp. 3308-3315.

amplification in CZE. *Analytical Chemistry*, Vol.64, No. 8, pp. 489A-496A

electrophoresis system. *Electrophoresis,* Vol.22, No.1, pp. 29-32.

*Chirality,* Vol.10, pp. 661–666.

*Chromatography A,* Vol.1184, No.1-2, pp. 191-219.

No.1-2, pp. 176-182.

No. 1-2, pp. 241–245.

*IL, 29 May-3 June 1994.* 

*Electrophoresis,* Vol.30, No.16, pp. 2905-2911.

*Current Organic Chemistry*, Vol.14, No.19, pp. 2337-2347.

*Science,* Vol.31, No.5, pp. 845-852.

Vol.120, No.46, pp. 12125-12126.


[46] Cardoso, C.D.; Jabor, V.A.P. & Bonato, P.S. (2006). Capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of

[47] Carlavilla, D.; Moreno-Arribas, M.V.; Fanali, S. & Cifuentes, A. (2006). Chiral MEKC-LIF of amino acids in foods: Analysis of vinegars. *Electrophoresis,* Vol.27, No.13, pp.

[48] Carlsson, Y.; Hedeland, M.; Bondesson, U. & Pettersson, C. (2001). Non-aqueous capillary electrophoretic separation of enantiomeric amines with (-)-2,3:4,6-di-Oisopropylidene-2-keto-L-gulonic acid as chiral counter ion. *Journal of Chromatography* 

[49] Caslavska, J. & Thormann, W. (2011). Stereoselective determination of drugs and metabolites in body fluids, tissues and microsomal preparations by capillary electrophoresis (2000-2010). *Journal of Chromatography A*, Vol. 1218, No.4 (Sp. Iss. SI),

[50] Castro-Puyana, M.; Salgado, A.; Hazen, R.M.; Crego, A.L. & Alegre, M.L.M. (2008). The first contribution of capillary electrophoresis to the study of abiotic origins of homochirality: Investigation of the enantioselective adsorption of 3-carboxy adipic

[51] Chang, H.T. & Yeung, E.S. (1995). Determination of catecholamines in single adrenal medullary cells by capillary electrophoresis and laser-induced native fluorescence.

[52] Chankvetadze, B.; Endresz, G. & Blaschke, G. (1994). About some aspects of the use of charged cyclodextrins for capillary electrophoresis enantioseparation. *Electrophoresis,*

[53] Chankvetadze, B. (1997). *Capillary Electrophoresis in Chiral Analysis.* Wiley, Chichester. [54] Chankvetadze, B.; Burjanadze, N.; Bergenthal, D. & Blaschke, G. (1999). Potential of flow-counterbalanced capillary electrophoresis for analytical and micropreparative

[55] Chankvetadze, B. & Blaschke, G. (2001). Enantioseparations in capillary electromigration techniques: Recent developments and future trends. *Journal of* 

[56] Chankvetadze, B.; Burjanadze, N. & Blaschke, G. (2001). Enantioseparation of the anticoagulant drug phenprocoumon in capillary electrophoresis with UV and laserinduced fluorescence detection and application of the method to urine samples.

[57] Chankvetadze, B. (2007). Enantioseparations by using capillary electrophoretic techniques. The story of 20 and a few more years. *Journal of Chromatography A,*

[58] Chankvetadze, B. (2008). *Cyclodextrins and their Complexes.* Dodziuk, H. (Ed.), pp. 119–

[59] Chaurasia, C.S. (1999). In vivo microdialysis sampling: theory and applications.

[60] Chen, D. & Dovichi, N.J. (1996). Single-molecule detection in capillary electrophoresis: molecular shot noise as a fundamental limit to chemical analysis. *Analytical Chemistry,*

liver homogenates. *Electrophoresis,* Vol.27, No.5-6, pp. 1248-1254.

acid on minerals. *Electrophoresis*, Vol.29, No.7, pp. 1548-1555.

*Analytical Chemistry,* Vol.67, No.6, pp. 1079-1083.

separations. *Electrophoresis,* Vol.20, No.13, pp. 2680-2685.

*Chromatography A,* Vol.906, No.1-2, pp. 309-363.

*Electrophoresis,* Vol.22, No.15, pp. 3281-3285.

146, Wiley-VCH, Weinheim, Germany.

*Biomedical Chromatography*, Vol.13, No. 5, pp. 317-332.

2551-2557.

pp. 588-601.

*A,* Vol.922, No.1-2, pp. 303-311.

Vol.15, No.6, pp. 804-807.

Vol.1168, No.1-2, pp. 45-70.

Vol.68, No.4, pp. 690-696.


determination of peptides and proteins. *Journal of Chromatography,* Vol.554, No.1-2, pp.

[92] Dhopeshwarkar, R.; Crooks, R.M.; Hlushkou, D. & Tallarek, U. (2008). Transient Effects on Microchannel Electrokinetic Filtering with an Ion-Permselective Membrane.

References 157

[93] Diode-array detection in capillary electrophoresis using Agilent Extended Light Path

[94] Du, Y.; Taga, A.; Suzuki, S.; Liu, W. & Honda, S. (2002). Colominic acid: a novel chiral selector for capillary electrophoresis of basic drugs. *Journal of Chromatography A*,

[95] Edmonds, C.G.; Loo, J.A. & Ogorzalek Loo, R.R. (1991). Application of Electrospray Ionization Mass-Spectrometry and Tandem Mass-Spectrometry in Combination with Capillary Electrophoresis for Biochemical Investigations. *Biochemical Society* 

[96] El-Enany, N.; El-Sherbiny, D. & Belal, F. (2007). Spectrophotometric, spectrofluorometric and HPLC determination of desloratadine in dosage forms and human plasma. *Chemical and Pharmaceutical Bulletin,* Vol.55, No.12, pp. 1662-1670. [97] El-Hady, D.A. & El-Maali, N.A. (2008). Determination of catechin isomers in human plasma subsequent to green tea ingestion using chiral capillary electrophoresis with a

[98] Esteban, J.; Pellín, M.D.L.C.; Gimeno, C.; Barril, J.; Mora, E.; Giménez, J. & Vilanova, E. (2004). Detection of clinical interactions between methadone and anti-retroviral compounds using an enantio selective capillary electrophoresis for methadone

[99] Ewing, A.G.; Wallingford, R.A. & Olefirowicz, T.M. (1989). Capillary Electrophoresis.

[100] Ewing, A.G.; Mesaros, J.M. & Gavin, P.F. (1994). Electrochemical Detection in Microcolumn Separations. *Analytical Chemistry*, Vol.66, No., pp. 527A-537A. [101] Fan, G.R.; Hong, Z.Y.; Lin, M.; Yin, X.P. & Wu, Y.T. (2004). Study of stereoselective pharmacokinetics of anisodamine enantiomers in rabbits by capillary electrophoresis.

[102] Fanali, S.; Desiderio, C.; Schulte, G.; Heitmeier, S.; Strickmann, D.; Chankvetadze, B. & Blaschke, G. (1998a). Chiral capillary electrophoresis–electrospray mass spectrometry coupling using vancomycin as chiral selector. *Journal of Chromatography A*, Vol.800,

[103] Fanali, S.; Aturki, Z. & Desiderio, C. (1998b). New strategies for chiral analysis of drugs by capillary electrophoresis. *Forensic Science International,* Vol.92, No.2-3, pp.

[104] Fanali, S.; Desiderio, C.; Ölvecká, E.; Kaniansky, D.; Vojtek, M. & Ferancová, A. (2000). Separation of enantiomers by on-line capillary isotachophoresis-capillary zone electrophoresis. *Journal of High Resolution Chromatography*, Vol.23, No.9, pp. 531-538. [105] Fanali, S.; Catarcini, P.; Blaschke, G. & Chankvetadze, B. (2001). Enantioseparations by capillary electrochromatography. *Electrophoresis*, Vol.22, No. 15, pp. 3131-3151. [106] Fanali, S. (2002). *Century of Separation Science,* H.J. Issaq (Ed.), pp. 579-588, Marcel

329-338.

*Analytical Chemistry*, Vol.80, pp.1039–1048.

capillaries, Agilent Technologies, 2009.

*Transactions,* Vol.19, No.4, pp. 943-947.

high-sensitivity cell. *Talanta,* Vol. 76, No.1, pp. 138-145.

analysis. *Toxicology Letters*, Vol.151, No.1, pp. 243-249.

*Journal of Chromatography B*, Vol.809, No.2, pp. 265-271.

*Analytical Chemistry*, Vol.61, No.4, pp. 292A-303A.

Vol.962, No.1-2, pp. 221-231.

No.1, pp. 69–76.

Dekker, New York.

137-155.


[77] Cserhati, T. (2008). New applications of cyclodextrins in electrically driven chromatographic systems: a review. *Biomedical Chromatography,* Vol.22, No.6, pp. 563-571. [78] Cucinotta, V.; Contino, A.; Giuffrida, A.; Maccarrone, G. & Messina, M. (2010). Application of charged single isomer derivatives of cyclodextrins in capillary electrophoresis for chiral analysis. *Journal of Chromatography A*, Vol.1217, No.7, pp. 953-

[79] Culbertson, C.T. & Jorgenson, J.W. (1994). Flow counterbalanced capillary

[80] Culbertson, C.T. & Jorgenson, J.W. (1999). Lowering the UV absorbance detection limit and increasing the sensitivity of capillary electrophoresis using a dual linear photodiode array detector and signal averaging. *Journal of Microcolumn Separations,*

[81] Danger, G. & Ross, D. (2008). Development of a temperature gradient focusing method for in situ extraterrestrial biomarker analysis. *Electrophoresis* Vol.29, No.15, pp. 3107-

[82] Danková, M.; Kaniansky, D.; Fanali, S. & Iványi, F. (1999). Capillary zone electrophoresis separations of enantiomers present in complex ionic matrices with online isotachophoretic sample pretreatment. *Journal of Chromatography A*, Vol.838, No. 1-

[83] da Silva, J.A.F.; Guzman, N. & de Lago, C.L. (2002). Contactless conductivity detection for capillary electrophoresis: Hardware improvements and optimization of the inputsignal amplitude and frequency. *Journal of Chromatography A*, Vol.942, No.1-2, pp. 249-

No.15, Gaitani,

[84] da Silva, J.A.F. (2003). Detecção Eletroquímica em eletroforese capliar. *Quimica Nova*,

[85] de Gaitani, M.C.; Martinez, A.S. & Bonato, P.S. (2003). Study on thioridazine 5 sulfoxide epimerization and degradation by capillary electrophoresis. *Electrophoresis*,

[86] de Jong, J.; Lammertink, R.G.H. & Wessling, M. (2006). Membranes and microfluidics:

[87] de Oliveira, A.R.M.; Cardoso, C.D. & Bonato, P.S. *(*2007). Stereoselective determination of hydroxychloroquine and its metabolites in human urine by liquid-phase

[88] de Santana, F.J.M.; Lanchote, V.C. & Bonato, P.S. (2008). Capillary electrophoretic chiral determination of mirtazapine and its main metabolites in human urine after

[89] Denola, N.L.; Quiming, N.S.; Saito, Y. & Jinno, K. (2007). Simultaneous enantioseparation and sensitivity enhancement of basic drugs using large-volume

[90] Desiderio, C.; Polcaro, C.M.; Padiglioni, P. & Fanali, S. (1997). Enantiomeric separation of acidic herbicides by capillary electrophoresis using vancomycin as chiral selector.

[91] Deterding, L.J.; Parker, C.E.; Perkins, J.R.; Moseley, M.A.; Jorgenson, J.W. & Tomer, K.B. (1991). Nanoscale separations. Capillary liquid chromatography-mass spectrometry and capillary zone electrophoresis-mass spectrometry for the

microextraction and CE. *Electrophoresis*, Vol.28, No.7, pp. 1081-1091.

enzymatic hydrolysis. *Electrophoresis*, Vol.29, No.18, pp. 3924-3932.

sample stacking. *Electrophoresis*, Vol.28, No.19, pp. 3542-3552.

*Journal of Chromatography A*, Vol.781, No.1-2, pp. 503-513.

electrophoresis. *Analytical Chemistry,* Vol.66, No.7, pp. 955-962.

967.

3114.

258.

2, pp. 31-43

Vol.11, No.9, pp. 652-662.

Vol.26, No.1, pp. 56-64.

Vol.24, No.15, pp. 2723-2730.

a review. *Lab Chip*, Vol.6, No.9, pp. 1125-1139.

determination of peptides and proteins. *Journal of Chromatography,* Vol.554, No.1-2, pp. 329-338.


[125] Glowka, F.K. & Karzniewicz, M. *(*2004a). High performance capillary electrophoresis for determination of the enantiomers of 2-arylpropionic acid derivatives in human serum: Pharmacokinetic studies of ketoprofen enantiomers following administration of standard and sustained release tablets. *Journal of Pharmaceutical and Biomedical* 

References 159

[126] Glowka, F.K. & Karzniewicz, M. *(*2004b). Resolution of indobufen enantiomers by capillary zone electrophoresis Pharmacokinetic studies of human serum. *Journal of* 

[127] Glowka, F.K. & Karazniewicz, M. (2007). Enantioselective CE method for pharmacokinetic studies on ibuprofen and its chiral metabolites with reference to

[128] Goel, T.V.; Nikelly, J.G.; Simpson, R.C. & Matuszewski, B.K. *(*2004). Chiral separation of labetalol stereoisomers in human plasma by capillary electrophoresis. *Journal of* 

[129] Gong, X.Y. & Hauser, P.C. (2006). Enantiomeric separation of underivatized small amines in conventional and on-chip capillary electrophoresis with contactless

[130] Goodlett, D.R.; Loo, R.R.O.; Loo, J.A.; Wahl, J.H.; Udseth H.R. & Smith, R.D. (1994). A study of the thermal denaturation of ribonuclease S by electrospray ionization mass spectrometry. *Journal of American Society for Mass Spectrometry.,* Vol.5, No.7, pp. 614-622. [131] Gotti, R.; Calleri, E.; Massolini, G.; Furlanetto, S. & Cavrini, V. (2006). Penicillin G acylase as chiral selector in CE using a pullulan-coated capillary. *Electrophoresis*,

[132] Grant, I.H. & Steuer, W. (1990). Extended path length UV absorbance detector for capillary zone electrophoresis. *Journal of Microcolumn Separations*, Vol.2, No.2, pp. 74-79. [133] Grard, S.; Morin, P. & Ribet, J.P. (2002). Application of capillary electrophoresis with field-amplified sample injection for the detection of new adrenoreceptor antagonist enantiomers in plasma in the low ng/mL concentration range. *Electrophoresis*, Vol.23,

[134] Gübitz, G. & Schmid, M.G. (2000a). Recent progress in chiral separation principles in

[135] Gübitz, G. & Schmid, M.G. (2000b). Chiral separation by capillary

[136] Gübitz, G. & Schmid, M.G. (2004). Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography. *Electrophoresis*, Vol.25,

[137] Gübitz, G. & Schmid, M.G. (2006). Chiral separation principles in chromatographic and electromigration techniques. *Molecular Biotechnology*, Vol.32, No.2, pp. 159-179. [138] Gübitz, G. & Schmid, M.G. (2007). Advances in chiral separation using capillary

[139] Gübitz, G. & Schmid, M.G. (2008). Chiral separation by capillary electromigration

[140] Guihen, E.; Hogan, A.M. & Glennon, J.D. (2009). High-Speed Microchip Electrophoresis Method for the Separation of (R,S)-Naproxen. *Chirality*, Vol.21, No.2, pp. 292-298.

electromigration techniques. *Electrophoresis*, Vol.28, No.1-2, pp. 114-126.

techniques. *Journal of Chromatography A*, Vol.1204, No.2, pp. 140-156.

capillary electrophoresis. *Electrophoresis*, Vol.21, No., pp. 4112-4135.

electrochromatography. E*nantiomer*, Vol.5, No.1, pp. 5-11.

genetic polymorphism. *Electrophoresis*, Vol.28, No.15, pp. 2726-2737.

conductivity detection. *Electrophoresis,* Vol.27, No.21, pp. 4375-4382.

*Analysis*, Vol.35, No.4, pp. 807-816.

Vol.27, No.23, pp. 4746-4754.

No.15, pp. 2399-2407.

No.23-24, pp. 3981-3996.

*Chromatography A,* Vol.1032, No.1-2, pp. 219-225.

*Chromatography A*, Vol.1027, No.1-2, pp. 213-221.


[107] Fanali, S. (2009). Chiral separations by CE employing CDs. *Electrophoresis.* Vol.30,

[108] Fang, L.; Kang, J.; Yin, X.-B.; Yang, X. & Wang, E. (2006). CE coupling with endcolumn electrochemiluminescence detection for chiral separation of disopyramide.

[109] Fang, H.F.; Zeng, Z.R. & Liu, L. (2006a). Centrifuge microextraction coupled with online back-extraction field-amplified sample injection method for the determination of trace ephedrine derivatives in the urine and serum. *Analytical Chemistry*, Vol.78, No.

[110] Fang, H.F.; Liu, M.M. & Zeng, Z.R. (2006b). Solid-phase microextraction coupled with capillary electrophoresis to determine ephedrine derivatives in water and urine using a sol-gel derived butyl methacrylate/silicone fiber. *Talanta*, Vol.68, No. 3, pp. 979-986. [111] Fenyvesi, E. *(*1988). Cyclodextrin polymers in the pharmaceutical industry. *Journal of* 

[112] Fillet, M.; Hubert, P. & Crommen, J. (2000). Enantiomeric separations of drugs using mixtures of charged and neutral cyclodextrins. *Journal of Chromatography A*, Vol.875,

[113] Frost, N.W.; Jing, M. & Bowser, M.T. (2010). Capillary Electrophoresis. *Analytical* 

[114] Gale, D.C. & Smith, R.D. (1993). Small volume and low flow-rate electrospray ionization mass spectrometry of aqueous samples. *Rapid Communications in Mass* 

[115] Gaš, B.; Demjanenko, M. & Vacík, J. (1980). High frequency contactless conductivity detection in isotachophoresis. *Journal of Chromatography,* Vol.192, No. 2, pp. 253-257. [116] Gaspar, A.; Englmann, M.; Fekete, A.; Harir, M. & Schmitt-Kopplin, P. (2008). Trends

[117] Gassmann, E.; Kuo, J.E. & Zare, R.N. (1985). Electrokinetic separation of chiral

[118] Gatti, R. & Gioia, M.G. (2008). Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinone. *Biomedical* 

[119] Gebauer, P.; Čáslavská, J.; Thormann, W. & Boček, P. (1997). Prediction of zone patterns in capillary zone electrophoresis with conductivity detection. Concept of the zone conductivity diagram. *Journal of Chromatography A*, Vol.772, No.1-2, pp. 63-71. [120] Gebauer, P.; Malá, Z. & Boček, P. (2009). Recent progress in analytical capillary ITP.

[121] Gebauer, P.; Malá, Z. & Boček, P. (2011). Recent progress in analytical capillary

[122] Gerhardt, G.C.; Cassidy, R.M. & Baranski, A.S. (2000). Adsorption-based electrochemical detection of nonelectrochemically active analytes for capillary

[123] Giddings, J.C. (1969). Generation of Variance, "Theoretical Plates," Resolution, and Peak Capacity in Electrophoresis and Sedimentation. *Separation Science,* Vol.4, No.3, pp. 181-189. [124] Gilpin, R.K. & Gilpin, C.S. (2009). Pharmaceuticals and related drugs. *Analytical* 

*Inclusion Phenomena and Macrocyclic Chemistry*, Vol.6, pp. 537-545.

in CE-MS 2005-2006. *Electrophoresis*, Vol.29, No.1, pp. 66–79.

compounds. *Science*, Vol.230, No.4727, pp. 813-814.

isotachophoresis. *Electrophoresis*, Vol.32, No.1, pp. 83-89.

electrophoresis. *Analytical Chemistry*, Vol.72, No.5, pp. 908–915.

No.SUPPL. 1, p. S203-S210.

17, pp. 6043-6049.

No.1-2, pp. 123-134.

*Electrophoresis*, Vol.27, No.22, pp. 4516–4522.

*Chemistry,* Vol.82, No.12, pp. 4682-4698.

*Spectrometry,* Vol.7, No.11, pp. 1017-1021.

*Chromatography*, Vol.22, No.2, 207-213.

*Electrophoresis*, Vol.30, No.1, pp. 29–35.

*Chemistry,* Vol.81, No.12, pp. 4679-4694.


[156] Henion, J.; Mordehai, A. & Cai, J. (1994). Quantitative capillary electrophoresis-ion spray mass spectrometry on a benchtop ion trap for the determination of isoquinoline

References 161

[157] Hernández, L.S.; Crego, A.L.; Marina, M.L. & Ruiz, C.G. (2008). Sensitive chiral

[158] Hernández, L.S.; Ruiz, C.G.; Marina, M.L. & Crego, A.L. (2010). Recent approaches for enhancing sensitivity in enantioseparations by CE. *Electrophoresis*, Vol.31, No.1, pp. 28-

[159] Hödl, H.; Koidl, J.; Schmid, M.G. & Gübitz, G. (2006). Chiral resolution of tryptophan derivatives by CE using canine serum albumin and bovine serum albumin as chiral

[160] Hödl, H.; Krainer, A.; Holzmuller, K.; Koidl, J.; Schmid, M.G. & Gübitz, G. (2007). Chiral separation of sympathomimetics and β-blockers by ligand-exchange CE using Cu(II) complexes of L-tartaric acid and L-threonine as chiral selectors. *Electrophoresis*,

[161] Hogan, B.L.; Lunte, S.M.; Stobaugh, J.F. & Lunte, C.E. (1994). Online coupling of invivo microdialysis sampling with capillary electrophoresis. *Analytical Chemistry*,

[162] Holtzel, A. & Tallarek, U. (2007). Ionic conductance of nanopores in microscale analysis systems: Where microfluidics meets nanofluidics. *Journal of Separation Science*,

[163] Hopfgartner, G.; Wachs, T.; Bean, K. & Henion, J. (1993). High-flow ion spray liquid chromatography/mass spectrometry. *Analytical Chemistry,* Vol.65, No.4, pp. 439-446. [164] Horáková, J.; Petr, J.; Maier, V.; Tesařová, E.; Veis, L.; Armstrong, D.W.; Gaš, B. & Ševčík, J. (2007). On-line preconcentration of weak electrolytes by electrokinetic accumulation in CE: Experiment and simulation. *Electrophoresis*, Vol.28, No.10, pp.

[165] Horstkötter, C. & Blaschke, G. (2001). Stereoselective determination of ofloxacin and its metabolites in human urine by capillary electrophoresis using laser-induced

[167] Hou, J.G.; Rizvi, S.A.A.; Zheng, J. & Shamsi, S.A. (2006). Application of polymeric surfactants in micellar electrokinetic chromatography-electrospray ionization mass spectrometry of benzodiazepines and benzoxazocine chiral drugs. *Electrophoresis*,

[168] Hou, J.; Zheng, J. & Shamsi, S.A. (2007). Separation and determination of warfarin enantiomers in human plasma using a novel polymeric surfactant for micellar electrokinetic chromatography-mass spectrometry. *Journal of Chromatography A*,

[169] Hsi, T.S.; Lin, J.N.; Kuo, K.Y. & Chin, J. (1997). Simultaneous Determination of Carbohydrates and Amino Acids by Capillary Zone Electrophoresis with Constant Potential Amperometric Detection at a Copper Electrode. *Journal of the Chinese Chemical* 

fluorescence detection. *Journal of Chromatography B*, Vol.754, No.1, pp. 169-178. [166] Hou, J.G.; He, T.X.; Mao, X.F.; Du, X.Z.; Deng, H.L. & Gao, J.Z. (2003). Enantiomeric separation using erythromycin as a new capillary zone electrophoresis chiral selector.

alkaloids. *Analytical Chemistry,* Vol.66, No.13, pp. 2103-2109.

selectors. *Electrophoresis*, Vol.27, No.23, pp. 4755-4762.

Vol.28, No.15, pp. 2675-2682.

Vol.66, No.5, pp. 596-602.

Vol.30, No.10, pp. 1398-1419.

Vol.27, No.5-6, pp. 1263–1275.

Vol.1159, No.1-2, pp. 208-216.

*Society*, Vol. 44, No.2, pp. 101–106.

*Analytical Letters*, Vol.36, No.7, pp. 1437-1499.

1540-1547.

43.

analysis by CE: An update. *Electrophoresis*, Vol.29, No.1, pp. 237-251.


[141] Ha, P.T.T.; Van Schepdael, A.; Van Vaeck, L.; Augistijns, P. & Hoogmartens, J. (2004a). Chiral capillary electrophoretic method for quantification of apomorphine. *Journal of* 

[142] Ha, P.T.T.; Van Schepdael, A.; Roets, E. & Hoogmartens, J. (2004b). Investigating the potential of erythromycin and derivatives as chiral selector in capillary electrophoresis.

[143] Ha, P.T.T.; Hoogmartens, J. & Van Schepdael, A. (2006). Recent advances in pharmaceutical applications of chiral capillary electrophoresis. *Journal of Pharmaceutical* 

[144] Haber, C.; Roosli, S.; Tsuda, T.; Scheidegger, D.; Müller, S. & Simon, W. (1990). The potentiometric microelectrode as a detector in fast LC and CZE. *12th Int. Symp.* 

[145] Haber, C.; Silvestri, I.; Roosli, S. & Simon, W. (1991). Potentiometric Detector for

[146] Haber, C.; VanSaun, R.J. & Jones, W.R. (1998). Quantitative Analysis of Anions at ppb/ppt Levels with Capillary Electrophoresis and Conductivity Detection: Enhancement of System Linearity and Precision Using an Internal Standard. *Analytical* 

[147] Hadwiger, M.E.; Torchia, S.R.; Park, S.; Biggin, M.E. & Lunte, C.E. (1996). Optimization of the separation and detection of the enantiomers of isoproterenol in microdialysis samples by cyclodextrin-modified capillary electrophoresis using electrochemical detection. *Journal of Chromatography B*, Vol.681, No.2, pp. 241-249. [148] Haginaka, J. (2000). Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors. *Journal of Chromatography A,* Vol.875, No.1-2, pp. 235-254. [149] Hashim, N.H.; Shafie, S. & Khan, S.J. (2010). Enantiomeric fraction as an indicator of pharmaceutical biotransformation during wastewater treatment and in the

environment - a review. *Environmental Technology*, Vol.31, No.12, pp. 1349-1370. [150] Hashimoto, A.; Nishikawa, T.; Oka, T.; Takahashi, K. & Hayashi, T. (1992). Determination of free amino-acid enantiomers in rat-brain and serum by high-performance liquidchromatography after derivatization with n-tert-butyloxycarbonyl-L-cysteine and O-

[151] Hashimoto, A.; Oka, T. & Nishikawa, T. (1995). Anatomical distribution and postnatal changes in endogenous free D-aspartate and D-serine in rat-brain and periphery.

[152] Hedeland, Y.; Lehtinen, J. & Pettersson, C. (2007). Ketopinic acid and diisoproylideneketogulonic acid as chiral ion-pair selectors in capillary electrophoresis - Enantiomeric impurity analysis of S-timolol and 1R,2S-ephedrine. *Journal of* 

[153] Heiger, D.N.; Kaltenbach, P. & Sievert, H.J.P. (1994). Diode array detection in capillary

[154] Heiger, D. (2000). *High performance capillary electrophoresis: An introduction,* Agilent

[155] Hempel, G. (2000). Strategies to improve the sensitivity in capillary electrophoresis for the analysis of drugs in biological fluids. *Electrophoresis*, Vol.21, No.4, pp. 691-698.

phthaldialdehyde. *Journal of Chromatography A*, Vol.582, No.1-2, pp. 41-48.

*European Journal of Neuroscience,* Vol. 7, No.8, pp. 1657-1663.

electrophoresis. *Electrophoresis*, Vol.15, No.10, pp. 1234-1247.

Technologies, Publication No. 5968-9963E, pp. 135, Germany.

*Chromatography A*, Vol.1141, No.2, pp. 287-294.

*Journal of Pharmaceutical and Biomedical Analysis*, Vol.34, No.5, pp. 861-870.

Capillary Zone Electrophoresis. *Chimia*, Vol.45, No.4, pp. 117-121.

*Chromatography A,* Vol.1049, No.1-2, pp. 195-203.

*and Biomedical Analysis*, Vol.41, No.1, pp. 1-11.

*Capillary Chromatography, Kobe, Japan*.

*Chemistry,* Vol.70, No.11, pp. 2261–2267.


[186] Kahle, K.A. & Foley, J.P. (2007c). Two-chiral component microemulsion EKC - Chiral surfactant and chiral oil. Part 2: Diethyl tartrate. *Electrophoresis*, Vol.28, No.15, pp.

References 163

[187] Kahle, K.A. & Foley, J.P. (2007d). Influence of microemulsion chirality on chromatographic figures of merit in EKC: Results with novel three-chiral-component microemulsions and comparison with one- and two-chiral-component

[189] Kaniansky, D. (1981). Kapilárna izotachoforéza, kapilárna zónová elektroforéza a ich

[190] Kaniansky, D.; Koval', M. & Stankoviansky, S. (1983). Simple cell for conductimetric detection in capillary isotachophoresis. *Journal of Chromatography B,* Vol.267, No.C, pp.

[191] Kaniansky, D. & Marák, J. (1990). On-line coupling of capillary isotachophoresis with capillary zone electrophoresis. *Journal of Chromatography*, Vol.498, pp. 191-204. [192] Kaniansky, D.; Marák, J.; Madajová, V. & Šimuničová, E. (1993). Capillary zone electrophoresis of complex ionic mixtures with on-line isotachophoretic sample

[193] Kaniansky, D.; Iványi, F. & Onuska, F.I. (1994a). Online isotachophoretic sample pretreatment in ultratrace determination of paraquat and diquat in water by capillary

[194] Kaniansky, D.; Zelenský, I.; Hybenová, A. & Onuska, F.I. (1994b). Determination of chloride, nitrate, sulfate, nitrite, fluoride, and phosphate by on line coupled capillary isotachophoresis-capillary zone electrophoresis with conductivity detection. *Analytical* 

[195] Kaniansky, D.; Havaši, P.; Iványi, F. & Marák, J. (1995). Galvanic decoupling of a postcolumn amperometric detector in capillary electrophoresis. *Journal of* 

[196] Kaniansky, D.; Marák, J.; Masár, M.; Iványi, F.; Madajová, V.; Šimuničová, E. & Zelenská, V. (1997). Capillary zone electrophoresis in a hydrodynamically closed separation system with enhanced sample loadability. J*ournal of Chromatography A*,

[197] Kaniansky, D.; Zelenská, V.; Masár, M.; Iványi, F. & Gazdíková, Š. (1999). Contactless conductivity detection in capillary zone electrophoresis. *Journal of Chromatography A*,

[198] Kang, J.W.; Wistuba, D. & Schurig, V. (2002). Recent progress in enantiomeric separation by capillary electrochromatography. *Electrophoresis*, Vol.23, No.22-23, pp. 4005-4021. [199] Karbaum, A. & Jira, T. (1999). Nonaqueous capillary electrophoresis: Application possibilities and suitability of various solvents for the separation of basic analytes.

[200] Kataoka, H. (2003). New trends in sample preparation for clinical and pharmaceutical

[201] Kavran-Belin, G.; Rudaz, S. & Veuthey, J.L. (2005). Enantioseparation of baclofen with highly sulfated β-cyclodextrin by capillary electrophoresis with laser-induced fluorescence detection. *Journal of Separation Science,* Vol.28, No.16, pp. 2187–2192.

analysis. *Trends of Analytical Chemistry*, Vol.22, No.4, pp. 232-244.

microemulsions. *Electrophoresis*, Vol.28, No.11, pp. 3024–3040. [188] Kaltenbach, P.; Ross, G. & Heiger, D.N. (1997). *HPCE*, Anaheim, CA.

on-line spojenie. Ph.D. Thesis, Comenius University, Bratislava.

pretreatment. *Journal of Chromatography*, Vol.638, No.2, pp. 137-146.

*Chemistry*, Vol.66, No.23, pp. 4258-4264.

Vol.772, No.1-2, pp. 103-114.

Vol.844, No.1-2, pp. 349–359.

*Chromatography A,* Vol.709, No.1, pp. 69-79.

*Electrophoresis*, Vol.20, No.17, pp. 3396-3401.

zone electrophoresis. *Analytical Chemistry*, Vol.66, No.11, pp. 1817-1824.

2644–2657.

67-73.


[170] Hu, T.; Zuo, H.; Riley, C.M.; Stobaugh, J.F. & Lunte, S.M. (1995). Determination of αdifluoromethylornithine in blood by microdialysis sampling and capillary electrophoresis

[171] Huang, Z. & Ivory, C.F. (1999). Digitally controlled electrophoretic focusing. *Analytical* 

[172] Huang, Y.S.; Liu, J.T.; Lin, L.C. & Lin, C.H. (2003). Chiral separation of 3,4 methylenedioxymeth-amphetamine and related compounds in clandestine tablets and urine samples by capillary electrophoresis/fluorescence spectroscopy. *Electrophoresis*,

[173] Huang, L.; Lin, J.M.; Yu, L.; Xu, L. & Chen, G. (2008). Field-amplified on-line sample stacking for simultaneous enantioseparation and determination of some beta-blockers

[175] Huck, C.W.; Bakry, R.; Huber, L.A. & Bonn, G.K. (2006). Progress in capillary electrophoresis coupled to matrix-assisted laser desorption/ionization time of flight

[176] Huo, Y. & Kok, W.T. (2008). Recent applications in CEC. *Electrophoresis*, Vol.29, No.1,

[177] Ibrahim, W.A.W.; Hermawan, D.; Sanagi, M.M. (2007). On-line preconcentration and chiral separation of propiconazole by cyclodextrin-modified micellar electrokinetic

[180] Ivory, C.F. (2000). A brief review of alternative electrofocusing techniques. *Separation* 

[181] Jiménez, J.R. & de Castro, M.D.L. (2008). Lab-on-valve for the automatic determination of the total content and individual profiles of linear alkylbenzene sulfonates in water

[182] Juvancz, Z.; Kendrovics, R.B.; Ivanyi, R. & Szente, L. (2008). The role of cyclodextrins in chiral capillary electrophoresis. *Electrophoresis*, Vol.29, No.8, pp. 1701-1712. [183] Kahle, K.A. & Foley, J.P. (2006). Chiral microemulsion electrokinetic chromatography with two chiral components: Improved separations *via* synergies between a chiral

surfactant and a chiral cosurfactant, *Electrophoresis*, Vol.27, No.4, pp. 896–904. [184] Kahle, K.A. & Foley, J.P. (2007a). Review of aqueous chiral electrokinetic chromatography (EKC) with an emphasis on chiral microemulsion EKC.

[185] Kahle, K.A. & Foley, J.P. (2007b). Two-chiral-component microemulsion electrokinetic chromatography-chiral surfactant and chiral oil: Part 1. Dibutyl tartrate. *Electrophoresis*,

chromatography. *Journal of Chromatography A*, Vol.1170, No.1-2, pp. 107-113. [178] Iio, R.; Chinaka, S.; Takayama, N. & Hayakawa, K. (2005). Simultaneous chiral analysis of methamphetamine and related compounds by capillary electrophoresis/mass spectrometry using anionic cyclodextrin. *Analytical Science*, Vol.21, No.1, pp. 15–19. [179] Ingelse, B.; Everaerts, F.M.; Desiderio, C. & Fanali, S. (1995). Enantiomeric separation by capillary electrophoresis using a soluble neutral β-cyclodextrin polymer. *Journal of* 

using capillary electrophoresis. *Electrophoresis*, Vol.29, No.17, pp. 3588-3594. [174] Huang, L.; Lin, Q.; Chen, Y.T. &Chen, G. (2011). Transient isotachophoresis with fieldamplified sample injection for on-line preconcentration and enantioseparation of some

beta-agonists. *Analytical Methods,* Vol.3, No.2, pp. 294-298.

*Chromatography A*, Vol.709, No.1, pp. 89-98.

*Science and Technology,* Vol.35, No.11, pp. 1777.

*Electrophoresis*, Vol.28, No.15, pp. 2503-2526.

Vol.28, No.11, pp., 1723–1734.

samples. *Electrophoresis,* Vol. 29, No. 3, pp. 590-596.

mass spectrometry. *Electrophoresis*, Vol.27, No.11, pp. 2063–2074.

with UV detection. *Journal of Chromatography A*, Vol.716, No.1-2, pp. 381-388.

*Chemistry,* Vol.71, No.8, pp. 1628-1632.

Vol.24, No.6, pp. 1097-1104.

pp. 80-93.


[216] Koegler, W.S. & Ivory, C.F. (1996b). Focusing proteins in an electric field gradient.

References 165

[217] Kohr, J. & Engelhardt, H. (1993). Characterization of quartz capillaries for capillary

[218] Kok, S.J.; Velthorst, N.H.; Gooijer, C. & Brinkman, U.A.T. (1998). Analyte identification in capillary electrophoretic separation techniques. *Electrophoresis*, Vol.19, No.16-17, pp.

[219] Konieczna, L.; Plenis, A.; Oledzka, I.; Kowalski, P. & Baczek, T. (2010). Rapid RP-LC Method with Fluorescence Detection for Analysis of Fexofenadine in Human Plasma.

[220] Kraly, J.; Fazal, M.A.; Schoenherr, R.M.; Bonn, R.; Harwood, M.M.; Turner, E.; Jones, M. & Dovichi, N.J. (2006). Bioanalytical applications of capillary electrophoresis.

[221] Kriger, M.S.; Ramsey, R.S. & Cook, K.D. (1994). Evaluation of a Sheathless Capillary Electrophoresis/Electrospray Ionization Interface on an Ion Trap Mass Spectrometer. *42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, 29 May-3 June* 

[222] Kubačák, P.; Mikuš, P.; Valášková, I. & Havránek, E. (2006a). Separation of dimetinden enantiomers in drugs by means of capillary isotachophoresis. *Česká a Slovenská* 

[223] Kubačák, P.; Mikuš, P.; Valášková, I. & Havránek, E. (2006b). Chiral separation of feniramine in medicaments with the use of capillary isotachophoresis. *Farmaceutický* 

[224] Kubačák, P.; Mikuš, P.; Valášková, I. & Havránek, E. (2007). Chiral separation of alkylamine antihistamines in pharmaceuticals by capillary Isotachophoresis with charged cyclodextrin. *Drug Development and Industrial Pharmacy*, Vol.33, No.11, pp.

[225] Kuhn, R. (1999). Enantiomeric separation by capillary electrophoresis using a crown

[226] Lacorte, S. & Fernandez-Albaz, A. R. (2006). Time of flight mass spectrometry applied to the liquid chromatographic analysis of pesticides in water and food. *Mass* 

[227] Lada, M.W. & Kennedy, R.T. (1995). Quantitative *in vivo* measurements using microdialysis on-line with capillary zone electrophoresis. *Journal of Neuroscience* 

[228] Lada, M.W. & Kennedy, R.T. (1996). Quantitative in vivo monitoring of primary amines in rat caudate nucleus using microdialysis coupled by a flow-gated interface to capillary electrophoresis with laser-induced fluorescence detection. *Analytical* 

[229] Lada, M.W. & Kennedy, R.T. (1997). In vivo monitoring of glutathione and cysteine in rat caudate nucleus using microdiolysis on-line with capillary zone electrophoresislaser induced fluorescence detection. *Journal of Neuroscience Methods,* Vol. 72, No.2, pp.

[230] Lada, M.W.; Vickroy, T.W. & Kennedy, R.T. (1997). High temporal resolution monitoring of glutamate and aspartate in vivo using microdialysis on-line with

ether as chiral selector. *Electrophoresis*, Vol.20, No.13, pp. 2605-2613.

*Spectrometry Reviews*, Vol.25, No.6, pp. 866–880.

electrophoresis. *Journal of Chromatography,* Vol. 652, No.2, pp. 309-316.

*Journal of Chromatography A,* Vol.726, No.1-2, pp. 229-236.

*Chromatographia*, Vol.71, No.11-12, pp. 1081-1086.

*Analytical Chemistry*, Vol.78, No.12, pp. 4097-4110.

*Farmacie*, Vol.55, No.1, pp. 32-35.

*Obzor*, Vol.75, No.2-3, pp. 48-51.

*Methods,* Vol.63, No.1-2, pp.147-.

*Chemistry,* Vol.68, No.17, pp. 2790-2797.

2753-2776.

*1994.* 

1199-1204.

153-159.


[202] Kelly, J.F; Thibault, P.; Locke, S. & Ramaley, L.R. (1994). Analysis of the Oligoaccharides isolated from Glycoproteins by Capillary Elecrrophoresis-Electrospray Mass Spectrometry with Discontinuous Buffer Systems. *42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, 29 May-3 June 1994.*  [203] Kim, J.B. & Terabe, S. (2003). On-line sample preconcentration techniques in micellar electrokinetic chromatography. *Journal of Pharmaceutical and Biomedical Analysis,* Vol.30,

[204] Kim, J.B.; Britz-McKibbin, P.; Hirokawa, T. & Terabe, S. (2003). Mechanistic study on analyte focusing by dynamic pH junction in capillary electrophoresis using computer

[205] Kim, M.S.; Cho, S.I.; Lee, K.N. & Kim, Y.K. (2005). Fabrication of microchip electrophoresis devices and effects of channel surface properties on separation

[206] Kirby, D.; Greve, K.F.; Foret, F.; Vouros, P.; Karger B.L. & Nashabeh, W. (1994). Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry Utilizing Background Electrolytes Containing Surfactants. *42nd ASMS Conference on Mass* 

[207] Kirschner, D.L.; Jaramillo, M. & Green, T.K. (2007). Enantioseparation and stacking of cyanobenz[f]isoindole-amino acids by reverse polarity capillary electrophoresis and

[208] Kitae, T.; Nakayama, T. & Kano, K. (1998). Chiral recognition of α-amino acids by charged cyclodextrins through cooperative effects of Coulomb interaction and inclusion. *Journal of Chemical Society. Perkin Transactions.,* Vol.2, No.2, pp. 207-212. [209] Kitagawa, F.; Tsuneka, T.; Akimoto, Y.; Sueyoshi, K.; Uchiyama, K.; Hattori, A. & Otsuka, K. (2006). Toward million-fold sensitivity enhancement by sweeping in capillary electrophoresis combined with thermal lens microscopic detection using an

[210] Kitagawa, F. & Otsuka, K. (2011). Recent progress in capillary electrophoretic analysis of amino acid enantiomers. *Journal of Chromatography B*, Vol.879, No.29, pp. 3078– 3095. [211] Knudsen, C.B. & Beattie, J.H. (1997). On-line solid-phase extraction capillary electrophoresis for enhanced detection sensitivity and selectivity: application to the analysis of metallothionein isoforms in sheep fetal liver. *Journal of Chromatography A,*

[212] Kodama, S.; Yamamoto, A.; Matsunaga, A. & Hayakawa, K. (2003). Direct chiral resolution of tartaric acid by ion-pair capillary electrophoresis using an aqueous background electrolyte with (1R,2R)-(-)-1,2-diaminocyclohexane as a chiral counterion.

[213] Kodama, S.; Yamamoto, A.; Iio, R.; Aizawa, S.I.; Nakagomi, K., Hayakawa, K. (2005). Chiral ligand exchange micellar electrokinetic chromatography using borate anion as a

[214] Kodama, S.; Aizawa, S.; Taga, A.; Yamashita, T. & Yamamoto, A. (2006). Chiral resolution of monosaccharides as 1-phenyl-3-methyl-5-pyrazolone derivatives by ligand-exchange CE using borate anion as a central ion of the chiral selector.

[215] Koegler, W.S. & Ivory, C.F. (1996a). Field gradient focusing: A novel method for

protein separation. *Biotechnology Progress,* Vol.12, No.6, pp. 822-836.

sulfated beta-cyclodextrin. *Analytical Chemistry*, Vol.79, No.2, pp. 736-743.

interface chip. *Journal of Chromatography A*, Vol.1106, No.1-2., pp. 36-42.

simulation. *Analytical Chemistry,* Vol.75, No.16, pp. 3986-3993.

efficiency. *Sensors and Actuators B*, Vol.107, No.2, pp. 818–824.

*Spectrometry and Allied Topics, Chicago, IL, 29 May-3 June 1994.* 

No.6, pp. 1625-1643.

Vol.792, No.1-2, pp. 463-473.

*Electrophoresis*, Vol.24, No.15, pp. 2711-2715.

*Electrophoresis*, Vol.27, No.23, pp. 4730-4734.

central ion. *Electrophoresis*, Vol.26, No.20, pp. 3884–3889.

	- [225] Kuhn, R. (1999). Enantiomeric separation by capillary electrophoresis using a crown ether as chiral selector. *Electrophoresis*, Vol.20, No.13, pp. 2605-2613.
	- [226] Lacorte, S. & Fernandez-Albaz, A. R. (2006). Time of flight mass spectrometry applied to the liquid chromatographic analysis of pesticides in water and food. *Mass Spectrometry Reviews*, Vol.25, No.6, pp. 866–880.
	- [227] Lada, M.W. & Kennedy, R.T. (1995). Quantitative *in vivo* measurements using microdialysis on-line with capillary zone electrophoresis. *Journal of Neuroscience Methods,* Vol.63, No.1-2, pp.147-.
	- [228] Lada, M.W. & Kennedy, R.T. (1996). Quantitative in vivo monitoring of primary amines in rat caudate nucleus using microdialysis coupled by a flow-gated interface to capillary electrophoresis with laser-induced fluorescence detection. *Analytical Chemistry,* Vol.68, No.17, pp. 2790-2797.
	- [229] Lada, M.W. & Kennedy, R.T. (1997). In vivo monitoring of glutathione and cysteine in rat caudate nucleus using microdiolysis on-line with capillary zone electrophoresislaser induced fluorescence detection. *Journal of Neuroscience Methods,* Vol. 72, No.2, pp. 153-159.
	- [230] Lada, M.W.; Vickroy, T.W. & Kennedy, R.T. (1997). High temporal resolution monitoring of glutamate and aspartate in vivo using microdialysis on-line with

[246] Long, Z.C., Liu, D.Y., Ye, N.N., Qin, J.H. & Lin, B.C. (2006). Integration of nanoporous membranes for sample filtration/preconcentration in microchip electrophoresis.

References 167

[247] Lord, H. & Pawliszyn, J. (2000). Evolution of solid-phase microextraction technology.

[248] Lü, W.J.; Chen, Y.L.; Zhu, J.H. & Chen, X.G. (2009). The combination of flow injection with electrophoresis using capillaries and chips. *Electrophoresis*, Vol.30, No.1, pp.83-91. [249] Lu, W.Z. & Cassidy, R.M. (1993). Pulsed amperometric detection of carbohydrates separated by capillary electrophoresis. *Analytical Chemistry*, Vol.65, No.20, pp. 2878-2881. [250] Lu, W. Z.; Poon, G. K.; Carmichael, P. L &, Cole, R. B. (1996). Analysis of tamoxifen and its metabolites by on-line capillary electrophoresis electrospray ionization mass spectrometry employing nonaqueous media containing surfactants. *Analytical* 

[251] Lu, W. & Cole, R. B. (1998). Determination of chiral pharmaceutical compounds, terbutaline, ketamine and propranolol, by on-line capillary electrophoresis-electrospray ionization mass spectrometry. *Journal of Chromatography B,* Vol.714, No.1, pp. 69–75. [252] Lu, H.A. & Chen, G.N. (2011). Recent advances of enantioseparations in capillary electrophoresis and capillary electrochromatography. *Analytical Methods*, Vol.3, No.3,

[253] Lurie, I.S. (1997). Separation selectivity in chiral and achiral capillary electrophoresis with mixed cyclodextrins. *Journal of Chromatography A,* Vol.792, No.1-2, pp. 297-307. [254] Maier, N. M.; Franco, P. & Lindner, W. (2001). Separation of enantiomers: Needs, challenges, perspectives. *Journal of Chromatography A,* Vol.906, No.1-2, pp. 3–33. [255] Mainka, A. & Bachmann, K. (1997). UV detection of derivatized carbonyl compounds in rain samples in capillary electrophoresis using sample stacking and a Z-shaped flow

[256] Malá, Z.; Křivánková, L.; Gebauer, P. & Boček, P. (2007). Contemporary sample stacking in CE: A sophisticated tool based on simple principles. *Electrophoresis*, Vol.28,

[257] Malá, Z.; Šlampová, A.; Gebauer, P. & Boček, P. (2009). Contemporary sample stacking

[258] Malá, Z.; Gebauer, P. & Boček, P. (2011). Contemporary sample stacking in analytical

[259] Mandrioli, R.; Pucci, V.; Sabbioni, C.; Bartoletti, C.; Fanali, S. & Raggi, M.A. (2004). Enantioselective determination of the novel antidepressant mirtazapine and its active demethylated metabolite in human plasma by means of capillary electrophoresis.

[260] Marák, J.; Mikuš, P.; Maráková, K.; Kaniansky, D.; Valášková, I. & Havránek, E. (2007). Enantioselective analysis of pheniramine in urine by charged CD-mediated CZE provided with a fiber-based DAD and an on-line sample pretreatment by capillary

[261] Marák, J.; Mikuš, P.; Maráková, K.; Kaniansky, D.; Valášková, I. & Havránek, E. (2008). Potentialities of ITP-CZE method with diode array detection for enantiometric purity control of dexbrompheniramine in pharmaceuticals. *Journal of Pharmaceutical and* 

cell. *Journal of Chromatography A,* Vol.767, No.1-2, pp. 241-247.

in CE. *Electrophoresis*, Vol.30, No.1, pp. 215–229.

ITP. *Electrophoresis*, Vol.28, No.15, pp. 2738-2747.

*Biomedical Analysis*, Vol.46, No.5, pp. 870-876.

electrophoresis*. Electrophoresis*, Vol.32, No.1, pp. 116-126.

*Journal of Chromatography A,* Vol.1051*,* No.1-2, pp. 253-260.

*Electrophoresis*, Vol.*27*, No.24, pp. 4927–4934.

*Chemistry*, Vol.68, No.4, pp. 668–674.

pp. 488-508.

No.1-2, pp. 243-253

*Journal of Chromatography A*, Vol.885, No.1-2, pp. 153–193.

capillary electrophoresis with laser-induced fluorescence detection. *Analytical Chemistry,* Vol.69, No.22, pp. 4560-4565.


[231] Lada, M.W.; Vickroy, T.W. & Kennedy, R.T. (1998). Evidence for neuronal origin and metabotropic receptor-mediated regulation of extracellular glutamate and aspartate in rat striatum in vivo following electrical stimulation of the prefrontal cortex. *Journal of* 

[232] Lämmerhofer, M.; Svec, F.; Frechet, J.M.J. & Lindner, W. (2000). Separation of enantiomers by capillary electrochromatography. *Trac-Trends in Analytical Chemistry*,

[234] Lapainis, T.; Scanlan, C.; Rubakhin, S.S. & Sweedler, J.V. (2007). A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons. *Analytical and Bioanalytical Chemistry,* Vol.387,

[235] Lee, J.; Lee, H.K.; Rasmussen, K.E. & Bjergaard, S.P. (2008). Environmental and bioanalytical applications of hollow fiber membrane liquid-phase microextraction: A

[236] Li, S.F.Y. & Kricka, L.J. (2006). Clinical Analysis by Microchip Capillary

[237] Li, O.L.; Liu, C. & Chen, Z.G. (2008). Application of capillary electrophoresis and microfluidics in drug screening*. Chinese Journal of New Drugs,* Vol.17, No.22, pp. 1910-

[238] Li, H.; Wang, P.H.; Li, C.; Wang, H. & Zhang, H.S. (2008). Steroselective determination of trihexyphenidyl using carboxylmethyl-beta-cyclodextrin by capillary electrophoresis

[239] Li, X.; Song, B.; Yuan, B.; Sun, J. & You, T. (2009). Enantioseparation of Dioxopromethazine Hydrochloride in Urine with Liquid-Liquid Extraction by CE-ECL

[240] Li, Y.; Song, C.; Zhang, L.; Zhang, W. & Fu, H. (2010). Fabrication and evaluation of chiral monolithic column modified by beta-cyclodextrin derivatives. *Talanta*, Vol.80,

[241] Lin, C.C.; Li, Y.T. & Chen, S.H. (2003). Recent progress in pharmacokinetic applications

[242] Lin, C.H. & Kaneta, T. (2004). On-line sample concentration techniques in capillary electrophoresis: Velocity gradient techniques and sample concentration techniques for

[243] Liu, H.C.; Yu, Y.; Wang, N.; Deng, M.; Liu, J.F. & Xue, H.Y. (2004). Pharmacokinetics of the Enantiomers of trans-Tramadol and its Active Metabolite, trans-O-Demethyltramadol, in Healthy Male and Female Chinese Volunteers. *Chirality*, Vol.16,

[244] Liu, Z. & Pawliszyn, J. (2006). Online coupling of solid-phase microextraction and capillary electrophoresis. *Journal of Chromatographic Science*, Vol.44, No.6, pp. 366–374. [245] Lloyd, D.K. & Wätzig, H. (1995). Sodium dodecyl-sulfate solution is an effective between-run rinse for capillary electrophoresis of samples in biological matrices.

with field-amplified sample stacking. *Microchem. J*., Vol.89, No.1, pp. 34-41.

of capillary electrophoresis. *Electrophoresis,* Vol.24, No.22-23, pp. 4106-4115.

[233] Landers, J.P. (1997). *Handbook of Capillary Electrophoresis,* CRC Press, New York.

*Chemistry,* Vol.69, No.22, pp. 4560-4565.

*Neurochemistry,* Vol.70, No.2, pp.617-625.

review. *Anal. Chim. Acta*, Vol.624, No.2, pp. 253-268.

Electrophoresis. *Clinical Chemistry*, Vol.52, No.1,pp. 37–45.

Detection. *Chromatographia*, Vol.70, No.7-8, pp. 1291-1293.

biomolecules. *Electrophoresis*, Vol.25, No.23-24, pp. 4058-4073.

*Journal of Chromatography B*, Vol.663, No.2, pp. 400-405.

Vol.19, No.11, pp. 676-698.

No.1, pp. 97-105.

No.3, pp. 1378-1384.

No.2, pp. 112-118.

1914.

capillary electrophoresis with laser-induced fluorescence detection. *Analytical* 


[277] Mikuš, P.; Valášková, I. & Havránek, E. (2005b). Enantioselective analysis of cetirizine in pharmaceuticals by cyclodextrin-mediated capillary electrophoresis. *Journal of* 

References 169

[278] Mikuš, P.; Kubačák, P.; Valášková, I. & Havránek, E. (2006a). Analysis of enantiomers in biological matrices by charged cyclodextrin-mediated capillary zone electrophoresis in column-coupling arrangement with capillary isotachophoresis. *Talanta*, Vol.70,

[279] Mikuš, P.; Kubačák, P.; Valášková, I. & Havránek, E. (2006b). Comparison of capillary zone electrophoresis and isotachophoresis determination of dimethindene enantiomers in pharmaceuticals using charged carboxyethyl-beta-cyclodextrin as a chiral selector. *Methods and Findings in Experimenatal and Clinical Pharmacology*, Vol.28,

[280] Mikuš, P. & Kaniansky, D. (2007). Capillary zone electrophoresis resolutions of 2,4 dinitrophenyl labeled amino acids enantiomers by N-methylated amino-β-

[281] Mikuš, P.; Maráková, K.; Marák, J.; Nemec, I.; Valášková, I. & Havránek, E. (2008a). Direct quantitative determination of amlodipine enantiomers in urine samples for pharmacokinetic study using on-line coupled isotachophoresis-capillary zone electrophoresis separation method with diode array detection. *Journal of* 

amlodipine

[282] Mikuš, P.; Maráková, K.; Marák, J.; Kaniansky, D.; Valášková, I. & Havránek, E. (2008b). Possibilities of column-coupling electrophoresis provided with a fiber-based diode array detection in enantioselective analysis of drugs in pharmaceutical and

[283] Mikuš, P. & Maráková, K. (2009). Advanced capillary electrophoresis for chiral analysis of drugs, metabolites and biomarkers in biological samples. *Electrophoresis*,

[284] Mikuš, P. & Maráková, K. (2010). Chiral capillary electrophoresis with on-line sample

[285] Mikuš, P. (2010). Chiral Capillary electrophoresis with on-line sample pretreatment and spectral detection in pharmaceutical and biomedical analysis. *Acta Facultatis* 

[286] Mikuš, P. & Maráková, K (2011). Column Coupling Electrophoresis in Biomedical Analysis, R. Fazel-Rezai, (Ed.), in: *Biomedical Engineering - From Theory to Applications*,

[287] Minard, R.D.; Chin-Fatt, D.; Carry, P.J. & Ewing, A.G. (1988). *36th ASMS Conference on* 

[288] Mohanty, A. & Dey, J. (2006). Enantioselectivity of vesicle-forming chiral surfactants in capillary electrophoresis. Role of the surfactant headgroup structure. *Journal of* 

[289] Mokhtari, B., Pourabdollah, K. & Dalali, N. (2011). Molecule and ion recognition of nano-baskets of calixarenes since 2005. *Journal of Coordination Chemistry*, Vol.64, No.5,

[290] Monton, M.R.N. & Soga, T. (2007). Metabolome analysis by capillary electrophoresismass spectrometry. *Journal of Chromatography A,* Vol.1168, No.1-2, pp. 237–246.

clinical samples. *Journal of Chromatography A*, Vol.1179, No.1, pp. 9-16.

preparation. *Current Pharmaceutical Analysis*, Vol.6, No.2, pp. 76-100.

*Mass Spectrometry and Allied Topics, San Francisco, CA, 5-10 June 1988*.

*Pharmaceuticae Universitatis Comenianae*, Vol.57, No.1, pp. 9-17.

pp. 81-130, InTech, ISBN 978-953-307-637-0, Rijeka.

*Chromatography A,* Vol.1128, No.1-2, pp. 259–266.

*Separation Science,* Vol.28, No.12, pp. 1278-1284.

cyclodextrins. *Analytical Letters,* Vol.40, No.2, pp. 335-347.

*Chromatography B*, Vol.875, No.1, pp. 266-272.

Vol.30, No.16, pp. 2773-2802.

pp. 743-794.

No.4, pp. 840-846.

No.9, pp. 595-599.


[262] Mardones, C.; Ríos, A.; Valcárcel, M. & Cicciarelli, R. (1999). Enantiomeric separation of D- and L-carnitine by integrating on-line derivatization with capillary zone

[263] Marsh, A.; Clark, B.; Broderick, M.; Power, J.; Donegan, S. & Altria, K. (2004). Recent advances in microemulsion electrokinetic chromatography. *Electrophoresis*, Vol.25,

[264] Martínez-Gómez, M.A.; Sagrado, S.; Villanueva-Camañas, R.M. & Medina-Hernández, M.J. (2007). Enantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector.

[265] Martins, L.F.; Yegles, M. & Wennig, R. (2008). Simultaneous enantioselective quantification of methadone and of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in oral fluid using capillary electrophoresis. *Journal of Chromatography B*,

[266] Masár, M.; Žúborová, M.; Bielčíková, J.; Kaniansky, D.; Jöhnck, M. & Stanislawski, B. (2001). Conductivity detection and quantitation of isotachophoretic analytes on a planar chip with on-line coupled separation channels. *Journal of Chromatography A,*

[267] McEvoy, E.; Marsh, A.; Altria, K.; Donegan, S. & Power, J. (2007). Recent advances in the development and application of microemulsion EKC. *Electrophoresis*, Vol.28, No.1-

[268] Mechref, Y. & El Rassi, Z. (1997). Capillary electrophoresis of herbicides .4. Evaluation of octylmaltopyranoside chiral surfactant in the enantiomeric separation of fluorescently labeled phenoxy acid herbicides and their laser-induced fluorescence

[269] Miao, H.; Rubakhin, S. S. & Sweedler, J. V. (2005). Subcellular analysis of D-Aspartate.

[270] Miao, H.; Rubakhin, S. S. & Sweedler, J. V. (2006). Confirmation of peak assignments in capillary electrophoresis using immunoprecipitation: Application to D-aspartate measurements in neurons. *Journal of Chromatography A,* Vol.1106, No.1-2, pp. 56–60. [271] Mikkers, F.E.P.; Everaerts, F.M. & Verheggen, T.P.E.M. (1979). Concentration distributions in free zone electrophoresis. *Journal of Chromatography,* Vol.169, No.C, pp. 1-10. [272] Mikuš, P.; Kaniansky, D.; Šebesta, R. & Sališová, M. (1999). Analytical characterizations of purities of alkyl- and arylamino derivatives of β-cyclodextrin by capillary zone electrophoresis with conductivity detection. *Enantiomer*, Vol.4, No.3-4, pp. 279-287. [273] Mikuš, P. & Kaniansky, D. (2000). Derivatization Reactions in Capillary Electrophoresis of Amino Acids [Derivatizačné reakcie v kapilárnej elektroforéze

[274] Mikuš, P.; Kaniansky, D. & Fanali, S. (2001). Separation of multicomponent mixtures of 2,4-dinitrophenyl labelled amino acids and their enantiomers by capillary zone

[275] Mikuš, P.; Kaniansky, D.; Šebesta, R. & Sališová, M. (2002). Cyclodextrins and Their Complexes - Structure and Interactions. *Chemické Listy*, Vol.96, No.8, pp.696-697. [276] Mikuš, P.; Valášková, I. & Havránek, E. (2005a). Enantioselective determination of pheniramine in pharmaceuticals by capillary electrophoresis with charged cyclodextrin. *Journal of Pharmaceutical and Biomedical Analysis,* Vol.38, No.3, pp. 442-448.

electrophoresis. *Journal of Chromatography A*, Vol.849, No.2, pp. 609-6166.

No.23-24, pp. 3970-3980.

Vol.862, No.1-2, pp. 79-85.

Vol.916, No.1-2, pp. 101–111.

2, pp. 193-207.

*Anal. Chim. Acta*, Vol.592, No.2, pp. 202-209.

detection. *Electrophoresis*, Vol.18, No.2, pp. 220-226.

*Analytical Chemistry*, Vol.77, No.22, pp. 7190–7194.

aminokyselín]. *Chemické Listy*, Vol.94, No.6, pp. 347-354.

electrophoresis. *Electrophoresis*, Vol.22, No.3, pp. 470-477.


[306] Nozal, L.; Arce, L.; Simonet, B.M.; Rios, A. & Valcárcel, M. (2007). In-line liquid-phase microextraction for selective enrichment and direct electrophoretic analysis of acidic

References 171

[307] Nzeadibe, K. & Vigh, G. (2007). Synthesis of mono-6-deoxy-6-N,N,N′,N′,N′ pentamethylethylenediammonio-cyclomaltoheptaose, a single-isomer, monosubstituted, permanently dicationic β-CD and its use for enantiomer separations

[308] O'Brien, K.B.; Esguerra, M.; Klug, C.T.; Miller, R.F. & Bowser, M.T. (2003). A highthroughput on-line microdialysis-capillary assay for D-serine. *Electrophoresis*, Vol.24,

[309] Ohnesorge, J.; Neusüß X, Ch. & Wätzig, H. (2005). Quantitation in capillary electrophoresis-mass spectrometry. *Electrophoresis*, Vol.26, No.21, pp. 3973–3987. [310] Ölvecká, E.; Masár, M.; Kaniansky, D.; Jöhnck, M. & Stanislawski, B. (2001). Isotachophoresis separations of enantiomers on a planar chip with coupled separation

[311] O'Shea, T.J.; Lunte, S.M. & LaCourse, W.R. (1993). Detection of carbohydrates by capillary electrophoresis with pulsed amperometric detection. *Analytical Chemistry*,

[312] Ossicini, L. & Fanali, S. (1997). Enantiomeric separations by electrophoretic

[313] Oswald, T.M. & Ward, T.J. (1999). Enantioseparations with the macrocyclic antibiotic ristocetin a using A countercurrent process in CE. *Chirality*, Vol.11, No.8, pp. 663-668. [314] Otsuka, K.; Matsumura, M.; Kim, J.B. & Terabe, S. (2003). On-line preconcentration and enantio selective separation of triadimenol by electrokinetic chromatography using cyclodextrins as chiral selectors. *Journal of Pharmaceutical and Biomedical Analysis*,

[315] Ouyang, G. & Pawliszyn, J. (2006). SPME in environmental analysis. *Analytical and* 

[316] Palcut, M. & Rabara, L. (2009). Host-guest Interactions of Coumarin 6 with betacyclodextrin in Polar Solvents. *Acta Chimica Slovenica*, Vol.56, No.4, pp. 845-851. [317] Pálmarsdóttir, S. & Edholm, L.H. (1995). Enhancement of selectivity and concentration sensitivity in capillary zone electrophoresis by online coupling with column liquidchromatography and utilizing a double stacking procedure allowing for microliter

[318] Pálmarsdóttir, S.; Mathiasson, L.; Jönsson, J.A. & Edholm, L.H. (1996). Micro-CLC as an interface between SLM extraction and CZE for enhancement of sensitivity and selectivity in bioanalysis of drugs. *Journal of Capillary Electrophoresis*, Vol.3, No.5, pp.

[319] Pálmarsdóttir, S.; Mathiasson, L.; Jönsson, J.A. & Edholm, L.H. (1997). Determination of a basic drug, bambuterol, in human plasma by capillary electrophoresis using double stacking for large volume injection and supported liquid membranes for

sample pretreatment. *Journal of Chromatography B*, Vol.688, No.1, pp. 127-134. [320] Palmer, J.; Munro, N.J. & Landers, J.P. (1999). A universal concept for stacking neutral analytes in micellar capillary electrophoresis. *Analytical Chemistry,* Vol.71, No.9, pp.

drugs. *Electrophoresis*, Vol.28, No.18, pp. 3284-3289.

by CE. *Electrophoresis*, Vol.28, No.15, pp. 2589-2605.

channels. *Electrophoresis*, Vol.22, No.15, pp. 3347-3353.

techniques. *Chromatographia*, Vol.45, pp. 428-432.

*Bioanalytical Chemistry*, Vol.386, No.4, pp.1059–1073.

injections. *Journal of Chromatography A*, Vol.693, No.1, pp. 131-143.

No.7-8, pp. 1227-1235.

Vol.65, No.7, pp. 948-951.

Vol.30, No.6, pp. 1861-1867.

255-260.

1679-1687.


[291] Moseley, M.A.; Deterding, L.J.; Tomer, K.B. & Jorgenson, J.W. (1989a). Capillary-zone electrophoresis/fast-atom bombardment mass spectrometry: design of an on-line coaxial continuous-flow interface. *Rapid Communications in Mass Spectrometry,* Vol.3,

[292] Moseley, M.A.; Deterding, L.J.; Tomer, K.B. & Jorgenson, J.W. (1989b). Coupling of capillary zone electrophoresis and capillary liquid chromatography with coaxial continuous-flow fast atom bombardment tandem sectro mass spectrometry. *Journal of* 

[293] Moseley, M.A. (1994). Preconcenrration Capillary Zone Electrophoresis Coupled with Electrospray Ionization Mass Spectrometry. *42nd ASMS Conference on Mass* 

[294] Muck, W.M. & Henion, J.D. (1989). Determination of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid by microbore high-performance liquid chromatography and capillary zone electrophoresis coupled to tandem mass

[295] Müller, D. & Blaschke, G. (2000). Enantioselective assay of chloroquine and its main metabolite deethyl chloroquine in human plasma by capillary electrophoresis. *Journal* 

[296] Natishan, T.K. (2005). Recent progress in the analysis of pharmaceuticals by capillary electrophoresis. *Journal of Liquid Chromatography and Related Technologies*, Vol.28, No.7-

[297] Nie, S.; Dadoo, R. & Zare, N. (1993). Ultrasensitive fluorescence detection of polycyclic aromatic hydrocarbons in capillary electrophoresis. *Analytical Chemistry*, Vol.65, No.24,

[298] Nilsson, J.; Spégel, P. & Nilsson, S. (2004) Molecularly imprinted polymer formats for capillary electrochromatography. *Journal of Chromatography B*, Vol.804, No.1, pp. 3-12 [299] Nishi, H.; Fukuyama, T. & Terabe, S. (1991). Chiral separation by cyclodextrinmodified micellar electrokinetic chromatography. *Journal of Chromatography*, Vol.553,

[300] Nishi, H.; Izumoto, S.; Nakamura, K.; Nakai, H. & Sato, T. (1996a). Dextran and dextrin as chiral selectors in capillary zone electrophoresis. *Chromatographia*, Vol.42,

[301] Nishi, H.S.; Nakamura, K.; Nakai, H. & Sato, T. (1996b). Enantiomer separation by capillary electrophoresis using DEAE-dextran and aminoglycosidic antibiotics.

[302] Nishi, H. (1997). Enantioselectivity in chiral capillary electrophoresis with polysaccharides. *Journal of Chromatography A*, Vol.792, No.1-2, pp. 327-347. [303] Nojavan, S. & Fakhari, A.R. (2010). Electro membrane extraction combined with capillary electrophoresis for the determination of amlodipine enantiomers in biological

[304] Nojavan, S. & Fakhari, A.R. (2011). Chiral separation and quantitation of cetirizine and hydroxyzine by maltodextrin-mediated CE in human plasma: Effect of zwitterionic property of cetirizine on enantioseparation. *Electrophoresis*, Vol.32, No.6-7, pp. 764-771. [305] Novotny, M.; Soini, H. & Stefansson, M. (1994). Chiral separation through capillary electromigration methods. *Analytical Chemistry,* Vol.66, No.11, pp. 646A-655A.

samples*. Journal of Separation Science,* Vol.33, No.20, pp. 3231-3238.

No.3, pp. 87-93.

8, pp. 1115-1160.

pp. 3571-3575.

No.1-2, pp. 503-516.

No.11-12, pp. 617-630.

*Chromatographia*, Vol.43, No.7-8, pp. 426-430.

*Chromatography,* Vol.480, No.C, pp. 197-209.

*Spectrometry and Allied Topics, Chicago, IL, 29 May-3 June 1994.* 

spectrometry. *Journal of Chromatography,* Vol.495, pp. 41-59.

*of Chromatographic Science*, Vol.38, No.10, pp. 435-440.


[337] Prost, F.; Čáslavská, J. & Thormann, W. (2002). Chiral analysis of albendazole sulfoxide enantiomers in human plasma and saliva using capillary electrophoresis with oncolumn absorption and fluorescence detection. *Journal of Separation Science*, Vol.25,

on-

References 173

[338] Ptolemy, A.S.; Le Bilhan, M. & Britz-McKibbin, P. (2005). On-line sample preconcentration with chemical derivatization of bacterial biomarkers by capillary electrophoresis: A dual strategy for integrating sample pretreatment with chemical

[339] Ptolemy, A.S.; Tran, L. & Britz-McKibbin, P. (2006). Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization. *Analytical Biochemistry*, Vol.354, No.2, pp. 192-204. [340] Ptolemy, A.S. & Britz-McKibbin, P. (2006). Sample preconcentration with chemical derivatization in capillary electrophoresis - Capillary as preconcentrator, microreactor and chiral selector for high throughput metabolite screening. *Journal of Chromatography* 

[341] Puig, P.; Borrull, F.; Calull, M. & Aguilar, C. (2007a). Recent advances in coupling solid-phase extraction and capillary electrophoresis (SPE-CE). *Trac-Trends in Analytical* 

[342] Puig, P.; Tempels, F.W.A.; Borrull, F.; Calull, M.; Aguilar, C.; Somsen, G.W & de Jong, G.J. (2007b). On-line coupling of solid-phase extraction and capillary electrophoresis for the determination of cefoperazone and ceftiofur in plasma. *Journal of* 

[343] Puig, P.; Borrull, F.; Calull, M. & Aguilar, C. (2008). Sorbent preconcentration procedures coupled to capillary electrophoresis for environmental and biological

[344] Quan, Z.; Song, Y.; Feng, Y.; LeBlanc, M.H. & Liu, Y.-M. (2005). Detection of D-serine in neural samples by saccharide enhanced chiral capillary electrophoresis. *Analytica* 

[345] Quirino, J.P. & Terabe, S. (1998). Exceeding 5000-fold concentration of dilute analytes in micellar electrokinetic chromatography. *Science*, Vol.282, No.5388, pp. 465-468. [346] Quirino, J.P. & Terabe, S. (1999). Sweeping of analyte zones in electrokinetic

[347] Quirino, J.P. & Terabe, S. (2000). Sample stacking of cationic and anionic analytes in capillary electrophoresis. *Journal of Chromatography A*, Vol.902, No.1, pp. 119-135. [348] Quirino, J.P.; Iwai, Y.; Otsuka, K. & Terabe, S. (2000). Determination of environmentally relevant aromatic amines in the ppt levels by cation selective exhaustive injection-sweeping-micellar electrokinetic chromatography. *Electrophoresis*,

[349] Ramautar, R.; Demirci, A. & De Jong, G. J. (2006). Capillary electrophoresis in

[350] Ramautar, R.; Somsen, G.W. & de Jong, G.J. (2009). CE-MS in metabolomics.

[351] Riekkola, M.L.; Wiedmer, S.K.; Valkó, I.E. & Sirén, H. (1997). Selectivity in capillary electrophoresis in the presence of micelles, chiral selectors and non-aqueous media.

metabolomics. *Trends in Analytical Chemistry*, Vol.25, No.5, pp. 455–466.

analysis. *Electrophoresis*, Vol.26, No.23-24, pp. 4206-4214.

No.15-17, pp. 1043-1054.

*A*, Vol.1106, No.1-2, pp. 7-18.

*Chemistry*, Vol.26, No.7, pp. 664-678.

*Chromatography B,* Vol. 856, No.1-2, pp. 365-370.

*Chimica Acta*, Vol.528, No.1, pp. 101–106.

*Electrophoresis*, Vol.30, No.1, pp. 276–291.

*Journal of Chromatography A*, Vol.792, No.1-2, pp. 13-35.

Vol.21, No.14, pp. 2899-2903.

applications. *Analytica Chimica Acta*, Vol.616, No.1, pp. 1-18.

chromatography. *Analytical Chemistry*, Vol.71, No.8, pp. 1638-1644.


[321] Palmer, J.; Burgi, D.S. & Landers, J.P. (2001). Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis. *Analytical Chemistry*,

[322] Palmer, C.P. & McCarney, J.P. (2004a). Developments in the use of soluble ionic polymers as pseudo-stationary phases for electrokinetic chromatography and stationary phases for

[324] Palmer, C.P. (2007). Recent progress in the use of ionic polymers as pseudostationary

[325] Pantučková, P.; Gebauer, P.; Boček, P. & Křivánková, L. (2009). Electrolyte systems for on-line CE-MS: Detection requirements and separation possibilities. *Electrophoresis*,

[326] Park, H.; Lee, S.; Kang, S.; Jung, Y.J. & Jung, S.H. (2004). Enantioseparation using sulfated cyclosophoraoses as a novel chiral additive in capillary electrophoresis.

[327] Pawliszyn, J. (1997). *Solid-phase Microextraction—Theory and Practice*, Wiley-VCH: New

[328] Peng, X.J.; Sternberg, E. & Dolphin, D. (2002). Aberrant expression of signaling-related proteins 14-3-3 gamma and RACK1 in fetal Down Syndrome brain (trisomy 21).

[329] Petersson, M.; Wahlund, K.G. & Nilsson S. (1999). Miniaturised on-line solid-phase extraction for enhancement of concentration sensitivity in capillary electrophoresis.

[330] Piehl, N.; Ludwig, M. & Belder, D. (2004). Subsecond chiral separations on a

[331] Phillips, T.M. (1998). Determination of in situ tissue neuropeptides by capillary immunoelectrophoresis. *Analytica Chimica Acta*, Vol.372, No.1-2, pp. 209-218. [332] Pleasance, S.; Thibault, P. & Kelly, J. (1992). Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry. *Journal of Chromatography,* Vol.591,

[333] Poppe, H. (1980). Characterization and design of liquid phase flow-through detector

[334] Powe, A.M.; Das, S.*;* Lowry, M.; El-Zahab, B.; Fakayode, S.O.; Geng, M.L.; Baker, G.A.; Wang, L.; McCarroll, M.E*.;* Patonay, G.; Li, M.; Aljarrah, M.; Neal, S. & Warner, I.M. (2010). Molecular Fluorescence, Phosphorescence, and Chemiluminescence

[335] Preinerstorfer, B.; Lubda, D.; Lindner, W. & Lämmerhofer, M. (2006). Monolithic silicabased capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography. *Journal of* 

[336] Preinerstorfer, B.; Lämmerhofer, M. & Lindner, W. (2009). Advances in enantioselective separations using electromigration capillary techniques.

electrochromatography. *Journal of Chromatography A,* Vol.1044, No.1-2, pp. 159-176. [323] Palmer, C.P. & McCarney, J.P. (2004b). Recent progress in the use of soluble ionic polymers as pseudostationary phases for electrokinetic chromatography.

Vol.73, No.4, pp. 725-731

Vol.30, No.1, pp. 203–214.

No.1-2, pp. 325-339.

York.

*Electrophoresis*, Vol.25, No.23-24, pp. 4086-4094.

*Electrophoresis,* Vol.25, No.16, pp. 2671-2674.

*Electrophoresis*, Vol.23, No.1, pp. 93-101.

*Journal of Chromatography A*, Vol.841, No.2, pp. 249-261.

microchip. *Electrophoresis*, Vol.25, No.21-22, pp. 3848-3852.

systems. *Analytica Chimica Acta*, Vol.114, No.C, pp. 59-70.

*Chromatography A*, Vol.1106, No.1-2, pp. 94–105.

*Electrophoresis,* Vol.30, No.1, pp. 100-132.

Spectrometry, *Analytical Chemistry,* Vol.82, No.12, pp. 4865-4894.

phases for EKC. *Electrophoresis*, Vol.28, No.1-2, pp. 164-173.


[368] Schmitt-Kopplin, P. & Frommberger, M. (2003). Capillary electrophoresis - Mass spectrometry: 15 years of developments and applications. *Electrophoresis*, Vol.24,

References 175

[369] Schmitt-Kopplin, P. & Englmann, M. (2005). Capillary electrophoresis - Mass spectrometry: Survey on developments and applications 2003-2004*. Electrophoresis*,

[370] Schulte, G.; Heitmeier, S.; Chankvetadze, B. & Blaschke, G. (1998). Chiral capillary electrophoresis-electrospray mass spectrometry coupling with charged cyclodextrin derivatives as chiral selectors. *Journal of Chromatography A*, Vol.800, No.1, pp. 77–82. [371] Schwarz, M.A. & Hauser, P.C. (2001). Rapid chiral on-chip separation with simplified amperometric detection. *Journal of Chromatography A*, Vol.928, No.2, pp. 225–232. [372] Schwarz; M.A. & Hauser, P.C. (2003). Chiral on-chip separations of neurotransmitters.

[373] Schwer, C.; Gaš, B.; Lottspeich, F. & Kenndler, E. (1993). Computer simulation and experimental evaluation of on-column sample preconcentration in capillary zone electrophoresis by discontinuous buffer systems. *Analytical Chemistry*, Vol.65, No.15,

[374] Scriba, G.K.E. (2002). Selected fundamental aspects of chiral electromigration techniques and their application to pharmaceutical and biomedical analysis. *Journal of* 

[375] Scriba, G.K.E. (2003). Pharmaceutical and biomedical applications of chiral capillary electrophoresis and capillary electrochromatography: An update. *Electrophoresis*,

[376] Scriba, G.K.E. (2007). Nonaqueous capillary electrophoresis-mass spectrometry. *Journal* 

[377] Scriba, G.K.E. (2008). Cyclodextrins in capillary electrophoresis enantioseparations - Recent developments and applications. *Journal of Separation Science,* Vol. 31, No.11, pp.

[378] Scriba, G.K.E. (2011). Fundamental aspects of chiral electromigration techniques and application in pharmaceutical and biomedical analysis. *Journal of Pharmaceutical and* 

[379] Ševčík, J.; Stránský, Z.; Ingelse, B. & Lemr, K. (1996). Capillary electrophoretic enantioseparation of selegiline, methamphetamine and ephedrine using a neutral βcyclodextrin epichlorhydrin polymer. *Journal of Pharmaceutical and Biomedical Analysis,* 

[380] Servais, A.C.; Chiap, P.; Hubert, P.; Crommen, J. & Fillet, M. (2004). Determination of salbutamol enantiomers in human urine using heptakis(2,3-di-O-acetyl-6-O-sulfo)-βcyclodextrin in nonaqueous capillary electrophoresis. *Electrophoresis*, Vol.25, No.10-11,

[381] Servais, A.C.; Fillet, M.; Mol, R.; Somsen, G.W.; Chiap, P.; Jong, G.J.D. & Crommen, J. (2006). On-line coupling of cyclodextrin mediated nonaqueous capillary electrophoresis to mass spectrometry for the determination of salbutamol enantiomers in urine. *Journal of Pharmaceutical and Biomedical Analysis,* Vol.40, No.3, pp. 752–757. [382] Severs, J.C. & Games, D.E. (1994). CZE and Transient CTTP-ES-MS Using H20 and D20 Based Electrolyte Systems. *42nd ASMS Conference on Mass Spectrometry and Allied* 

*Pharmaceutical and Biomedical Analysis,* Vol.27, No.3-4, pp. 373-399.

No.22-23, pp. 3837–3867.

pp. 2108-2115.

1991-2011.

pp. 1632-1640.

Vol.26, No.7-8, pp. 1209-1220.

Vol.24, No.15, pp. 2409-2421.

Vol.14, No.8-10, pp. 1089-1094.

*Topics, Chicago, IL, 29 May-3 June 1994.* 

*Analytical Chemistry*, Vol.75, No.17, pp. 4691-4695.

*of Chromatography A*, Vol.1159, No.1-2, pp. 28–41.

*Biomedical Analysis,* Vol.55, No.4, pp. 688-701.


[352] Rizkov, D.; Mizrahi, S.; Cohen, S. & Lev, O. (2010). beta-Amino alcohol selectors for enantioselective separation of amino acids by ligand-exchange capillary zone electrophoresis in a low molecular weight organogel*. Electrophoresis,* Vol.31, No.23-24,

[353] Rizvi, S.A.A.; Akbay, C. & Shamsi, S.A. (2004). Polymeric alkenoxy amino acid surfactants: II. Chiral separations of -blockers with multiple stereogenic centers.

[354] Rizvi, S.A.A. & Shamsi, S.A. (2006). Synthesis, characterization, and application of chiral ionic liquids and their polymers in micellar electrokinetic chromatography.

[355] Rizvi, S.A.A.; Zheng, J.; Apkarian, R.P.; Dublin, S.N., & Shamsi, S.A. (2007). Polymeric sulfated amino acid surfactants: A class of versatile chiral selectors for micellar electrokinetic chromatography (MEKC) and MEKC-MS. *Analytical Chemistry*, Vol.79,

[356] Rizzi, A. (2001). Fundamental aspects of chiral separations by capillary

[357] Robinson, T.E. & Justice, J.B. (1991). *Microdialysis in the Neurosciences*, Elsevier: Amsterdam. [358] Ross, D. & Locascio, L.E. (2002). Microfluidic temperature gradient focusing. *Analytical* 

[359] Rudaz, S.; Cherkaoui, S.; Dayer, P.; Fanali, S. & Veuthey, J.L. (2000). Simultaneous stereoselective analysis of tramadol and its main phase I metabolites by on-line capillary zone electrophoresis-electrospray ionization mass spectrometry. *Journal of* 

[360] Rudaz, S.; Geiser, L.; Souverain, S.; Prat, J. & Veuthey, J.L. (2005). Rapid stereoselective separations of amphetamine derivatives with highly sulfated γ-cyclodextrin.

[361] Ruiz, C.G. & Marina, M.L. (2006). Sensitive chiral analysis by capillary electrophoresis.

[362] Rundlett, K.L. & Armstrong, D.W. (1995). Effect of micelles and mixed micelles on efficiency and selectivity of antibiotic-based capillary electrophoretic

[363] Ryan, R.; Donegan, S.; Power, J.; McEvoy, E. & Altria, K. (2009). Recent advances in the methodology, optimisation and application of MEEKC. *Electrophoresis*, Vol.30, No.1,

[364] Saito, Y. & Jinno, K. (2003). Miniaturized sample preparation combined with liquid phase separations. *Journal of Chromatography A*, Vol.1000, No.1-2, pp. 53–67. [365] Santos, B.; Simonet, B.M.; Rios, A. & Valcarcel, M. (2007). On-line coupling of solidphase microextraction to commercial CE-MS equipment. *Electrophoresis,* Vol.28*,* No*.*9*,*

[366] Schappler, J.; Guillarme, D.; Prat, J.; Veuthey, J.L. & Rudaz, S. (2006). Enhanced method performances for conventional and chiral CE-ESI/MS analyses in plasma.

[367] Schappler, J.; Guillarme, D.; Prat, J.; Veuthey, J.L. & Rudaz, S. (2008). Validation of chiral capillary electrophoresis-electrospray ionization-mass spectrometry methods for

ecstasy and methadone in plasma. *Electrophoresis*, Vol.29, No.10, pp. 2193-2202.

enantioseparations. *Analytical Chemistry*, Vol.67, No.13, pp. 2088-2095.

pp. 3921-3927.

No.3, pp. 879–898.

pp. 65–82.

pp. 1312–1318.

*Electrophoresis*, Vol.25, No.6, pp. 853–860.

*Chemistry*, Vol.74, No.11, pp. 2556-2564.

*Chromatography A*, Vol.868, No.2, pp. 295-303.

*Electrophoresis*, Vol.26, No.20, pp. 3910–3920.

*Electrophoresis,* Vol.27, No.1, pp. 195-212.

*Electrophoresis*, Vol.27, No.8, pp. 1537–1546.

*Analytical Chemistry*, Vol.78, No.19, pp. 7061-7069.

electrophoresis. *Electrophoresis*, Vol.22, No.15, pp. 3079–3106.


ephedrine, amphetamine and methamphetamine by capillary electrophoresis in uncoated and coated capillaries with native beta-cyclodextrin as the chiral selector: Preliminary

application to the analysis of urine and hair. *Electrophoresis*, Vol.19, No.1, pp. 42-50. [400] Tan, L.; Wang, Y.; Liu, X.; Ju, H. & Li, J. (2005). Simultaneous determination of L- and D-lactic acid in plasma by capillary electrophoresis. *Journal of Chromatography B*,

References 177

[401] Tanaka, Y. & Terabe, S. (1995). Partial separation zone technique for the separation of enantiomers by affinity electrokinetic chromatography with proteins as chiral pseudo-

[402] Taylor, J.A. & Yeung, E.S. (1991). Axial-beam absorbance detection for capillary

[403] Tekeľ, J. & Mikuš, P. (2005). *Analysis of Substances in Biological Systems*; Jahnátková; A.

[404] Tempels, F.W.A.; Wiese, G.; Underberg, W.J.M; Somsen, G.W. & de Jong, G.J. (2006). On-line coupling of size exclusion chromatography and capillary electrophoresis via solid-phase extraction and a Tee-split interface. *Journal of Chromatography B,* Vol. 839,

[405] Tempels, F.W.A.; Underberg, W.J.M.; Somsen, G.W. & de Jong, G.J. (2007). On-line coupling of SPE and CE-MS for peptide analysis. *Electrophoresis*, Vol.28, No.9, pp.1319–

[406] Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A. & Ando, T. (1984). Electrokinetic separations with micellar solutions and open-tubular capillaries [2]. *Analytical* 

[408] Theurillat, R.; Knobloch, M.; Levionnois, O.; Larenza, P.; Mevissen, M. & Thormann, W. (2005). Characterization of the stereoselective biotransformation of ketamine to norketamine via determination of their enantiomers in equine plasma by capillary

[409] Theurillat, R.; Knobloch, M.; Schmitz, A.; Lassahn, P.G.; Mevissen, M. & Thormann, W. (2007). Enantioselective analysis of ketamine and its metabolites in equine plasma and urine by CE with multiple isomer sulfated β-CD. *Electrophoresis*, Vol.28, No.15, pp.

[410] Theurillat, R. & Thormann, W. (2008). Capillary electrophoresis evidence of the stereoselective ketoreduction of mebendazole and aminomebendazole in

echinococcosis patients. *Journal of Separation Science.*, Vol.31, No.1, pp. 188-194. [411] Thiele, C.; Auerbach, D.; Jung, G. & Wenz, G. (2011). Inclusion of chemotherapeutic agents in substituted beta-cyclodextrin derivatives, Conference Information: 1st European Cyclodextrin Conference, OCT 11-13, 2009 Aalborg, DENMARK, *Journal of Inclusion Phenomena and Macrocyclic Chemistry*, Vol.69, No.3-4 (Sp. Iss.), pp. SI 303-307. [412] Thibault, P.; Paris, C. & Pleasance, S. (1991). Analysis of peptides and proteins by capillary electrophoresis/mass spectrometry using acidic buffers and coated capillaries. *Rapid Communications in Mass Spectrometry,* Vol.5, No.10, pp. 484-490. [413] Thompson, T.J.; Foret, F.; Vouros, E. & Karger, B.L. (1993). Capillary electrophoresis/electrospray ionization mass spectrometry: Improvement of protein detection limits using on-column transient isotachophoretic sample preconcentration.

[407] Terabe, S. (1992). *Micellar Electrokinetic Chromatography*, Beckman., California

electrophoresis. *Electrophoresis*, Vol.26, No.20, pp. 3942-3951.

*Analytical Chemistry*, Vol.65, No.7, pp. 900-906.

stationary phases. *Journal of Chromatography A*, Vol.694, No.1, pp. 277-284.

electrophoresis. *Journal of Chromatography*, Vol.550, No.1-2, pp. 831-837.

(Ed.), pp. 135-171, Comenius University, Bratislava.

Vol.814, No.2, pp. 393-398.

No.1-2, pp. 30-35.

*Chemistry*, Vol.56, No.1, pp*.* 111-113

1326.

2748-2757.


[383] Shackman, J.G. & Ross, D. (2007). Counter-flow gradient electrofocusing.

[384] Shamsi, S.A. (2002). Chiral capillary electrophoresis-mass spectrometry: Modes and

[385] Sheeley, S.A.; Miao, H.; Ewing, M.A.; Rubakhin, S.S. & Sweedler, J.V. (2005). Measuring D-amino acid-containing neuropeptides with capillary electrophoresis.

[386] Shihabi, Z.K. (2002). Transient pseudo-isotachophoresis for sample concentration in

[387] Shou, C.Q., Kang, J.F. & Song, N.J. (2008). Preparation and evaluation of capillary electrophoresis column bonded by carbosilane dendrimer. *Chinese Journal of Analytical* 

[388] Silva, M. (2009). Micellar electrokinetic chromatography: Methodological and instrumental

[389] Simpson, S.L.; Quirino, J.P. & Terabe, S. (2008). On-line preconcentration in capillary electrophoresis Fundamentals and applications. *Journal of Chromatography A*, Vol.1184,

[390] Skoog, D.A.; Holler, F.J. & Crouch, S.R (2007). *Principles of Instrumental Analysis* 6th ed.

[391] Smyth, W.F. & Rodriguez, V. (2007). Recent studies of the electrospray ionisation behaviour of selected drugs and their application in capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry. *Journal of* 

[392] Soetebeer, U.B.; Schierenberg, M.O.; Schulz, H.; Andresen, P. & Blaschke, G. (2001). Direct chiral assay of tramadol and detection of the phase II metabolite O-demethyltramadol glucuronide in human urine using capillary electrophoresis with laser-induced

native fluorescence detection. *Journal of Chromatography B*, Vol.765, No.1,pp. 3-13. [393] Soga, T.; Ohashi, Y.; Ueno, Y.; Naraoka, H.; Tomita, M. & Nishioka, T. (2003). Quantitative Metabolome Analysis Using Capillary Electrophoresis Mass

[394] Soga, T. (2007). Capillary electrophoresis-mass spectrometry for metabolomics.

[395] Strašík, S.; Danková, M.; Molnárová, M.; Ölvecká, E. & Kaniansky, D. (2003). Capillary zone electrophoresis in wide bore capillary tubes with fiber-coupled diode array

[396] Suntornsuk, L. (2010). Recent advances of capillary electrophoresis in pharmaceutical

[397] Swinney, K. & Bornhop, D.J. (2000). Detection in capillary electrophoresis.

[398] Szökö, E.; Tábi, T.; Halasz, A.S.; Palfi, M.; Kalasz, M. & Magyar, K. (2004). Chiral characterization and quantification of deprenyl-N-oxide and other deprenyl metabolites in rat urine by capillary electrophoresis. *Chromatographia*, Vol.60,

[399] Tagliaro, F.; Manetto, G.; Bellini, S.; Scarcella, D.; Smith, F.P. & Marigo, M. (1998). Simultaneous chiral separation of 3,4-methylenedioxymethamphetamine (MDMA), 3-4 methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDE),

Spectrometry. *Journal of Proteome Research*, Vol.2, No.5, pp. 488–494.

detection. *Journal of Chromatography A,* Vol.990, No.1-2, pp. 23-33.

analysis. *Analytical and Bioanalytical Chemistry,* Vol.398, No.1, pp. 29-52.

advances focused on practical aspects. *Electrophoresis*, Vol.30, No.1, pp. 50–64.

capillary electrophoresis. *Electrophoresis*, Vol.23, No.11, pp. 1612-1617.

applications. *Electrophoresis*, Vol.23, No.22-23, pp. 4036–4051.

*Electrophoresis*, Vol.28, No.4, pp. 556-571.

*Analyst*, Vol.130, No.8, pp. 1198–1203.

*Chemistry*, Vol.36, No.3, pp. 297-300.

Thomson Brooks/Cole Publishing: Belmont, CA.

*Chromatography A*, Vol.1159, No.1-2, pp. 159–174.

*Methods in Molecular Biology*, Vol.358, pp. 129–137.

*Electrophoresis,* Vol.21, no. 7, p. 1239-1250.

No.SUPPL, pp. S245-S251.

No.1-2, pp. 504 – 541.

ephedrine, amphetamine and methamphetamine by capillary electrophoresis in uncoated and coated capillaries with native beta-cyclodextrin as the chiral selector: Preliminary application to the analysis of urine and hair. *Electrophoresis*, Vol.19, No.1, pp. 42-50.


[429] Vespalec, R. & Boček, P. (1999). Chiral separations in capillary electrophoresis.

References 179

[430] Vespalec, R. & Boček, P. (2000). Chiral separations in capillary electrophoresis.

[431] von Brocke, A.; Nicholson, G. & Bayer, E. (2001). Recent advances in capillary electrophoresis/electrospray-mass spectrometry. *Electrophoresis*, Vol.22, No.7, pp.

[441] Wahl, J.H.; Goodlett, D.R.; Udseth, H.R. & Smith, R.D. (1992). Attomole level capillary electrophoresis-mass spectrometric protein analysis using 5-μm-i.d. capillaries.

[442] Wahl, J.H.; Goodlett, D.R. & Udseth, H.R. (1993). Use of small-diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis-mass

[443] Wahl, J.H.; Gale, D.C. & Smith, R.D. (1994). Sheathless capillary electrophoresiselectrospray ionization mass spectrometry using 10 μm I.D. capillaries: Analyses of tryptic digests of cytochrome c. *Journal of Chromatography* A, Vol.659, No.1, pp. 217-222. [444] Wang, T.; Aiken, J.H.; Huie, C.W. & Hartwick, R.A. (1991). Nanoliter-scale multireflection cell for absorption detection in capillary electrophoresis. *Analytical* 

[445] Wang, F. & Khaledi, M.G. (1996). Chiral Separations by Nonaquaous Capillary

[446] Wang, C.Y.; Shang, Z.C.; Mei, J.H. & Yu, Q.S. (2003). Synthesis of a new chiral receptor containing 1,7-diaza-12-crown-4 and its application in chiral separation. *Synthetic* 

[447] Wang, Z.; Wang, J.; Hu, Z. & Kang, J. (2007). Enantioseparation by CE with vacomycin as chiral selector: Improving the separation performance by dynamic coating of the capillary with poly(dimethylacrylamide). *Electrophoresis*, Vol.28, No.6, pp. 938-943. [448] Wang, R.; Jia, Z.P.; Fan, J.J.; Chen, L.R.; Xie, H.; Ma, J.; G, X. & Wang, J. (2007). CE, with hydroxypropyl-β-cyclodextrin as chiral selector, for separation and determination of the enantiomers of amlodipine in the serum of hypertension patients. *Chromatographia*,

[449] Wang, L.J.; Hu, S.Q.; Guo, Q.L., Yang, G.L. & Chen, X.G. (2011). Di-n-amyl L-tartrateboric acid complex chiral selector in situ synthesis and its application in chiral nonaqueous capillary electrophoresis. *Journal of Chromatography A,* Vol.1218, No.9, pp.

Electrophoresis. *Analytical Chemistry*, Vol.68, No.19, pp. 3460-3467.

*Electrophoresis*, Vol.20, No.13, pp. 2579-2591.

1251–1266

[437] www.ks.uiuc.edu

[440] www.sigmaaldrich.com

[439] www.rsc.org

[433] www.chm.bris.ac.uk/ms/theory

[435] www.csi.chemie.tu-darmstadt.de [436] www.kore.co.uk/tutorial.htm

*Chemical Reviews*, Vol.100, No.10, pp. 3715-3753.

[432] www.chm.bris.ac.uk/ms/theory/maldi-ionisation.html

[434] www.chm.bris.ac.uk/ms/theory/qit-massspec.html

[438] www.noble.org/PlantBio/Sumner/metabolomics.html

*Analytical Chemistry*, Vol.64, No.24, pp. 3194-3196.

*Chemistry*, Vol.63, No.14, pp. 1372-1376.

Vol.65, No.9-10, pp. 575-579.

1300-1309.

*Communications*, Vol.33, No.19, pp. 3381-3386.

spectrometry. *Electrophoresis,* Vol.14, No.5-6, pp. 448-457.


[414] Thompson, J.E.; Vickroy, T.W. & Kennedy, R.T. (1999). Rapid determination of aspartate enantiomers in tissue samples by microdialysis coupled on-line with

[416] Tomlinson, A.J.; Nevala, W.K.; Braddock, W.D.; Strausbauch, M.A.; Wettstein, P.J. & Naylor, S. (1994). Drug Metabolism Studies by On-Line CE-ESI-MS/MS. *42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, 29 May-3 June 1994.*  [417] Toussaint, B.; Hubert, P.; Tjaden, U.R.; Van Der Greef, J. & Crommen, J. (2000). Enantiomeric separation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection - New optimization technique for transient

[419] Trojanowicz, M. (2009). Recent developments in electrochemical flow detections—A review, Part I. Flow analysis and capillary electrophoresis. *Analytica Chimica Acta,*

[420] Tsimachidis, D.; Česla, P.; Hájek, T.; Theodoridis, G. & Jandera, P. (2008). Capillary electrophoretic chiral separation of Cinchona alkaloids using a cyclodextrin selector.

[421] Tsuda, T.; Sweedler, J.V. & Zare, R.N. (1990). Rectangular capillaries for capillary zone

[422] Turiel, E. & Martin-Esteban, A. (2004). Molecularly imprinted polymers: towards highly selective stationary phases in liquid chromatography and capillary electrophoresis. *Analytical and Bioanalytical Chemistry,* Vol.378, No.8, pp. 1876-1886 [423] Tzanavaras, P.D. (2010). Recent Advances in the Analysis of Organic Impurities of Active Pharmaceutical Ingredients and Formulations: A Review. *Current Organic* 

[424] Urbánek, M.; Křivánková, L. & Boček, P. (2003). Stacking phenomena in electromigration: From basic principles to practical procedures. *Electrophoresis,* Vol.24,

[425] Vacík, J.; Zuska, J. & Muselasová, I. (1985). Improvement of the performance of a highfrequency contactless conductivity detector for isotachophoresis. *Journal of* 

[426] Valkó, I.E.; Sirén, H. & Riekkola, M.L. (1996). Chiral separation of dansyl amino acids by capillary electrophoresis: Comparison of formamide and N-methylformamide as

[427] Van Eeckhaut, A. & Michotte, Y. (2006). Chiral separations by capillary electrophoresis: Recent developments and applications. *Electrophoresis*, Vol.27, No.14,

[428] Varghese, J.V. & Cole, R.B. (1993). Cetyltrimethylammonium chloride as a surfactant buffer additive for reversed polarity capillary electrophoresis-electrospray mass

background electrolytes. *Chromatographia*, Vol.43, No.5-6, pp. 242-246.

spectrometry. *Journal of Chromatography A,* Vol.652, No.2, pp. 369-372

capillary electrophoresis. *Analytical Chemistry,* Vol.71, No.13, pp.2379-2384. [415] Tomáš, R.; Koval, M. & Foret, F. (2010). Coupling of hydronynamically closed large bore capillary isotachophoresis with electrospray mass spectrometry. *Journal of* 

isotachophoresis *Journal of Chromatography A*, Vol.871, No.1-2, pp. 173-180. [418] Toussaint, B.; Palmer, M. & Chiap, P. (2001)*.* On-line coupling of partial fillingcapillary zone electrophoresis with mass spectrometry for the separation of

clenbuterol enantiomers. *Electrophoresis*, Vol.22, No.7, pp. 1363-1372.

*Journal of Separation Science,* Vol.31, No.6-7, pp. 1130-1136.

*Chemistry,* Vol.14, No.19, pp. 2348-2634.

*Chromatography,* Vol.320, No.1, pp. 233-240.

electrophoresis. *Analytical Chemistry*, Vol.62, No.19, pp. 2149-2152.

*Chromatography A*, Vol.1217, No.25, pp. 4144-4149.

Vol.653, No.1, pp. 36–58.

No.3, pp. 466.

pp. 2880-2895.


[467] Yang, L.; Harrata, K. & Lee, S. (1997). On-Line Micellar Electrokinetic Chromatography-Electrospray Ionization Mass Spectrometry Using Anodically

References 181

[468] Yeung, E.S. (1989). Indirect detection methods: Looking for what is not there. *Acc.* 

[469] Yu, C.J.; Chang, H.C. & Tseng, W.L. (2008). On-line concentration of proteins by SDS-

[470] Zandkarimi, M.; Shafaati, A.; Foroutan, S.M. & Lucy, Charles A. (2009). Rapid enantioseparation of amlodipine by highly sulfated cyclodextrins using short-end injection capillary electrophoresis. *Daru-Journal of Faculty of Pharmacy,* Vol.17, No.4, pp. 269-276. [471] Zeng, A.G.; Wang, C.H.; Wang, L. & Fu, Q. (2010). Study on absorptive difference between S-amlodipine and RS-amlodipine in rat intestine. *Chinese Pharmaceutical* 

[472] Zhang, L.H. & Wu, X.Z. (2007). Capillary electrophoresis with in-capillary solid-phase extraction sample cleanup. *Analytical Chemistry*, Vol.79, No.6, pp. 2562–2569. [473] Zhao, J.; Hooker, T. & Jorgenson, J.W. (1999). Synchronous cyclic capillary electrophoresis using conventional capillaries: System design and preliminary results.

[474] Zhao, J. & Jorgenson, J.W. (1999). Application of synchronous cyclic capillary electrophoresis: Isotopic and chiral separations. *Journal of Microcolumn Separations,*

[475] Zhao, S.L.; Yuan, H.Y. & Xiao, D. (2005a). Detection of D-Serine in rat brain by capillary electrophoresis with laser induced fluorescence detection. *Journal of* 

[476] Zhao, S.L.; Song, Y.R. & Liu, Y.M. (2005b). A novel capillary electrophoresis method for the determination of d-serine in neural samples. *Talanta*, Vol.67, No.1, pp. 212–216. [477] Zhao, S.; Wang, H.; Zhang, R.; Tang, L. & Liu, Y.M. (2006a). Degradingdehydroabietylisothiocyanate as a chiral derivatizing reagent for enantiomeric separations by capillary electrophoresis. *Electrophoresis*, Vol.27, No.17,

[478] Zhao, S.; Zhang, R.; Wang, H.; Tang, L. & Pan, Y. (2006b). Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection. *Journal of Chromatography B*,

[479] Zhao, S.; Wang, H.; Pan, Y.; He, M. & Zhao, Z. (2007). 3-[(3- Dehydroabietamidopropyl)dimethylammonio]-1-propane-sulfonate as a new type of chiral surfactant for enantiomer separation in micellar electrokinetic chromatography.

[480] Zheng, J. & Shamsi, S. A. (2006). Simultaneous enantioseparation and sensitive detection of eight β-blockers using capillary electrochromatography-electrospray

ionization-mass spectrometry. *Electrophoresis*, Vol.27, No.11, pp. 2139–2151. [481] Zheng, J.; Norton, D. & Shamsi, S. A. (2006). Fabrication of internally tapered capillaries for capillary electrochromatography electrospray ionization mass

Migrating Micelles. *Analytical Chemistry*, Vol.69*,* No.10, pp. 1820–1826.

CGE with LIF detection. *Electrophoresis*, Vol.29, No.2, pp. 483-490.

*Journal of Microcolumn Separations,* Vol.11, No.6, pp. 431-437.

*Journal of Chromatography A*, Vol.1145, No.1-2, pp. 246-249.

spectrometry. *Analytical Chemistry*, Vol.78, No.4, pp. 1323–1330.

*Chromatography B*, Vol.822, No.1-2, pp. 334–338.

*Chem. Res.*, Vol.22, No.4, pp. 125-130.

*Journal*, Vol.45, No.13, pp. 991-996.

Vol.11, No.6, pp. 439-449.

Vol.833, No.2, pp. 186-190.

pp. 3428-3433.


[450] Wang, H.Y.; Chu, X.; Zhao, Z.X.; He, X.S. & Guo, Y.L. (2011). Analysis of low molecular weight compounds by MALDI-FTICR-MS. *Journal of Chromatography* B,

[451] Wang, Z.Y.; Liu, C. & Kang, J.W. (2011). A highly sensitive method for enantioseparation of fenoprofen and amino acid derivatives by capillary electrophoresis with on-line sample preconcentration. *Journal of Chromatography A,* 

[452] Ward, T.J. (1994). For capillary electrophoresis. *Analytical Chemistry*, Vol.66, No.11, pp.

[453] Ward, T.J.; Baker, B.A. (2008). Chiral separations. *Analytical Chemistry*, Vol.80, No.12,

[454] Ward, T.J. & Ward, K.D. (2010). Chiral Separations: Fundamental Review 2010.

[455] Weiss, D.J.; Saunders, K. & Lunte, C.E. (2001). Determination of nucleosides natural Cordyceps sinensis and cultured Cordyceps mycelia by capillary electrophoresis.

[456] Weng, X.; Bi, H.; Liu, B. & Kong, J. (2006). On-chip chiral separation based on bovine serum albumin-conjugated carbon nanotubes as stationary phase in a microchannel.

[457] Wenz, G.; Strassnig, C.; Thiele, C.; Engelke, A.; Morgenstern, B. & Hegetschweiler, K. (2008). Recognition of ionic guests by ionic beta-cyclodextrin derivatives. *Chemistry - A* 

[458] Wistuba, D. & Schurig, V. (2000a). Recent progress in enantiomer separation by capillary electrochromatography. *Electrophoresis,* Vol.21, No.18, pp. 4136-4158. [459] Wistuba, D. & Schurig, V. (2000b). Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography. *Journal of Chromatography A*, Vol.875, No.1-2, pp. 255-276. [460] Wistuba, D.; Bogdanski, A.; Larsen, K.L. & Schurig, V. (2006). δ-Cyclodextrin as novel chiral probe for enantiomeric separation by electromigration methods. *Electrophoresis*,

[461] Whitehouse, C.M.; Dreyer, R.N.; Yamashita, M. & Fenn, J.B. (1985). Electrospray interface for liquid chromatographs and mass spectrometers. *Analytical Chemistry*,

[462] Wren, S.A.C. & Rowe, R.C. (1992). Theoretical aspects of chiral separation in capillary electrophoresis. I. Initial evaluation of a model. *Journal of Chromatography*, Vol.603,

[463] Wren, S.A.C. & Rowe, R.C. (1993). Theoretical aspects of chiral separation in capillary electrophoresis. III. Application to β-blockers. *Journal of Chromatography*, Vol.635, No.1-

[464] Wu, X.Z. (2003). New approaches to sample preparation for capillary electrophoresis.

[465] Xu, L.; Basheer, C. & Lee, H.K. (2007). Developments in single-drop microextraction.

[466] Xue, Q. & Yeung, E.S. (1995). Differences in the chemical reactivity of individual

Vol.879, No.17-18, pp. 1166–1179.

Vol.1218, No.13, pp. 1775-1779.

*Analytical Chemistry*, Vol.82, No.12, p. 4712-4722

*Electrophoresis,* Vol.22, No1. pp. 144-150.

*Electrophoresis*, Vol.27, No.15, pp. 3129-3135

*European Journal*, Vol.14, No.24, pp. 7202-7211.

*Trends in Analytical Chemistry*, Vol.22, No.1, pp. 48-58.

*Journal of Chromatography A*, Vol.1152, No.1-2, pp. 184-192.

molecules of an enzyme. *Nature*, Vol.373, No.6516, pp. 681-683.

Vol.27, No.21, pp. 4359-4363.

Vol.57, No.3, pp. 675-679.

No.1-2, pp. 235-241.

2, pp. 113-118.

633A-640A.

pp. 4363-4372.


**8** 

**Abbreviations** 

ACN acetonitrile AML amlodipine

CBI cyanobenz[*f*]isoindole CC column coupling CDs cyclodextrins

BGE/BGS background electrolyte/background system

CM--CD carboxymethyl-- cyclodextrin HP--CD hydroxypropyl-- cyclodextrin HTM--CD hydroxytrimethyl -- cyclodextrin HDAS--CD heptakisdiacethylsulfo-- cyclodextrin SBE--CD sulfobuthylether-- cyclodextrin ODAS--CD oktakisdiacethylsulfo-- cyclodextrin

HDM--CD hydroxydimethyl-- cyclodextrin HS--CD highly sulphated-- cyclodextrin SB--CD sulfobuthyl-- cyclodextrin

CDEKC cyclodextrin mediated electrokinetic chromatography

DM--CD dimethyl-- cyclodextrin

CID collision-induced dissociation CE capillary electrophoresis

CEC capillary electrochromatography

CFGF counter-flow gradient focusing CGE capillary gel electrophoresis CME centrifuge microextraction

CSP chiral stationary phase

CV coefficients of variance

DAD diode array detection DMR *N*-desmethylmirtazapine DNA deoxyribonucleic acid DNP 6-O-desmethylnaproxen

CWEs crown ethers

CSEI cation-selective exhaustive injection

CTAC cetyltrimethylammonium chloride

CZE capillary zone electrophoresis

CF-FAB continuous-flow fast atom bombardment



### **Abbreviations**


182 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

[482] Zhou, S.Y.; Zuo, H.; Stobaugh, J.F.; Lunte, C.E. & Lunte, S.M. (1995). Continuous invivo monitoring of amino-acid neurotransmitters by microdialysis sampling with online derivatization and capillary electrophoresis separation. *Analytical Chemistry*,

[483] Zhou, S.Y.; Heckert, D.M.; Zuo, H.; Lunte, C.E. & Lunte, S.M. (1999). Online coupling of in vivo microdialysis with capillary electrophoresis/electrochemistry. *Analytica* 

[484] Zhou, Z.*;* Li, X.*;* Chen, X. & Hao, X. (2010). Synthesis of ionic liquids functionalized βcyclodextrin-bonded chiral stationary phases and their applications in highperformance liquid chromatography*. Analytica Chimica Acta*, Vol.678, No.2, pp. 208-

[485] Zhu, Z.; Zhang, L.; Marimuthu, A. & Yang, Z. (2003). Large-volume sample stacking combined with separation by 2-hydroxypropyl-b-cyclodextrin for analysis of isoxyzolylpenicillins by capillary electrophoresis. *Electrophoresis*, Vol.24, No.17, pp.

Vol.67, No.3, pp. 594-599.

214.

3089-3096.

*Chimica Acta*, Vol.379, No.3, pp.307-317.


MS mass spectrometry NA noradrenaline

NMDA N-methyl-D-aspartate NMR nuclear magnetic resonance NSM normal stacking mode

PMT photomultiplier tube PP protein precipitation PVA poly(vinyl alcohol) QC quality control RE relative error

RSD relative standard deviation

SDC sodium deoxycholate

SDS sodium dodecyl sulfate SI sulindac sulfide SO sulindac sulfone

SPE solid-phase extraction SPME solid-phase microextraction

SPD (5S)-pinandiol

SU sulindac SWP sweeping

TOF time-of-flight TQ triple quadrupole UV ultraviolet VIS visible

ZE zone electrophoresis

PHM pheniramine

NACE non-aqueous capillary electrophoresis NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole NDA naphthalene-2,3-dicarboxaldehyde

Abbreviations 185

OPA/NAC o-phthalaldehyde/N-acetyl-L-cysteine

SCCE synchronous cyclic capillary electrophoresis

SDME SDME=single drop microextraction

SRMP stacking with reverse migrating phase

T/TE terminator/terminating electrolyte

t-ITP transient isotachophoresis TGF temperature gradient focusing

SDLIL sodium *N*-[4-*n-*dodecyloxybenzoyl]-L-isoleucinate SDLL sodium *N*-[4-*n*-dodecyloxybenzoyl]-L-leucinate

SPCD sample preconcentration with chemical derivatization

LVSEP-ASEI large volume sample stacking with EOF as a pump plus anion-selective

DPJ dynamic pH junction EC electrochemical detection

EH enzymatic hydrolysis EK electrokinetic injection

EOF electroosmotic flow ESI electro spray ionization

FIA flow injection analysis FITC fluorescein isothiocyanate FMN flavin mononucleotide

GABA amino-*n*butyric acid HD hydrodynamic injection

IEF isoelectric focusing

ITP isotachophoresis ISP Ion spray

MRT mirtazapine

IT ion trap

FI flow injection

ECL electrochemiluminiscent detection EFGF electric-field gradient focusing

EKC electrokinetic chromatography EKS electrokinetic supercharging EME electro membrane extraction

FESI field-enhanced sample injection FESS field-enhanced sample stacking

FMOC-C1 9-fluorenylmethyl chloroformate

HS-Cys highly sulfated cyclosophoraoses

LIF laser induced fluorescence detection

exhaustive injection

MALDI matrix assisted laser desorption ionisation MCE microchip capillary electrophoresis MDA 3-4-methylenedioxyamphetamine MDE 3,4-methalenedioxyethylamphetamine MDMA 3,4-ethylenedioxymethamphetamine

MEEKC microemulsion electrokinetic chromatography MEKC micellar electrokinetic chromatography MLC microcolumn liquid chromatography

LVSS large volume sample stacking

L/LE leading/leading electrolyte

LLE liquid-liquid extraction LOD limit of detection LOQ limit of quantification LPME liquid-phase microextraction

FT-ICR Fourier transform ion cyclotron resonance

HPLC high performance liquid chromatography

FCCE flow counterbalanced capillary electrophoresis


**A**

accuracy, 91‐93, 95, 99, 137

AD, *see* amperometric detection achiral additive(s), 18, 21, 143

amphiphilic aminosaccharide(s), 41

automatization, 3, 51, 52, 91

baclofen, 9, 10, 32, 44, 55, 141

BGE, *see* background electrolyte BGS, *see* background electrolyte

biomarker(s), 1‐3, 5, 52, 89, 97, 142

biopolymer(s), 13, 14, 84

brompheniramine, 56, 108

blood, 2, 68, 89 body fluid(s), 16

biological fluid(s), 3, 71, 81, 96, 111, 146

biological matrix(‐ces), 1, 3, 11, 45, 46, 58, 62, 89, 90

biologically active compound(s), 11, 16, 58, 90, 92, 141

104, 105, 108, 112, 117, 128, 129, 139, 143, 145, 146

119, 120, 124, 127, 130, 137, 141, 142, 144‐146

bambuterol, 56, 108

ACN, *see* acetonitrile

AML, *see* amlodipine

anisodamine, 8 apomorfine, 8

**B**

144‐146

acetonitrile, 7, 47, 59, 65, 71, 103, 104, 128, 146

amlodipine, 9, 31, 54, 99, 103, 104, 116, 141, 144 amperometric detection, 8‐11, 50, 120‐122, 142

amino acid(s), 7, 15, 17, 32, 34‐36, 38‐40, 54, 55, 77, 78, 89, 90, 95‐98, 141, 142

background electrolyte(s), 13, 14, 16, 48, 60, 64, 66‐69, 71‐76, 79, 95, 104, 112, 123, 127, 131,

biological sample(s), 1‐3, 5, 7, 29, 31, 32, 34‐36, 39, 41, 42, 44, 47, 52, 73, 90, 97, 99, 103, 106,

buffer(s), 16, 19, 21, 24, 26, 28, 39, 43‐45, 47, 48, 50, 66‐69, 72, 74, 76, 78, 80, 82, 92, 96, 98, 101,

**9** 

**Index** 

**Index** 

### **A**

accuracy, 91‐93, 95, 99, 137 acetonitrile, 7, 47, 59, 65, 71, 103, 104, 128, 146 ACN, *see* acetonitrile AD, *see* amperometric detection achiral additive(s), 18, 21, 143 amino acid(s), 7, 15, 17, 32, 34‐36, 38‐40, 54, 55, 77, 78, 89, 90, 95‐98, 141, 142 AML, *see* amlodipine amlodipine, 9, 31, 54, 99, 103, 104, 116, 141, 144 amperometric detection, 8‐11, 50, 120‐122, 142 amphiphilic aminosaccharide(s), 41 anisodamine, 8 apomorfine, 8 automatization, 3, 51, 52, 91

### **B**

background electrolyte(s), 13, 14, 16, 48, 60, 64, 66‐69, 71‐76, 79, 95, 104, 112, 123, 127, 131, 144‐146 baclofen, 9, 10, 32, 44, 55, 141 bambuterol, 56, 108 BGE, *see* background electrolyte BGS, *see* background electrolyte biological fluid(s), 3, 71, 81, 96, 111, 146 biological matrix(‐ces), 1, 3, 11, 45, 46, 58, 62, 89, 90 biological sample(s), 1‐3, 5, 7, 29, 31, 32, 34‐36, 39, 41, 42, 44, 47, 52, 73, 90, 97, 99, 103, 106, 119, 120, 124, 127, 130, 137, 141, 142, 144‐146 biologically active compound(s), 11, 16, 58, 90, 92, 141 biomarker(s), 1‐3, 5, 52, 89, 97, 142 biopolymer(s), 13, 14, 84 blood, 2, 68, 89 body fluid(s), 16 brompheniramine, 56, 108 buffer(s), 16, 19, 21, 24, 26, 28, 39, 43‐45, 47, 48, 50, 66‐69, 72, 74, 76, 78, 80, 82, 92, 96, 98, 101, 104, 105, 108, 112, 117, 128, 129, 139, 143, 145, 146

chiral stationary phase(s), 5, 7, 10, 23, 26, 43, 47

clean‐up, 2, 54‐58, 62, 74, 77, 80, 83, 89, 90, 92, 101, 128, 130, 146

column(s), 11, 18, 24, 25, 27, 47, 48, 52, 61, 62, 75, 76, 78‐81, 83, 84, 91, 92, 102, 106, 112, 114,

Index 189

cyclodextrin(s), 7‐11, 13‐15, 17, 18, 21, 23, 25, 26, 28‐33, 35, 37, 38, 41, 43‐50, 54‐58, 73, 77, 82,

derivatization, 3, 5, 11, 31, 55‐58, 78, 79, 90‐92, 97, 108, 109, 113, 114, 117, 123, 141, 143, 147

detection, 1‐3, 7, 11, 16, 21, 23, 24, 30, 35‐37, 39, 43, 47‐51, 54, 56, 58, 60, 61, 68, 73, 75, 77, 80‐82, 84, 89, 90, 93, 94, 96‐98, 100, 101, 103, 104, 106, 111‐131, 133, 135, 137‐139, 141‐145, 147 detector(s), 18, 21, 24, 47, 51, 81, 85, 89, 96, 102, 112, 113, 115‐117, 120, 122, 123‐125, 126, 131,

complex matrix(‐ces), 2, 3, 25, 48, 52, 60, 81‐83, 92, 100, 113, 116, 130, 140, 141, 145

column coupled electrophoretic technique(s), 80, 99

conductivity, 59, 64, 66‐71, 73, 91, 114, 120, 124‐126 conductivity detection, 30, 120, 122‐125, 143, 144 conductivity detector(s), 102, 123‐126, 131

continuous‐flow fast atom bombardment, 131, 133

CSEI, *see* cation selective exhaustive injection

CSP(s), *see* chiral stationary phase(s)

CZE, *see* capilary zone electrophoresis

DAD, *see* diode array detection

countercurrent migration, 2, 20, 43, 44, 61, 112, 139, 145 counter‐flow gradient focusing, 53, 55, 58, 60, 63, 79, 98

97, 100, 103‐105, 109, 116, 122, 123, 128, 131, 139, 140‐146 cyclodextrin mediated electrokinetic chromatography, 7‐11, 64 cyclodextrin mediated micellar electrokinetic chromatography, 11, 18

column coupling, 2, 80, 81, 91, 102, 110, 126

115, 117, 118, 120‐124, 127, 129, 139, 144

chromatographic method(s), 1, 19, 128, 129 chromatographic technique(s), 2, 19, 61, 83

chloroquine, 11

cimaterol, 54, 97 circular dichroism, 112

clenbuterol, 10, 54, 58, 97

correlation coefficient, 96

crown ether(s), 13, 33, 49, 58

cyclosphoraose(s), 41

desalting, 83, 105, 130

138, 139, 141‐143, 145, 146

**D**

deprenyl, 8

CME, *see* centrifuge microextraction collision‐induced dissociation, 138

citalopram, 7

### **C**

calixarene(s), 41, 42 capillary(‐ies), 2, 17, 22, 24, 25, 27, 35, 36, 45‐47, 51, 52, 59‐66, 68, 69, 71‐74, 76‐81, 83‐88, 91, 92, 96‐98, 102, 103, 106, 108, 111‐115, 118, 120, 122‐125, 128, 130, 131, 133, 135, 136, 139, 142, 144‐146 capillary electrochromatography, 3, 5‐7, 10‐12, 23‐26, 36, 42, 47, 48, 51, 61, 63, 111, 139 capillary electrokinetic chromatography, 6 capillary electrophoresis, 1‐3, 5, 6, 8, 11‐14, 16‐22, 25‐28, 30, 32, 34‐38, 40, 41, 44, 46, 47, 49, 51‐55, 58, 73‐78, 80‐94, 96‐98, 103‐109, 111, 112, 115‐123, 126‐137, 139, 143, 145, 146 capillary gel electrophoresis, 6 capillary zone electrophoresis, 6, 12, 36, 64, 66, 68, 69, 71, 72, 76, 80, 81, 123‐125, 128‐130 carrier migration mode(s), 21 carvedilol, 11, 54 catechin, 7 cation selective exhaustive injection, 75 CC, *see* column coupling CD(s), *see* cyclodextrin(s) CDEKC, *see* cyclodextrin mediated electrokinetic chromatography CDMEKC, *see* cyclodextrin mediated micellar electrokinetic chromatography CE, *see* capillary electrophoresis CEC, *see* capillary electrochromatography cell(s), 7, 8, 62, 89, 96, 113 centrifuge microextraction, 58, 105 cetirizine, 7, 9, 34 CF‐FAB, *see* continuous‐flow fast atom bombardment CFGF, *see* counter‐flow gradient focusing CGE, *see* capillary gel electrophoresis channel(s), 25‐28, 52, 54, 63, 73, 80, 82, 84, 101, 102, 118, 122, 125, 144 charged cyclodextrin(s), 73, 143 charged chiral selector(s), 14, 16, 20, 21, 82, 112, 145 chip(s), 25, 26, 51, 52, 82, 91, 102, 114, 126 chiral additive(s), 28, 41, 91 chiral analysis, 1, 3, 5, 34, 53, 69, 82, 86, 101, 111, 112, 139, 144 chiral bioanalysis, 1, 42 chiral compound(s), 1, 13, 20, 29, 40, 46 chiral drug(s), 1, 21, 44, 83, 92, 108, 141, 146 chiral mobile phase additive(s), 5 chiral pseudostationary phase(s), 12, 21 chiral selector(s), 1, 3, 5, 7, 11‐14, 16‐24, 27‐30, 34‐38, 41‐49, 51, 53, 54, 64, 65, 82, 96, 97, 100, 101, 109, 112, 113, 116, 121, 124, 125, 139, 140‐142, 145, 146 chiral separation, 1, 3, 5, 6, 12, 26‐28, 37, 40‐42, 44, 46, 48, 49, 51, 64, 69, 76, 79, 91, 97, 101, 112, 121, 127, 130, 141, 144, 145

capillary(‐ies), 2, 17, 22, 24, 25, 27, 35, 36, 45‐47, 51, 52, 59‐66, 68, 69, 71‐74, 76‐81, 83‐88, 91, 92, 96‐98, 102, 103, 106, 108, 111‐115, 118, 120, 122‐125, 128, 130, 131, 133, 135, 136, 139, 142,

capillary electrophoresis, 1‐3, 5, 6, 8, 11‐14, 16‐22, 25‐28, 30, 32, 34‐38, 40, 41, 44, 46, 47, 49, 51‐55, 58, 73‐78, 80‐94, 96‐98, 103‐109, 111, 112, 115‐123, 126‐137, 139, 143, 145, 146

capillary zone electrophoresis, 6, 12, 36, 64, 66, 68, 69, 71, 72, 76, 80, 81, 123‐125, 128‐130

CDEKC, *see* cyclodextrin mediated electrokinetic chromatography

channel(s), 25‐28, 52, 54, 63, 73, 80, 82, 84, 101, 102, 118, 122, 125, 144

chiral selector(s), 1, 3, 5, 7, 11‐14, 16‐24, 27‐30, 34‐38, 41‐49, 51, 53, 54, 64, 65, 82, 96, 97, 100,

chiral separation, 1, 3, 5, 6, 12, 26‐28, 37, 40‐42, 44, 46, 48, 49, 51, 64, 69, 76, 79, 91, 97, 101, 112,

chiral analysis, 1, 3, 5, 34, 53, 69, 82, 86, 101, 111, 112, 139, 144

101, 109, 112, 113, 116, 121, 124, 125, 139, 140‐142, 145, 146

CDMEKC, *see* cyclodextrin mediated micellar electrokinetic chromatography

capillary electrochromatography, 3, 5‐7, 10‐12, 23‐26, 36, 42, 47, 48, 51, 61, 63, 111, 139

**C**

144‐146

calixarene(s), 41, 42

capillary electrokinetic chromatography, 6

cation selective exhaustive injection, 75

CEC, *see* capillary electrochromatography

CFGF, *see* counter‐flow gradient focusing CGE, *see* capillary gel electrophoresis

chip(s), 25, 26, 51, 52, 82, 91, 102, 114, 126

chiral compound(s), 1, 13, 20, 29, 40, 46 chiral drug(s), 1, 21, 44, 83, 92, 108, 141, 146

chiral mobile phase additive(s), 5 chiral pseudostationary phase(s), 12, 21

CF‐FAB, *see* continuous‐flow fast atom bombardment

charged chiral selector(s), 14, 16, 20, 21, 82, 112, 145

capillary gel electrophoresis, 6

carrier migration mode(s), 21

CC, *see* column coupling CD(s), *see* cyclodextrin(s)

cell(s), 7, 8, 62, 89, 96, 113

cetirizine, 7, 9, 34

CE, *see* capillary electrophoresis

centrifuge microextraction, 58, 105

charged cyclodextrin(s), 73, 143

chiral additive(s), 28, 41, 91

chiral bioanalysis, 1, 42

121, 127, 130, 141, 144, 145

carvedilol, 11, 54 catechin, 7

chiral stationary phase(s), 5, 7, 10, 23, 26, 43, 47 chloroquine, 11 chromatographic method(s), 1, 19, 128, 129 chromatographic technique(s), 2, 19, 61, 83 cimaterol, 54, 97 circular dichroism, 112 citalopram, 7 clean‐up, 2, 54‐58, 62, 74, 77, 80, 83, 89, 90, 92, 101, 128, 130, 146 clenbuterol, 10, 54, 58, 97 CME, *see* centrifuge microextraction collision‐induced dissociation, 138 column coupled electrophoretic technique(s), 80, 99 column coupling, 2, 80, 81, 91, 102, 110, 126 column(s), 11, 18, 24, 25, 27, 47, 48, 52, 61, 62, 75, 76, 78‐81, 83, 84, 91, 92, 102, 106, 112, 114, 115, 117, 118, 120‐124, 127, 129, 139, 144 complex matrix(‐ces), 2, 3, 25, 48, 52, 60, 81‐83, 92, 100, 113, 116, 130, 140, 141, 145 conductivity, 59, 64, 66‐71, 73, 91, 114, 120, 124‐126 conductivity detection, 30, 120, 122‐125, 143, 144 conductivity detector(s), 102, 123‐126, 131 continuous‐flow fast atom bombardment, 131, 133 correlation coefficient, 96 countercurrent migration, 2, 20, 43, 44, 61, 112, 139, 145 counter‐flow gradient focusing, 53, 55, 58, 60, 63, 79, 98 crown ether(s), 13, 33, 49, 58 CSEI, *see* cation selective exhaustive injection CSP(s), *see* chiral stationary phase(s) cyclodextrin(s), 7‐11, 13‐15, 17, 18, 21, 23, 25, 26, 28‐33, 35, 37, 38, 41, 43‐50, 54‐58, 73, 77, 82, 97, 100, 103‐105, 109, 116, 122, 123, 128, 131, 139, 140‐146 cyclodextrin mediated electrokinetic chromatography, 7‐11, 64 cyclodextrin mediated micellar electrokinetic chromatography, 11, 18 cyclosphoraose(s), 41 CZE, *see* capilary zone electrophoresis

### **D**

DAD, *see* diode array detection deprenyl, 8 derivatization, 3, 5, 11, 31, 55‐58, 78, 79, 90‐92, 97, 108, 109, 113, 114, 117, 123, 141, 143, 147 desalting, 83, 105, 130 detection, 1‐3, 7, 11, 16, 21, 23, 24, 30, 35‐37, 39, 43, 47‐51, 54, 56, 58, 60, 61, 68, 73, 75, 77, 80‐82, 84, 89, 90, 93, 94, 96‐98, 100, 101, 103, 104, 106, 111‐131, 133, 135, 137‐139, 141‐145, 147 detector(s), 18, 21, 24, 47, 51, 81, 85, 89, 96, 102, 112, 113, 115‐117, 120, 122, 123‐125, 126, 131, 138, 139, 141‐143, 145, 146

eluent, 135

**F**

**G**

**H**

fenoprofen, 54

FI, *see* flow injection

flavin(s), 57, 96, 97

filtration, 2, 62, 82, 89, 106

gemifloxacin, 34, 48, 54, 101

HD, *see* hydrodynamic injection hemispherodextrin(s), 41

glycopeptide(s), 36 guanosine gel(s), 41

enantioselective agent(s), 5

97‐104, 106, 108, 112, 116, 124, 125, 130, 139‐147

enantioselectivity, 16, 23, 27, 41, 46, 139

96, 97, 100, 108, 124, 140, 142, 144, 145 enzymatic hydrolysis 8, 11, 55, 58 EOF, *see* electroosmotic flow

FESI, *see* field enhanced sample injection FESS, *see* field enhanced sample stacking

flow injection, 2, 57, 58, 91, 108, 109, 120, 147 fluorescence detection, 101, 111, 117, 118, 141 Fourier transform ion cyclotron resonance , 137 FT‐ICR, *see* Fourier transform ion cyclotron resonance

ESI, *see* electrospray ionization exhaustive sample injection, 66

enantioresolution, 12‐16, 18‐21, 23, 37, 43, 44, 46, 48, 94, 139

ephedrine, 41, 43, 55‐57, 94, 95, 104, 105, 108, 142, 146

FCCE, *see* flow counterbalanced capillary electrophoresis

flow counterbalanced capillary electrophoresis, 22, 23, 46

enantiomer(s), 1, 3, 5, 12‐14, 18‐21, 25, 28, 30‐32, 34‐36, 38, 44‐52, 55, 57, 78, 79, 81, 91‐95,

Index 191

enantioseparation(s), 5, 6, 13, 14, 16, 18, 19, 21‐23, 28, 32, 34, 35, 38, 42‐47, 49, 51, 65, 77, 81,

extraction, 2, 11, 48, 52, 58, 62, 63, 66, 79, 82‐84, 86‐88, 90, 91, 102, 103‐105, 147

field enhanced sample injection, 54‐59, 63, 66, 71, 75, 77, 87, 92, 93, 97, 104, 105 field enhanced sample stacking, 11, 53, 54, 57‐59, 63, 64, 66, 68, 75, 91‐93

high performance liquid chromatography, 1, 5, 12, 16, 21, 23, 25, 51, 89, 127, 129

dextran(s), 34 dextrin(s), 34 dialysis, 52, 89, 90, 106, 143 dimethindene, 32, 44, 54, 99, 139, 144 diode array detection, 7‐11, 54‐58, 93, 94, 99, 105, 115‐117, 140, 141 dioxopromethazine, 7, 32, 44, 54, 99, 139, 144 disopyramide, 9, 32, 44 dispersion, 46, 60, 73 distribution, 1, 21, 61, 62, 82, 135 DPJ, *see* dynamic pH junction drug(s), 1, 3, 5, 21, 29, 30, 32, 34‐38, 41‐44, 46, 48, 52, 54, 77, 82, 83, 86, 88, 92, 93, 99, 105, 108, 111, 112, 116, 127, 132, 137, 139, 141, 142, 144‐147 dynamic pH junction, 55, 58, 60, 63, 72, 73, 75‐77, 91, 96, 97

### **E**

ECL, *see* electrochemiluminiscent detection efficiency, 1, 2, 12, 19, 25, 34, 35, 39, 47, 52, 60, 61, 68, 73, 77, 85, 86, 90, 103, 112, 113, 128, 130, 138, 139, 146 EFGF, *see* electric field gradient focusing EH, *see* enzymatic hydrolysis EK, *see* electrokinetic injection EKC, *see* electrokinetic chromatography electric field, 22, 23, 25, 46, 52, 60, 64, 65, 73, 79, 80, 120, 137 electric field gradient focusing, 46, 79, 80 electrocapture, 53, 76 electrochemical detection, 106, 112, 120, 123, 142, 143, 148 electrochemiluminiscent detection, 7, 9, 11 electrokinetic focusing, 78, 79 electrokinetic chromatography, 6, 7, 11, 13, 14, 21, 35, 38, 42‐44, 51, 54, 55, 58, 64, 65, 71, 80‐82, 96, 99, 100, 105, 106, 116, 124, 125, 139, 140‐142, 144, 145 electrokinetic injection, 2, 7, 8, 10, 11, 54, 55, 58, 60, 64, 66, 68, 73, 75, 84, 87, 92‐94, 97, 107, 144, 145 electrolyte(s), 5, 12, 13, 15‐17, 26, 27, 34, 38, 43, 44, 59‐61, 70‐72, 76, 83‐85, 94, 95, 100, 102, 104, 113, 122‐125, 127, 127, 128, 131, 140, 145 electromigration effects, 2, 19, 20, 52, 53, 112 electromigration technique(s), 6, 19, 25, 51 electroosmosis, 27 electroosmotic flow, 2, 14, 20‐22, 25, 44, 58, 59, 61, 63‐69, 72, 74, 77, 100, 112, 130, 134, 139, 140 electroosmotic migration, 21 electrophoretic methods, 120 electrophoretic mobility, 23, 59, 66, 70‐72, 79 electrospray ionization, 55, 75, 127‐131, 133‐136, 139, 144‐146

drug(s), 1, 3, 5, 21, 29, 30, 32, 34‐38, 41‐44, 46, 48, 52, 54, 77, 82, 83, 86, 88, 92, 93, 99, 105, 108,

efficiency, 1, 2, 12, 19, 25, 34, 35, 39, 47, 52, 60, 61, 68, 73, 77, 85, 86, 90, 103, 112, 113, 128, 130,

electrokinetic chromatography, 6, 7, 11, 13, 14, 21, 35, 38, 42‐44, 51, 54, 55, 58, 64, 65, 71,

electrokinetic injection, 2, 7, 8, 10, 11, 54, 55, 58, 60, 64, 66, 68, 73, 75, 84, 87, 92‐94, 97, 107,

electrolyte(s), 5, 12, 13, 15‐17, 26, 27, 34, 38, 43, 44, 59‐61, 70‐72, 76, 83‐85, 94, 95, 100, 102,

electroosmotic flow, 2, 14, 20‐22, 25, 44, 58, 59, 61, 63‐69, 72, 74, 77, 100, 112, 130, 134, 139, 140

dextran(s), 34 dextrin(s), 34

**E**

138, 139, 146

dialysis, 52, 89, 90, 106, 143

disopyramide, 9, 32, 44 dispersion, 46, 60, 73

dimethindene, 32, 44, 54, 99, 139, 144

distribution, 1, 21, 61, 62, 82, 135 DPJ, *see* dynamic pH junction

dioxopromethazine, 7, 32, 44, 54, 99, 139, 144

111, 112, 116, 127, 132, 137, 139, 141, 142, 144‐147

ECL, *see* electrochemiluminiscent detection

EFGF, *see* electric field gradient focusing

EKC, *see* electrokinetic chromatography

electric field gradient focusing, 46, 79, 80

electrochemiluminiscent detection, 7, 9, 11

104, 113, 122‐125, 127, 127, 128, 131, 140, 145 electromigration effects, 2, 19, 20, 52, 53, 112 electromigration technique(s), 6, 19, 25, 51

electrophoretic mobility, 23, 59, 66, 70‐72, 79

EH, *see* enzymatic hydrolysis EK, *see* electrokinetic injection

electrokinetic focusing, 78, 79

electrocapture, 53, 76

144, 145

electroosmosis, 27

electroosmotic migration, 21 electrophoretic methods, 120

dynamic pH junction, 55, 58, 60, 63, 72, 73, 75‐77, 91, 96, 97

electric field, 22, 23, 25, 46, 52, 60, 64, 65, 73, 79, 80, 120, 137

electrochemical detection, 106, 112, 120, 123, 142, 143, 148

80‐82, 96, 99, 100, 105, 106, 116, 124, 125, 139, 140‐142, 144, 145

electrospray ionization, 55, 75, 127‐131, 133‐136, 139, 144‐146

diode array detection, 7‐11, 54‐58, 93, 94, 99, 105, 115‐117, 140, 141

eluent, 135

enantiomer(s), 1, 3, 5, 12‐14, 18‐21, 25, 28, 30‐32, 34‐36, 38, 44‐52, 55, 57, 78, 79, 81, 91‐95, 97‐104, 106, 108, 112, 116, 124, 125, 130, 139‐147 enantioresolution, 12‐16, 18‐21, 23, 37, 43, 44, 46, 48, 94, 139 enantioselective agent(s), 5 enantioselectivity, 16, 23, 27, 41, 46, 139 enantioseparation(s), 5, 6, 13, 14, 16, 18, 19, 21‐23, 28, 32, 34, 35, 38, 42‐47, 49, 51, 65, 77, 81, 96, 97, 100, 108, 124, 140, 142, 144, 145 enzymatic hydrolysis 8, 11, 55, 58 EOF, *see* electroosmotic flow ephedrine, 41, 43, 55‐57, 94, 95, 104, 105, 108, 142, 146 ESI, *see* electrospray ionization exhaustive sample injection, 66 extraction, 2, 11, 48, 52, 58, 62, 63, 66, 79, 82‐84, 86‐88, 90, 91, 102, 103‐105, 147

### **F**

FCCE, *see* flow counterbalanced capillary electrophoresis fenoprofen, 54 FESI, *see* field enhanced sample injection FESS, *see* field enhanced sample stacking FI, *see* flow injection field enhanced sample injection, 54‐59, 63, 66, 71, 75, 77, 87, 92, 93, 97, 104, 105 field enhanced sample stacking, 11, 53, 54, 57‐59, 63, 64, 66, 68, 75, 91‐93 filtration, 2, 62, 82, 89, 106 flavin(s), 57, 96, 97 flow counterbalanced capillary electrophoresis, 22, 23, 46 flow injection, 2, 57, 58, 91, 108, 109, 120, 147 fluorescence detection, 101, 111, 117, 118, 141 Fourier transform ion cyclotron resonance , 137 FT‐ICR, *see* Fourier transform ion cyclotron resonance

#### **G**

gemifloxacin, 34, 48, 54, 101 glycopeptide(s), 36 guanosine gel(s), 41

### **H**

HD, *see* hydrodynamic injection hemispherodextrin(s), 41 high performance liquid chromatography, 1, 5, 12, 16, 21, 23, 25, 51, 89, 127, 129

large volume sample stacking, 11, 54, 55, 57‐59, 63, 65, 68, 77, 93, 95

leading electrolyte, 13,71, 95, 100, 122, 125, 126, 129, 131, 140

limit(s) of detection, 1, 2, 7, 9‐11, 54, 58, 81, 86, 95, 141, 142, 146

mass spectrometry, 2, 11, 24, 58, 111, 114, 126, 132, 136, 137, 145

98‐100, 101, 105, 113, 116, 119, 124, 125, 130, 132, 139‐142, 144, 145

MEEKC, *see* microemulsion electrokinetic chromatography membrane(s), 2, 58, 61, 62, 80, 82, 87, 89, 103, 104, 108, 122

matrix(‐ces), 1‐3, 11, 21, 23, 25, 44‐46, 48, 51‐53, 55, 57‐60, 62, 64‐66, 68‐73, 81‐83, 86, 89‐93, 96,

metabolite(s), 1, 3, 5, 7‐11, 21, 34, 44, 48, 52‐55, 57, 75, 93, 94, 97, 99, 127, 132, 137, 140, 141,

micelle(s), 14, 16, 18, 19, 21, 38, 39, 41, 43, 45, 55, 57, 60, 64, 65, 67, 69, 73, 74, 76, 77, 139, 146, 147

liquid‐liquid extraction, 7‐11, 54, 56‐58, 62, 63, 83, 87, 88, 105, 144, 145

121, 141, 142, 147

linearity, 93, 96

lorazepam, 31, 55, 94

**M**

MALDI, 132

144, 147

maltodextrin(s), 7, 34

metabolomics, 127, 137 metamphetamine, 8, 10 methadone, 8, 10, 11, 144

metoxamine, 54 mexiletine, 7, 48

LC, *see* liquid chromatography LE, *see* leading electrolyte

LIF, *see* laser induced fluorescent detection

limit(s) of quantification, 7, 11, 145, 146

liquid chromatography, 1, 83, 128, 130

LPME, *see* liquid phase microextraction LVSS, *see* large volume sample stacking

macrocyclic antibiotic(s), 13, 23, 35, 47

MCE, *see* microchip capillary electrophoresis

metabolic study, 3, 8, 10, 11, 44, 54, 55, 99, 139, 141

microcolumn liquid chromatography, 56, 83, 108 microdialysis, 2, 8, 56, 62, 82, 89, 90, 105‐107, 143, 147

microemulsion(s), 14, 16, 38, 41, 73

LLE, *see* liquid‐liquid extraction LOD(s), *see* limit(s) of detection LOQ(s), *see* limit(s) of quantification

liquid phase microextraction, 7‐9, 11, 58, 87, 88

laser induced fluorescent detection, 2, 8‐11, 44, 54‐58, 77, 89, 90, 95‐97, 105, 111, 114, 117‐119,

Index 193

homogenate(s), 2, 7, 46, 56, 89, 106, 142 HPLC, *see* high performance liquid chromatography hydrodynamic flow, 25, 80, 134 hydrodynamic injection, 10, 11, 55, 58, 68, 77, 79, 92‐94, 96, 97 hydroxychloroquine, 7 hydroxyzine, 7, 34 hyphenation, 2, 62, 111, 112, 127, 129‐132, 139, 141

### **I**

ibuprofen, 9 IEF, *see* isoelectric focusing in‐capillary, 64, 78‐80, 97, 106 indobufen, 8 injection technique, 90 in‐line, 83, 84, 86, 88 interface(s), 83, 85‐87, 90, 91, 108, 114, 127, 131‐136, 145 ion spray, 131, 133 ion trap, 137, 138 ionic strength, 68‐70, 75, 80, 92, 128 ionization, 35, 60, 66, 72, 127, 128, 130‐135, 146 ion‐pairing reagent(s), 37 isoelectric focusing, 6, 12, 13, 53, 79 isomer(s), 7, 10, 75, 102, 128 isoproterenol, 31, 56, 106, 107, 143 isotachophoresis, 6, 11‐13, 51, 53‐55, 58, 59, 63, 70, 71, 80‐82, 94, 97, 99, 100, 102, 116, 124‐126, 128‐130, 139‐141, 143, 144 isoxyzolylpeniciline(s), 58 ITP, *see* isotachophoresis

### **J**

Joule heating, 17, 25

### **K**

ketamine, 9, 10, 75 ketoprofen, 8

### **L**

labetalol, 8 lactic acid, 8

isotachophoresis, 6, 11‐13, 51, 53‐55, 58, 59, 63, 70, 71, 80‐82, 94, 97, 99, 100, 102, 116, 124‐126,

homogenate(s), 2, 7, 46, 56, 89, 106, 142

hydrodynamic flow, 25, 80, 134

hydroxychloroquine, 7 hydroxyzine, 7, 34

IEF, *see* isoelectric focusing in‐capillary, 64, 78‐80, 97, 106

ionic strength, 68‐70, 75, 80, 92, 128

isoelectric focusing, 6, 12, 13, 53, 79

ionization, 35, 60, 66, 72, 127, 128, 130‐135, 146

injection technique, 90 in‐line, 83, 84, 86, 88

ion‐pairing reagent(s), 37

isomer(s), 7, 10, 75, 102, 128 isoproterenol, 31, 56, 106, 107, 143

128‐130, 139‐141, 143, 144 isoxyzolylpeniciline(s), 58 ITP, *see* isotachophoresis

Joule heating, 17, 25

ketamine, 9, 10, 75 ketoprofen, 8

ion spray, 131, 133 ion trap, 137, 138

**I**

**J**

**K**

**L**

labetalol, 8 lactic acid, 8

ibuprofen, 9

indobufen, 8

HPLC, *see* high performance liquid chromatography

hyphenation, 2, 62, 111, 112, 127, 129‐132, 139, 141

interface(s), 83, 85‐87, 90, 91, 108, 114, 127, 131‐136, 145

hydrodynamic injection, 10, 11, 55, 58, 68, 77, 79, 92‐94, 96, 97

large volume sample stacking, 11, 54, 55, 57‐59, 63, 65, 68, 77, 93, 95 laser induced fluorescent detection, 2, 8‐11, 44, 54‐58, 77, 89, 90, 95‐97, 105, 111, 114, 117‐119, 121, 141, 142, 147 LC, *see* liquid chromatography LE, *see* leading electrolyte leading electrolyte, 13,71, 95, 100, 122, 125, 126, 129, 131, 140 LIF, *see* laser induced fluorescent detection limit(s) of detection, 1, 2, 7, 9‐11, 54, 58, 81, 86, 95, 141, 142, 146 limit(s) of quantification, 7, 11, 145, 146 linearity, 93, 96 liquid chromatography, 1, 83, 128, 130 liquid‐liquid extraction, 7‐11, 54, 56‐58, 62, 63, 83, 87, 88, 105, 144, 145 liquid phase microextraction, 7‐9, 11, 58, 87, 88 LLE, *see* liquid‐liquid extraction LOD(s), *see* limit(s) of detection LOQ(s), *see* limit(s) of quantification lorazepam, 31, 55, 94 LPME, *see* liquid phase microextraction LVSS, *see* large volume sample stacking

### **M**

macrocyclic antibiotic(s), 13, 23, 35, 47 MALDI, 132 maltodextrin(s), 7, 34 mass spectrometry, 2, 11, 24, 58, 111, 114, 126, 132, 136, 137, 145 matrix(‐ces), 1‐3, 11, 21, 23, 25, 44‐46, 48, 51‐53, 55, 57‐60, 62, 64‐66, 68‐73, 81‐83, 86, 89‐93, 96, 98‐100, 101, 105, 113, 116, 119, 124, 125, 130, 132, 139‐142, 144, 145 MCE, *see* microchip capillary electrophoresis MEEKC, *see* microemulsion electrokinetic chromatography membrane(s), 2, 58, 61, 62, 80, 82, 87, 89, 103, 104, 108, 122 metabolic study, 3, 8, 10, 11, 44, 54, 55, 99, 139, 141 metabolite(s), 1, 3, 5, 7‐11, 21, 34, 44, 48, 52‐55, 57, 75, 93, 94, 97, 99, 127, 132, 137, 140, 141, 144, 147 metabolomics, 127, 137 metamphetamine, 8, 10 methadone, 8, 10, 11, 144 metoxamine, 54 mexiletine, 7, 48 micelle(s), 14, 16, 18, 19, 21, 38, 39, 41, 43, 45, 55, 57, 60, 64, 65, 67, 69, 73, 74, 76, 77, 139, 146, 147 microcolumn liquid chromatography, 56, 83, 108 microdialysis, 2, 8, 56, 62, 82, 89, 90, 105‐107, 143, 147 microemulsion(s), 14, 16, 38, 41, 73

precision, 68, 91‐95, 97, 99, 106, 108

106, 112, 113, 119, 120, 129, 139, 141 preseparation, 12, 124, 130, 131

pretreatment, 3, 5, 25, 46, 48, 68, 74, 91, 92, 120, 143, 147

protein(s), 23, 35, 36, 47, 62, 89, 96, 103, 130, 131, 136

relative standard deviation, 92‐94, 99, 105, 106, 108, 146

sample injection, 51, 58, 63, 66, 76, 78, 81, 97, 98, 104, 122, 130, 139

sample preconcentration with chemical derivatization, 55, 58, 78, 79, 97, 98

reproducibility, 25, 30, 59, 61‐63, 66, 68, 80, 92, 93, 96, 97, 108, 121, 125, 128, 130

resolution, 1, 2, 12‐16, 18‐21, 23, 27, 28, 34, 37, 41, 43, 44, 46‐49, 75, 94, 95, 102, 103, 108, 113,

sample preparation, 2, 3, 5, 7, 11, 21, 51‐55, 58, 75, 78, 80‐83, 90‐92, 100, 106, 112, 113, 119, 129,

selectivity, 2, 14, 16, 17, 19, 23, 24, 27, 28, 34, 35, 40, 41, 46‐48, 51, 61, 62, 66, 75, 83, 86, 92, 99,

purification, 3, 21, 52, 53, 59, 60, 62, 66, 80, 83, 87, 89, 120, 141, 147

protein precipitation, 7‐11, 54, 57, 58, 71, 145 pseudostationary phase(s), 12, 16, 21, 38, 60, 69, 73

111, 114, 123, 130, 139, 141‐143

primaquine, 7, 34 propaphenone, 7 propiconazol, 57

**R**

propranolol, 47, 56, 93, 108

radioisotope detection, 112 Raman‐based detection, 112 recovery, 62, 92‐94, 99, 105, 106 refractive index detection, 112

relative error, 93, 99

repeatability, 94

137, 139, 141‐143 retention time(s), 17, 92

robustness, 69

139, 141, 147, 148

sample self‐stacking, 71

105, 118, 134, 137, 139, 141

**S**

RSD, *see* relative standard deviation

salbutamol, 8, 38, 44, 145, 146

preconcentration, 2, 3, 11, 52‐59, 62, 64, 66, 70, 71, 73‐77, 79‐81, 89‐92, 94‐97, 100, 105, 106, 108,

Index 195

preparation, 2, 3, 5, 7, 11, 21, 30, 51‐55, 57‐59, 61, 63, 65, 67, 69, 71, 73, 75, 78, 80‐83, 90‐92, 100,

microemulsion electrokinetic chromatography, 5, 6, 16, 41 microextraction, 11, 58, 84, 86‐88, 103, 105, 147 microchip(s), 6, 11, 25‐28, 52, 80, 82, 86, 89, 101, 125 microchip capillary electrophoresis, 5, 6, 11, 25‐28, 42, 48‐50, 52, 54, 58, 60, 73, 84, 86, 89, 111, 118, 120, 142, 147 migration time(s), 19, 23, 25, 48, 77, 93, 94, 96, 97, 99, 108, 119, 124, 128, 141 miniaturization, 3, 51, 87 mirtazapine, 8, 10, 48 MLC, *see* microcolumn liquid chromatography mobility, 5, 12, 13, 19‐23, 32, 59, 66, 70‐72, 79, 113, 124, 126, 129 MS, *see* mass spectrometry

### **N**

NACE, *see* non‐aqueous capillary electrophoresis NMR, *see* nuclear magnetic resonance non‐aqueous capillary electrophoresis, 8, 9, 11, 17, 44, 55, 58, 128, 145, 146 normal stacking mode, 63, 64 nuclear magnetic resonance, 16, 31‐33, 111, 114, 116, 117

### **O**

ofloxacin, 7, 11 oligosaccharides, 28 on‐line, 2, 3, 5, 18, 24, 51‐53, 58, 66, 68, 70, 73‐75, 77, 79‐83, 85‐87, 89‐93, 97, 100, 102, 103, 105, 106, 108, 111‐113, 116‐119, 123, 124, 126‐132, 139‐144, 146 on‐line sample preparation, 51‐53, 58, 81, 82, 90‐92, 100, 106, 112, 113, 129, 139, 141 oral fluids, 10

### **P**

performance parameter(s), 92, 99, 105, 148 pharmaceutical analysis, 35, 52 pharmaceutical(s), 1, 3, 27, 35, 52, 55, 80, 92, 117, 127 pharmacokinetic study (‐ies), 54, 56, 99, 142 pheniramine, 14, 32, 44, 46, 54, 99, 100, 139‐141, 144 phenprocoumone, 10 PHM, *see* pheniramine pH‐mediated stacking, 69, 77, 95 photothermal refraction, 112 plasma, 7‐11, 31, 32, 34, 39, 43, 44, 48, 54, 55‐57, 77, 92, 93, 96, 102‐104, 106, 108, 132, 141, 143‐145 polysaccharide(s), 23, 34 PP, *see* protein precipitation

microchip capillary electrophoresis, 5, 6, 11, 25‐28, 42, 48‐50, 52, 54, 58, 60, 73, 84, 86, 89, 111,

on‐line, 2, 3, 5, 18, 24, 51‐53, 58, 66, 68, 70, 73‐75, 77, 79‐83, 85‐87, 89‐93, 97, 100, 102, 103, 105,

plasma, 7‐11, 31, 32, 34, 39, 43, 44, 48, 54, 55‐57, 77, 92, 93, 96, 102‐104, 106, 108, 132, 141, 143‐145

on‐line sample preparation, 51‐53, 58, 81, 82, 90‐92, 100, 106, 112, 113, 129, 139, 141

migration time(s), 19, 23, 25, 48, 77, 93, 94, 96, 97, 99, 108, 119, 124, 128, 141

non‐aqueous capillary electrophoresis, 8, 9, 11, 17, 44, 55, 58, 128, 145, 146

microemulsion electrokinetic chromatography, 5, 6, 16, 41

mobility, 5, 12, 13, 19‐23, 32, 59, 66, 70‐72, 79, 113, 124, 126, 129

microextraction, 11, 58, 84, 86‐88, 103, 105, 147 microchip(s), 6, 11, 25‐28, 52, 80, 82, 86, 89, 101, 125

MLC, *see* microcolumn liquid chromatography

NACE, *see* non‐aqueous capillary electrophoresis

nuclear magnetic resonance, 16, 31‐33, 111, 114, 116, 117

106, 108, 111‐113, 116‐119, 123, 124, 126‐132, 139‐144, 146

performance parameter(s), 92, 99, 105, 148

pharmacokinetic study (‐ies), 54, 56, 99, 142

pharmaceutical(s), 1, 3, 27, 35, 52, 55, 80, 92, 117, 127

pheniramine, 14, 32, 44, 46, 54, 99, 100, 139‐141, 144

pharmaceutical analysis, 35, 52

pH‐mediated stacking, 69, 77, 95 photothermal refraction, 112

phenprocoumone, 10 PHM, *see* pheniramine

polysaccharide(s), 23, 34 PP, *see* protein precipitation

NMR, *see* nuclear magnetic resonance

normal stacking mode, 63, 64

118, 120, 142, 147

**N**

**O**

**P**

ofloxacin, 7, 11 oligosaccharides, 28

oral fluids, 10

miniaturization, 3, 51, 87 mirtazapine, 8, 10, 48

MS, *see* mass spectrometry

precision, 68, 91‐95, 97, 99, 106, 108 preconcentration, 2, 3, 11, 52‐59, 62, 64, 66, 70, 71, 73‐77, 79‐81, 89‐92, 94‐97, 100, 105, 106, 108, 111, 114, 123, 130, 139, 141‐143 preparation, 2, 3, 5, 7, 11, 21, 30, 51‐55, 57‐59, 61, 63, 65, 67, 69, 71, 73, 75, 78, 80‐83, 90‐92, 100, 106, 112, 113, 119, 120, 129, 139, 141 preseparation, 12, 124, 130, 131 pretreatment, 3, 5, 25, 46, 48, 68, 74, 91, 92, 120, 143, 147 primaquine, 7, 34 propaphenone, 7 propiconazol, 57 propranolol, 47, 56, 93, 108 protein(s), 23, 35, 36, 47, 62, 89, 96, 103, 130, 131, 136 protein precipitation, 7‐11, 54, 57, 58, 71, 145 pseudostationary phase(s), 12, 16, 21, 38, 60, 69, 73 purification, 3, 21, 52, 53, 59, 60, 62, 66, 80, 83, 87, 89, 120, 141, 147

### **R**

radioisotope detection, 112 Raman‐based detection, 112 recovery, 62, 92‐94, 99, 105, 106 refractive index detection, 112 relative error, 93, 99 relative standard deviation, 92‐94, 99, 105, 106, 108, 146 repeatability, 94 reproducibility, 25, 30, 59, 61‐63, 66, 68, 80, 92, 93, 96, 97, 108, 121, 125, 128, 130 resolution, 1, 2, 12‐16, 18‐21, 23, 27, 28, 34, 37, 41, 43, 44, 46‐49, 75, 94, 95, 102, 103, 108, 113, 137, 139, 141‐143 retention time(s), 17, 92 robustness, 69 RSD, *see* relative standard deviation

#### **S**

salbutamol, 8, 38, 44, 145, 146 sample injection, 51, 58, 63, 66, 76, 78, 81, 97, 98, 104, 122, 130, 139 sample preparation, 2, 3, 5, 7, 11, 21, 51‐55, 58, 75, 78, 80‐83, 90‐92, 100, 106, 112, 113, 119, 129, 139, 141, 147, 148 sample preconcentration with chemical derivatization, 55, 58, 78, 79, 97, 98 sample self‐stacking, 71 selectivity, 2, 14, 16, 17, 19, 23, 24, 27, 28, 34, 35, 40, 41, 46‐48, 51, 61, 62, 66, 75, 83, 86, 92, 99, 105, 118, 134, 137, 139, 141

**U**

**V**

**W**

**Z**

130, 139‐141, 144‐146 UV‐VIS, 111‐117, 148

warfarin, 9, 39, 47

zwitterionic, 73

ZE, *see* zone electrophoresis

zone electrophoresis, 6, 13, 36, 65, 69, 71, 143

vancomycin, 10, 18, 19, 36, 47, 54, 57

urine, 2, 7‐11, 31, 32, 34, 38, 39, 43, 44, 47, 48, 54‐58, 68, 75, 77, 86, 88, 96‐101, 104, 105, 116,

Index 197

selector(s), 1—3, 5, 7, 11‐14, 16‐24, 27‐30, 34‐39, 41‐49, 51, 53, 54, 61, 64, 82, 96, 97, 100, 101, 109, 112, 113, 116, 121, 124, 125, 139, 140‐142, 145, 146 sensitivity, 1, 2, 7, 23, 43, 51, 69, 71, 73‐75, 83‐87, 92, 93, 95‐97, 102, 103, 105, 106, 108, 111‐114, 116‐119, 121, 123, 125, 127‐130, 134‐135, 137, 139, 141‐143, 145, 146 separation efficiency, 1, 12, 34, 35, 47, 60, 61, 63, 86, 90, 103, 113, 128, 130 serine, 8, 31, 32, 45, 46, 56, 89, 105, 142 serum, 7‐9, 11, 35, 38, 54, 56, 57, 93, 105 single column, 18, 61, 62, 64, 75, 76, 78‐81, 91, 102 sodium cholate, 10, 11, 38, 69 solid phase extraction, 7‐11, 48, 52, 55, 57, 58, 62, 63, 79, 83‐86, 91, 92, 96, 102, 103, 145, 146 solid‐phase microextraction, 55, 86, 87, 104, 105 solubility, 17, 24, 28, 30‐32 spacer, 122 SPCD, *see* sample preconcentration with chemical derivatization SPE, *see* solid phase extraction SPME, *see* solid‐phase microextraction stacking, 2, 11, 52, 53, 56, 58‐60, 63‐65, 67‐71, 73‐77, 90‐93, 95‐97, 105, 106, 108, 123, 128, 143 stationary phase(s), 5, 12, 16, 21, 23, 24, 26, 36, 38, 40, 43, 47, 60, 61, 69, 73 sulindac, 57, 93 surfactant(s), 39‐41, 74‐76, 128, 146 sweeping, 53, 55, 57, 60, 63, 73‐77, 94‐97 switching, 2, 65, 80, 124 SWP, *see* sweeping

### **T**

TE, *see* terminating electrolyte(s) temperature gradient focusing, 46, 79, 80, 98, 99 terbutaline, 54‐56, 85, 93, 97, 102, 103, 108 tergurides, 41 terminating electrolyte(s), 13, 71, 95, 100, 122, 126, 129, 131, 140 TGF, *see* temperature gradient focusing theoretical plates, 139 tITP, *see* transient isotachophoresis tissue(s), 2, 3, 45, 56, 77, 89, 106, 119, 142 tramadol, 8, 10, 144 transient isotachophoresis, 53, 55, 58, 59, 63, 70, 71, 75, 77, 78, 94, 95, 97 triadimenol, 57 trihexyphenidyl, 54 triple quadrupole, 137 tryptophan, 26, 58, 75, 102, 144

### **U**

196 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

selector(s), 1—3, 5, 7, 11‐14, 16‐24, 27‐30, 34‐39, 41‐49, 51, 53, 54, 61, 64, 82, 96, 97, 100, 101,

sensitivity, 1, 2, 7, 23, 43, 51, 69, 71, 73‐75, 83‐87, 92, 93, 95‐97, 102, 103, 105, 106, 108, 111‐114,

solid phase extraction, 7‐11, 48, 52, 55, 57, 58, 62, 63, 79, 83‐86, 91, 92, 96, 102, 103, 145, 146

stacking, 2, 11, 52, 53, 56, 58‐60, 63‐65, 67‐71, 73‐77, 90‐93, 95‐97, 105, 106, 108, 123,

stationary phase(s), 5, 12, 16, 21, 23, 24, 26, 36, 38, 40, 43, 47, 60, 61, 69, 73

109, 112, 113, 116, 121, 124, 125, 139, 140‐142, 145, 146

single column, 18, 61, 62, 64, 75, 76, 78‐81, 91, 102

solid‐phase microextraction, 55, 86, 87, 104, 105

serine, 8, 31, 32, 45, 46, 56, 89, 105, 142 serum, 7‐9, 11, 35, 38, 54, 56, 57, 93, 105

sodium cholate, 10, 11, 38, 69

solubility, 17, 24, 28, 30‐32

SPE, *see* solid phase extraction

SPME, *see* solid‐phase microextraction

surfactant(s), 39‐41, 74‐76, 128, 146 sweeping, 53, 55, 57, 60, 63, 73‐77, 94‐97

TE, *see* terminating electrolyte(s)

TGF, *see* temperature gradient focusing

tITP, *see* transient isotachophoresis tissue(s), 2, 3, 45, 56, 77, 89, 106, 119, 142

tryptophan, 26, 58, 75, 102, 144

temperature gradient focusing, 46, 79, 80, 98, 99 terbutaline, 54‐56, 85, 93, 97, 102, 103, 108

terminating electrolyte(s), 13, 71, 95, 100, 122, 126, 129, 131, 140

transient isotachophoresis, 53, 55, 58, 59, 63, 70, 71, 75, 77, 78, 94, 95, 97

spacer, 122

128, 143

**T**

sulindac, 57, 93

tergurides, 41

theoretical plates, 139

tramadol, 8, 10, 144

triadimenol, 57 trihexyphenidyl, 54 triple quadrupole, 137

switching, 2, 65, 80, 124 SWP, *see* sweeping

116‐119, 121, 123, 125, 127‐130, 134‐135, 137, 139, 141‐143, 145, 146 separation efficiency, 1, 12, 34, 35, 47, 60, 61, 63, 86, 90, 103, 113, 128, 130

SPCD, *see* sample preconcentration with chemical derivatization

urine, 2, 7‐11, 31, 32, 34, 38, 39, 43, 44, 47, 48, 54‐58, 68, 75, 77, 86, 88, 96‐101, 104, 105, 116, 130, 139‐141, 144‐146 UV‐VIS, 111‐117, 148

### **V**

vancomycin, 10, 18, 19, 36, 47, 54, 57

### **W**

warfarin, 9, 39, 47

### **Z**

ZE, *see* zone electrophoresis zone electrophoresis, 6, 13, 36, 65, 69, 71, 143 zwitterionic, 73

### *Authored by Peter Mikus*

The scientific monograph by the author Peter Mikus entitled "Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis" provides a comprehensive view on the advanced capillary electrophoresis techniques aimed to current chiral bioanalysis. The advances in the chiral electrophoresis analytical approaches are divided and theoretically described in three sections involving (i) advanced chiral separations for the optimization of chiral resolution (separation mechanisms; electrophoresis techniques in capillary and microchip format; electrophoretic modes such as ITP, CZE/EKC, CEC; chiral additives / pseudophases / phases), (ii) advanced sample preparation for the on-line preconcentration, sample clean-up and analyte derivatization (implementation of electrophoretic effects such as stacking; non-electrophoretic effects such as SPE, chromatography, dialysis; combinations of these effects; multidimensional CE systems; instrumental schemes), (iii) advanced combinations of detection and electrophoresis for the optimization in qualitative and quantitative evaluation (the most important universal as well as selective detection approaches such as absorption and fluorescence spectrophotometry, electrochemical detection, mass spectrometry vs. (i) and/ or (ii)). Real analytical potential (benefits and limitations) of these advanced analytical approaches is emphasized by selected performance parameters of the methods and illustrated by many current practical applications including chiral analyses of drugs, their (bio)degradation products and biomarkers in pharmaceutical and biological matrices. The author wishes the readers many inspirations in the creation of new innovative approaches in the field of advanced chiral electrophoresis techniques with the aim to overcome capabilities of the current analytical techniques.

Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Chiral Capillary

Electrophoresis in Current

Pharmaceutical and

Biomedical Analysis

*Authored by Peter Mikus*

Photo by RomoloTavani / iStock