Analytical Instrumentation and Methods

*Recent Advances in Analytical Chemistry*

2013;**5**:2509-2520

2017;**1041-1042**:19-26

2017;**89**:5193-5200

2018;**10**:1129-1141

2005;**44**:349-366

[28] Rochat B, Peduzzi D, McMullen J, Favre A, Kottelat E, Favrat B, et al. Validation of hepcidin quantification in plasma using LC-HRMS and discovery of a new hepcidin isoform. Bioanalysis.

[35] Partridge E, Trobbiani S, Stockham P, Scott T, Kostakis C. A validated method for the screening of 320 forensically significant compounds in blood by LC/QTOF, with simultaneous quantification of selected compounds. Journal of Analytical Toxicology.

[36] Li D, Cao Z, Liao X, Yang P, Liu L. The development of a quantitative and qualitative method based on UHPLC-QTOF MS/MS for evaluation paclitaxel-tetrandrine interaction and its application to a pharmacokinetic study. Talanta. 2016;**160**:256-267

[37] Qu L, Fan Y, Wang W, Ma K, Yin Z. Development, validation and clinical application of an online-SPE-LC-HRMS/MS for simultaneous quantification of phenobarbital, phenytoin, carbamazepine, and its active metabolite carbamazepine 10,11-epoxide. Talanta. 2016;**158**:77-88

[38] Tebani A, Afonso C, Marre S, Bekri S. Omics-based strategies in precision medicine: Toward a paradigm shift in inborn errors of metabolism investigations. International Journal of Molecular Sciences. 2016;**17**:E1555

[39] Dénes J, Szabó E, Robinette SL, Szatmári I, Szőnyi L, Kreuder JG, et al. Metabonomics of newborn screening dried blood spot samples: A novel approach in the screening and diagnostics of inborn errors of metabolism. Analytical Chemistry.

2012;**84**:0113-10120

2018;**42**:220-231

[29] Primikyri A, Papanastasiou M, Sarigiannis Y, Koutsogiannaki S, Reis ES, Tuplano JV, et al. Method development and validation for the quantitation of the complement inhibitor Cp40 in human and

cynomolgus monkey plasma by UPLC-ESI-MS. Journal of Chromatography B.

[30] Zhou X, Meng X, Cheng L, Su C, Sun Y, Sun L, et al. Development and application of an MSALL-based approach for the quantitative analysis of linear polyethylene glycols in rat plasma by liquid chromatography triple-quadrupole/time-of-flight mass spectrometry. Analytical Chemistry.

[31] Kellmann M, Muenster H, Zomer P, Mol JGJ. Full scan MS in comprehensive qualitative and quantitative residue analysis in food and feed matrices: How much resolving power is required? Journal of the American Society for Mass Spectrometry. 2009;**20**:1464-1476

[32] Rochat B. Fully-automated systems and the need for global approaches should exhort clinical labs to reinvent routine MS analysis? Bioanalysis.

[33] Rochat B. Role of cytochrome P450 activity in the fate of anticancer agents and in drug resistance: Focus on tamoxifen, paclitaxel and imatinib metabolism. Clinical Pharmacokinetics.

[34] Tonoli D, Varesio E, Hopfgartner G. Mass spectrometric QUAL/QUAN approaches for drug metabolism and metabolomics. Chimia. 2012;**66**:218-222

**20**

**23**

**Chapter 2**

**Abstract**

Animal Origin

pre-concentration, miniaturization

**1. Introduction**

food products.

Modern Extraction and Cleanup

 Extensive research on the presence of veterinary drug residues in food samples has been conducted and is still underway. The inappropriate or excessive use of veterinary drugs in food producing animals may result in trace quantities of these drugs or their metabolites in food samples. Food contamination by veterinary drug residues is one of the main challenges worldwide to public health with drug resistance being the biggest threat. One of the challenges in veterinary drug residue analysis is their occurrence in trace amounts that are normally below limits of detection of most analytical instruments. Various efficient, economical, miniaturized and environmentally friendly extraction methods have been developed in recent years to pre-concentrate these analytes before instrumental analysis to enhance their detection and also to overcome the limitations of traditional extraction methods such as liquid-liquid extraction and solid phase extraction. These methods include quick, easy, cheap, effective, rugged and safe (QuEChERS), molecularly imprinted polymers, dispersive liquid-liquid microextraction and hollow fiber liquid-phase microextraction, and they will be discussed in this chapter.

**Keywords:** veterinary drug residues, food samples, modern extraction methods,

Food is an indispensable part of human life and supplies the energy and nutrients needed for the development and growth of the neonate [1]. However, food safety is an important issue regarding residues of veterinary drugs in foods from food producing animals. Veterinary drugs are used to prevent and treat bacterial infections as well as improve feed efficiency and to promote animal growth worldwide [2]. The use of veterinary drugs in food producing animals may result in residues of the drugs or their metabolites being present in food samples, and this might be due to the inappropriate or excessive use of these drugs [3]. Various veterinary drugs have been reported to be retained in meat and milk of food producing animals [4–6] and this might be a health problem to humans who consume these

Methods of Veterinary Drug

Residues in Food Samples of

*Babra Moyo and Nikita Tawanda Tavengwa*

#### **Chapter 2**

## Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples of Animal Origin

*Babra Moyo and Nikita Tawanda Tavengwa*

## **Abstract**

 Extensive research on the presence of veterinary drug residues in food samples has been conducted and is still underway. The inappropriate or excessive use of veterinary drugs in food producing animals may result in trace quantities of these drugs or their metabolites in food samples. Food contamination by veterinary drug residues is one of the main challenges worldwide to public health with drug resistance being the biggest threat. One of the challenges in veterinary drug residue analysis is their occurrence in trace amounts that are normally below limits of detection of most analytical instruments. Various efficient, economical, miniaturized and environmentally friendly extraction methods have been developed in recent years to pre-concentrate these analytes before instrumental analysis to enhance their detection and also to overcome the limitations of traditional extraction methods such as liquid-liquid extraction and solid phase extraction. These methods include quick, easy, cheap, effective, rugged and safe (QuEChERS), molecularly imprinted polymers, dispersive liquid-liquid microextraction and hollow fiber liquid-phase microextraction, and they will be discussed in this chapter.

**Keywords:** veterinary drug residues, food samples, modern extraction methods, pre-concentration, miniaturization

#### **1. Introduction**

Food is an indispensable part of human life and supplies the energy and nutrients needed for the development and growth of the neonate [1]. However, food safety is an important issue regarding residues of veterinary drugs in foods from food producing animals. Veterinary drugs are used to prevent and treat bacterial infections as well as improve feed efficiency and to promote animal growth worldwide [2]. The use of veterinary drugs in food producing animals may result in residues of the drugs or their metabolites being present in food samples, and this might be due to the inappropriate or excessive use of these drugs [3]. Various veterinary drugs have been reported to be retained in meat and milk of food producing animals [4–6] and this might be a health problem to humans who consume these food products.

Various pre-treatment methods have been described for the extraction of veterinary drug residues in food samples, such as liquid-liquid extraction (LLE) [7–9], solid-phase extraction (SPE) [10], solid-phase micro extraction [11–14]. Pre-concentration is necessary because veterinary drug residues often occur in trace amounts. However, some of these methods are laborious and time consuming, like LLE and SPE. It is very important to develop simple, rapid and efficient methods for the determination of veterinary drug residues in foods samples. In recent years, extraction and pre-concentration techniques that are compliant to the green chemistry methods have been developed and they will be discussed in Section 6. Moreover, several countries and different international organizations such as the World Health Organization (WHO), the Food Agriculture Organization (FAO) and the European Union (EU) have set maximum residue limits (MRLs) of veterinary drug residues in food to ensure food safety.

#### **2. Physicochemical properties and uses of veterinary drugs**

Physicochemical properties and uses of different veterinary drug classes are described below. A few examples of the physicochemical properties of selected veterinary drugs are shown on **Table 1**. Sulfonamides (SAs) show impartially low sorption capacity to solids compared to other veterinary drugs. These are used for the treatment of bacterial infections in animal husbandry and also act as growth promotants. Sulphonamides are also used in farm animal feeds and fish cultures [15]. Examples include sulfadiazine, sulfamethazine, sulfamethoxazole and sulfaquinoxaline.

Tetracyclines (TCs), including tetracycline, oxytetracycline, chlortetracycline and doxycycline are broad-spectrum veterinary drugs with broad use in animal husbandry. They are amphoteric compounds. Generally, TCs are more stable in acidic conditions.

Quinolones (QNs) are synthetic veterinary drugs with broad-spectrum antibacterial effects. This veterinary drug class consists of plain quinolones, such as oxolinic acid and nalidixic acid and fluorinated quinolones, known as fluoroquinolones (FQs), such as ciprofloxacin, flumequine and sarafloxacin.

Amphenicols are a broad-spectrum veterinary drug group that include chloramphenicol and its metabolites, thiamphenicol and florfenicol. Florfenicol has its own metabolite, florfenicol amine. The most common member of this veterinary drug class is chloramphenicol which is effective against many bacterial strains. Its toxicity and unwanted effects have restricted its use over the past years [16, 17].

Macrolides are a class of semi-synthetic medium-spectrum veterinary drugs. The most commonly used macrolides have 12–16 membered structures. Erythromycin is the most common veterinary drug in this class. Generally, macrolides have weak characteristics and thus are unstable under acidic conditions. Examples include erythromycin, tylosin, spiramycin, tilmicosin and tulathromycin.

Beta lactam veterinary drugs consist of several groups of compounds with cephalosporins and penicillins among the most important. Penicillins are commonly used for their microbial activity against both gram-positive and gram-negative organisms. The main clean-up method of penicillins for their analysis is pre- and postcolumn derivatization and the commonly used detection methods are LC-MS and LC-UV. Examples of penicillins are amoxicillin, ampicillin, oxacillin and cloxacillin, and examples of cephalosporins are cephapyin, ceftiofur and cefadroxil.

Aminoglycosides are broad spectrum veterinary drugs with antibacterial and antifungal activities produced by *Streptomyces* and *Micromospora*. The use of aminoglycosides has been clinically limited to severe infections because of its toxicity. More toxic veterinary drugs in this class have been restricted to topical or oral use for the treatment of infections caused by *Enterobacteriaceae*. Less toxic aminoglycosides are

**25**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

Tetracyclines Tetracycline 231 3.3

Sulphonamides Sulfadiazine 77 6.5

Macrolides Erythromycin 2000 8.8

Quinolones Ciprofloxacin Insoluble —

**Name of antibiotic Solubility in water (mg L<sup>−</sup><sup>1</sup>**

Chlortetracycline ~8600 3.3 Doxycycline 50,000 — Oxytetracycline 1 × 106 3.27

Sulfamethoxazole 500 8.8 Sulfaquinoxaline 7.5 — Sulfamethazine 1500 8.43

Tylosin 5000 — Azithromycin <1000 —

Enrofloxacin 146 — Oxolinic acid Insoluble 6.87

**) pKa**

used for treatment of severe sepsis caused by gram-negative aerobes. Examples of aminoglycosides are streptomycin, kanamycin, tobramycin and gentamicin.

*Physicochemical properties of selected veterinary drugs from different classes.*

Nitrofurans are synthetic chemotherapeutic agents used in the treatment and prevention of gastrointestinal infections caused by *Escherichia coli* and *Salmonella*. They are broadly used in cattle, cows, pigs and poultry and are administered orally or as feed additives. Examples of nitrofurans include are furazolidine, furaltadone,

In summary of this section, generally, veterinary drugs are compounds characterized by a complex chemical structure that have very variable water solubilities, low volatilization potential, several ionizable functional groups (amphoteric molecules) and different pKa values hence they have a low bioaccumulation potential [18]. Veterinary drugs may have different functionalities within the same molecule, making them either neutral, cationic, anionic, or zwitterionic under different pH conditions. Different functionalities within a single molecule may result in its physicochemical and biological characteristics such as, sorption behavior, photo reactivity and toxicity changing with pH. Solubility and hydrophobicity are also are pH dependent. The pH dependency of antibiotic solubility can affect the extraction

The use of veterinary drugs in food producing animals can result in the presence of residues in animal derived products such meat, milk, eggs and honey. This poses a health hazard to the consumers [3]. Veterinary drugs such as macrolides, tetracyclines, sulfonamides and penicillins are also used as antibiotics in humans [20, 21]. Physicochemical properties of drugs, pharmacokinetic characteristics or biological processes of animals are factors that affect the presence of drug residues in food of animal origin. Improper drug usage and failure to observe withdrawal periods may be a reason for the occurrence of veterinary drug residues in foods derived from animals.

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

**Class of veterinary antibiotic** 

**drugs**

*–, Not available.*

**Table 1.**

nitrofurantoin and nitrofurazone.

and quantification by analytical techniques [19].

**3. Contamination of food by veterinary drugs**


*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

#### **Table 1.**

*Recent Advances in Analytical Chemistry*

drug residues in food to ensure food safety.

**2. Physicochemical properties and uses of veterinary drugs**

lones (FQs), such as ciprofloxacin, flumequine and sarafloxacin.

ity and unwanted effects have restricted its use over the past years [16, 17]. Macrolides are a class of semi-synthetic medium-spectrum veterinary drugs. The most commonly used macrolides have 12–16 membered structures. Erythromycin is the most common veterinary drug in this class. Generally, macrolides have weak characteristics and thus are unstable under acidic conditions. Examples include erythromycin, tylosin, spiramycin, tilmicosin and tulathromycin. Beta lactam veterinary drugs consist of several groups of compounds with cephalosporins and penicillins among the most important. Penicillins are commonly used for their microbial activity against both gram-positive and gram-negative organisms. The main clean-up method of penicillins for their analysis is pre- and postcolumn derivatization and the commonly used detection methods are LC-MS and LC-UV. Examples of penicillins are amoxicillin, ampicillin, oxacillin and cloxacillin,

and examples of cephalosporins are cephapyin, ceftiofur and cefadroxil.

Aminoglycosides are broad spectrum veterinary drugs with antibacterial and antifungal activities produced by *Streptomyces* and *Micromospora*. The use of aminoglycosides has been clinically limited to severe infections because of its toxicity. More toxic veterinary drugs in this class have been restricted to topical or oral use for the treatment of infections caused by *Enterobacteriaceae*. Less toxic aminoglycosides are

Physicochemical properties and uses of different veterinary drug classes are described below. A few examples of the physicochemical properties of selected veterinary drugs are shown on **Table 1**. Sulfonamides (SAs) show impartially low sorption capacity to solids compared to other veterinary drugs. These are used for the treatment of bacterial infections in animal husbandry and also act as growth promotants. Sulphonamides are also used in farm animal feeds and fish cultures [15]. Examples include sulfadiazine, sulfamethazine, sulfamethoxazole and sulfaquinoxaline.

Tetracyclines (TCs), including tetracycline, oxytetracycline, chlortetracycline and doxycycline are broad-spectrum veterinary drugs with broad use in animal husbandry. They are amphoteric compounds. Generally, TCs are more stable in acidic conditions. Quinolones (QNs) are synthetic veterinary drugs with broad-spectrum antibacterial effects. This veterinary drug class consists of plain quinolones, such as oxolinic acid and nalidixic acid and fluorinated quinolones, known as fluoroquino-

Amphenicols are a broad-spectrum veterinary drug group that include chloramphenicol and its metabolites, thiamphenicol and florfenicol. Florfenicol has its own metabolite, florfenicol amine. The most common member of this veterinary drug class is chloramphenicol which is effective against many bacterial strains. Its toxic-

Various pre-treatment methods have been described for the extraction of veterinary drug residues in food samples, such as liquid-liquid extraction (LLE) [7–9], solid-phase extraction (SPE) [10], solid-phase micro extraction [11–14]. Pre-concentration is necessary because veterinary drug residues often occur in trace amounts. However, some of these methods are laborious and time consuming, like LLE and SPE. It is very important to develop simple, rapid and efficient methods for the determination of veterinary drug residues in foods samples. In recent years, extraction and pre-concentration techniques that are compliant to the green chemistry methods have been developed and they will be discussed in Section 6. Moreover, several countries and different international organizations such as the World Health Organization (WHO), the Food Agriculture Organization (FAO) and the European Union (EU) have set maximum residue limits (MRLs) of veterinary

**24**

*Physicochemical properties of selected veterinary drugs from different classes.*

used for treatment of severe sepsis caused by gram-negative aerobes. Examples of aminoglycosides are streptomycin, kanamycin, tobramycin and gentamicin.

Nitrofurans are synthetic chemotherapeutic agents used in the treatment and prevention of gastrointestinal infections caused by *Escherichia coli* and *Salmonella*. They are broadly used in cattle, cows, pigs and poultry and are administered orally or as feed additives. Examples of nitrofurans include are furazolidine, furaltadone, nitrofurantoin and nitrofurazone.

In summary of this section, generally, veterinary drugs are compounds characterized by a complex chemical structure that have very variable water solubilities, low volatilization potential, several ionizable functional groups (amphoteric molecules) and different pKa values hence they have a low bioaccumulation potential [18]. Veterinary drugs may have different functionalities within the same molecule, making them either neutral, cationic, anionic, or zwitterionic under different pH conditions. Different functionalities within a single molecule may result in its physicochemical and biological characteristics such as, sorption behavior, photo reactivity and toxicity changing with pH. Solubility and hydrophobicity are also are pH dependent. The pH dependency of antibiotic solubility can affect the extraction and quantification by analytical techniques [19].

#### **3. Contamination of food by veterinary drugs**

The use of veterinary drugs in food producing animals can result in the presence of residues in animal derived products such meat, milk, eggs and honey. This poses a health hazard to the consumers [3]. Veterinary drugs such as macrolides, tetracyclines, sulfonamides and penicillins are also used as antibiotics in humans [20, 21]. Physicochemical properties of drugs, pharmacokinetic characteristics or biological processes of animals are factors that affect the presence of drug residues in food of animal origin. Improper drug usage and failure to observe withdrawal periods may be a reason for the occurrence of veterinary drug residues in foods derived from animals.

#### **4. Health effects**

The threat of food contamination by veterinary drug residues is one of the major challenges to public health worldwide [3]. The presence of low levels of veterinary drug residues may not have a negative impact on public health. However, the substantial use of drugs may increase the risk of adverse effects of these residues to humans [3, 22, 23]. Continuous ingestion of veterinary drug residues can promote the development of drug resistance bacterial strains in an individual, resulting in resistance to treatment with the same antibiotics when need arises [24–26]. Veterinary drug traces also have harmful effects on humans, such as allergic reactions, liver damage, yellowing of teeth and gastrointestinal disturbance [27]. Sulphonamides can cause drug intoxication and hypersensitivity. Signs of hypersensitivity and intoxication are fever and anemia respectively.

Manuring, treatment of animals and disposal of carcasses, offals, urine, feces and unused products can contaminate the environment with veterinary drugs [28]. An excessive use of antibiotics in commercially reared animals does not only affect humans, it can also affect the food chain leading to ecological imbalances. For example, a deficient management of the livestock carcasses can lead to antibiotic resistance in the scavengers that ingest them, like vultures [24–26]. The disposal of medicated animals should be regulated to minimize the risk in scavenger birds.

#### **5. Maximum residual limits**

The MRL values for food products result from calculations based upon the acceptable daily intake. MRL values depend on chronic toxicity of the antibiotic in question. More toxic drugs have lower MRL values compared to moderately toxic drugs. Prohibited substances are pharmacologically active substances for which an MRL cannot be established because of their toxicity and these include substances such as chloramphenicol, nitrofurans and nitroimidazoles. The kidney is the most important organ of drug excretion and that might be the reason why for most drugs it is allocated a higher MRL. For example, in the European Union (EU), countries have established a MRL of 200, 100, and 300 μg kg<sup>−</sup><sup>1</sup> for liver, muscle and kidney tissues, respectively for enrofloxacin and ciprofloxacin. The MRL set by the EU Committee for veterinary medicinal products is 200 μg kg<sup>−</sup><sup>1</sup> in muscles, liver and kidneys of animal origin, 40 μg kg<sup>−</sup><sup>1</sup> in milk, and 150 μg kg<sup>−</sup><sup>1</sup> in eggs for the macrolide drugs. **Table 2** shows some MRL values for different foods of animal origin.


**27**

required.

**6.1 QuEChERS**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

Tetracycline, oxytetracycline, chlortetracycline and doxycycline

Tetracycline, oxytetracycline, chlortetracycline and doxycycline

*Maximum residue limit for veterinary drug residues in food samples according to European Community,* 

**Target veterinary drug Matrix MRL** 

Macrolides Milk 40 Macrolides Eggs 150

**(μg kg<sup>−</sup><sup>1</sup> )**

Muscle, milk 100

Eggs 200

Veterinary drug residues in food of animal origin are of great concern to regulatory agencies and consumers, hence reliable extraction methods for rapid, selective and sensitive detection of these residues are necessary to ensure food safety [29]. There are various extraction methods that have been used in veterinary drug residues analysis in food samples, such as liquid-liquid extraction (LLE) [30–32] and solid phase extraction (SPE) [33, 34]. These methods suffer a number of drawbacks even though they perform their tasks adequately. Both LLE and SPE are environmentally unfriendly due to the large amounts of organic solvents they use, they are time consuming and labor intensive. Another disadvantage of SPE is that cartridges

Promising extraction and pre-concentration techniques for veterinary drug residues that have been explored recently by many researchers include dispersive liquid-liquid microextraction (DLLME) [5, 6, 35, 36], hollow fiber based liquidphase microextraction (HFLPME) [37–40] and quick, easy, cheap, effective, rugged and safe (QuEChERS) [4, 41–43] where the general trend is compliance with green chemistry principles. Veterinary drug residues occur at trace levels as low nanogram per gram [4, 37] hence the need to pre-concentrate. The application of QuEChERS, DLLME, HFLPME and molecularly imprinted polymers (MIPs) for the extraction and pre-concentration of veterinary drug residues in food samples will be discussed

The food industry also needs the development of new methods that are fast, easy and cheap for routine analysis of residues in food samples. The latest trend in drug residue analysis is the development of generic methods that are capable of monitoring a wide variety of compounds, belonging to different veterinary drug classes. This has proven to be challenge due to the varying chemistries and physicochemical properties of veterinary drugs from different classes, as a result, multi-class methods for veterinary drugs are still not so widespread although they are strongly

The quick easy cheap effective rugged safe (QuEChERS) method is an extraction technique that employs an organic solvent and phase separation using high salt content, in some cases followed by dispersive solid phase extraction (d-SPE) for sample clean up. The QuEChERS method, which was originally developed for pesticide analysis in fruits and vegetables [44, 45], has recently been proposed for the analysis of veterinary drugs in different food matrices [4, 41, 43, 46]. Recent

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

**Class of veterinary** 

**drugs**

Tetracyclines (single/total)

Tetracyclines (single/total)

**Table 2.**

**6. Pre-concentration techniques**

*Commission Regulation (EU) No. 37/2010.*

below and summarized in **Table 3**.

applications of this method are discussed below.

are costly.

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*


**Table 2.**

*Recent Advances in Analytical Chemistry*

**4. Health effects**

**26**

**Class of veterinary** 

**5. Maximum residual limits**

kidneys of animal origin, 40 μg kg<sup>−</sup><sup>1</sup>

**Target veterinary drug Matrix MRL** 

in milk, and 150 μg kg<sup>−</sup><sup>1</sup>

Sulphonamides Eggs Not

Enrofloxacin and ciprofloxacin Eggs Not

Enrofloxacin and ciprofloxacin Liver 200 Enrofloxacin and ciprofloxacin Kidney 300

other seafood

for liver, muscle and kidney

in muscles, liver and

in eggs for the macro-

and kidneys

Muscle 100

Sulphonamides Sulphonamides Milk, fish and

The threat of food contamination by veterinary drug residues is one of the major challenges to public health worldwide [3]. The presence of low levels of veterinary drug residues may not have a negative impact on public health. However, the substantial use of drugs may increase the risk of adverse effects of these residues to humans [3, 22, 23]. Continuous ingestion of veterinary drug residues can promote the development of drug resistance bacterial strains in an individual, resulting in resistance to treatment with the same antibiotics when need arises [24–26]. Veterinary drug traces also have harmful effects on humans, such as allergic reactions, liver damage, yellowing of teeth and gastrointestinal disturbance [27]. Sulphonamides can cause drug intoxication and hypersensitivity. Signs of hyper-

Manuring, treatment of animals and disposal of carcasses, offals, urine, feces and unused products can contaminate the environment with veterinary drugs [28]. An excessive use of antibiotics in commercially reared animals does not only affect humans, it can also affect the food chain leading to ecological imbalances. For example, a deficient management of the livestock carcasses can lead to antibiotic resistance in the scavengers that ingest them, like vultures [24–26]. The disposal of medicated animals should be regulated to minimize the risk in scavenger birds.

The MRL values for food products result from calculations based upon the acceptable daily intake. MRL values depend on chronic toxicity of the antibiotic in question. More toxic drugs have lower MRL values compared to moderately toxic drugs. Prohibited substances are pharmacologically active substances for which an MRL cannot be established because of their toxicity and these include substances such as chloramphenicol, nitrofurans and nitroimidazoles. The kidney is the most important organ of drug excretion and that might be the reason why for most drugs it is allocated a higher MRL. For example, in the European Union (EU), countries

tissues, respectively for enrofloxacin and ciprofloxacin. The MRL set by the EU

lide drugs. **Table 2** shows some MRL values for different foods of animal origin.

sensitivity and intoxication are fever and anemia respectively.

and oxolinic acid

Macrolides Macrolides Muscle, liver

Quinolones Danofloxacin, enrofloxacin-ciprofloxacin

have established a MRL of 200, 100, and 300 μg kg<sup>−</sup><sup>1</sup>

Committee for veterinary medicinal products is 200 μg kg<sup>−</sup><sup>1</sup>

**(μg kg<sup>−</sup><sup>1</sup> )**

100

allowed

allowed

200

**drugs**

*Maximum residue limit for veterinary drug residues in food samples according to European Community, Commission Regulation (EU) No. 37/2010.*

#### **6. Pre-concentration techniques**

Veterinary drug residues in food of animal origin are of great concern to regulatory agencies and consumers, hence reliable extraction methods for rapid, selective and sensitive detection of these residues are necessary to ensure food safety [29]. There are various extraction methods that have been used in veterinary drug residues analysis in food samples, such as liquid-liquid extraction (LLE) [30–32] and solid phase extraction (SPE) [33, 34]. These methods suffer a number of drawbacks even though they perform their tasks adequately. Both LLE and SPE are environmentally unfriendly due to the large amounts of organic solvents they use, they are time consuming and labor intensive. Another disadvantage of SPE is that cartridges are costly.

Promising extraction and pre-concentration techniques for veterinary drug residues that have been explored recently by many researchers include dispersive liquid-liquid microextraction (DLLME) [5, 6, 35, 36], hollow fiber based liquidphase microextraction (HFLPME) [37–40] and quick, easy, cheap, effective, rugged and safe (QuEChERS) [4, 41–43] where the general trend is compliance with green chemistry principles. Veterinary drug residues occur at trace levels as low nanogram per gram [4, 37] hence the need to pre-concentrate. The application of QuEChERS, DLLME, HFLPME and molecularly imprinted polymers (MIPs) for the extraction and pre-concentration of veterinary drug residues in food samples will be discussed below and summarized in **Table 3**.

The food industry also needs the development of new methods that are fast, easy and cheap for routine analysis of residues in food samples. The latest trend in drug residue analysis is the development of generic methods that are capable of monitoring a wide variety of compounds, belonging to different veterinary drug classes. This has proven to be challenge due to the varying chemistries and physicochemical properties of veterinary drugs from different classes, as a result, multi-class methods for veterinary drugs are still not so widespread although they are strongly required.

#### **6.1 QuEChERS**

The quick easy cheap effective rugged safe (QuEChERS) method is an extraction technique that employs an organic solvent and phase separation using high salt content, in some cases followed by dispersive solid phase extraction (d-SPE) for sample clean up. The QuEChERS method, which was originally developed for pesticide analysis in fruits and vegetables [44, 45], has recently been proposed for the analysis of veterinary drugs in different food matrices [4, 41, 43, 46]. Recent applications of this method are discussed below.


**29**

**Target antibiotic**

Florfenicol and Chloramphenicol

Pasteurized Milk

HPLC-UV

Traditional DLLME (chloroform as an extracting solvent and water as a dispenser solvent)

HFLPME (0.1 mol L−1 nitric

24. 8 ng g−1

0.5–20 ng g−1

1.25–40 ng g−1

—

[37]

danofloxacin

37.5 ng g−1

tetracycline

—

0.95–3.6 μg L−1

5–15 μg L−1

92.38–

[38]

107.3

acid and sodium chloride was

the acceptor phase, 10% w/v

Aliquat-336 in 1-octanol

HF-DLLME (chloroform as an

extracting solvent and water as a

dispenser solvent)

Carrier mediated three phase

6.0–27.4 μg L−1

0.5–1.0 μg L−1

0.5–1.0 μg L−1


[39]

HFLPME (0.1 M phosphoric

acid, 1.0 M sodium chloride with

pH = 1.6 as an acceptor phase,

0.05 M disodium hydrogen

phosphate (pH between 9.1 and 9.5)

as donor phase and 10% (w/v) of

Aliquat-336 in octanol as an SLM.

HFLPME-TiO2 (TiO2 was dispersed

—

0.21 μg L−1

—

89–99

[40]

in 1-octanol)

Mixed template MIP-MSPD (0.15 g

—

0.5–3.0 ng g−1

1.5–6.0 ng g−1

92–99

[57]

MMIP, methanol/water (2:8, v/v) as

a washing solvent, methanol/acetic

acid (9:1, v/v) as an eluting solvent)

Tylosin Eight FQs, eight

Pork

UPLC-PDA

SAs and four TCs

Milk

UV/Vis

Five QNs and four

Milk, honey,

HPLC-DAD

fish, liver and

muscles of

lamb

Milk

HPLC-UV

Four TCs Three TCs

Bovine milk

HPLC-UV

TCs

**Food matrix**

**Analytical technique**

**Extraction technique**

**Concentration of antibiotic detected**

62.4 μg kg−1 florfenicol

12.2 and 12.5 μg kg−1

36.6 and 37.5 μg kg−1

—

[53]

**LOD**

**LOQ**

**Recovery (%)**

**References**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

#### *Recent Advances in Analytical Chemistry*


#### *Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

*Recent Advances in Analytical Chemistry*

**28**

**Target antibiotic**

Seven macrolides

Six multi-residues

Sixteen

Preserved

UHPLC-MS/

MS

eggs

multi-residues

Three SAs Seven TCs Several SAs

Six FQs Four TCs

Milk Milk and eggs

HPLC-UV

IL-DLLME ([C

MIM][PF

6] as an

—

6 extraction solvent, FIL-NOSM a

disperser solvent)

HPLC-UV

Milk

HPLC-FD

Beef

LC-MS/MS

Chicken

HPLC-DAD

breast

Bovine milk

LC-MS/MS

Milk

LC-MS/MS

QuEChERS based on acetonitrile

extraction + a mixture of salts

(sodium sulfate, sodium chloride

and potassium carbonate)

QuEChERS based on acetonitrile

—

—

—

84.18–

[42]

115.99

followed by a cleanup with d-SPE

based C18, PSA and sodium acetate

QuEChERS based on water,

—

0.1–0.9 μg kg−1

0.3–3.0 μg kg−1

73.8–127.4

[43]

acetonitrile with 1% acetic acid

followed by a cleanup using d-SPE

with C18 and PSA as sorbents

QuEChERS based on acetonitrile

—

10 and

25–30 μg kg−1

75.4–98.7

[46]

13 μg kg−1

and water with 1% CH

 CO 3 H 2 followed by a cleanup using d-SPE

Oasis HLB as a sorbent.

DLLME, methanol was a disperser

38.4 and

2.2–3.6 μg kg−1

7.4–11.5 μg kg−1

80–105

[5]

82.3 μg kg−1

solvent and dichloromethane was

an extracting solvent

Traditional DLLME (extraction

—

0.73–1.21 μg L−1

—

92.9–104.7

[36]

solvent (1 mL chloroform)

and dispersive solvent (1.9 mL

acetonitrile)

DLLME was coupled to QuEChERS

—

—

0.08–

—

94.1–102.1

[6]

1.12 μg kg−1

2.5–15 μg kg−1

74.1–101.4

[35]

**Food matrix**

**Analytical** 

**Extraction technique**

**Concentration** 

**LOD**

**LOQ**

**Recovery** 

**References**

**(%)**

**of antibiotic** 

**detected**

—

0.84 μg kg−1

2.79 μg kg−1

89–97

[41]

**technique**


*Recent Advances in Analytical Chemistry*

**Table 3.**

**31**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

da Costa et al. [41] developed a modified QuEChERS extraction technique using acetonitrile, followed by the addition of a mixture of salts (sodium sulfate, sodium chloride and potassium carbonate) for the extraction of seven macrolide drugs in milk followed by analysis on liquid chromatography and tandem mass spectrometry (LC-MS/MS). Sodium sulfate and sodium chloride removed water from samples promoting the salting out effect while acetonitrile was used for deproteination. Potassium carbonate salt was included to elevate the extraction pH to around 9.5 promoting an increase in the recovery, since macrolides have a pKa between 6.6 and 9.2. The limit of detection (LOD) and limit of quantification (LOQ ) were 0.84

further clean-up step such as an additional d-SPE step was required, hence reducing

In another study by Wang et al. [42], a modified QuEChERS extraction technique based on octadecylsilane (C18), primary secondary amine (PSA) and sodium acetate for six multi-residue veterinary drugs in bovine milk followed by analysis using LC-MS/MS. The QuEChERS method was optimized for use in the determination of multi-class veterinary drug residues in fatty foods (milk) using response surface methodology. The amounts of C18, PSA, and sodium acetate used in this study were determined by the response surface methodology variables. PSA, C18 and sodium acetate have a dissolving effect on milk-fat globules and hence, resulting in higher recoveries (84.18–115.99%) compared to da Costa et al. [41]. Organic solvents, such as acetonitrile, methanol and ethanol, are commonly employed in the precipitation of proteins in biological matrices. For all residues, the LOQs were low

Li et al. [43] employed the QuEChERS method followed by d-SPE coupled to ultrahigh-performance liquid chromatography tandem mass spectrometry for the multi-residue analysis of 16 veterinary drugs belonging to three classes (macrolides, quinolones, and sulfonamides) in preserved eggs. Graphitized carbon black was used as a comparative d-SPE sorbent. The recoveries of all veterinary drugs decreased with the addition of graphitized carbon black, while purification with a conjugation of PSA and C18 in the presence of magnesium sulfate resulted in better

) which indicated that the proposed

while recoveries

results. The results demonstrated good linearity, accuracy, precision, LOD

method was highly sensitive and could efficiently determine trace amounts.

and the LOQs ranged between 25 and 30 μg kg<sup>−</sup><sup>1</sup>

Machado et al. [46] developed a QuEChERS method followed by analysis on the high performance liquid chromatography (HPLC) with a diode array detector (DAD) for the simultaneous determination of sulfadiazine, sulfamethoxazole and sulfamethoxypyridazine in chicken breast samples. The LODs ranged between 10

ranged between 75.4 and 98.7%. SPE was done for comparison and recoveries lower than 70% were obtained. However, SPE, proved to reduce the matrix effect com-

Traditional sample preparation techniques such as liquid-liquid extraction (LLE) have drawbacks in spite of the substantial use of this method over the years. The LLE method is tedious, time consuming and uses large amounts of toxic organic solvents which are non-compliant to the green analytical chemistry (GAC) principles. In order to overcome these drawbacks, new extraction techniques that are simple, rapid and inexpensive, miniaturized and have the ability of automation have been developed in recent years [47]. The efforts of various researchers in this area have resulted in the development of a new extraction technique known as

) and LOQ (0.3–3.0 μg kg<sup>−</sup><sup>1</sup>

respectively and recoveries were ranging between 89 and 97%. No

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

enough to quantify the analytes below their MRLs.

and 2.79 μg kg<sup>−</sup><sup>1</sup>

(0.1–0.9 μg kg<sup>−</sup><sup>1</sup>

and 13 μg kg<sup>−</sup><sup>1</sup>

pared to the QuEChERS method.

**6.2 Liquid phase microextraction**

time, cost and labor.

 *Modern analytical techniques in the analysis of antibiotic residues in food samples.*

#### *Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

da Costa et al. [41] developed a modified QuEChERS extraction technique using acetonitrile, followed by the addition of a mixture of salts (sodium sulfate, sodium chloride and potassium carbonate) for the extraction of seven macrolide drugs in milk followed by analysis on liquid chromatography and tandem mass spectrometry (LC-MS/MS). Sodium sulfate and sodium chloride removed water from samples promoting the salting out effect while acetonitrile was used for deproteination. Potassium carbonate salt was included to elevate the extraction pH to around 9.5 promoting an increase in the recovery, since macrolides have a pKa between 6.6 and 9.2. The limit of detection (LOD) and limit of quantification (LOQ ) were 0.84 and 2.79 μg kg<sup>−</sup><sup>1</sup> respectively and recoveries were ranging between 89 and 97%. No further clean-up step such as an additional d-SPE step was required, hence reducing time, cost and labor.

In another study by Wang et al. [42], a modified QuEChERS extraction technique based on octadecylsilane (C18), primary secondary amine (PSA) and sodium acetate for six multi-residue veterinary drugs in bovine milk followed by analysis using LC-MS/MS. The QuEChERS method was optimized for use in the determination of multi-class veterinary drug residues in fatty foods (milk) using response surface methodology. The amounts of C18, PSA, and sodium acetate used in this study were determined by the response surface methodology variables. PSA, C18 and sodium acetate have a dissolving effect on milk-fat globules and hence, resulting in higher recoveries (84.18–115.99%) compared to da Costa et al. [41]. Organic solvents, such as acetonitrile, methanol and ethanol, are commonly employed in the precipitation of proteins in biological matrices. For all residues, the LOQs were low enough to quantify the analytes below their MRLs.

Li et al. [43] employed the QuEChERS method followed by d-SPE coupled to ultrahigh-performance liquid chromatography tandem mass spectrometry for the multi-residue analysis of 16 veterinary drugs belonging to three classes (macrolides, quinolones, and sulfonamides) in preserved eggs. Graphitized carbon black was used as a comparative d-SPE sorbent. The recoveries of all veterinary drugs decreased with the addition of graphitized carbon black, while purification with a conjugation of PSA and C18 in the presence of magnesium sulfate resulted in better results. The results demonstrated good linearity, accuracy, precision, LOD (0.1–0.9 μg kg<sup>−</sup><sup>1</sup> ) and LOQ (0.3–3.0 μg kg<sup>−</sup><sup>1</sup> ) which indicated that the proposed method was highly sensitive and could efficiently determine trace amounts.

Machado et al. [46] developed a QuEChERS method followed by analysis on the high performance liquid chromatography (HPLC) with a diode array detector (DAD) for the simultaneous determination of sulfadiazine, sulfamethoxazole and sulfamethoxypyridazine in chicken breast samples. The LODs ranged between 10 and 13 μg kg<sup>−</sup><sup>1</sup> and the LOQs ranged between 25 and 30 μg kg<sup>−</sup><sup>1</sup> while recoveries ranged between 75.4 and 98.7%. SPE was done for comparison and recoveries lower than 70% were obtained. However, SPE, proved to reduce the matrix effect compared to the QuEChERS method.

#### **6.2 Liquid phase microextraction**

Traditional sample preparation techniques such as liquid-liquid extraction (LLE) have drawbacks in spite of the substantial use of this method over the years. The LLE method is tedious, time consuming and uses large amounts of toxic organic solvents which are non-compliant to the green analytical chemistry (GAC) principles. In order to overcome these drawbacks, new extraction techniques that are simple, rapid and inexpensive, miniaturized and have the ability of automation have been developed in recent years [47]. The efforts of various researchers in this area have resulted in the development of a new extraction technique known as

*Recent Advances in Analytical Chemistry*

**30**

**Target antibiotic**

Four TCs Ten macrolides

Swine, cattle

LC-MS/MS

and chicken

muscles

Ten FQs Three FQs

TCs *–, Not mentioned; ND, not detected.*

**Table 3.**

*Modern analytical techniques in the analysis of antibiotic residues in food samples.*

Milk

Fluorescent

sensing

Milk

HPLC-UV

Fish

HPLC-FLD

Pork, milk

HPLC-PDA

MIP-SPE (30 mg MIP particles,

0.01 mol L−1 trifluoroacetic acid,

pH 3.0 as the loading solvent,

methanol/acetic acid (9:1, v/v) as

the elution solvent)

MIP-SPE (20 mg MIP particles,

—

0.1–0.4 μg kg−1

0.3–1.0 μg kg−1

60.7–

[56]

100.3

10% methanol in water as the

washing solvent, 5% ammonia in

methanol as the elution solvent)

DMIP-MSPD (50 mg MIP particles,

—

0.06–

—

64.4–102.7

[59]

0.22 ng g−1

20% methanol in water as the

washing solvent, 1% trifluoroacetic

acid in acetonitrile as the elution

solvent)

Mini-MISPE (40 mg MIP particles,

Ciprofloxacin:

1.5–2.3 ng mL−1

5.0–7.5 ng mL−1

87.2–106.1

[60]

0.21 and

0.25 g mL−1

water as the washing solvent,

methanol-acetic acid (19:1, v/v) as

the elution solvent)

CDs@MIPs (40 mg MIP particles,

ND

5.48 nM

—

97.3–105.3

[61]

1% (v/v) trichloroacetic acid

solution was a solvent)

and eggs

**Food matrix**

**Analytical** 

**Extraction technique**

**Concentration** 

**LOD**

**LOQ**

**Recovery** 

**References**

**(%)**

**of antibiotic** 

**detected**

52 ng mL−1: TC

20–40 ng mL−1

50–80 ng mL−1

74–93

[58]

87 ng mL−1:

oxytetracycline

in milk only

**technique**

liquid-phase microextraction (LPME). LPME offers an alternative to SPME [48]. LPME can be divided into three main modes which are single-drop microextraction (SD-LPME), hollow fiber liquid phase microextraction (HFLPME) and dispersive liquid-liquid microextraction (DLLME). Among these modes of LPME, HFLPME and DLLME have been the most used because of the advantages that they offer [47]. SD-LPME is the least used mode because excessive stirring tends to break up the droplet, extraction is time consuming and reaching equilibrium can be a challenge. This disadvantage overrides the advantage that this method has, which is the enormous reduction of volumes of organic solvent it uses [49].

These methods are cheap and do not have sample carryover problems that are associated with SPME [48]. LPME offers advantages such as high recovery and high enrichment factors, simplicity of operation, rapidity and they are also environment friendly [50]. Below is a summary of some studies that have used DLLME and HFLPME for the extraction of veterinary drug residues from food samples.

#### *6.2.1 Dispersive liquid-liquid microextraction*

Rezaee and co-workers [51] introduced DLLME as a new LLE technique for the determination of polyaromatic hydrocarbons and pesticides. The application of DLLME in the extraction of veterinary drugs in literature has increased over the years [5, 6, 35, 36]. This technique is based on a ternary component solvent system including an extraction solvent, disperser solvent and an aqueous sample and is known as traditional DLLME. The advantages of traditional DLLME are the microliter-level volumes required for extraction and dispersive solvents and short extraction times. However, the disadvantage of traditional DLLME is the use of organic solvents as the extraction and dispersive solvents.

Modified modes of DLLME have been invented recently and they include, low-density solvent based DLLME (LDS-DLLME), solidified floating organic drop DLLME (SFO-DLLME), effervescence assisted DLLME, air assisted dispersive liquid-liquid microextraction (AA-DLLME), surfactant assisted DLLME (SA-DLLME) and cloud point DLLME (CP-DLLME), ionic liquid DLLME (IL-DLLME) [6] to address the disadvantages associated with traditional DLLME. Despite these disadvantages, DLLME is more advantageous in terms of short total time, low cost and feasibility compared with other liquid-phase microextraction techniques [52]. Below is research that has been done recently on veterinary drugs in food samples using DLLME.

Mookantsa et al. [5] employed traditional DLLME for the extraction of seven tetracyclines from beef where methanol was a disperser solvent and dichloromethane was an extracting solvent followed by LC-MS/MS. Recoveries of spiked blank muscle samples at three levels (50, 100 and 150 μg kg<sup>−</sup><sup>1</sup> ) ranged from 80–105%. LODs and LOQs ranged from 2.2 to 3.6 μg kg<sup>−</sup><sup>1</sup> and from 7.4 to 11.5 μg kg<sup>−</sup><sup>1</sup> respectively. Concentrations of chlortetracycline and oxytetracycline were detected in bovine muscle samples to be between 38.4 and 82.3 μg kg<sup>−</sup><sup>1</sup> which is lower than the stipulated European Union MRL of 100 μg kg<sup>−</sup><sup>1</sup> . DLLME was compared to a South African National Accreditation System accredited d-SPE method and the t-test showed that the results obtained by the methods had no significant difference. However, DLLME was simple, fast, inexpensive and uses very low volumes of organic solvents hence more greener compared to d-SPE.

In a study done by Karami-Osboo et al. [35], DLLME was coupled to QuEChERS for the determination of six fluoroquinolones using HPLC with ultra-violet (UV) detection. The dried supernatant from the QuEChERS method was resuspended in 1.0 mL of a 10% acetic acid-acetonitrile mixture, combined with 200 μL of chloroform and rapidly injected into 4 mL of deionized water. The cloudy solution

**33**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

was centrifuged for 5 minutes at 4500 rpm. By coupling QuEChERS to DLLME, the authors removed matrix interference, which is a common problem with the detection of fluoroquinolones. The method showed good recoveries (74.1–101.4% for all

Arroyo-Manzanares et al. [36] used traditional DLLME for the determination of several sulfonamides in milk. The analytes were detected by HPLC with fluorescence detection. The authors also compared their optimized DLLME procedure to QuEChERS. Proteins were precipitated using trichloroacetic acid and then filtered. The DLLME extraction procedure was optimized using a central composite design. The optimum volumes for chloroform as an extraction solvent and acetonitrile as a dispersive solvent were 1 and 1.9 mL, respectively. DLLME resulted in lower LODs

104.7% compared to 83.6–97.1%, when samples were spiked with sulfonamides at

milk producing animals from which milk is produced for human consumption, it

Ionic liquids (ILs), consisting of organic cations and inorganic or organic anions with melting points at or below 100°C, have been widely applied as green solvents to improve extraction and enrichment performance as compared to the traditional use of organic solvents. A significant advantage of this method is that the metathesis reaction and extraction are accomplished in one step making it rapid and suitable for high-throughput analysis. Gao et al. [6] used functionalized ionic liquid-based non-organic solvent microextraction (FIL-NOSM) based on 1-butyl-3-methylimidazolium naphthoic acid salt ([C4MIM][NPA]) with strong acidity for the determination of TCs in milk and eggs. The use of [C4MIM][NPA] in the FIL-NOSM method eliminated the pH adjustment step because of its strong acidity which saves as a pH regulator. This proposed method provided high extraction efficiency, less pretreatment time and requires non-organic solvents for determination of trace TC concentrations in complex animal-based food matrices. Moreover, no organic solvent was utilized in this IL-based DLLME procedure making this method more environmentally friendly. The LODs were between 0.08 and 1.12 μg kg<sup>−</sup><sup>1</sup>

was detected in one of the samples at a concentration of 62.4 μg kg<sup>−</sup><sup>1</sup>

milk and egg samples. The recoveries ranged from 94.1 to 102.1%.

Hollow fiber liquid phase microextraction is a mode of LPME that uses a porous polypropylene hollow fiber for immobilization of organic solvent in its pores. The development of HFLPME provides a way to stabilize the extraction droplet in SD-LPME by placing it in a hollow fiber [54]. The main consumable material is the hollow fiber membrane, which is lower than other methods in cost and sample consumption [38]. The different modes of HFLPME are static, dynamic, two and three phase. The advantages of HFLPME are high enrichment, high degree of sample clean-up and low solvent consumption. The disadvantage

*6.2.2 Hollow fiber liquid phase microextraction*

lower relative standard deviation values of 2.9–7.1 and 3.0–9.7%, respectively. In another study by Karami-Osboo et al. [53], traditional DLLME coupled to HPLC- UV was used for the determination of chloramphenicol and florfenicol residues in milk samples where chloroform was used as extraction solvent and the deproteinized milk as a disperser solvent. The blank milk samples were spiked at three levels, 150, 300 and 600 μg of each chloramphenicol and florfenicol per kg of milk and recoveries were between 69.1 and 79.4%. The LODs for chloramphenicol

. However, QuEChERS proved to be more reproducible than DLLME with

respectively. Despite the use of florfenicol not being permitted for

) than QuEChERS (1.15–2.73 μg L<sup>−</sup><sup>1</sup>

for danofloxacin and below 15 μg kg<sup>−</sup><sup>1</sup>

respectively whereas the LOQs were 37.5

.

in

) and higher recoveries (92.9–

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

analytes) and low LOQs (below 2.5 μg kg<sup>−</sup><sup>1</sup>

and florfenicol were 12.5 and 12.2 μg kg<sup>−</sup><sup>1</sup>

for all other FQs).

(0.73–1.21 μg L<sup>−</sup><sup>1</sup>

and 36.6 μg kg<sup>−</sup><sup>1</sup>

150 μg L<sup>−</sup><sup>1</sup>

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

was centrifuged for 5 minutes at 4500 rpm. By coupling QuEChERS to DLLME, the authors removed matrix interference, which is a common problem with the detection of fluoroquinolones. The method showed good recoveries (74.1–101.4% for all analytes) and low LOQs (below 2.5 μg kg<sup>−</sup><sup>1</sup> for danofloxacin and below 15 μg kg<sup>−</sup><sup>1</sup> for all other FQs).

Arroyo-Manzanares et al. [36] used traditional DLLME for the determination of several sulfonamides in milk. The analytes were detected by HPLC with fluorescence detection. The authors also compared their optimized DLLME procedure to QuEChERS. Proteins were precipitated using trichloroacetic acid and then filtered. The DLLME extraction procedure was optimized using a central composite design. The optimum volumes for chloroform as an extraction solvent and acetonitrile as a dispersive solvent were 1 and 1.9 mL, respectively. DLLME resulted in lower LODs (0.73–1.21 μg L<sup>−</sup><sup>1</sup> ) than QuEChERS (1.15–2.73 μg L<sup>−</sup><sup>1</sup> ) and higher recoveries (92.9– 104.7% compared to 83.6–97.1%, when samples were spiked with sulfonamides at 150 μg L<sup>−</sup><sup>1</sup> . However, QuEChERS proved to be more reproducible than DLLME with lower relative standard deviation values of 2.9–7.1 and 3.0–9.7%, respectively.

In another study by Karami-Osboo et al. [53], traditional DLLME coupled to HPLC- UV was used for the determination of chloramphenicol and florfenicol residues in milk samples where chloroform was used as extraction solvent and the deproteinized milk as a disperser solvent. The blank milk samples were spiked at three levels, 150, 300 and 600 μg of each chloramphenicol and florfenicol per kg of milk and recoveries were between 69.1 and 79.4%. The LODs for chloramphenicol and florfenicol were 12.5 and 12.2 μg kg<sup>−</sup><sup>1</sup> respectively whereas the LOQs were 37.5 and 36.6 μg kg<sup>−</sup><sup>1</sup> respectively. Despite the use of florfenicol not being permitted for milk producing animals from which milk is produced for human consumption, it was detected in one of the samples at a concentration of 62.4 μg kg<sup>−</sup><sup>1</sup> .

Ionic liquids (ILs), consisting of organic cations and inorganic or organic anions with melting points at or below 100°C, have been widely applied as green solvents to improve extraction and enrichment performance as compared to the traditional use of organic solvents. A significant advantage of this method is that the metathesis reaction and extraction are accomplished in one step making it rapid and suitable for high-throughput analysis. Gao et al. [6] used functionalized ionic liquid-based non-organic solvent microextraction (FIL-NOSM) based on 1-butyl-3-methylimidazolium naphthoic acid salt ([C4MIM][NPA]) with strong acidity for the determination of TCs in milk and eggs. The use of [C4MIM][NPA] in the FIL-NOSM method eliminated the pH adjustment step because of its strong acidity which saves as a pH regulator. This proposed method provided high extraction efficiency, less pretreatment time and requires non-organic solvents for determination of trace TC concentrations in complex animal-based food matrices. Moreover, no organic solvent was utilized in this IL-based DLLME procedure making this method more environmentally friendly. The LODs were between 0.08 and 1.12 μg kg<sup>−</sup><sup>1</sup> in milk and egg samples. The recoveries ranged from 94.1 to 102.1%.

#### *6.2.2 Hollow fiber liquid phase microextraction*

Hollow fiber liquid phase microextraction is a mode of LPME that uses a porous polypropylene hollow fiber for immobilization of organic solvent in its pores. The development of HFLPME provides a way to stabilize the extraction droplet in SD-LPME by placing it in a hollow fiber [54]. The main consumable material is the hollow fiber membrane, which is lower than other methods in cost and sample consumption [38]. The different modes of HFLPME are static, dynamic, two and three phase. The advantages of HFLPME are high enrichment, high degree of sample clean-up and low solvent consumption. The disadvantage

*Recent Advances in Analytical Chemistry*

liquid-phase microextraction (LPME). LPME offers an alternative to SPME [48]. LPME can be divided into three main modes which are single-drop microextraction (SD-LPME), hollow fiber liquid phase microextraction (HFLPME) and dispersive liquid-liquid microextraction (DLLME). Among these modes of LPME, HFLPME and DLLME have been the most used because of the advantages that they offer [47]. SD-LPME is the least used mode because excessive stirring tends to break up the droplet, extraction is time consuming and reaching equilibrium can be a challenge. This disadvantage overrides the advantage that this method has, which is the

These methods are cheap and do not have sample carryover problems that are associated with SPME [48]. LPME offers advantages such as high recovery and high enrichment factors, simplicity of operation, rapidity and they are also environment friendly [50]. Below is a summary of some studies that have used DLLME and HFLPME for the extraction of veterinary drug residues from food samples.

Rezaee and co-workers [51] introduced DLLME as a new LLE technique for the determination of polyaromatic hydrocarbons and pesticides. The application of DLLME in the extraction of veterinary drugs in literature has increased over the years [5, 6, 35, 36]. This technique is based on a ternary component solvent system including an extraction solvent, disperser solvent and an aqueous sample and is known as traditional DLLME. The advantages of traditional DLLME are the microliter-level volumes required for extraction and dispersive solvents and short extraction times. However, the disadvantage of traditional DLLME is the use of

Modified modes of DLLME have been invented recently and they include, low-density solvent based DLLME (LDS-DLLME), solidified floating organic drop DLLME (SFO-DLLME), effervescence assisted DLLME, air assisted dispersive liquid-liquid microextraction (AA-DLLME), surfactant assisted DLLME (SA-DLLME) and cloud point DLLME (CP-DLLME), ionic liquid DLLME (IL-DLLME) [6] to address the disadvantages associated with traditional DLLME. Despite these disadvantages, DLLME is more advantageous in terms of short total time, low cost and feasibility compared with other liquid-phase microextraction techniques [52]. Below is research that has been done recently on veterinary drugs in food samples

Mookantsa et al. [5] employed traditional DLLME for the extraction of seven tetracyclines from beef where methanol was a disperser solvent and dichloromethane was an extracting solvent followed by LC-MS/MS. Recoveries of spiked blank

tively. Concentrations of chlortetracycline and oxytetracycline were detected in

South African National Accreditation System accredited d-SPE method and the t-test showed that the results obtained by the methods had no significant difference. However, DLLME was simple, fast, inexpensive and uses very low volumes of

In a study done by Karami-Osboo et al. [35], DLLME was coupled to QuEChERS for the determination of six fluoroquinolones using HPLC with ultra-violet (UV) detection. The dried supernatant from the QuEChERS method was resuspended in 1.0 mL of a 10% acetic acid-acetonitrile mixture, combined with 200 μL of chloroform and rapidly injected into 4 mL of deionized water. The cloudy solution

) ranged from 80–105%.

which is lower than

. DLLME was compared to a

respec-

and from 7.4 to 11.5 μg kg<sup>−</sup><sup>1</sup>

enormous reduction of volumes of organic solvent it uses [49].

organic solvents as the extraction and dispersive solvents.

muscle samples at three levels (50, 100 and 150 μg kg<sup>−</sup><sup>1</sup>

bovine muscle samples to be between 38.4 and 82.3 μg kg<sup>−</sup><sup>1</sup>

organic solvents hence more greener compared to d-SPE.

LODs and LOQs ranged from 2.2 to 3.6 μg kg<sup>−</sup><sup>1</sup>

the stipulated European Union MRL of 100 μg kg<sup>−</sup><sup>1</sup>

*6.2.1 Dispersive liquid-liquid microextraction*

**32**

using DLLME.

of HFLPME procedure is that it is slow with extraction times ranging from 15 to 45 minutes and target analytes may partly be trapped in the supporting liquid membrane (SLM) [39]. Another disadvantage is that there is no complete setup commercially available for this method although hollow fibers are commercially available [55]. Below are some recent studies on veterinary drug residues that have been carried out using HFLPME.

Tajabadi et al. [37] used a carrier mediated three phase HLFPME prior to analysis on the HPLC-DAD for the simultaneous determination of the veterinary drug residues of four TCs and five QNs in a wide range of animal source food samples such as fish, milk and honey as well as the liver and muscles of lamb and chicken. Multivariate curve resolution-alternative least squares was used for resolving some overlapped peaks in multivariate data of HPLC-DAD and made possible the simultaneous analysis of nine TCs and QNs in minimum time. LODs and LOQs for the different veterinary drugs ranged between 0.5–20 and 1.25–40 ng g<sup>−</sup><sup>1</sup> . Danofloxacin was detected at a concentration of 24.8 ng g<sup>−</sup><sup>1</sup> in chicken liver, tetracycline was detected at 37.5 ng g<sup>−</sup><sup>1</sup> in lamb liver which are less than the stipulated MRLs according to EU 37/2010 and the rest of the veterinary drugs were not detected.

Xu et al. [38] employed a carrier mediated three phase hollow fiber membrane based dynamic liquid-liquid microextraction coupled with HPLC-UV detection for the residue analysis of TCs in milk samples without deproteinization and defatting, but the milk samples were diluted five folds. A peristaltic pump was used to promote mass transfer between the carrier and the operated solution. The standard addition method was used to eliminate the matrix effect. Octanol containing 20% (w/w) Aliquat-336 was used as a SLM, 0.05 M disodium hydrogen phosphate, pH 9.0 containing the sample was a donor phase and solutions of 1.0 mol L<sup>−</sup><sup>1</sup> sodium chloride and phosphoric acid (pH = 1.0) were used as the acceptor solvent. The LOD and LOQ were in the range of 0.95–3.6 and 5–15 μg L<sup>−</sup><sup>1</sup> respectively. The recoveries in spiked samples ranged from 92.38 to 107.3%.

A similar study was carried out by Shariati et al. [39] where tetracycline, oxytetracycline and doxycycline were extracted from bovine milk, human plasma and water samples using a carrier mediated three phase HFLPME prior analysis on the HPLC-UV. The acceptor solvent was 0.1 M phosphoric acid, 1.0 M sodium chloride with pH = 1.6, 0.05 M disodium hydrogen phosphate (pH between 9.1 and 9.5) containing the sample as the donor phase and 10% (w/v) of Aliquat-336 in octanol as a SLM. The LOD and LOQ were 0.5–1.0 and 0.5–1.0 μg L<sup>−</sup><sup>1</sup> respectively which are lower compared to the ones obtained by Xu et al. [38] proving that fiber membranebased dynamic liquid-liquid microextraction is a more efficient extraction method. All the milk samples contained TCs in the range of 6.0–27.4 μg L<sup>−</sup><sup>1</sup> that was below the MRL as set by the EU.

From the two studies that are above it can be concluded that passive transport of TCs in the absence of the carrier is difficult because of existence of TCs as zwitterionic forms (at the studied conditions) in solution and hence they have a very small tendency to pass through the impregnated organic solvent. A unique advantage of the carrier mediator Aliquat-336 is that it stays in a cationic form in all pH ranges.

Sehati et al. [40] coupled HFLPME to nanomaterials, where TiO2 nanomaterials were dispersed in 1-octanol and used it to fill the lumen of a HF. Then, they sealed the two ends of the HF with orthodontic stainless steel wires. The LPME took place by putting the HF into the milk samples for the extraction of tylosin. This method allowed obtaining recovery percentages in the range 89–99% and despite using an ultraviolet- visible spectrophotometer for the determination of tylosin, an LOD of 0.21 mg L<sup>−</sup><sup>1</sup> was achieved which proves the efficiency of the extraction method that was used.

**35**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

Molecularly imprinted polymers (MIPs) are synthesized using a template, functional monomer, cross-linker and an initiator. MIPs are selective towards the target molecules, allowing them to be eluted from the SPE cartridge almost free of co-extracted compounds compared to classical sorbents used for clean-up procedures [56]. SPE sorbents such as C18, hydrophilic lipophilic balanced (HLB) material, diatomite, N-propylethylenediamine, alumina and Florisil are susceptible to interferences by impurities in biological samples and the cartridges can only be used once [57]. Therefore, it is important to develop simple, rapid and environmentally friendly methods. MIPs overcome the above-mentioned drawbacks of traditional SPE sorbents. MIPs are stable under different harsh conditions (extreme pH, high pressures and high temperatures) and can be reused several times [58]. Below are a few studies where MIPs were applied in the solid phase extraction of veterinary

In a study conducted by Song et al. [56], a MIP-SPE method combining LC-MS/ MS was developed to determine the residues of macrolide drugs in animal derived foods. Tylosin was used as a virtual template and the synthesized MIPs were used as the selective sorbent for packing SPE cartridge. A system of sodium borate buffer solution (pH = 10.0) and ethyl acetate was selected for the extraction of the residues of macrolides from muscle samples. Mean recoveries of 10 target analytes were in the range of 60.7–100.3%. Compared with the conventional SPE cartridges (approximately 60–90%), the MISPE cartridge was highly selective and obtained higher recoveries for the 10 macrolides drugs. The LOD and LOQ values ranged

sensitivity of the proposed method for the determination of 10 macrolide drugs

MMIPs were compared to C18 and diatomaceous earth dispersing sorbents. The obtained chromatograms showed that the two sorbents were able to achieve the satisfactory purification effects, but the recoveries of the 20 drugs from the two

In another study by Feng et al. [58], a MIP-SPE method combining HPLC was developed to determine the residues of TC drugs in animal derived foods. A template for MIP synthesis was selected among doxycycline, oxytetracycline and chlortetracycline for enhanced enrichment factors. Results showed that

negative. In order to compare the purification effect of MIP-SPE with conven-

with three commercial SPE cartridges containing different sorbents (strong cation exchange phase, HLB and C18) and there were different interfering peaks around TCs peaks in the chromatograms, revealing inferior purification performances of these sorbents. MIP-SPE proved to be specific, sensitive and accurate

Dummy molecularly imprinted polymers (DMIPs) based on the matrix solid phase dispersion method for the extraction of FQs from fish prior to analysis on the HPLC with fluorescence detection were used by Sun et al. [59]. The use of

tional SPE, the extracts of TCs fortified blank milk (100 ng mL<sup>−</sup><sup>1</sup>

Wang et al. [57] used a mixed-template molecularly imprinted polymer (MMIP) coupled with matrix solid phase dispersion (MSPD) to recognize eight FQs, eight SAs and four TCs from pork samples following analysis with ultraperformance liquid chromatography with a photo diode array detector. The LOD and LOQ were

respectively. The results indicated that the

respectively. The recoveries ranged between 92 and 99%.

) and another milk sample

) were purified

), but the residue levels were

). Results of other samples were

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

**6.3 Molecularly imprinted polymers**

drug residues in food samples.

between 0.1–0.4 and 0.3–1.0 μg kg<sup>−</sup><sup>1</sup>

0.5–3.0 and 1.5–6.0 ng g<sup>−</sup><sup>1</sup>

residues in animal muscle samples was acceptable.

sorbents (70–95%) were lower than that from MMIP.

one milk sample contained TC residue (52 ng mL<sup>−</sup><sup>1</sup>

contained oxytetracycline residue (87 ng mL<sup>−</sup><sup>1</sup>

lower than their MRLs in milk (100 ng mL<sup>−</sup><sup>1</sup>

for the extraction of TCs residues.

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

#### **6.3 Molecularly imprinted polymers**

*Recent Advances in Analytical Chemistry*

been carried out using HFLPME.

detected at 37.5 ng g<sup>−</sup><sup>1</sup>

the MRL as set by the EU.

all pH ranges.

0.21 mg L<sup>−</sup><sup>1</sup>

was used.

was detected at a concentration of 24.8 ng g<sup>−</sup><sup>1</sup>

of HFLPME procedure is that it is slow with extraction times ranging from 15 to 45 minutes and target analytes may partly be trapped in the supporting liquid membrane (SLM) [39]. Another disadvantage is that there is no complete setup commercially available for this method although hollow fibers are commercially available [55]. Below are some recent studies on veterinary drug residues that have

Tajabadi et al. [37] used a carrier mediated three phase HLFPME prior to analysis on the HPLC-DAD for the simultaneous determination of the veterinary drug residues of four TCs and five QNs in a wide range of animal source food samples such as fish, milk and honey as well as the liver and muscles of lamb and chicken. Multivariate curve resolution-alternative least squares was used for resolving some overlapped peaks in multivariate data of HPLC-DAD and made possible the simultaneous analysis of nine TCs and QNs in minimum time. LODs and LOQs for the

Xu et al. [38] employed a carrier mediated three phase hollow fiber membrane based dynamic liquid-liquid microextraction coupled with HPLC-UV detection for the residue analysis of TCs in milk samples without deproteinization and defatting, but the milk samples were diluted five folds. A peristaltic pump was used to promote mass transfer between the carrier and the operated solution. The standard addition method was used to eliminate the matrix effect. Octanol containing 20% (w/w) Aliquat-336 was used as a SLM, 0.05 M disodium hydrogen phosphate, pH 9.0 containing the sample was a donor phase and solutions of 1.0 mol L<sup>−</sup><sup>1</sup> sodium chloride and phosphoric acid (pH = 1.0) were used as the acceptor solvent.

A similar study was carried out by Shariati et al. [39] where tetracycline, oxytetracycline and doxycycline were extracted from bovine milk, human plasma and water samples using a carrier mediated three phase HFLPME prior analysis on the HPLC-UV. The acceptor solvent was 0.1 M phosphoric acid, 1.0 M sodium chloride with pH = 1.6, 0.05 M disodium hydrogen phosphate (pH between 9.1 and 9.5) containing the sample as the donor phase and 10% (w/v) of Aliquat-336 in octanol

lower compared to the ones obtained by Xu et al. [38] proving that fiber membranebased dynamic liquid-liquid microextraction is a more efficient extraction method.

From the two studies that are above it can be concluded that passive transport

Sehati et al. [40] coupled HFLPME to nanomaterials, where TiO2 nanomaterials were dispersed in 1-octanol and used it to fill the lumen of a HF. Then, they sealed the two ends of the HF with orthodontic stainless steel wires. The LPME took place by putting the HF into the milk samples for the extraction of tylosin. This method allowed obtaining recovery percentages in the range 89–99% and despite using an ultraviolet- visible spectrophotometer for the determination of tylosin, an LOD of

was achieved which proves the efficiency of the extraction method that

of TCs in the absence of the carrier is difficult because of existence of TCs as zwitterionic forms (at the studied conditions) in solution and hence they have a very small tendency to pass through the impregnated organic solvent. A unique advantage of the carrier mediator Aliquat-336 is that it stays in a cationic form in

. Danofloxacin

respectively. The

respectively which are

that was below

in chicken liver, tetracycline was

in lamb liver which are less than the stipulated MRLs accord-

different veterinary drugs ranged between 0.5–20 and 1.25–40 ng g<sup>−</sup><sup>1</sup>

ing to EU 37/2010 and the rest of the veterinary drugs were not detected.

The LOD and LOQ were in the range of 0.95–3.6 and 5–15 μg L<sup>−</sup><sup>1</sup>

recoveries in spiked samples ranged from 92.38 to 107.3%.

as a SLM. The LOD and LOQ were 0.5–1.0 and 0.5–1.0 μg L<sup>−</sup><sup>1</sup>

All the milk samples contained TCs in the range of 6.0–27.4 μg L<sup>−</sup><sup>1</sup>

**34**

Molecularly imprinted polymers (MIPs) are synthesized using a template, functional monomer, cross-linker and an initiator. MIPs are selective towards the target molecules, allowing them to be eluted from the SPE cartridge almost free of co-extracted compounds compared to classical sorbents used for clean-up procedures [56]. SPE sorbents such as C18, hydrophilic lipophilic balanced (HLB) material, diatomite, N-propylethylenediamine, alumina and Florisil are susceptible to interferences by impurities in biological samples and the cartridges can only be used once [57]. Therefore, it is important to develop simple, rapid and environmentally friendly methods. MIPs overcome the above-mentioned drawbacks of traditional SPE sorbents. MIPs are stable under different harsh conditions (extreme pH, high pressures and high temperatures) and can be reused several times [58]. Below are a few studies where MIPs were applied in the solid phase extraction of veterinary drug residues in food samples.

In a study conducted by Song et al. [56], a MIP-SPE method combining LC-MS/ MS was developed to determine the residues of macrolide drugs in animal derived foods. Tylosin was used as a virtual template and the synthesized MIPs were used as the selective sorbent for packing SPE cartridge. A system of sodium borate buffer solution (pH = 10.0) and ethyl acetate was selected for the extraction of the residues of macrolides from muscle samples. Mean recoveries of 10 target analytes were in the range of 60.7–100.3%. Compared with the conventional SPE cartridges (approximately 60–90%), the MISPE cartridge was highly selective and obtained higher recoveries for the 10 macrolides drugs. The LOD and LOQ values ranged between 0.1–0.4 and 0.3–1.0 μg kg<sup>−</sup><sup>1</sup> respectively. The results indicated that the sensitivity of the proposed method for the determination of 10 macrolide drugs residues in animal muscle samples was acceptable.

Wang et al. [57] used a mixed-template molecularly imprinted polymer (MMIP) coupled with matrix solid phase dispersion (MSPD) to recognize eight FQs, eight SAs and four TCs from pork samples following analysis with ultraperformance liquid chromatography with a photo diode array detector. The LOD and LOQ were 0.5–3.0 and 1.5–6.0 ng g<sup>−</sup><sup>1</sup> respectively. The recoveries ranged between 92 and 99%. MMIPs were compared to C18 and diatomaceous earth dispersing sorbents. The obtained chromatograms showed that the two sorbents were able to achieve the satisfactory purification effects, but the recoveries of the 20 drugs from the two sorbents (70–95%) were lower than that from MMIP.

In another study by Feng et al. [58], a MIP-SPE method combining HPLC was developed to determine the residues of TC drugs in animal derived foods. A template for MIP synthesis was selected among doxycycline, oxytetracycline and chlortetracycline for enhanced enrichment factors. Results showed that one milk sample contained TC residue (52 ng mL<sup>−</sup><sup>1</sup> ) and another milk sample contained oxytetracycline residue (87 ng mL<sup>−</sup><sup>1</sup> ), but the residue levels were lower than their MRLs in milk (100 ng mL<sup>−</sup><sup>1</sup> ). Results of other samples were negative. In order to compare the purification effect of MIP-SPE with conventional SPE, the extracts of TCs fortified blank milk (100 ng mL<sup>−</sup><sup>1</sup> ) were purified with three commercial SPE cartridges containing different sorbents (strong cation exchange phase, HLB and C18) and there were different interfering peaks around TCs peaks in the chromatograms, revealing inferior purification performances of these sorbents. MIP-SPE proved to be specific, sensitive and accurate for the extraction of TCs residues.

Dummy molecularly imprinted polymers (DMIPs) based on the matrix solid phase dispersion method for the extraction of FQs from fish prior to analysis on the HPLC with fluorescence detection were used by Sun et al. [59]. The use of

DMIPs was to prevent any possible template leakage which could still happen even after thorough washing steps. Template leakage could have a serious impact on the accuracy of the analytical method or made it not suitable for simultaneous analysis of the whole class of FQs. This problem has become one of the major area of concern in sample pre-treatment methods of MIPs. Good recoveries, low LODs and excellent accuracy demonstrated the suitability of the DMIP sorbent for pre-treatment of FQs in fish samples. The use of DMIP resulted in less matrix interferences compared to directly extracted samples and no co-eluted peaks were observed in the chromatogram. The LOD was 0.06–0.22 ng g<sup>−</sup><sup>1</sup> and recoveries ranged between 64.4 and 102.7%.

Wang et al. [60] used an inorganic-organic co-functional monomer, methacrylic acid-vinyltriethoxysilane (MAA-VTES) for the synthesis of molecularly imprinted microspheres (MIMs). The obtained MAA-VTES based MIMs exhibited good recognition and selectivity to FQs and were successfully applied as selective sorbents of a miniaturized home-made solid phase extraction device for the determination of ofloxacin, lomefloxacin and ciprofloxacin in milk samples. The LODs and the LOQs of FQs were 1.5–2.3 and 5.0–7.5 ng mL<sup>−</sup><sup>1</sup> , respectively. The average recoveries for the analyte were in the range of 87.2–106.1%. Ciprofloxacin was detected in two samples as 0.21 and 0.25 ng mL<sup>−</sup><sup>1</sup> which were below the MRL established by EU (100 g kg<sup>−</sup><sup>1</sup> ). Due to the efficiency of the developed co-functional monomer based mini-MISPE-HPLC method, it was possible to analyze the target compounds in milk samples at ng mL<sup>−</sup><sup>1</sup> level.

A selective and eco-friendly sensor for the detection of tetracycline by grafting imprinted polymers onto the surface of carbon quantum dots was used by Hou et al. [61]. A simple microwave-assisted approach was utilized to fabricate the fluorescent imprinted composites rapidly for the first time, which could shorten the polymerization time which normally takes 8–24 hours and simplify the experimental procedure. In this study polymerization took about 1 hour. The development of fluorescent molecularly imprinted composites might be a promising method for rapid analysis in complex samples in future. TCs were not detected in milk samples. Recoveries ranged from 97.3 to 105.3%.

#### **7. Challenges and future trends**

In high-fat foods like milk and meat, veterinary drug residues may bind to lipoproteins and extraction solvents forming emulsions and foam, especially polar veterinary drugs which may decrease recoveries and hence, affecting separation and analysis [56, 62]. Extracting analytes from biological samples using modern extraction techniques like DLLME has some challenges. In traditional DLLME, prior to a DLLME procedure on a complex matrix such as milk, lipids and proteins must be eliminated since they can act like surfactants and disrupt the interfacial tension at the droplet surface, constraining phase separation [63]. During the sample pretreatment step, salts are added for analyte partitioning, phase separation, buffering and for reducing the amounts of co-extracted matrix that could hinder the transfer of analytes from the aqueous phase to the organic phase [5].

TCs are challenging drugs for analytical analysis because they are hydrophilic compounds with high solubility in aqueous media. They have both acidic and basic functionalities, and therefore exist in various forms at different pH conditions [39]. Moreover, they can form complexes with divalent metal ions and silanolic groups on the HPLC column which may result in severe peak tailing [64]. Reverse phase-HPLC with mobile phases containing acids such as phosphoric, acetic and tartaric acids can be used to reduce peak tailing or an RP-amide

**37**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

column can be used. The ability of the RP-amide column to separate TCs might be explained by the hydrogen bonding between the amide functionality of the column and the hydroxyl functionality of TCs. Another challenge is that TCs are

Overlapping peaks during multi-residual analysis when using HPLC-DAD is a challenge. Multivariate curve-resolution coupled to alternating least squares to calculate the exact peak area of overlapping compounds was used by Tajabadi et al. [37], hence more sensitive analytical instruments such as the LC-MS/MS are required for multi-residual analysis. Moreover, the solubilization procedure of veterinary drug residues is a rate-limiting step in multi-residual analysis.

The matrix effect still remains an issue when extracting veterinary drug residues

The world is moving towards the use of greener solvents and hence promoting the principles of GAC, therefore, it can be envisioned that most extraction methods still making use of organic solvents may be completely eliminated in future. Currently greener solvents such ionic liquids are widely used in microextraction procedures as dispersive or extraction solvents according to their different solubili-

Electrochemical sensors and their relative detection strategies, with the advantages of high sensitivity, simplicity and rapid response, have attracted considerable attention in recent years. Among them, aptasensors are considered as one of the most promising research directions owing to the employment of an aptamer. Aptamers, with the advantage of high affinity and specificity to targets, low price and easy to be synthetic in vitro, have provided a broad prospect for developing

Expanding agriculture, aquaculture and apiculture practices have resulted in increased levels of infections among species. Various classes of veterinary drugs including QNs, TCs, β-lactams, SAs and others exhibit activity against both gram-positive and gram-negative bacteria, hence they are widely used to treat or prevent diseases. However, extended use of these veterinary drugs has led to food safety issues worldwide and hence a need for developing sensitive methods for their determination. The focus of this chapter has been to present the trends in modern extraction and clean-up techniques of veterinary drug residues from food samples of animal origin, with milk being the most studied matrix because of its importance on the diet of humans and one of the most consumed foods in the world. Even though some of these veterinary drugs such as chloramphenicols have been banned in some countries due to their dangerous side effects on humans they are still detected in food samples because farmers are not adhering to EU regulations. Generally, in most studies these veterinary drug residues are below stipulated MRLs. Although most extraction methods that are emerging are promising, multi-

using the QuEChERS method from complex samples such as meat, and hence

reducing the sensitivity of chromatographic instruments [46].

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

prone to photo-degradation.

**8. Future trends**

ties in DLLME.

**9. Conclusion**

electrochemical sensing system.

residual analysis is still a challenge.

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

column can be used. The ability of the RP-amide column to separate TCs might be explained by the hydrogen bonding between the amide functionality of the column and the hydroxyl functionality of TCs. Another challenge is that TCs are prone to photo-degradation.

Overlapping peaks during multi-residual analysis when using HPLC-DAD is a challenge. Multivariate curve-resolution coupled to alternating least squares to calculate the exact peak area of overlapping compounds was used by Tajabadi et al. [37], hence more sensitive analytical instruments such as the LC-MS/MS are required for multi-residual analysis. Moreover, the solubilization procedure of veterinary drug residues is a rate-limiting step in multi-residual analysis.

The matrix effect still remains an issue when extracting veterinary drug residues using the QuEChERS method from complex samples such as meat, and hence reducing the sensitivity of chromatographic instruments [46].

#### **8. Future trends**

*Recent Advances in Analytical Chemistry*

ranged between 64.4 and 102.7%.

samples as 0.21 and 0.25 ng mL<sup>−</sup><sup>1</sup>

(100 g kg<sup>−</sup><sup>1</sup>

samples at ng mL<sup>−</sup><sup>1</sup>

LOQs of FQs were 1.5–2.3 and 5.0–7.5 ng mL<sup>−</sup><sup>1</sup>

level.

Recoveries ranged from 97.3 to 105.3%.

**7. Challenges and future trends**

DMIPs was to prevent any possible template leakage which could still happen even after thorough washing steps. Template leakage could have a serious impact on the accuracy of the analytical method or made it not suitable for simultaneous analysis of the whole class of FQs. This problem has become one of the major area of concern in sample pre-treatment methods of MIPs. Good recoveries, low LODs and excellent accuracy demonstrated the suitability of the DMIP sorbent for pre-treatment of FQs in fish samples. The use of DMIP resulted in less matrix interferences compared to directly extracted samples and no co-eluted peaks were

Wang et al. [60] used an inorganic-organic co-functional monomer, methacrylic acid-vinyltriethoxysilane (MAA-VTES) for the synthesis of molecularly imprinted microspheres (MIMs). The obtained MAA-VTES based MIMs exhibited good recognition and selectivity to FQs and were successfully applied as selective sorbents of a miniaturized home-made solid phase extraction device for the determination of ofloxacin, lomefloxacin and ciprofloxacin in milk samples. The LODs and the

for the analyte were in the range of 87.2–106.1%. Ciprofloxacin was detected in two

mini-MISPE-HPLC method, it was possible to analyze the target compounds in milk

A selective and eco-friendly sensor for the detection of tetracycline by grafting imprinted polymers onto the surface of carbon quantum dots was used by Hou et al. [61]. A simple microwave-assisted approach was utilized to fabricate the fluorescent imprinted composites rapidly for the first time, which could shorten the polymerization time which normally takes 8–24 hours and simplify the experimental procedure. In this study polymerization took about 1 hour. The development of fluorescent molecularly imprinted composites might be a promising method for rapid analysis in complex samples in future. TCs were not detected in milk samples.

In high-fat foods like milk and meat, veterinary drug residues may bind to lipoproteins and extraction solvents forming emulsions and foam, especially polar veterinary drugs which may decrease recoveries and hence, affecting separation and analysis [56, 62]. Extracting analytes from biological samples using modern extraction techniques like DLLME has some challenges. In traditional DLLME, prior to a DLLME procedure on a complex matrix such as milk, lipids and proteins must be eliminated since they can act like surfactants and disrupt the interfacial tension at the droplet surface, constraining phase separation [63]. During the sample pretreatment step, salts are added for analyte partitioning, phase separation, buffering and for reducing the amounts of co-extracted matrix that could hinder the transfer

TCs are challenging drugs for analytical analysis because they are hydrophilic compounds with high solubility in aqueous media. They have both acidic and basic functionalities, and therefore exist in various forms at different pH conditions [39]. Moreover, they can form complexes with divalent metal ions and silanolic groups on the HPLC column which may result in severe peak tailing [64]. Reverse phase-HPLC with mobile phases containing acids such as phosphoric, acetic and tartaric acids can be used to reduce peak tailing or an RP-amide

of analytes from the aqueous phase to the organic phase [5].

). Due to the efficiency of the developed co-functional monomer based

and recoveries

, respectively. The average recoveries

which were below the MRL established by EU

observed in the chromatogram. The LOD was 0.06–0.22 ng g<sup>−</sup><sup>1</sup>

**36**

The world is moving towards the use of greener solvents and hence promoting the principles of GAC, therefore, it can be envisioned that most extraction methods still making use of organic solvents may be completely eliminated in future. Currently greener solvents such ionic liquids are widely used in microextraction procedures as dispersive or extraction solvents according to their different solubilities in DLLME.

Electrochemical sensors and their relative detection strategies, with the advantages of high sensitivity, simplicity and rapid response, have attracted considerable attention in recent years. Among them, aptasensors are considered as one of the most promising research directions owing to the employment of an aptamer. Aptamers, with the advantage of high affinity and specificity to targets, low price and easy to be synthetic in vitro, have provided a broad prospect for developing electrochemical sensing system.

#### **9. Conclusion**

Expanding agriculture, aquaculture and apiculture practices have resulted in increased levels of infections among species. Various classes of veterinary drugs including QNs, TCs, β-lactams, SAs and others exhibit activity against both gram-positive and gram-negative bacteria, hence they are widely used to treat or prevent diseases. However, extended use of these veterinary drugs has led to food safety issues worldwide and hence a need for developing sensitive methods for their determination. The focus of this chapter has been to present the trends in modern extraction and clean-up techniques of veterinary drug residues from food samples of animal origin, with milk being the most studied matrix because of its importance on the diet of humans and one of the most consumed foods in the world. Even though some of these veterinary drugs such as chloramphenicols have been banned in some countries due to their dangerous side effects on humans they are still detected in food samples because farmers are not adhering to EU regulations. Generally, in most studies these veterinary drug residues are below stipulated MRLs. Although most extraction methods that are emerging are promising, multiresidual analysis is still a challenge.

*Recent Advances in Analytical Chemistry*

#### **Author details**

Babra Moyo and Nikita Tawanda Tavengwa\* Department of Chemistry, School of Mathematical and Natural Sciences, University of Venda, Thohoyandou, South Africa

\*Address all correspondence to: nikita.tavengwa@univen.ac.za

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**39**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

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[8] Poole CF. New trends in solidphase extraction. Trends in Analytical

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Chemistry. 2003;**22**:362-373

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[13] Ravelo-Pérez LM, Herrera-Herrera AV, Hernández-Borges J, Rodríguez-Delgado MA. Carbon nanotubes: Solid-phase extraction. Journal of Chromatography A.

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*DOI: http://dx.doi.org/10.5772/intechopen.82656*

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Performance Liquid Chromatography using pressurized liquid extraction for the determination of seven tetracyclines in egg, fish and shrimp. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences.

[3] Beyene T. Veterinary drug residues in food-animal products its risk factors and potential effects on public health. Journal of Veterinary Science

supplementary feeding of endangered

[5] Mookantsa SOS, Dube S, Nindi MM. Development and application

Technology. 2016;**7**(1):1-7

[4] Gómez-Ramírez P, Jiménez-Montalbán PJ, Delgado D, Martínez-López E, María-Mojica P, Godino A, et al. Development of a QuEChERS method for simultaneous analysis of antibiotics in carcasses for

vultures. Science of the Total Environment. 2018;**626**:319-327

of a dispersive liquid-liquid micro-extraction method for the determination of tetracyclines in beef by liquid chromatography mass spectrometry. Talanta.

[6] Gao J, Wang H, Qu J, Wang H, Wang X. Development and optimization of a naphthoic acidbased ionic liquid as a "non-organic solvent micro-extraction" for the determination of tetracycline antibiotics in milk and chicken eggs. Food Chemistry. 2017;**215**:138-148

2016;**148**:321-328

2012;**52**:321-333

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*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

#### **References**

*Recent Advances in Analytical Chemistry*

**38**

**Author details**

provided the original work is properly cited.

Babra Moyo and Nikita Tawanda Tavengwa\*

University of Venda, Thohoyandou, South Africa

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Chemistry, School of Mathematical and Natural Sciences,

\*Address all correspondence to: nikita.tavengwa@univen.ac.za

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[17] Manivasagan P, Venkatesan J, Sivakumar K, Kim SK. Marine actinobacterial metabolites: Current status and future perspectives. Microbiological Research. 2013;**168**:311-332

[18] Thiele-Bruhn S, Seibicke T, Schulten HR, Leinweber P. Sorption of pharmaceutical antibiotics on whole soils and particle size fractions. Journal of Environmental Quality. 2004;**33**:1331-1342

[19] Fatta-Kassinos D, Meric S, Nikolaou A. Pharmaceutical residues in environmental waters and wastewater: Current state of knowledge and future research. Analytical and Bioanalytical Chemistry. 2011;**399**:251-275

[20] Shao B, Chen D, Zhang J, Wu Y, Sun C. Determination of 76 pharmaceutical drugs by liquid chromatography-tandem mass spectrometry in slaughterhouse wastewater. Journal of Chromatography A. 2009;**1216**:47

[21] Zhang G, Fang B, Liu Y, Wang X, Xu L, Zhang Y. Development of a multi-residue method for fast screening and confirmation of 20 prohibited veterinary drugs in feedstuffs by liquid chromatography tandem mass spectrometry. Journal of Chromatography B. 2013;**936**:10

[22] Beyene T, Tesega B. Rational veterinary drug use: Its significance in public health. Journal of Veterinary Medicine and Animal Health. 2014;**6**:302-308

[23] Samanidou V, Nisyriou S. Multiresidue methods for confirmatory determination of antibiotics in milk. Journal of Separation Science. 2008;**31**:2068-2090

[24] Blanco G, Junza A, Barrón D. Food safety in scavenger conservation: Diet-associated exposure to livestock pharmaceuticals and opportunist mycoses in threatened Cinereous and Egyptian vultures. Ecotoxicology and Environmental Safety. 2017;**135**:292-301

[25] Blanco G, Junza A, Barrón D. Occurrence of veterinary pharmaceuticals in golden eagle nestlings: Unnoticed scavenging on livestock carcasses and other potential exposure routes. Science of the Total Environment. 2017;**586**:355-361

[26] Pitarch A, Gil C, Blanco G. Oral mycoses in avian scavengers exposed to antibiotics from livestock farming. Science of the Total Environment. 2017;**605-606**:139-146

[27] Jing T, Gaol XD, Wang P, Wang Y, Lin YF, Hu XZ, et al. Determination of trace tetracycline antibiotics in foodstuffs by liquid chromatography-tandem mass spectrometry coupled with selective molecular-imprinted solid-phase extraction. Analytical and Bioanalytical Chemistry. 2009;**393**:2009-2018

[28] Margalida A, Bogliani G, Bowden CGR, Donázar JA, Genero F, Gilbert M, et al. Science and regulation, One health approach to use of veterinary pharmaceuticals. Science. 2014;**346**:1296-1298

[29] Lu YK, Zhang JQ , Guo YZ, Zhang W, Sun HW. Determination of tetracyclines residues in egg, milk, and milk powder by online coupling of a precolumn packed with molecular imprinted hybrid composite materials to RPHPLC-UV. Journal of Liquid Chromatography & Related Technologies. 2015;**38**:1-7

**41**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

[36] Arroyo-Manzanares N, Gamiz-Gracia L, Garcia Campana AM. Alternative sample treatments for the determination of sulfonamides in milk by HPLC with fluorescence

detection. Food Chemistry.

[37] Tajabadi F, Ghambarian M, Yamini Y, Yazdanfar N. Combination of hollow fiber liquid phase microextraction followed by HPLC-DAD and multivariate curve resolution to determine antibacterial residues in foods of animal origin. Talanta.

[38] Xu H, Mi HY, Guan MM, Shan HY, Fei Q , Huan YF, et al. Residue analysis of tetracyclines in milk by HPLC coupled with hollow fiber membranes-

based dynamic liquid-liquid micro-extraction. Food Chemistry.

[39] Shariati S, Yamini Y, Esrafili A. Carrier mediated hollow fiber liquid phase micro-extraction combined with HPLC-UV for preconcentration and determination of some tetracycline antibiotics. Journal of Chromatography B.

[40] Sehati N, Dalali N, Soltanpour S, Dorraji MSS. Extraction and preconcentration of tylosin from milk samples through functionalized TiO2 nanoparticles reinforced with a hollow fiber membrane as a novel solid/liquid phase micro-extraction technique. Journal of Separation Science.

[41] da Costa RP, Spisso BF, Pereira MU, Monteiro MA, Ferreira RG, da Nobrega AW. Innovative mixture of salts in the quick, easy, cheap, effective, rugged, and safe method for the extraction of residual macrolides in milk followed by analysis with liquid chromatography and tandem mass spectrometry.

2014;**143**:459-464

2016;**160**:400-409

2017;**232**:198-202

2009;**877**:393-400

2014;**37**:2025-2031

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

[30] Hernández-Mesa M, Carbonell-Rozas L, Cruces-Blanco C, García-Campaña AM. A high-throughput UHPLC method for the analysis of 5-nitroimidazole residues in milk based on salting-out assisted liquid-liquid extraction. Journal of Chromatography

B. 2017;**1068-1069**:125-130

[31] Jank L, Martins MT, Arsand JB, Motta TMC, Hoff RB, Barreto F, et al. High-throughput method for macrolides and lincosamides antibiotics residues analysis in milk and muscle using a simple liquid-liquid extraction technique and liquid chromatography-electrospraytandem mass spectrometry analysis (LC-MS/MS). Talanta. 2015;**144**:686-695

[32] Moreno-González D, Rodríguez-Ramírez R, del Olmo-Iruela M, GarcíaCampaña AM. Validation of a new method based on salting-out assisted liquid-liquid extraction and UHPLC-MS/MS for the determination of betalactam antibiotics in infant dairy products. Talanta. 2017;**167**:493-498

[33] Yu Y, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high performance liquid chromatography method and a liquid chromatographytandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin. Journal of Chromatography B.

[34] Sichilongo KF, Muckoya VA, Nindi MM. A rapid and sensitive LC-MS/ MS method for the determination of multi-class residues of antibiotics in chicken liver. South African Journal of

[35] Karami-Osboo R, Hossein Shojaee M, Miri R, Kobarfard F, Javidnia K. Simultaneous determination of six fluoroquinolones in milk by validated QuEChERS-DLLME HPLCFLD. Analytical Methods.

2011;**879**:2653-2662

Chemistry. 2015;**68**:1-6

2014;**6**(15):5632-5638

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

[30] Hernández-Mesa M, Carbonell-Rozas L, Cruces-Blanco C, García-Campaña AM. A high-throughput UHPLC method for the analysis of 5-nitroimidazole residues in milk based on salting-out assisted liquid-liquid extraction. Journal of Chromatography B. 2017;**1068-1069**:125-130

*Recent Advances in Analytical Chemistry*

[15] Nollet LML. Food Analysis by HPLC. 2nd revised and expanded ed. Marcel Dekker, Inc.; pp. 621-670

[23] Samanidou V, Nisyriou S. Multiresidue methods for confirmatory determination of antibiotics in milk. Journal of Separation Science.

[24] Blanco G, Junza A, Barrón D. Food safety in scavenger conservation: Diet-associated exposure to livestock pharmaceuticals and opportunist mycoses in threatened Cinereous and Egyptian vultures. Ecotoxicology and Environmental Safety. 2017;**135**:292-301

[25] Blanco G, Junza A, Barrón D.

[26] Pitarch A, Gil C, Blanco G. Oral mycoses in avian scavengers exposed to antibiotics from livestock farming. Science of the Total Environment.

[27] Jing T, Gaol XD, Wang P, Wang Y, Lin YF, Hu XZ, et al. Determination of trace tetracycline antibiotics in foodstuffs by liquid chromatography-tandem mass spectrometry coupled with selective molecular-imprinted solid-phase extraction. Analytical and Bioanalytical

Chemistry. 2009;**393**:2009-2018

[29] Lu YK, Zhang JQ , Guo YZ, Zhang W, Sun HW. Determination of tetracyclines residues in egg, milk, and milk powder by online coupling of a precolumn packed with molecular imprinted hybrid composite materials to RPHPLC-UV. Journal of Liquid Chromatography & Related

Technologies. 2015;**38**:1-7

2014;**346**:1296-1298

[28] Margalida A, Bogliani G, Bowden CGR, Donázar JA, Genero F, Gilbert M, et al. Science and regulation, One health approach to use of veterinary pharmaceuticals. Science.

Occurrence of veterinary pharmaceuticals in golden eagle nestlings: Unnoticed scavenging on livestock carcasses and other potential exposure routes. Science of the Total Environment. 2017;**586**:355-361

2017;**605-606**:139-146

2008;**31**:2068-2090

[16] Hauser AR. Antibiotics Basics for Clinicians: The ABCs of Choosing the Right Antibacterial Agent. 2nd ed. Philadelphia, PA, USA: Lippincott

Williams & Wilkins; 2007

2013;**168**:311-332

2004;**33**:1331-1342

A. 2009;**1216**:47

[17] Manivasagan P, Venkatesan J, Sivakumar K, Kim SK. Marine actinobacterial metabolites: Current status and future perspectives. Microbiological Research.

[18] Thiele-Bruhn S, Seibicke T, Schulten HR, Leinweber P. Sorption of pharmaceutical antibiotics on whole soils and particle size fractions. Journal of Environmental Quality.

[19] Fatta-Kassinos D, Meric S,

Chemistry. 2011;**399**:251-275

Nikolaou A. Pharmaceutical residues in environmental waters and wastewater: Current state of knowledge and future research. Analytical and Bioanalytical

[20] Shao B, Chen D, Zhang J, Wu Y, Sun C. Determination of 76 pharmaceutical drugs by liquid chromatography-tandem mass spectrometry in slaughterhouse wastewater. Journal of Chromatography

[21] Zhang G, Fang B, Liu Y, Wang X, Xu L, Zhang Y. Development of a multi-residue method for fast screening and confirmation of 20 prohibited veterinary drugs in feedstuffs by liquid chromatography tandem mass spectrometry. Journal of Chromatography B. 2013;**936**:10

[22] Beyene T, Tesega B. Rational veterinary drug use: Its significance in public health. Journal of Veterinary

Medicine and Animal Health.

**40**

2014;**6**:302-308

[31] Jank L, Martins MT, Arsand JB, Motta TMC, Hoff RB, Barreto F, et al. High-throughput method for macrolides and lincosamides antibiotics residues analysis in milk and muscle using a simple liquid-liquid extraction technique and liquid chromatography-electrospraytandem mass spectrometry analysis (LC-MS/MS). Talanta. 2015;**144**:686-695

[32] Moreno-González D, Rodríguez-Ramírez R, del Olmo-Iruela M, GarcíaCampaña AM. Validation of a new method based on salting-out assisted liquid-liquid extraction and UHPLC-MS/MS for the determination of betalactam antibiotics in infant dairy products. Talanta. 2017;**167**:493-498

[33] Yu Y, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high performance liquid chromatography method and a liquid chromatographytandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin. Journal of Chromatography B. 2011;**879**:2653-2662

[34] Sichilongo KF, Muckoya VA, Nindi MM. A rapid and sensitive LC-MS/ MS method for the determination of multi-class residues of antibiotics in chicken liver. South African Journal of Chemistry. 2015;**68**:1-6

[35] Karami-Osboo R, Hossein Shojaee M, Miri R, Kobarfard F, Javidnia K. Simultaneous determination of six fluoroquinolones in milk by validated QuEChERS-DLLME HPLCFLD. Analytical Methods. 2014;**6**(15):5632-5638

[36] Arroyo-Manzanares N, Gamiz-Gracia L, Garcia Campana AM. Alternative sample treatments for the determination of sulfonamides in milk by HPLC with fluorescence detection. Food Chemistry. 2014;**143**:459-464

[37] Tajabadi F, Ghambarian M, Yamini Y, Yazdanfar N. Combination of hollow fiber liquid phase microextraction followed by HPLC-DAD and multivariate curve resolution to determine antibacterial residues in foods of animal origin. Talanta. 2016;**160**:400-409

[38] Xu H, Mi HY, Guan MM, Shan HY, Fei Q , Huan YF, et al. Residue analysis of tetracyclines in milk by HPLC coupled with hollow fiber membranesbased dynamic liquid-liquid micro-extraction. Food Chemistry. 2017;**232**:198-202

[39] Shariati S, Yamini Y, Esrafili A. Carrier mediated hollow fiber liquid phase micro-extraction combined with HPLC-UV for preconcentration and determination of some tetracycline antibiotics. Journal of Chromatography B. 2009;**877**:393-400

[40] Sehati N, Dalali N, Soltanpour S, Dorraji MSS. Extraction and preconcentration of tylosin from milk samples through functionalized TiO2 nanoparticles reinforced with a hollow fiber membrane as a novel solid/liquid phase micro-extraction technique. Journal of Separation Science. 2014;**37**:2025-2031

[41] da Costa RP, Spisso BF, Pereira MU, Monteiro MA, Ferreira RG, da Nobrega AW. Innovative mixture of salts in the quick, easy, cheap, effective, rugged, and safe method for the extraction of residual macrolides in milk followed by analysis with liquid chromatography and tandem mass spectrometry.

Journal of Separation Science. 2015;**38**:3743-3749

[42] Wang YL, Liu Z, Ren J, Guo BH. Development of a method for the analysis of multiclass antibiotic residues in milk using QuEChERS and liquid chromatography-tandem mass spectrometry. Foodborne Pathogens and Disease. 2015;**12**(8):693-703

[43] Li Y, Chen Z, Wen S, Hou X, Zhang R, Ma M. Multiresidue Determination of Antibiotics in Preserved Eggs Using a QuEChERS-Based Procedure by Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry. Acta Chromatographica; 2018;**30**(1):9-16

[44] Anastassiades M, Lehotay S. Fast and easy multiresidue method employing acetonitrile extraction/ partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce. Journal of AOAC International. 2003;**86**:412-431

[45] Berendsen BJA, Stolker LAAM, Nielen MWF. Selectivity in the sample preparation for the analysis of drug residues in products of animal origin using LC-MS. TrAC Trends in Analytical Chemistry. 2013;**43**:229-239

[46] Machado SC, Landin-Silva M, Mai PP, Rath S, Martins I. QuEChERS-HPLC-DAD method for sulphonamides in chicken breast. Brazilian Journal of Pharmaceutical Sciences. 2013;**49**(1):155-166

[47] Sharifi V, Abbasi A, Nosrati A. Application of hollow fiber liquid phase micro-extraction and dispersive liquid-liquid micro-extraction techniques in analytical toxicology. Journal of Food and Drug Analysis. 2016;**24**:264-276

[48] Quigley A, Cummins W, Connolly D. Dispersive liquid-liquid microextraction in the analysis of milk and

dairy products: A review. Journal of Chemistry. 2016;**2016**:12

[49] Lopez-Darias J, German-Hernandez M, Pino V, Afonso AM. Dispersive liquid-liquid micro-extraction versus single-drop micro-extraction for the determination of several endocrinedisrupting phenols from seawaters. Talanta. 2010;**80**(5):1611-1618

[50] Hadjmohammadi M, Karimiyan H, Sharifi V. Hollow fibre-based liquid phase micro-extraction combined with high performance liquid chromatography for the analysis of flavonoids in *Echinophora platyloba* DC and *Mentha piperita*. Food Chemistry. 2013;**141**:731-735

[51] Rezaee M, Assadi Y, Milani Hosseini MR, Aghaee E, Ahmadi F, Berijani S. Determination of organic compounds in water using dispersive liquid-liquid microextraction. Journal of Chromatography A. 2006;**1116**:1-9

[52] Al-Saidi HM, Emara AA. The recent developments in dispersive liquid-liquid micro-extraction for pre-concentration and determination of inorganic analytes. Journal of Chemistry. 2013;**11**:1343-1351

[53] Karami-Osboo R, Miria R, Javidnia K, Kobarfard F. Simultaneous chloramphenicol and florfenicol determination by a validated DLLME-HPLC-UV method in pasteurized Milk. Iranian Journal of Pharmaceutical Research. 2016;**15**:361-368

[54] Ghambarian M, Yamini Y, Esrafili A. Developments in hollow fiber based liquid-phase microextraction: Principles and applications. Microchimica Acta. 2012;**177**:271-294

[55] Gjelstad A, Pedersen BS. Perspective: Hollow fibre liquidphase micro-extraction—Principles, performance, applicability, and future directions. Scientia Chromatographica. 2013;**5**:181-189

**43**

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples…*

liquid chromatography. Analytical and Bioanalytical Chemistry.

[63] Vinas P, Campillo N, López-García I, Hernández-Córdoba M. Dispersive liquid-liquid microextraction in food analysis. A critical review

microextraction techniques. Analytical

and Bioanalytical Chemistry.

[64] Zhu J, Snow DD, Cassada DA, Monson SJ, Spalding RF. Analysis of oxytetracycline, tetracycline, and chlortetracycline in water using solid-phase extraction and liquid chromatography-tandem mass spectrometry. Journal of Chromatography. A. 2001;**928**:177

2006;**384**:1228-1235

2014;**406**:2067-2099

*DOI: http://dx.doi.org/10.5772/intechopen.82656*

[56] Song X, Zhou T, Liu Q , Zhang M, Meng C, Li J, et al. Molecularly imprinted solid-phase extraction for the determination of ten macrolide drugs residues in animal muscles by liquid chromatography-tandem mass spectrometry. Food Chemistry.

[57] Wang GN, Zhang L, Song YP, Liu JX, Wang JP. Application of molecularly imprinted polymer based matrix solid phase dispersion for determination of fluoroquinolones, tetracyclines and sulfonamides in meat. Journal of Chromatography B.

[58] Feng MX, Wang GN, Yang K, Liu HZ, Wang JP. Molecularly imprinted polymer-high performance liquid chromatography for the determination of tetracycline drugs in animal derived foods. Food Control. 2016;**69**:171-176

[59] Sun X, Wang J, Li Y, Yang J, Jina J, Shaha SM, et al. Novel dummy molecularly imprinted polymers for matrix solid-phase dispersion extraction of eight fluoroquinolones from fish samples. Journal of Chromatography A.

[60] Wang H, Wang R, Han Y. Preparation of molecular imprinted microspheres based on inorganicorganic co-functional monomer for miniaturized solid-phase extraction of fluoroquinolones in milk. Journal of Chromatography B. 2014;**949-950**:24-29

[61] Hou J, Li H, Wang L, Zhang P, Zhou T, Ding H, et al. Rapid microwave-assisted synthesis of molecularly imprinted polymers on carbon quantum dots for fluorescent sensing of tetracycline in milk. Talanta.

[62] Huang JF, Lin B, Yu QW, Feng YQ. Determination of fluoroquinolones in eggs using in-tube solid-phase microextraction coupled to high-performance

2016;**208**:169-176

2017;**1065-1066**:104-111

2014;**1359**:1-7

2016;**146**:34-40

*Modern Extraction and Cleanup Methods of Veterinary Drug Residues in Food Samples… DOI: http://dx.doi.org/10.5772/intechopen.82656*

[56] Song X, Zhou T, Liu Q , Zhang M, Meng C, Li J, et al. Molecularly imprinted solid-phase extraction for the determination of ten macrolide drugs residues in animal muscles by liquid chromatography-tandem mass spectrometry. Food Chemistry. 2016;**208**:169-176

*Recent Advances in Analytical Chemistry*

[42] Wang YL, Liu Z, Ren J, Guo BH. Development of a method for the analysis of multiclass antibiotic residues in milk using QuEChERS and liquid chromatography-tandem mass spectrometry. Foodborne Pathogens and dairy products: A review. Journal of

[49] Lopez-Darias J, German-Hernandez M, Pino V, Afonso AM. Dispersive liquid-liquid micro-extraction versus single-drop micro-extraction for the determination of several endocrinedisrupting phenols from seawaters. Talanta. 2010;**80**(5):1611-1618

[50] Hadjmohammadi M, Karimiyan H, Sharifi V. Hollow fibre-based liquid phase micro-extraction combined with high performance liquid chromatography for the analysis of flavonoids in *Echinophora platyloba* DC and *Mentha piperita*. Food

Chemistry. 2013;**141**:731-735

[51] Rezaee M, Assadi Y, Milani Hosseini MR, Aghaee E, Ahmadi F, Berijani S. Determination of organic compounds in water using dispersive liquid-liquid microextraction. Journal of Chromatography A. 2006;**1116**:1-9

and determination of inorganic analytes. Journal of Chemistry.

[53] Karami-Osboo R, Miria R,

Research. 2016;**15**:361-368

[54] Ghambarian M, Yamini Y, Esrafili A. Developments in hollow fiber based liquid-phase micro-

[55] Gjelstad A, Pedersen BS. Perspective: Hollow fibre liquidphase micro-extraction—Principles, performance, applicability, and future directions. Scientia Chromatographica.

2013;**5**:181-189

Javidnia K, Kobarfard F. Simultaneous chloramphenicol and florfenicol determination by a validated DLLME-HPLC-UV method in pasteurized Milk. Iranian Journal of Pharmaceutical

extraction: Principles and applications. Microchimica Acta. 2012;**177**:271-294

2013;**11**:1343-1351

[52] Al-Saidi HM, Emara AA. The recent developments in dispersive liquid-liquid micro-extraction for pre-concentration

Chemistry. 2016;**2016**:12

[43] Li Y, Chen Z, Wen S, Hou X, Zhang R, Ma M. Multiresidue Determination of Antibiotics in Preserved Eggs Using a QuEChERS-Based Procedure by Ultrahigh-Performance Liquid Chromatography Tandem Mass

Spectrometry. Acta Chromatographica;

[44] Anastassiades M, Lehotay S. Fast and easy multiresidue method employing acetonitrile extraction/ partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce. Journal of AOAC International. 2003;**86**:412-431

[45] Berendsen BJA, Stolker LAAM, Nielen MWF. Selectivity in the sample preparation for the analysis of drug residues in products of animal origin using LC-MS. TrAC Trends in Analytical Chemistry. 2013;**43**:229-239

[46] Machado SC, Landin-Silva M, Mai PP, Rath S, Martins I. QuEChERS-HPLC-DAD method for sulphonamides in chicken breast. Brazilian Journal of Pharmaceutical Sciences.

[47] Sharifi V, Abbasi A, Nosrati A. Application of hollow fiber liquid phase micro-extraction and dispersive

liquid-liquid micro-extraction techniques in analytical toxicology. Journal of Food and Drug Analysis.

[48] Quigley A, Cummins W, Connolly D. Dispersive liquid-liquid microextraction in the analysis of milk and

2013;**49**(1):155-166

2016;**24**:264-276

Journal of Separation Science.

Disease. 2015;**12**(8):693-703

2015;**38**:3743-3749

2018;**30**(1):9-16

**42**

[57] Wang GN, Zhang L, Song YP, Liu JX, Wang JP. Application of molecularly imprinted polymer based matrix solid phase dispersion for determination of fluoroquinolones, tetracyclines and sulfonamides in meat. Journal of Chromatography B. 2017;**1065-1066**:104-111

[58] Feng MX, Wang GN, Yang K, Liu HZ, Wang JP. Molecularly imprinted polymer-high performance liquid chromatography for the determination of tetracycline drugs in animal derived foods. Food Control. 2016;**69**:171-176

[59] Sun X, Wang J, Li Y, Yang J, Jina J, Shaha SM, et al. Novel dummy molecularly imprinted polymers for matrix solid-phase dispersion extraction of eight fluoroquinolones from fish samples. Journal of Chromatography A. 2014;**1359**:1-7

[60] Wang H, Wang R, Han Y. Preparation of molecular imprinted microspheres based on inorganicorganic co-functional monomer for miniaturized solid-phase extraction of fluoroquinolones in milk. Journal of Chromatography B. 2014;**949-950**:24-29

[61] Hou J, Li H, Wang L, Zhang P, Zhou T, Ding H, et al. Rapid microwave-assisted synthesis of molecularly imprinted polymers on carbon quantum dots for fluorescent sensing of tetracycline in milk. Talanta. 2016;**146**:34-40

[62] Huang JF, Lin B, Yu QW, Feng YQ. Determination of fluoroquinolones in eggs using in-tube solid-phase microextraction coupled to high-performance liquid chromatography. Analytical and Bioanalytical Chemistry. 2006;**384**:1228-1235

[63] Vinas P, Campillo N, López-García I, Hernández-Córdoba M. Dispersive liquid-liquid microextraction in food analysis. A critical review microextraction techniques. Analytical and Bioanalytical Chemistry. 2014;**406**:2067-2099

[64] Zhu J, Snow DD, Cassada DA, Monson SJ, Spalding RF. Analysis of oxytetracycline, tetracycline, and chlortetracycline in water using solid-phase extraction and liquid chromatography-tandem mass spectrometry. Journal of Chromatography. A. 2001;**928**:177

**45**

**Chapter 3**

(Cl NCS)

**Abstract**

4000 cm<sup>−</sup><sup>1</sup>

modes of [C2H10N2]

DFT calculations

**1. Introduction**

group P1 (Ci). The entities [C2H10N2]

Contribution of Infrared

*Sahel Karoui and Slaheddine Kamoun*

mental infrared spectrum showed good agreement.

Spectroscopy to the Vibrational

Study of Ethylenediammonium

Chloride Thiocyanate: (C2H10N2)

The C2H10N2 Cl NCS (EDCT) compound is characterized by using infrared spectroscopy. The infrared spectrum of the title compound was recorded (400–

thiocyanate chloride crystallizes, at room temperature, in the triclinic system, space

(C1). Several ground state thermodynamic parameters were calculated using the ab initio Hartree-Fock (HF) and DFT (B3LYP) methods with 6-31++G (d, p) and 6-311++G (d, p) basic sets such as vibration frequencies, rotation constants, and optimized molecular geometry. The comparison between the theoretical and experi-

**Keywords:** ethylenediammonium chloride thiocyanate, IR, vibrational spectra,

This chapter is devoted to the characterization of C2H10N2 Cl NCS by infrared vibrational spectroscopy. These studies make it possible to highlight the structural analogies and to possibly provide some additional information to those obtained by X-ray diffraction. In this chapter, we used group theory; indeed, this valuable tool allows both to count the normal vibration modes of vibration of a crystal and to describe these vibrations in symmetrical coordinate terms. In addition, an attempt is made to assign the various modes of vibration to all the bands that have appeared. It is based on predictions theories and previous work carried out on similar compounds. Infrared is a research tool can also provide exquisite structural insights into the molecule and characterizes the vibrational modes of the molecules and has enfolded within it much information on chemical structure [1]. The combined use of FT-IR spectroscopy extracts most of the obtainable information and these are the popular tools in the chemist and physicist. Amine, amino acid and Schiff bases [2–6] have recently been the focus of coordination chemists due to their

) at room temperature and discussed, essentially in terms of vibrational

2+ cations and [SCN]<sup>−</sup> and [Cl]<sup>−</sup> anions. Ethylenediammonium

2+, [SCN]<sup>−</sup> and [Cl]<sup>−</sup> occupy sites of symmetry

#### **Chapter 3**

## Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium Chloride Thiocyanate: (C2H10N2) (Cl NCS)

*Sahel Karoui and Slaheddine Kamoun*

#### **Abstract**

The C2H10N2 Cl NCS (EDCT) compound is characterized by using infrared spectroscopy. The infrared spectrum of the title compound was recorded (400– 4000 cm<sup>−</sup><sup>1</sup> ) at room temperature and discussed, essentially in terms of vibrational modes of [C2H10N2] 2+ cations and [SCN]<sup>−</sup> and [Cl]<sup>−</sup> anions. Ethylenediammonium thiocyanate chloride crystallizes, at room temperature, in the triclinic system, space group P1 (Ci). The entities [C2H10N2] 2+, [SCN]<sup>−</sup> and [Cl]<sup>−</sup> occupy sites of symmetry (C1). Several ground state thermodynamic parameters were calculated using the ab initio Hartree-Fock (HF) and DFT (B3LYP) methods with 6-31++G (d, p) and 6-311++G (d, p) basic sets such as vibration frequencies, rotation constants, and optimized molecular geometry. The comparison between the theoretical and experimental infrared spectrum showed good agreement.

**Keywords:** ethylenediammonium chloride thiocyanate, IR, vibrational spectra, DFT calculations

#### **1. Introduction**

This chapter is devoted to the characterization of C2H10N2 Cl NCS by infrared vibrational spectroscopy. These studies make it possible to highlight the structural analogies and to possibly provide some additional information to those obtained by X-ray diffraction. In this chapter, we used group theory; indeed, this valuable tool allows both to count the normal vibration modes of vibration of a crystal and to describe these vibrations in symmetrical coordinate terms. In addition, an attempt is made to assign the various modes of vibration to all the bands that have appeared. It is based on predictions theories and previous work carried out on similar compounds. Infrared is a research tool can also provide exquisite structural insights into the molecule and characterizes the vibrational modes of the molecules and has enfolded within it much information on chemical structure [1]. The combined use of FT-IR spectroscopy extracts most of the obtainable information and these are the popular tools in the chemist and physicist. Amine, amino acid and Schiff bases [2–6] have recently been the focus of coordination chemists due to their

preparative accessibilities, structural varieties, and varied denticities. With those purposes, first, the EDCT was synthesized [7] and then characterized it by Infrared Spectroscopy. Simultaneously, to obtain the ground state optimized geometries and the vibrational wavenumbers of the different normal modes, we carried out the ab initio HF and DFT calculations. Here, the hybrid B3LYP method was used together with the 6-31++G (d, p) and 6-311++G (d, p) basis sets [8].

#### **2. Experimental details**

#### **2.1 Synthesis**

The title compound has been obtained by mixing, in stoichiometric proportions, a solution of ethylenediamine, a freshly prepared solution of thiocyanic acid HSCN and a solution of potassium halide. KX (X = Cl) [7].

#### **2.2 IR spectroscopy**

Infrared absorption spectrum was recorded at room temperature in the 400–4000 cm<sup>−</sup><sup>1</sup> frequency range on a Perkin-Elmer spectrometer equipped with a Universal ATR Accessory (UATR).

#### **2.3 Computational details**

Numerous studies [9–12] have shown that the method DFT-B3LYP in combination with the bases 6-31++G (d, p) and 6-311++G (d, p) allowed to determine with precision energies, molecular structures and infrared vibratory frequencies. In the ground state the molecular structure of the C2H10N2Cl NCS (EDCT) phase calculated was optimized by the use of the DFT/B3LYP methods with the methods 6-31++G (d, p) and 6-311++G (d, p) base set, and the calculated optimized structure was used in vibrational frequency calculations. The calculated harmonic vibratory frequencies and the minimal energy of the geometric structure were scaled by (B3LYP) with the base set 6-31++G (d, p) and 6-311++G (d, p). HF/DFT calculations for EDCT are performed using GAUSSIAN 03W program [13, 14]. On the other hand, the energies of the frontier orbital's were used to calculate the gap energy values and some interesting descriptors in order to predict their reactivities an behaviors at the same level of theory [14–17].

#### **3. Results and discussion**

#### **3.1 Molecular geometry**

The structure of the EDCT belongs to Ci point group symmetry and its molecular structure is obtained from GAUSSAN 03W and GAUSSVIEW programs are shown in **Figure 1**. The molecule contains one diprotonated ethylenediammonium cation, one Cl<sup>−</sup> and one SCN<sup>−</sup> anions. The comparative optimized structural parameters such as bond lengths and bond angles are presented in **Table 1**. The comparative graphs of bond lengths and bond angles of ethylenediammonium chloride thiocyanate for two sets are presented in **Figures 2** and **3** respectively. Most of the optimized bond lengths are slightly higher than the experimental values, depending on the theoretical values, because the theoretical calculations belong to isolated molecules in the gas phase and the experimental results to solid state molecules. The

**47**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

**Methods**

**B3LYP/6- 311++G (d. p)** **Experimental value [12]**

**B3LYP/6- 31++G (d. p)**

S(1)–C(1) 1.6314 1.6324 1.6324 1.6358(12) C(1)–N(1) 1.1651 1.1687 1.1651 1.1573(16) C(2)–N(2) 1.4847 1.4284 1.4847 1.4798(14) C(3)–N(3) 1.4817 1.5107 1.4817 1.4834(15) C(2)–C(3) 1.5066 1.5334 1.5066 1.5054(15) C(2)–H(1C2) 0.9763 1.0926 0.9763 0.9700 C(2)–H(2C2) 1.0059 1.0927 1.0059 0.9700 C(3)–H(1C3) 0.9694 1.0971 0.9694 0.9700 C(3)–H(2C3) 1.0299 1.0930 1.0299 0.9700 N(2)–H(1 N2) 0.9048 1.0333 0.9048 0.8900 N(2)–H(2N2) 0.8967 1.0357 0.8967 0.8900 N(2)–H(3N2) 0.8660 1.0264 0.8660 0.8900 N(3)–H(1N3) 0.8665 1.0202 0.8665 0.8900 N(3)–H(2N3) 0.8639 1.0540 0.8639 0.8900 N(3)–H(3N3) 0.8715 1.0219 0.8715 0.8900

N(1)–C(1)–S(1) 173.60 170.71 171.76 178.48(11) N(2)–C(2)–C(3) 114.00 115.16 114.16 113.06(9) N(3)–C(3)–C(2) 113.46 113.75 113.72 112.98(9) C(2)–N(2)–H(1N2) 109.86 111.45 111.44 109.5 C(2)–N(2)–H(2N2) 107.14 111.99 112.00 109.5 C(2)–N(2)–H(3N2) 110.63 112.81 112.83 109.5

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

*Molecular structure of ethylenediammonium chloride thiocyanate.*

**HF/6-311++G (d. p)**

**Figure 1.**

**Geometrical parameters**

**Bond length (Å)**

**Bond angle (°)**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*

**Figure 1.**

*Recent Advances in Analytical Chemistry*

**2. Experimental details**

**2.1 Synthesis**

**2.2 IR spectroscopy**

Universal ATR Accessory (UATR).

**2.3 Computational details**

**3. Results and discussion**

**3.1 Molecular geometry**

400–4000 cm<sup>−</sup><sup>1</sup>

preparative accessibilities, structural varieties, and varied denticities. With those purposes, first, the EDCT was synthesized [7] and then characterized it by Infrared Spectroscopy. Simultaneously, to obtain the ground state optimized geometries and the vibrational wavenumbers of the different normal modes, we carried out the ab initio HF and DFT calculations. Here, the hybrid B3LYP method was used together

The title compound has been obtained by mixing, in stoichiometric proportions, a solution of ethylenediamine, a freshly prepared solution of thiocyanic acid HSCN

frequency range on a Perkin-Elmer spectrometer equipped with a

Infrared absorption spectrum was recorded at room temperature in the

Numerous studies [9–12] have shown that the method DFT-B3LYP in combination with the bases 6-31++G (d, p) and 6-311++G (d, p) allowed to determine with precision energies, molecular structures and infrared vibratory frequencies. In the ground state the molecular structure of the C2H10N2Cl NCS (EDCT) phase calculated was optimized by the use of the DFT/B3LYP methods with the methods 6-31++G (d, p) and 6-311++G (d, p) base set, and the calculated optimized structure was used in vibrational frequency calculations. The calculated harmonic vibratory frequencies and the minimal energy of the geometric structure were scaled by (B3LYP) with the base set 6-31++G (d, p) and 6-311++G (d, p). HF/DFT calculations for EDCT are performed using GAUSSIAN 03W program [13, 14]. On the other hand, the energies of the frontier orbital's were used to calculate the gap energy values and some interesting descriptors in order to predict their reactivities

The structure of the EDCT belongs to Ci point group symmetry and its molecular structure is obtained from GAUSSAN 03W and GAUSSVIEW programs are shown in **Figure 1**. The molecule contains one diprotonated ethylenediammonium cation, one Cl<sup>−</sup> and one SCN<sup>−</sup> anions. The comparative optimized structural parameters such as bond lengths and bond angles are presented in **Table 1**. The comparative graphs of bond lengths and bond angles of ethylenediammonium chloride thiocyanate for two sets are presented in **Figures 2** and **3** respectively. Most of the optimized bond lengths are slightly higher than the experimental values, depending on the theoretical values, because the theoretical calculations belong to isolated molecules in the gas phase and the experimental results to solid state molecules. The

with the 6-31++G (d, p) and 6-311++G (d, p) basis sets [8].

and a solution of potassium halide. KX (X = Cl) [7].

an behaviors at the same level of theory [14–17].

**46**

*Molecular structure of ethylenediammonium chloride thiocyanate.*



**Table 1.**

*Optimized geometrical parameters for ethylenediammonium chloride thiocyanate computed at HF/6-311++G (d. p), B3LYP/6-31++G (d. p) and B3LYP/6-311++G(d. p) basis sets.*

**49**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

angles and binding lengths of B3LYP are compared with those of HF, the formers are generally larger than later and the values calculated by B3LYP are well correlated with the experimental data. The parameters (the vibration frequencies and the thermodynamic properties) represent a good approximation. The data presented in **Table 1** show that the theoretical HF and DFT levels (B3LYP/6-311++G (d, p)) generally estimate the same values for some link lengths and angles. The calculated C▬N bond lengths are found same at two positions (C2▬N2 and C3▬N3) is 1.4847 and 1.5066 Å (HF and DFT), 0.0049 and 0.0012 Å, respectively, differed from the

2+ dica-

2+ cat-

experimental value 1.4798(14) and 1.5054(15) Å [15–17]. The [C2H10N2]

1.6358 (12) and 1.1573 (16) Å [7], respectively.

*Bond angle differences between theoretical (HF and DFT) approaches.*

*3.2.1.1 Theoretical analysis of C2H10N2 Cl NCS vibrations*

**3.2 Vibrational analysis**

**Figure 3.**

tion shows an eclipsed conformation. The calculated N▬C▬C▬N torsion angle is 74.53° (HF and DFT), 2.44° differed from the experimental value 72.09(12)° [7]. The thiocyanate ion, present as a monodentate ligand, is almost linear. The calculated angle is 173.60° but the experimental value 178.48 (11)° and an average calculated and experimental C▬S and C▬N bond lengths are 1.6314 and 1.1651,

*3.2.1 Contribution of IR spectrometry to the vibrational study of C2H10N2 Cl NCS*

The factor group method of classifying fundamental vibrational modes of crystals, as developed by Bhagavantam and Venkatarayudu [18], is certainly the most powerful method of treating C2H10N2 Cl NCS crystal structure. The unit cell of C2H10N2 Cl NCS contains 18 atoms which correspond to 54 degrees of vibrational freedom. To simplify the discussion of the IR data, the vibrational modes will be considered in two groups: the internal modes of SCN<sup>−</sup> anions and (C2H10N2)

ions. Ethylenediammonium thiocyanate chloride crystallizes, at room temperature,

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

**Figure 2.** *Bond length differences between theoretical (HF and DFT) approaches.*

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*

**Figure 3.** *Bond angle differences between theoretical (HF and DFT) approaches.*

angles and binding lengths of B3LYP are compared with those of HF, the formers are generally larger than later and the values calculated by B3LYP are well correlated with the experimental data. The parameters (the vibration frequencies and the thermodynamic properties) represent a good approximation. The data presented in **Table 1** show that the theoretical HF and DFT levels (B3LYP/6-311++G (d, p)) generally estimate the same values for some link lengths and angles. The calculated C▬N bond lengths are found same at two positions (C2▬N2 and C3▬N3) is 1.4847 and 1.5066 Å (HF and DFT), 0.0049 and 0.0012 Å, respectively, differed from the experimental value 1.4798(14) and 1.5054(15) Å [15–17]. The [C2H10N2] 2+ dication shows an eclipsed conformation. The calculated N▬C▬C▬N torsion angle is 74.53° (HF and DFT), 2.44° differed from the experimental value 72.09(12)° [7]. The thiocyanate ion, present as a monodentate ligand, is almost linear. The calculated angle is 173.60° but the experimental value 178.48 (11)° and an average calculated and experimental C▬S and C▬N bond lengths are 1.6314 and 1.1651, 1.6358 (12) and 1.1573 (16) Å [7], respectively.

#### **3.2 Vibrational analysis**

#### *3.2.1 Contribution of IR spectrometry to the vibrational study of C2H10N2 Cl NCS*

#### *3.2.1.1 Theoretical analysis of C2H10N2 Cl NCS vibrations*

The factor group method of classifying fundamental vibrational modes of crystals, as developed by Bhagavantam and Venkatarayudu [18], is certainly the most powerful method of treating C2H10N2 Cl NCS crystal structure. The unit cell of C2H10N2 Cl NCS contains 18 atoms which correspond to 54 degrees of vibrational freedom. To simplify the discussion of the IR data, the vibrational modes will be considered in two groups: the internal modes of SCN<sup>−</sup> anions and (C2H10N2) 2+ cations. Ethylenediammonium thiocyanate chloride crystallizes, at room temperature,

*Recent Advances in Analytical Chemistry*

**HF/6-311++G (d. p)**

**Methods**

**B3LYP/6- 311++G (d. p)** **Experimental value [12]**

**B3LYP/6- 31++G (d. p)**

C(3)–N(3)–H(1N3) 112.06 113.78 113.77 109.5 C(3)–N(3)–H(2N3) 111.40 112.73 112.77 109.5 C(3)–N(3)–H(3N3) 107.78 107.37 107.38 109.5 N(2)–C(2)–H(1C2) 107.63 106.91 106.89 109.0 N(2)–C(2)–H(2C2) 105.68 105.49 105.65 109.0 N(3)–C(3)–H(2C3) 106.94 107.64 107.64 109.0 N(3)–C(3)–H(1C3) 107.95 105.96 105.94 109.0 C(2)–C(3)–H(1C3) 107.81 108.17 108.14 109.0 C(2)–C(3)–H(2C3) 111.33 111.69 111.62 109.0 C(3)–C(2)–H(1C2) 111.34 111.77 111.73 109.0 C(3)–C(2)–H(2C2) 107.22 108.54 108.49 109.0 H(1C2)–C(2)–H(2C2) 109.25 107.86 107.81 107.8 H(2C3)–C(3)–H(1C3) 110.86 108.27 108.30 107.8 H(1N2)–N(2)–H(2N2) 106.29 103.41 103.45 109.5 H(1N2)–N(2)–H(3N2) 108.76 108.24 108.27 109.5 H(2N2)–N(2)–H(3N2) 108.54 108.53 108.59 109.5 H(1N3)–N(3)–H(2N3) 112.15 112.78 112.80 109.5 H(1N3)–N(3)–H(3N3) 106.29 103.84 103.89 109.5 H(2N3)–N(3)–H(3N3) 107.41 107.83 107.88 109.5 N(2)–C(2)–C(3)–N(3) 74.53 74.32 74.36 72.09 (12)

*Optimized geometrical parameters for ethylenediammonium chloride thiocyanate computed at HF/6-311++G* 

*(d. p), B3LYP/6-31++G (d. p) and B3LYP/6-311++G(d. p) basis sets.*

*Bond length differences between theoretical (HF and DFT) approaches.*

**Geometrical parameters**

**48**

**Figure 2.**

**Table 1.**

in the triclinic system, space group P1 (Ci). The entities [C2H10N2] 2+, [SCN]<sup>−</sup> and [Cl]<sup>−</sup> occupy sites of symmetry (C1).

#### *3.2.1.2 Counting by the factor group method*

The number of normal modes of vibration of the group SCN<sup>−</sup> isolated of ideal symmetry C∞v is given by the representation:

$$
\Gamma\_{\text{SCN}} = \mathsf{ZA}\_1 \mathsf{+} \mathsf{E}\_1 \tag{1}
$$

While that of an isolated group [C2H10N2] 2+ of symmetry C2v is given by the irreducible representation:

$$
\Gamma\_{\text{[C2H10N2]}\text{2}\_{\ast}} = \mathbf{11A}\_1 + \mathbf{8A}\_2 + \mathbf{9B}\_1 + \mathbf{8B}\_2 \tag{2}
$$

The correlation diagram is given in **Table 2**. The counting of the main vibrations of this compound by the factor group method leads to the following results:


The analysis in terms of internal vibrations, rotation R' and translation T', is given in **Table 3** with their activities in IR.

#### *3.2.1.3 Enumeration by the site group method*

This method was used in order to have a detailed description of the symmetry and the nature of the internal vibrations (deformation in the plane or out of the plane, symmetrical or asymmetrical elongation, torsion, etc.).

*3.2.1.3.1 Vibrations of [C2H10N2] 2+ in group (C1)*

To describe the vibrations of the organic cation, we considered separately the vibrations of the groups (-NH3) and (-CH2-) and the skeleton (C2N2) 2+.

a.Description of the normal modes of vibration of the grouping (▬NH3)

The group (▬NH3) supposed free, has the symmetry 3 m (C3v), it presents nine internal vibrations schematized in **Figure 4**.

$$\text{2A1} \star \text{A2} \star \text{3E} \tag{3}$$

Each group (▬NH3) occupies a site (C1) in the cation. The use of correlation tables allows us to describe the symmetry of these vibrations in the molecular group of the cation (**Table 4**). The result is:

$$
\Gamma\_{\text{NHB}} = \mathbf{ 18Ag} + \mathbf{18Au} \tag{4}
$$

**51**

**Table 3.**

**Table 2.**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

b.Description of the normal modes of vibration of the grouping (▬CH2▬)

internal vibrations schematized in **Figure 5**.

*Internal mode correlation diagrams of C2H10N2 Cl NCS in Ci.*

cation (**Table 5**). The result is:

**EDA (C2v)**

**SCN<sup>−</sup> (C∞v)**

**Cl<sup>−</sup> (C1)**

*Enumeration of internal and external modes of C2H10N2 Cl NCS in Ci.*

**EDA (C2v)**

The group (▬CH2▬) supposed free, has the symmetry mm2 (C2v), it has six

2A1 + A2 + B1 + 2B2 (5)

The (▬CH2▬) groups occupy E(C1) sites in the cation, the correlation method allows us to determine their vibrational symmetry in the C1 molecular group of the

ГCH2 = 12Ag + 12Au (6)

**Ci ni n'i R' T' Activity**

**SCN<sup>−</sup> (C∞v)**

**Ag** 54 36 4 0 3 2 0 3 3 3 - + **Au** 54 36 4 0 3 2 0 3 3 3 + -

**Cl<sup>−</sup> (C1)**

**EDA (C2v)**

**SCN<sup>−</sup> (C∞v)**

**Cl<sup>−</sup> (C1)** **IR R**

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*

**Table 2.** *Internal mode correlation diagrams of C2H10N2 Cl NCS in Ci.*

b.Description of the normal modes of vibration of the grouping (▬CH2▬)

The group (▬CH2▬) supposed free, has the symmetry mm2 (C2v), it has six internal vibrations schematized in **Figure 5**.

$$\text{2A}\_1 + \text{A}\_2 + \text{B}\_1 + \text{2B}\_2 \tag{5}$$

The (▬CH2▬) groups occupy E(C1) sites in the cation, the correlation method allows us to determine their vibrational symmetry in the C1 molecular group of the cation (**Table 5**). The result is:

$$
\Gamma\_{\rm CH2} = \ 12\mathbf{A}\mathbf{g} + 12\mathbf{A}\mathbf{u} \tag{6}
$$


**Table 3.**

*Enumeration of internal and external modes of C2H10N2 Cl NCS in Ci.*

*Recent Advances in Analytical Chemistry*

[Cl]<sup>−</sup> occupy sites of symmetry (C1).

irreducible representation:

*3.2.1.2 Counting by the factor group method*

symmetry C∞v is given by the representation:

While that of an isolated group [C2H10N2]

• Translation modes: Г(T') = 9Ag + 9Au

• Rotation mode: Г(R') = 5Ag + 5Au

given in **Table 3** with their activities in IR.

*3.2.1.3 Enumeration by the site group method*

internal vibrations schematized in **Figure 4**.

of the cation (**Table 4**). The result is:

*3.2.1.3.1 Vibrations of [C2H10N2]*

in the triclinic system, space group P1 (Ci). The entities [C2H10N2]

The number of normal modes of vibration of the group SCN<sup>−</sup> isolated of ideal

ГSCN = 2A1 + E1 (1)

Г[C2H10N2]2+ = 11A1 + 8A2 + 9B1 + 8B2 (2)

of this compound by the factor group method leads to the following results:

• The representation of the internal vibrations is: Г(n'i) = 40Ag + 40Au

The analysis in terms of internal vibrations, rotation R' and translation T', is

This method was used in order to have a detailed description of the symmetry and the nature of the internal vibrations (deformation in the plane or out of the

To describe the vibrations of the organic cation, we considered separately the

The group (▬NH3) supposed free, has the symmetry 3 m (C3v), it presents nine

2A1 + A2 + 3E (3)

Each group (▬NH3) occupies a site (C1) in the cation. The use of correlation tables allows us to describe the symmetry of these vibrations in the molecular group

ГNH3 = 18Ag + 18Au (4)

*2+ in group (C1)*

a.Description of the normal modes of vibration of the grouping (▬NH3)

• Overall vibration representation: Г(ni) = 54Ag + 54Au

plane, symmetrical or asymmetrical elongation, torsion, etc.).

vibrations of the groups (-NH3) and (-CH2-) and the skeleton (C2N2)

The correlation diagram is given in **Table 2**. The counting of the main vibrations

2+, [SCN]<sup>−</sup> and

2+ of symmetry C2v is given by the

2+.

**50**

#### **Figure 4.**

*Normal modes of vibration of groups (-NH3) of symmetry 3m (C3v).*

c.Description of the vibration modes of the skeleton (NC2N)

To describe the vibrations of the skeleton (NC2N), the corresponding symmetrical coordinates as a function of the internal coordinates have been calculated as follows:


**53**

**Table 5.**

*Internal modes of (-CH2-) in (Ci).*

**Figure 5.**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

The number of coordinates is 6 = 3 N-6 (N: number of atoms in the backbone, here N = 4). Using the transforms of each coordinate under the symmetry operations of the point group Cs corresponding to the cation, six symmetrized coordinates were calculated (**Table 6**). At each coordinate a vibration mode has been assigned. These vibrations are shown schematically in **Figure 6**. The description of the normal modes of the NC2N backbone and their activities in IR are shown in **Table 7**. The irreducible

Skeletal = 6Ag + 6Au (7)

The internal vibrations of the SCN<sup>−</sup> anion have already been studied [2], they are described in terms of symmetrized coordinates as a function of the internal

) + E1(Π) (8)

representation of the internal vibration modes of the skeleton in Ci is:

coordinates. These modes are divided in the group C∞v as follows:

d.Description of the vibration modes of SCN<sup>−</sup>

*Normal modes of vibration of (-CH2-) of symmetry mm2, (C2v).*

Г(SCN−) = 2A1(Σ<sup>+</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*

#### **Figure 5.**

*Recent Advances in Analytical Chemistry*

c.Description of the vibration modes of the skeleton (NC2N)

• Increased C▬N bonds: Δri (i = 1, 2)

• Increase of the CCN bond angles: Δφi (i = 1, 2)

*Normal modes of vibration of groups (-NH3) of symmetry 3m (C3v).*

• Increased C▬C bonds: Δr3

• Torsion of DC links: τCC

To describe the vibrations of the skeleton (NC2N), the corresponding symmetrical coordinates as a function of the internal coordinates have been calculated

**52**

**Table 4.**

*Internal modes of (-NH3) in (Ci).*

as follows:

**Figure 4.**

*Normal modes of vibration of (-CH2-) of symmetry mm2, (C2v).*

The number of coordinates is 6 = 3 N-6 (N: number of atoms in the backbone, here N = 4). Using the transforms of each coordinate under the symmetry operations of the point group Cs corresponding to the cation, six symmetrized coordinates were calculated (**Table 6**). At each coordinate a vibration mode has been assigned. These vibrations are shown schematically in **Figure 6**. The description of the normal modes of the NC2N backbone and their activities in IR are shown in **Table 7**. The irreducible representation of the internal vibration modes of the skeleton in Ci is:

$$\text{Skeletal} \quad = \text{ } \mathsf{6Ag} \text{ } \mathsf{+6Au} \tag{7}$$

d.Description of the vibration modes of SCN<sup>−</sup>

The internal vibrations of the SCN<sup>−</sup> anion have already been studied [2], they are described in terms of symmetrized coordinates as a function of the internal coordinates. These modes are divided in the group C∞v as follows:

$$
\Gamma\_{\text{(SCN}^-)} = \text{ } \text{2A}\_1 \text{(}\Sigma^\*\text{)} \text{ } \text{+E}\_1 \text{(II)}\tag{8}
$$

**Table 5.** *Internal modes of (-CH2-) in (Ci).*


**Table 6.**

*Symmetric vibrational coordinates of NC2N in (C2v).*

These vibrations are shown schematically in **Figure 7**. The vibrational analysis in terms of internal vibrations is given in **Table 8**. The distribution of normal SCN group modes and their IR activity are shown in **Table 9**. The irreducible representation of the internal vibration modes of SCN<sup>−</sup> in Ci is:

$$
\Gamma\_{\text{SCN}}{}^{-} = \,^{4}\text{Ag} + \text{4Au} \tag{9}
$$

**55**

**Figure 7.**

*Normal modes of vibration of the anion SCN<sup>−</sup> in (C*∞*v).*

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

**Description of mnv C2v C1 (site group) Ci (factor group)**

νs(CN) A1(IR, R) A(IR, R) Ag(R) + Au(IR) νas(CN) B1(IR, R) A(IR, R) Ag(R) + Au(IR) νs (CC) A1(IR, R) A(IR, R) Ag(R) + Au(IR)

δs (CCN) A1(IR, R) A(IR, R) Ag(R) + Au(IR) δas (CCN) B1(IR, R) A(IR, R) Ag(R) + Au(IR)

τCC B2(IR, R) A(IR, R) Ag(R) + Au(IR)

DFT methods to HF/6-311++G (d, p), B3LYP/6-31++G (d, p) and B3LYP/6-311++G (d, p). The basic sets for the EDCT are shown in **Figure 10**. It appears from the figure that the frequencies calculated by B3LYP with 6-31++G (d, p) of basis sets are closer to the experimental frequencies as HF method with 6-311++G (d, p) base set.

The asymmetric stretching νas(NH3) of symmetries (Ag + Au) are observed in IR

twisting modes are assigned as predicted by the calculations and in accordance with the expected regions for similar compounds [7, 8, 19, 20], as observed in **Table 10**.

. The symmetric stretching νs(NH3) of symmetries (Ag + Au)

. The asymmetric deformation δas(NH3) of sym-

. The symmetric deformation

. The Rocking

. The rocking and

. The

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

**3.4 Bands assignments**

at 3325 and 3326 cm<sup>−</sup><sup>1</sup>

are observed in IR at 3210 cm<sup>−</sup><sup>1</sup>

metry (Ag + Au) observed IR at 1500 and 1570 cm<sup>−</sup><sup>1</sup>

*Distribution of normal skeleton vibration modes in C2H10N2 Cl NCS.*

δs(NH3) of symmetries (Ag + Au) are observed in IR at 1467 cm<sup>−</sup><sup>1</sup>

torsion δτ(NH3) of symmetries (Au) observed in IR at 483 cm<sup>−</sup><sup>1</sup>

δρ(NH3) of symmetries (Au) are observed only in IR at 493 and 498 cm<sup>−</sup><sup>1</sup>

*3.4.1 NH3 modes*

**Valence**

**Deformation**

**Torsion**

**Table 7.**

#### **3.3 Group theory analysis**

The comparisons of the experimental infrared spectra for EDCT, by using HF/6-311++G (d, p), (B3LYP) 311++G (d, p) and (B3LYP)/6-31++G (d, p) theory level, with the corresponding average predicted demonstrate good correlations as observed in **Figure 8**. Using the split triple valence base as well as the diffuse and polarization functions for computed harmonic vibratory frequencies of EDCT, 6-31++G (d, p) and 6-311++G (d, p), the frequencies FT-IR observed for various vibration modes were presented in **Table 10**. The comparative values of IR intensities activities are presented in **Table 11** and their corresponding graph given in **Figure 9**. The comparative graph of vibratory frequencies calculated by the HF and

**Figure 6.** *Normal modes of NC2N skeleton vibration in (C2V).*

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*


**Table 7.**

*Recent Advances in Analytical Chemistry*

A 1 = \_\_1 √ \_\_ <sup>2</sup> (∆r1 + ∆r2)

S2 A 1 = ∆r3

S3 A 1 = \_\_1 √ \_\_ 2

B

B 1 = \_\_1 √ \_\_ 2

*Symmetric vibrational coordinates of NC2N in (C2v).*

S5 B 1 = \_\_1 √ \_\_ 2

A1 S1

B1 S6

B2 S4

**3.3 Group theory analysis**

**Table 6.**

**Class Symmetric coordinate Vibration modes**

Symmetrical elongation C-N: νs (C-N) Symmetrical elongation C-C: νs (C-C)

Asymmetrical elongation C-N: νas (C-N)

Symmetrical deformation in the plane CCN: δs (CCN)

Asymmetrical deformation in the plane CCN: δas(CCN)

(∆φ1 + ∆φ 2)

(∆r1 − ∆r2)

(∆φ<sup>1</sup> − ∆φ2)

tion of the internal vibration modes of SCN<sup>−</sup> in Ci is:

2 = τcc Twist out of the plane CCN: τcc

These vibrations are shown schematically in **Figure 7**. The vibrational analysis in terms of internal vibrations is given in **Table 8**. The distribution of normal SCN group modes and their IR activity are shown in **Table 9**. The irreducible representa-

ГSCN<sup>−</sup> = 4Ag + 4Au (9)

The comparisons of the experimental infrared spectra for EDCT, by using HF/6-311++G (d, p), (B3LYP) 311++G (d, p) and (B3LYP)/6-31++G (d, p) theory level, with the corresponding average predicted demonstrate good correlations as observed in **Figure 8**. Using the split triple valence base as well as the diffuse and polarization functions for computed harmonic vibratory frequencies of EDCT, 6-31++G (d, p) and 6-311++G (d, p), the frequencies FT-IR observed for various vibration modes were presented in **Table 10**. The comparative values of IR intensities activities are presented in **Table 11** and their corresponding graph given in **Figure 9**. The comparative graph of vibratory frequencies calculated by the HF and

**54**

**Figure 6.**

*Normal modes of NC2N skeleton vibration in (C2V).*

*Distribution of normal skeleton vibration modes in C2H10N2 Cl NCS.*

DFT methods to HF/6-311++G (d, p), B3LYP/6-31++G (d, p) and B3LYP/6-311++G (d, p). The basic sets for the EDCT are shown in **Figure 10**. It appears from the figure that the frequencies calculated by B3LYP with 6-31++G (d, p) of basis sets are closer to the experimental frequencies as HF method with 6-311++G (d, p) base set.

#### **3.4 Bands assignments**

#### *3.4.1 NH3 modes*

The asymmetric stretching νas(NH3) of symmetries (Ag + Au) are observed in IR at 3325 and 3326 cm<sup>−</sup><sup>1</sup> . The symmetric stretching νs(NH3) of symmetries (Ag + Au) are observed in IR at 3210 cm<sup>−</sup><sup>1</sup> . The asymmetric deformation δas(NH3) of symmetry (Ag + Au) observed IR at 1500 and 1570 cm<sup>−</sup><sup>1</sup> . The symmetric deformation δs(NH3) of symmetries (Ag + Au) are observed in IR at 1467 cm<sup>−</sup><sup>1</sup> . The Rocking δρ(NH3) of symmetries (Au) are observed only in IR at 493 and 498 cm<sup>−</sup><sup>1</sup> . The torsion δτ(NH3) of symmetries (Au) observed in IR at 483 cm<sup>−</sup><sup>1</sup> . The rocking and twisting modes are assigned as predicted by the calculations and in accordance with the expected regions for similar compounds [7, 8, 19, 20], as observed in **Table 10**.

**Figure 7.** *Normal modes of vibration of the anion SCN<sup>−</sup> in (C*∞*v).*


#### **Table 8.**

*Internal modes of (SCN)<sup>−</sup> in (C*∞*v).*

#### *3.4.2 CH2 modes*

By comparison with previous works reported on similar compounds containing [C2H10N2] 2+ [21], we have attributed the bands observed in IR at 3222 and 2427 cm<sup>−</sup><sup>1</sup> to asymmetric stretching νas(CH2) and symmetric νs(CH2) of symmetries (Ag + Au), respectively. The asymmetric deformation δas(CH2) and symmetric δs(CH2) is observed at 1452 and 1200 cm<sup>−</sup><sup>1</sup> in IR spectrum at 1341 and 1454 cm<sup>−</sup><sup>1</sup> . The calculated frequencies of B3LYP/6-31++G (d, p) and B3LYP/6-311++G (d, p) methods for CH2 asymmetric and as asymmetric vibrations showed excellent agreement with recorded spectrum as well as literature data. The Rocking δρ (CH2) of symmetries (Ag + Au) are observed in IR at 1000 cm<sup>−</sup><sup>1</sup> . The torsion δτ(CH2) of symmetry (Ag + Au) observed in IR at 1124 cm<sup>−</sup><sup>1</sup> . The rocking and twisting modes are assigned as predicted by calculations, as indicated in **Table 10**.

#### *3.4.3 Skeletal modes*

The NCCN skeleton gives six normal modes of vibration that may be described as three skeleton stretching (2νCN + 1νCC), two NCCN deformation modes and one torsional mode around the C▬C bond. The symmetrical elongations of the symmetry skeleton νs (CC) of symmetries (Ag + Au) appear in IR at 750 cm<sup>−</sup><sup>1</sup> . The asymmetric stretching νas(CN) of symmetries (Ag + Au) observed in IR at 544 cm<sup>−</sup><sup>1</sup> . The symmetric stretching νs(CN) of symmetries (Au) observed in IR at 532 cm<sup>−</sup><sup>1</sup> . The asymmetric deformation δas(CCN) of symmetry (Ag + Au), is observed in IR at 435 cm<sup>−</sup><sup>1</sup> . The symmetric deformation δs(CCN) of symmetry (Au) observed only in IR at 430 cm<sup>−</sup><sup>1</sup> .

#### *3.4.4 Internal modes of the thiocyanate group (SCN<sup>−</sup>)*

The thiocyanate group (SCN<sup>−</sup> ) has four vibrations in the C∞v group: two of valence denoted [ν1(CS), ν2(CN)] of symmetry (Σ<sup>+</sup> ) and a doubly degenerate deformation


**57**

**Figure 8.**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

*(A) Experimental infrared spectrum of C2H10N2 Cl NCS in the solid phase compared with the calculated with:* 

*(B) (HF)/6-311++G (d, p), (C) B3LYP/6-31++G (d, p) and (D) B3LYP/6-311++G (d, p).*

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

**Table 9.**

*Description of the normal modes of SCN<sup>−</sup> in C2H10N2 Cl NCS.*

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*

#### **Figure 8.**

*Recent Advances in Analytical Chemistry*

*3.4.2 CH2 modes*

*Internal modes of (SCN)<sup>−</sup> in (C*∞*v).*

ing [C2H10N2]

*3.4.3 Skeletal modes*

δs(CH2) is observed at 1452 and 1200 cm<sup>−</sup><sup>1</sup>

of symmetries (Ag + Au) are observed in IR at 1000 cm<sup>−</sup><sup>1</sup>

are assigned as predicted by calculations, as indicated in **Table 10**.

skeleton νs (CC) of symmetries (Ag + Au) appear in IR at 750 cm<sup>−</sup><sup>1</sup>

ric stretching νs(CN) of symmetries (Au) observed in IR at 532 cm<sup>−</sup><sup>1</sup>

stretching νas(CN) of symmetries (Ag + Au) observed in IR at 544 cm<sup>−</sup><sup>1</sup>

deformation δas(CCN) of symmetry (Ag + Au), is observed in IR at 435 cm<sup>−</sup><sup>1</sup>

symmetric deformation δs(CCN) of symmetry (Au) observed only in IR at 430 cm<sup>−</sup><sup>1</sup>

**Description of mnv C∞<sup>v</sup> G.S (C1) C.G (G.F)**

ν1(CS) A1(IR, R) A(IR, R) 2Ag(R) + 2Au(IR) ν2(CN) A1(IR, R) A(IR, R) 2Ag(R) + 2Au(IR)

δ(SCN) E1 A(IR, R) 2Ag(R) + 2Au(IR)

symmetry (Ag + Au) observed in IR at 1124 cm<sup>−</sup><sup>1</sup>

*3.4.4 Internal modes of the thiocyanate group (SCN<sup>−</sup>)*

The thiocyanate group (SCN<sup>−</sup>

denoted [ν1(CS), ν2(CN)] of symmetry (Σ<sup>+</sup>

*Description of the normal modes of SCN<sup>−</sup> in C2H10N2 Cl NCS.*

2427 cm<sup>−</sup><sup>1</sup>

**Table 8.**

By comparison with previous works reported on similar compounds contain-

(Ag + Au), respectively. The asymmetric deformation δas(CH2) and symmetric

The calculated frequencies of B3LYP/6-31++G (d, p) and B3LYP/6-311++G (d, p) methods for CH2 asymmetric and as asymmetric vibrations showed excellent agreement with recorded spectrum as well as literature data. The Rocking δρ (CH2)

The NCCN skeleton gives six normal modes of vibration that may be described as three skeleton stretching (2νCN + 1νCC), two NCCN deformation modes and one torsional mode around the C▬C bond. The symmetrical elongations of the symmetry

2+ [21], we have attributed the bands observed in IR at 3222 and

to asymmetric stretching νas(CH2) and symmetric νs(CH2) of symmetries

in IR spectrum at 1341 and 1454 cm<sup>−</sup><sup>1</sup>

) has four vibrations in the C∞v group: two of valence

) and a doubly degenerate deformation

. The torsion δτ(CH2) of

. The asymmetric

. The symmet-

. The

.

. The asymmetric

. The rocking and twisting modes

.

**56**

**Table 9.**

**Valence**

**Deformation**

*(A) Experimental infrared spectrum of C2H10N2 Cl NCS in the solid phase compared with the calculated with: (B) (HF)/6-311++G (d, p), (C) B3LYP/6-31++G (d, p) and (D) B3LYP/6-311++G (d, p).*



#### **Table 10.**

*Observed, HF/6-31++G (d. p), B3LYP/6-31++G (d. p) and B3LYP/6-311++G (d. p) level calculated vibrational frequency of ethylenediammonium chloride thiocyanate.*

vibration denoted δ1 (SCN) of symmetry (π). From the bibliographic results [22–28] and the analysis by group theory, an attempt to attribute these vibrations observed in IR is illustrated in **Table 10**. The deformation δ1 (SCN) of symmetry (1Au) is observed in IR at 409 and 424 cm<sup>−</sup><sup>1</sup> . The calculated frequencies of B3LYP/6-31++G (d, p) and

**59**

**Figure 9.**

**Table 11.**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

**B3LYP/6-31++G (d. p)**

**IR intensity**

**IR intensity**

 14.48 25 12.72 13.50 19.57 15.30 14.59 15.73 26 24.56 9.27 18.75 10.22 22.15 7.17 27 32.62 6.33 36.54 4.37 34.14 20.26 28 30.24 12.70 33.84 10.60 31.04 13.87 29 0.11 13.85 1.40 17.22 0.40 9.54 30 23.90 9.61 131.09 7.91 129.89 28.49 31 12.19 27.24 9.80 27.24 9.09 45.53 32 176.12 73.75 149.02 70.95 141.72 111.58 33 199.40 87.48 190.70 87.08 188.76 29.05 34 82.23 6.61 48.96 8.11 42.56 2.12 35 13.39 166.12 62.76 186.03 62.76 6.16 36 65.01 12.53 99.11 13.63 99.11 31.58 37 64.34 3.41 161.00 5.11 159.11 12.29 38 194.80 37.30 83.40 35.20 83.40 18.08 39 955.17 5.35 398.06 3.15 518.06 2.11 40 595.05 7.87 17.3604 8.07 102.36 0.73 41 5.95 3.34 2.72 1.04 1.79 25.97 42 190.52 17.59 189.07 12.09 199.17 41.46 43 2.82 9.63 4.62 10.03 4.32 2.43 44 0.69 67.12 523.90 61.02 503.60 55.16 45 1.88 12.97 118.46 10.17 111.16 66.06 46 9.01 42.44 380.52 41.01 370.12 93.86 47 121.09 79.33 117.71 89.83 115.51 28.19 48 111.24 32.91 90.63 27.11 77.83

*Comparative values of IR intensities activities between HF/6-31++G (d. p), B3LYP/6-31++G (d. p) and* 

*B3LYP/6-311++ G (d. p) of ethylenediammonium chloride thiocyanate.*

*Comparative graph of IR intensities by HF and DFT (B3LYP).*

**Calculated with B3LYP/6-311++G (d. p)**

> **IR intensity**

**IR intensity**

**Calculated with HF/6-311++G (d. p) Calculated with** 

**IR intensity**

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

**Mode nos.**

**IR intensity**

**Mode nos.**


*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*

#### **Table 11.**

*Recent Advances in Analytical Chemistry*

**58**

**Table 10.**

in IR at 409 and 424 cm<sup>−</sup><sup>1</sup>

vibration denoted δ1 (SCN) of symmetry (π). From the bibliographic results [22–28] and the analysis by group theory, an attempt to attribute these vibrations observed in IR is illustrated in **Table 10**. The deformation δ1 (SCN) of symmetry (1Au) is observed

*Observed, HF/6-31++G (d. p), B3LYP/6-31++G (d. p) and B3LYP/6-311++G (d. p) level calculated* 

*vibrational frequency of ethylenediammonium chloride thiocyanate.*

. The calculated frequencies of B3LYP/6-31++G (d, p) and

*Comparative values of IR intensities activities between HF/6-31++G (d. p), B3LYP/6-31++G (d. p) and B3LYP/6-311++ G (d. p) of ethylenediammonium chloride thiocyanate.*

**Figure 9.** *Comparative graph of IR intensities by HF and DFT (B3LYP).*

#### **Figure 10.**

*Comparative graphs of computed frequencies (HF and DFT) with experimental frequencies.*

B3LYP/6-311++G (d, p) methods for SCN deformation symmetric vibrations showed excellent agreement with recorded spectrum as well as literature data. We note a rise of degeneracy of the symmetry π of δ1 (SCN) with a burst of 33 cm<sup>−</sup><sup>1</sup> . The symmetric stretching ν1 (C〓S) of symmetries (1Ag + 1Au) are observed in IR at 450 and 484 cm<sup>−</sup><sup>1</sup> . The calculated frequencies of B3LYP/6-31++G (d, p) and B3LYP/6-311++G (d, p) methods for C〓S symmetric vibrations showed excellent agreement with recorded spectrum as well as literature data. The symmetric stretching ν2 (C〓N) of symmetries (1Ag + 1Au), predicted by the group theory, are observed in IR at 1616 cm<sup>−</sup><sup>1</sup> .

#### **3.5 Other molecular properties**

Several calculated thermodynamic parameters are presented in **Table 12**. Scale factors have been recommended [29] for an accurate prediction in determining the


**61**

provided the original work is properly cited.

Sahel Karoui\* and Slaheddine Kamoun

National School of Engineering, Sfax, Tunisia

\*Address all correspondence to: karouisahel@yahoo.fr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Laboratory of Material Engineering and Environment (LR11ES46),

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

**Parameters HF/6-311++ (d, p) B3LYP/6-31++(d, p) B3LYP/6-311++G (d. p)**

*), rotational constants (GHz), rotational* 

 *K<sup>−</sup><sup>1</sup>*

*) entropies* 

*), molar capacity at constant volume (cal mol<sup>−</sup><sup>1</sup>*

Translational 41.025 41.025 41.025 Rotational 31.426 31.426 31.074 Vibrational 15.540 15.594 15.589 Total 87.792 87.546 87.587 Dipole moment 39.4045 38.6462 38.6426

*) and dipole moment (Debye) for ethylenediammonium chloride thiocyanate.*

zero-point vibration energies, and the entropy. It can be seen that the total energies decrease with the increase of the size of the basic set. Changes in the total entropy of EDCT at room temperature and in different basic sets are only marginal.

The present document attempts to define the appropriate frequency assignments for the thiocyanate ethylenediammonium chloride compound from the FT-IR spectrum. Vibrational frequencies and infrared intensities are calculated and analyzed by the theoretical HF and DFT (B3LYP) levels, using the 6-31++G (d, p) and 6-311++G (d, p)base sets.. The comparison between the calculated vibrational frequencies and the experimental values indicates that both methods can predict the FT-IR spectra of the title compound. The results of DFT-B3LYP method indicate better fit to experimental ones than ab initio HF upon evaluation of vibrational frequencies. Several thermodynamic parameters of the title molecule are comparatively discussed. The observed and the calculated wavenumbers

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

*Theoretically computed zero point vibrational energy (kcal mol<sup>−</sup><sup>1</sup>*

are found to be in good agreement with majority modes.

*temperature (K), thermal energy (kcal mol<sup>−</sup><sup>1</sup>*

**Entropy**

**Table 12.**

*(cal mol<sup>−</sup><sup>1</sup>*

 *K<sup>−</sup><sup>1</sup>*

**4. Conclusions**

**Author details**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*


#### **Table 12.**

*Recent Advances in Analytical Chemistry*

**3.5 Other molecular properties**

**Molar capacity at constant volume**

**Figure 10.**

B3LYP/6-311++G (d, p) methods for SCN deformation symmetric vibrations showed excellent agreement with recorded spectrum as well as literature data. We note a rise

stretching ν1 (C〓S) of symmetries (1Ag + 1Au) are observed in IR at 450 and 484 cm<sup>−</sup><sup>1</sup>

Several calculated thermodynamic parameters are presented in **Table 12**. Scale factors have been recommended [29] for an accurate prediction in determining the

**Parameters HF/6-311++ (d, p) B3LYP/6-31++(d, p) B3LYP/6-311++G (d. p)** Zero point vibration energy 105.84889 100.34649 98.57158 Rotational constants 1.55409 1.63393 1.55409

Rotational temperature 0.07458 0.07842 0.07458

Translational 0.889 0.889 0.889 Rotational 0.889 0.889 0.889 Vibrational 108.982 105.524 105.017 Total 110.759 107.302 107.394

Translational 2.981 2.981 2.981 Rotational 2.981 2.981 2.981 Vibrational 19.092 29.415 17.227 Total 25.054 25.377 23.189

0.47626 0.52535 0.47626 0.37522 0.46146 0.37522

0.02286 0.02521 0.02286 0.01801 0.02215 0.01801

The calculated frequencies of B3LYP/6-31++G (d, p) and B3LYP/6-311++G (d, p) methods for C〓S symmetric vibrations showed excellent agreement with recorded spectrum as well as literature data. The symmetric stretching ν2 (C〓N) of symmetries

(1Ag + 1Au), predicted by the group theory, are observed in IR at 1616 cm<sup>−</sup><sup>1</sup>

. The symmetric

.

.

of degeneracy of the symmetry π of δ1 (SCN) with a burst of 33 cm<sup>−</sup><sup>1</sup>

*Comparative graphs of computed frequencies (HF and DFT) with experimental frequencies.*

**60**

**Energy**

*Theoretically computed zero point vibrational energy (kcal mol<sup>−</sup><sup>1</sup> ), rotational constants (GHz), rotational temperature (K), thermal energy (kcal mol<sup>−</sup><sup>1</sup> ), molar capacity at constant volume (cal mol<sup>−</sup><sup>1</sup> K<sup>−</sup><sup>1</sup> ) entropies (cal mol<sup>−</sup><sup>1</sup> K<sup>−</sup><sup>1</sup> ) and dipole moment (Debye) for ethylenediammonium chloride thiocyanate.*

zero-point vibration energies, and the entropy. It can be seen that the total energies decrease with the increase of the size of the basic set. Changes in the total entropy of EDCT at room temperature and in different basic sets are only marginal.

#### **4. Conclusions**

The present document attempts to define the appropriate frequency assignments for the thiocyanate ethylenediammonium chloride compound from the FT-IR spectrum. Vibrational frequencies and infrared intensities are calculated and analyzed by the theoretical HF and DFT (B3LYP) levels, using the 6-31++G (d, p) and 6-311++G (d, p)base sets.. The comparison between the calculated vibrational frequencies and the experimental values indicates that both methods can predict the FT-IR spectra of the title compound. The results of DFT-B3LYP method indicate better fit to experimental ones than ab initio HF upon evaluation of vibrational frequencies. Several thermodynamic parameters of the title molecule are comparatively discussed. The observed and the calculated wavenumbers are found to be in good agreement with majority modes.

#### **Author details**

Sahel Karoui\* and Slaheddine Kamoun Laboratory of Material Engineering and Environment (LR11ES46), National School of Engineering, Sfax, Tunisia

\*Address all correspondence to: karouisahel@yahoo.fr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Konig JL, Antoon MK. Recent applications of FT-lR spectroscopy to polymer systems. Applied Optics. 1978;**17**:1374

[2] Neumann T, Werner J, Jess I, Näther C. Poly[(l-1,3-thiocyanatojN,S)(isonicotinato-jN,O)(ethanol-jO) cadmium(II)]. Acta Crystallographica. 2012;**68**:1338

[3] Wohlert S, Jess I, Näther C. Crystal structure of di-aqua-bis-(2,6-di-methylpyrazine-κN)bis-(thio-cyanato-κN) cobalt(II) 2,5-di-methyl-pyrazine tris-olvate. Acta Crystallographica. 2013;**69**:195

[4] Reinert T, Jess I, Näther C. Bis(3-tert-butylpyridine-jN)bis(4 tertbutylpyridine-jN)bis(thiocyanatojN)-cadmium. Acta Crystallographica. 2012;**68**:1372

[5] Werner J, Boeckmann J, Jess I, Näther C. Catena-Poly[[bis(3 acetylpyridine-jN)-cadmium]-di-lselenocyanatoj2N:Se;j2Se:N]. Acta Crystallographica. 2012;**68**:704

[6] Jan B, Näther C. Catena-Poly[[bis[[bis(3-aminopropyl)-amine-j3 N, N', N''](thiocyanato-jN)-cadmium] l4-sulfato-j4 O,O:O0,O0] methanol hemisolvate]. Acta Crystallographica. 2011;**67**:1201-1202

[7] Karoui S, Kamoun S, Michaud F. Ethylenediammonium chloride thiocyanate. Acta Crystallographica. 2013;**69**:669

[8] Jornet D, Bartovsky P, Domingo LR, Tormos R, Miranda MA. A characterization of the raman modes in a j-aggregate-forming dye: A comparison between theory and experiment. The Journal of Physical Chemistry. 2010;**114B**:11920

[9] Bartlett HE, Jurriaanse A, De Haas K. Activity coefficients of aqueous thiocyanic acid solutions from electromotive force, transference number, and freezing- point depression measurements. Canadian Journal of Chemistry. 1969;**47**(16):2981-2986

[10] Zhang J, Xiao HM. Computational studies on the infrared vibrational spectra, thermodynamic properties, detonation properties, and pyrolysis mechanism of octanitrocubane. The Journal of Chemical Physics. 2002;**116**:10674

[11] Xu XJ, Xiao HM, Ju XH, Gong XD, Zhu WH. Computational studies on polynitrohexaazaadmantanes as potential high energy density materials. The Journal of Physical Chemistry A. 2006;**110**:5929

[12] Chen ZX, Xiao JM, Xiao HM, Chiu YN. Studies on heats of formation for tetrazole derivatives with density functional theory B3LYP method. The Journal of Physical Chemistry A. 1999;**103**:8062

[13] Basis sets density functional (DFT) methods. Gaussian 03 Program. Wallingford, CT: Gaussian Inc.; 2000

[14] Frisch MJ, Nielsen AB, Holder AJ. Gauss View Users Manual. Pittsburgh, PA: Gaussian Inc; 2000

[15] Kamoun S, Jouini A, Kamoun M, Daoud A. Structure of ethylenediammonium bis(dihydrogenmonophosphate). Acta Crystallographica. 1989;**C45**:481-482

[16] Karoui S, Kamoun S, Jouini A. Synthesis, structural and electrical properties of [C2H10N2][(SnCl(NCS)2]2. Journal of Solid State Chemistry. 2013;**197**:60-68

**63**

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium…*

Spectrochimica Acta Part A.

[26] Kniezo L, Kristian P. Synthesis, structure, and properties of β-styryl isothiocyanate. Chemical Papers: Chemicke Zvesti. 1974;**28**:848-853

[27] Kabesova M, Gazo J. Structure and classification of thiocyanates and the mutual influence of their ligands. Chemical Papers: Chemicke Zvesti.

[28] Kohout J, Kabesova M, Gazo J. Synthesis and antiproliferative activity of cyclic arylidene ketones: A direct comparison of monobenzylidene and dibenzylidene derivatives. Monatshefte

für Chemie. 1977;**108**:1011-1018

[29] Palafox MA. Scaling factors for the prediction of vibrational spectra. I. Benzene molecule. International Journal of Quantum Chemistry. 2000;**77**:661

1959;**13**:296-299

1980;**34**:800-841

*DOI: http://dx.doi.org/10.5772/intechopen.82661*

NH,(CH2),NH,(H,P04). Spectrochimica

[18] Bhagavantam S, Venkatarayudu T. Raman effect in relation to crystal structure. Proceedings of the Indiana Academy of Sciences. 1939;**9**:224

[19] Ouasri A, Jeghnou H, Rhandour A, Dhamelincourt MC, Dhamelincourt P, Mazzah A, et al. Structural phase transition in [NH3(CH2)5NH3]BiCl5: Thermal and vibrational studies. Journal of Raman Specroscopy. 2005;**36**:791-796

[20] Jeghnou H, Ouasri A, Rhandour A, Dhamelincourt MC, Dhamelincourt P, Mazzah A, et al. Structural phase transition in (n-C4H9NH3)2SiF6: DSC and Raman studies. Journal of Raman Specroscopy. 2005;**36**:1023-1028

[21] Durig JR, Panikar SS, Iwata T, Gounev TK. Conformational stability of ethylenediamine from temperature

[22] Oden LL, Decius JC. The infrared spectrum of ammonium thiocyanate from 90 to 300°K. Spectrochimica Acta

[23] Duriq JR, Pate CB. Spectrochimica

[24] Wickleder C, Larsen P. Ca(SCN)2 and Ca(SCN)2 ∙ 2H2O: Crystal structure, thermal behavior and

vibrational spectroscopy. Zeitschrift für Naturforschung. 2002;**57**:1419-1426

[25] Lieber E, Rao CNR, Ramachandran J. The infrared spectra of organic thiocyanates and isothiocyanates.

Acta Part A. 1972;**28**:1031-1038

dependent infrared spectra of liquid xenon solutions, r0 structural parameters, ab initio calculations, and vibrational assignments. Journal of Molecular Structure. 2010;**984**:58-67

Part A. 1964;**20**:667-874

[17] Kamoun S, Kamoun M, Daoud A. Etude par spectroscopic (IR et Raman) de l%thyl&ne diammonium bis dihydrog&nomonophosphate

Acta Part A. 1991;**47**:1051-1059

*Contribution of Infrared Spectroscopy to the Vibrational Study of Ethylenediammonium… DOI: http://dx.doi.org/10.5772/intechopen.82661*

[17] Kamoun S, Kamoun M, Daoud A. Etude par spectroscopic (IR et Raman) de l%thyl&ne diammonium bis dihydrog&nomonophosphate NH,(CH2),NH,(H,P04). Spectrochimica Acta Part A. 1991;**47**:1051-1059

[18] Bhagavantam S, Venkatarayudu T. Raman effect in relation to crystal structure. Proceedings of the Indiana Academy of Sciences. 1939;**9**:224

[19] Ouasri A, Jeghnou H, Rhandour A, Dhamelincourt MC, Dhamelincourt P, Mazzah A, et al. Structural phase transition in [NH3(CH2)5NH3]BiCl5: Thermal and vibrational studies. Journal of Raman Specroscopy. 2005;**36**:791-796

[20] Jeghnou H, Ouasri A, Rhandour A, Dhamelincourt MC, Dhamelincourt P, Mazzah A, et al. Structural phase transition in (n-C4H9NH3)2SiF6: DSC and Raman studies. Journal of Raman Specroscopy. 2005;**36**:1023-1028

[21] Durig JR, Panikar SS, Iwata T, Gounev TK. Conformational stability of ethylenediamine from temperature dependent infrared spectra of liquid xenon solutions, r0 structural parameters, ab initio calculations, and vibrational assignments. Journal of Molecular Structure. 2010;**984**:58-67

[22] Oden LL, Decius JC. The infrared spectrum of ammonium thiocyanate from 90 to 300°K. Spectrochimica Acta Part A. 1964;**20**:667-874

[23] Duriq JR, Pate CB. Spectrochimica Acta Part A. 1972;**28**:1031-1038

[24] Wickleder C, Larsen P. Ca(SCN)2 and Ca(SCN)2 ∙ 2H2O: Crystal structure, thermal behavior and vibrational spectroscopy. Zeitschrift für Naturforschung. 2002;**57**:1419-1426

[25] Lieber E, Rao CNR, Ramachandran J. The infrared spectra of organic thiocyanates and isothiocyanates.

Spectrochimica Acta Part A. 1959;**13**:296-299

[26] Kniezo L, Kristian P. Synthesis, structure, and properties of β-styryl isothiocyanate. Chemical Papers: Chemicke Zvesti. 1974;**28**:848-853

[27] Kabesova M, Gazo J. Structure and classification of thiocyanates and the mutual influence of their ligands. Chemical Papers: Chemicke Zvesti. 1980;**34**:800-841

[28] Kohout J, Kabesova M, Gazo J. Synthesis and antiproliferative activity of cyclic arylidene ketones: A direct comparison of monobenzylidene and dibenzylidene derivatives. Monatshefte für Chemie. 1977;**108**:1011-1018

[29] Palafox MA. Scaling factors for the prediction of vibrational spectra. I. Benzene molecule. International Journal of Quantum Chemistry. 2000;**77**:661

**62**

*Recent Advances in Analytical Chemistry*

[1] Konig JL, Antoon MK. Recent applications of FT-lR spectroscopy to polymer systems. Applied Optics. [9] Bartlett HE, Jurriaanse A, De Haas K.

[10] Zhang J, Xiao HM. Computational studies on the infrared vibrational spectra, thermodynamic properties, detonation properties, and pyrolysis mechanism of octanitrocubane. The Journal of Chemical Physics.

[11] Xu XJ, Xiao HM, Ju XH, Gong XD, Zhu WH. Computational studies on polynitrohexaazaadmantanes as potential high energy density materials. The Journal of Physical Chemistry A.

[12] Chen ZX, Xiao JM, Xiao HM, Chiu YN. Studies on heats of formation for tetrazole derivatives with density functional theory B3LYP method. The Journal of Physical Chemistry A.

[13] Basis sets density functional

PA: Gaussian Inc; 2000

[15] Kamoun S, Jouini A,

of ethylenediammonium

2013;**197**:60-68

Kamoun M, Daoud A. Structure

bis(dihydrogenmonophosphate). Acta Crystallographica. 1989;**C45**:481-482

[16] Karoui S, Kamoun S, Jouini A. Synthesis, structural and electrical properties of [C2H10N2][(SnCl(NCS)2]2. Journal of Solid State Chemistry.

(DFT) methods. Gaussian 03 Program. Wallingford, CT: Gaussian Inc.; 2000

[14] Frisch MJ, Nielsen AB, Holder AJ. Gauss View Users Manual. Pittsburgh,

2002;**116**:10674

2006;**110**:5929

1999;**103**:8062

Activity coefficients of aqueous thiocyanic acid solutions from electromotive force, transference number, and freezing- point depression measurements. Canadian Journal of Chemistry. 1969;**47**(16):2981-2986

[2] Neumann T, Werner J, Jess I, Näther C. Poly[(l-1,3-thiocyanatojN,S)(isonicotinato-jN,O)(ethanol-jO) cadmium(II)]. Acta Crystallographica.

[3] Wohlert S, Jess I, Näther C. Crystal structure of di-aqua-bis-(2,6-di-methylpyrazine-κN)bis-(thio-cyanato-κN) cobalt(II) 2,5-di-methyl-pyrazine tris-olvate. Acta Crystallographica.

[4] Reinert T, Jess I, Näther C. Bis(3-tert-butylpyridine-jN)bis(4 tertbutylpyridine-jN)bis(thiocyanatojN)-cadmium. Acta Crystallographica.

[5] Werner J, Boeckmann J, Jess I, Näther C. Catena-Poly[[bis(3 acetylpyridine-jN)-cadmium]-di-lselenocyanatoj2N:Se;j2Se:N]. Acta Crystallographica. 2012;**68**:704

[6] Jan B, Näther C. Catena-

2011;**67**:1201-1202

2013;**69**:669

Poly[[bis[[bis(3-aminopropyl)-amine-j3 N, N', N''](thiocyanato-jN)-cadmium] l4-sulfato-j4 O,O:O0,O0] methanol hemisolvate]. Acta Crystallographica.

[7] Karoui S, Kamoun S, Michaud F. Ethylenediammonium chloride thiocyanate. Acta Crystallographica.

[8] Jornet D, Bartovsky P, Domingo LR, Tormos R, Miranda MA. A characterization of the raman modes in a j-aggregate-forming dye: A comparison between theory and experiment. The Journal of Physical

Chemistry. 2010;**114B**:11920

**References**

1978;**17**:1374

2012;**68**:1338

2013;**69**:195

2012;**68**:1372

**65**

database search or manually.

glycoproteomics

**1. Introduction**

**Chapter 4**

IgG

**Abstract**

Characterization of Whole and

Fragmented Wild-Type Porcine

*Evelyn Ang, Haley Neustaeter, Emy Komatsu, Oleg Krokhin,* 

Glycoproteomic analyses of tryptic (glyco)peptides from wild-type (WT) porcine IgG were performed. In a first protocol, intact antibody was digested with trypsin, followed by glycopeptide enrichment and liquid chromatographytandem MS (HPLC–MS/MS). This procedure allowed to detect *N*-glycopeptides observed previously (Lopez, P. G. et al., *Glycoconj. J.* 2016, *33* (1), 79), plus other non-reported *N*-glycopeptides. The method provided useful information but did not allow to discern between Fab (antigen-binding region) and Fc (constant region, fragment crystallizable) peptides/glycopeptides. In a second scheme, glycoproteomic analysis was attempted for Fab and Fc fragments obtained by papain and Fabulous™ hydrolysis. Usually employed for milligram amounts of antibodies, the papain and Fabulous™ protocols were adapted to 200 μg of WT IgG. Fab and Fc fragments were separated by size-exclusion (SEC) HPLC. Fractions collected were reanalyzed by gel electrophoresis (SDS-PAGE). Bands were excised, and fragments digested in-gel, followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and HPLC/MS–MS. In the protocol no glycopeptide enrichment was involved, that is, whole tryptic digests were analyzed. Fc *N*-glycopeptides were identified, and greater numbers of non-glycosylated peptides were tabulated. Very few peptides overlapped between Fc and Fab, as most peptides were clearly from Fc or Fab. HPLC-MS/MS detected more sialylated glycoforms than MALDI-TOF-MS. Sections of Fab and Fc were assigned de novo, through a

**Keywords:** porcine IgG, papain, enzymatic fragmentation, Fabulous™,

There have been reports on the mass spectrometric (MS) analysis of pig immunoglobulins (IgG) in relationship with use in a xenotransplantation context [1–4]. These studies have explored the amino acid composition and glycosylation of pig IgG according to glycoproteomic [2, 3] and glycomic [1] workflows involving the enzymatic digestion of whole antibodies. Glycoproteomic workflows resulted in the

*Claudia Nelson, Raymond Bacala, Baylie Gigolyk,* 

*Dave Hatcher and Hélène Perreault*

#### **Chapter 4**

## Characterization of Whole and Fragmented Wild-Type Porcine IgG

*Claudia Nelson, Raymond Bacala, Baylie Gigolyk, Evelyn Ang, Haley Neustaeter, Emy Komatsu, Oleg Krokhin, Dave Hatcher and Hélène Perreault*

## **Abstract**

Glycoproteomic analyses of tryptic (glyco)peptides from wild-type (WT) porcine IgG were performed. In a first protocol, intact antibody was digested with trypsin, followed by glycopeptide enrichment and liquid chromatographytandem MS (HPLC–MS/MS). This procedure allowed to detect *N*-glycopeptides observed previously (Lopez, P. G. et al., *Glycoconj. J.* 2016, *33* (1), 79), plus other non-reported *N*-glycopeptides. The method provided useful information but did not allow to discern between Fab (antigen-binding region) and Fc (constant region, fragment crystallizable) peptides/glycopeptides. In a second scheme, glycoproteomic analysis was attempted for Fab and Fc fragments obtained by papain and Fabulous™ hydrolysis. Usually employed for milligram amounts of antibodies, the papain and Fabulous™ protocols were adapted to 200 μg of WT IgG. Fab and Fc fragments were separated by size-exclusion (SEC) HPLC. Fractions collected were reanalyzed by gel electrophoresis (SDS-PAGE). Bands were excised, and fragments digested in-gel, followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and HPLC/MS–MS. In the protocol no glycopeptide enrichment was involved, that is, whole tryptic digests were analyzed. Fc *N*-glycopeptides were identified, and greater numbers of non-glycosylated peptides were tabulated. Very few peptides overlapped between Fc and Fab, as most peptides were clearly from Fc or Fab. HPLC-MS/MS detected more sialylated glycoforms than MALDI-TOF-MS. Sections of Fab and Fc were assigned de novo, through a database search or manually.

**Keywords:** porcine IgG, papain, enzymatic fragmentation, Fabulous™, glycoproteomics

#### **1. Introduction**

There have been reports on the mass spectrometric (MS) analysis of pig immunoglobulins (IgG) in relationship with use in a xenotransplantation context [1–4]. These studies have explored the amino acid composition and glycosylation of pig IgG according to glycoproteomic [2, 3] and glycomic [1] workflows involving the enzymatic digestion of whole antibodies. Glycoproteomic workflows resulted in the identification of many peptides that could be matched with the conserved gamma portion of the heavy chains of some of the 11 subtypes of pig IgG [5], including *N*-glycopeptides EEQFNSTYR and EAQFNSTYR [3]. No specific information was given on the variable portions of neither Fab nor Fc components, as most of such assignments would have had to be attributed *de novo*. The conserved Fc glycosylation site is often described as the only IgG site glycosylated at 100%, in spite of the fact that 10–15% of wild-type antibodies have glycosylation also in their variable region [6], and reports have shown that even higher glycosylation levels (up to 30–40%) can exist in the variable regions of polyclonal IgGs [7, 8].

For more specificity, the analysis of antibodies by MS can take great advantage of enzymatic fragmentation with papain [9, 10] or new enzymes produced by recombinant methods and available on the market [11]. This type of procedure has not been reported for the fragmentation of porcine IgG, to the authors' knowledge. For instance, procedures have been published for mouse [12, 13], chicken [14], rabbit [15], sheep [16], and human [17, 18] IgGs. The two groups of antibody fragments of primary interest are the antigen-binding fragments (Fab) and class-defining crystallizable fragments (Fc). The hinge region of immunoglobulins (IgGs) is readily accessible to proteolytic attack by enzymes [9, 10], and cleavage at that point produces F(ab')2 or Fab fragments and the Fc fragment. Papain is a nonspecific thiol-endopeptidase and has a sulfhydryl group in its active site, which must be reduced for activity. When IgG molecules are incubated with papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc fragment [9, 10].

Fabulous™ enzyme is a recombinant cysteine protease that under reduced conditions digests in the hinge region of antibodies from many species and subclasses, including human, mouse, rat, and goat, yielding Fab and Fc fragments [11]. As a reducing agent is present during digestion, it is likely that interchain thiols will be reduced. Fabulous™ and papain have typically been used for the production of relatively large amounts of antibody fragments (10 mg of starting material), whereas methods adapted to MS require much less, about 50–200 μg. There is a need for downscaling these workflows, especially for porcine IgG, which has not been previously studied by fragmentation followed by MS.

Reports on post-fragmentation MS analyses of antibodies have demonstrated that detailed fragment characterization allows for the identification of more glycosylation sites than bottom-up approaches [19, 20]. It is also important to study amino acid sequences of the variable portions of IgG for therapeutic purposes [21], and the information obtained after fragmentation is much more specific than data generated through the tryptic digestion of whole intact antibodies.

The interest of the present study is to compare results from two main workflows aimed at characterizing wild-type porcine IgG's glycosylation and amino acid sequence features. In the first workflow, porcine IgG is digested with trypsin, followed by glycopeptide enrichment. Reversed-phase high-performance liquid chromatography (RPLC) coupled with electrospray ionization (ESI) MS and tandem MS (MS/MS) is used to characterize the glycopeptide-rich fraction. The second workflow involves subjecting porcine IgG to fragmentation by one of two enzymes, papain or Fabulous™. The steps necessary between fragmentation and MS included size-exclusion chromatography (SEC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and tryptic digestion. This is the first attempt to characterize porcine IgGs in small amounts (sub-mg) using a combination of these methods. Two different MS techniques were used for the analysis of tryptic products of antibody fragments: matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and ESI/MS–MS coupled with RPLC.

**67**

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

As the tryptic digestion products of whole wild-type porcine IgG antibodies have been characterized by MALDI-MS [2, 3] and ESI-MS and MS/MS (new results presented in this report), data from these different workflows serve as comparative benchmarks between intact and fragmented IgG samples. Overlaps and differences allow to identify peptides and glycopeptides as originating from either the Fc or Fab portions, and database searches [22] can verify if these sequences are already available in the literature or need to be determined *de novo*. Porcine IgG is a complex mixture of subtypes, and the complementarity of MALDI- and LC/ESI-MS–MS brings a considerable amount of information to document the identification of these IgG components.

Wild-type porcine IgGs were obtained from Université de Nantes (Dr. Jean-Paul Soulillou's Laboratory). Trypsin Ultra™ was purchased from Promega (Wisconsin, USA). The Fabulous™ enzyme was kindly provided by Genovis (Cambridge, MA). Dihydroxybenzoic acid (DHB), sinapinic acid, ammonium bicarbonate, dithiothreitol (DTT), L-cysteine, iodoacetamide (IAA), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). Mini-Protean TGX precast gels (4–15%), Precision Plus™ protein standard, 2-mercaptoethanol, and 4x Laemmli sample buffer were obtained from Bio-Rad (Hercules, CA). Imperial™ protein stain, tris base (2-amino-2-(hydroxymethyl)-1,3-propanediol) and immobilized papain-cross linked and 6% in beaded agarose supplied as 50% glycerol in sodium acetate pH 4.5 were purchased from Thermo Scientific (Rockford, IL). Strata-X C-18 cartridges were obtained from Phenomenex (Torrance, CA). Acetonitrile (ACN) was purchased from EMD-Millipore (Darmstadt, Germany). Sodium phosphate dibasic anhydrous was purchased from McArthur Chemical Co. Ltd. (Montreal, Canada). Hydrochloric acid was purchased from Anachemia (Vancouver, Canada) and deion-

ized water was obtained from an adapted filtration system (Millipore).

Porcine IgG (200 μg) was dissolved in 100 mM ammonium bicarbonate (pH~8) and vortexed. A DTT solution (10 mM) was added to the sample, which was then vortexed and left to react at 56°C for 40 min, then cooled to room temperature. After 500 mM IA was added, and the sample was left to react in the dark for 45 min. The excess of IA was quenched with the addition of 100 mM DTT, and the sample was left to react for 10 min in the dark. Trypsin was added and proteolysis took place at 37°C for ~18 h. To deactivate trypsin, the sample was frozen and dried under vacuum. Glycopeptide enrichment was then performed using a ProteoExtract™ glycopeptide enrichment kit (Millipore-Sigma, Etobicoke, ON)

Just before use, 20 mM sodium phosphate digestion buffer was prepared with a 10 mM cysteine content, and the pH was adjusted to 7 Bead-immobilized papain slurry (20 μL, 50%) was added to an Eppendorf™ tube. The beads were washed twice with 160 μL of digestion buffer and then re-suspended in the buffer. Porcine IgG (200 μg) was dissolved in the digestion buffer. This was added to the tube containing the washed immobilized papain and digestion buffer. The sample was

**2.2 Tryptic digestion of whole porcine IgG**

according to the manufacturer's procedure [23].

**2.3 Papain digestion of porcine IgG**

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

**2. Experimental**

**2.1 Materials**

*Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

As the tryptic digestion products of whole wild-type porcine IgG antibodies have been characterized by MALDI-MS [2, 3] and ESI-MS and MS/MS (new results presented in this report), data from these different workflows serve as comparative benchmarks between intact and fragmented IgG samples. Overlaps and differences allow to identify peptides and glycopeptides as originating from either the Fc or Fab portions, and database searches [22] can verify if these sequences are already available in the literature or need to be determined *de novo*. Porcine IgG is a complex mixture of subtypes, and the complementarity of MALDI- and LC/ESI-MS–MS brings a considerable amount of information to document the identification of these IgG components.

#### **2. Experimental**

#### **2.1 Materials**

*Recent Advances in Analytical Chemistry*

fragment [9, 10].

identification of many peptides that could be matched with the conserved gamma portion of the heavy chains of some of the 11 subtypes of pig IgG [5], including *N*-glycopeptides EEQFNSTYR and EAQFNSTYR [3]. No specific information was given on the variable portions of neither Fab nor Fc components, as most of such assignments would have had to be attributed *de novo*. The conserved Fc glycosylation site is often described as the only IgG site glycosylated at 100%, in spite of the fact that 10–15% of wild-type antibodies have glycosylation also in their variable region [6], and reports have shown that even higher glycosylation levels (up to

For more specificity, the analysis of antibodies by MS can take great advantage

class-defining crystallizable fragments (Fc). The hinge region of immunoglobulins (IgGs) is readily accessible to proteolytic attack by enzymes [9, 10], and cleavage at that point produces F(ab')2 or Fab fragments and the Fc fragment. Papain is a nonspecific thiol-endopeptidase and has a sulfhydryl group in its active site, which must be reduced for activity. When IgG molecules are incubated with papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc

Fabulous™ enzyme is a recombinant cysteine protease that under reduced conditions digests in the hinge region of antibodies from many species and subclasses, including human, mouse, rat, and goat, yielding Fab and Fc fragments [11]. As a reducing agent is present during digestion, it is likely that interchain thiols will be reduced. Fabulous™ and papain have typically been used for the production of relatively large amounts of antibody fragments (10 mg of starting material), whereas methods adapted to MS require much less, about 50–200 μg. There is a need for downscaling these workflows, especially for porcine IgG, which has not

Reports on post-fragmentation MS analyses of antibodies have demonstrated

The interest of the present study is to compare results from two main workflows aimed at characterizing wild-type porcine IgG's glycosylation and amino acid sequence features. In the first workflow, porcine IgG is digested with trypsin, followed by glycopeptide enrichment. Reversed-phase high-performance liquid chromatography (RPLC) coupled with electrospray ionization (ESI) MS and tandem MS (MS/MS) is used to characterize the glycopeptide-rich fraction. The second workflow involves subjecting porcine IgG to fragmentation by one of two enzymes, papain or Fabulous™. The steps necessary between fragmentation and MS included size-exclusion chromatography (SEC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and tryptic digestion. This is the first attempt to characterize porcine IgGs in small amounts (sub-mg) using a combination of these methods. Two different MS techniques were used for the analysis of tryptic products of antibody fragments: matrix-assisted laser desorption/ionization

that detailed fragment characterization allows for the identification of more glycosylation sites than bottom-up approaches [19, 20]. It is also important to study amino acid sequences of the variable portions of IgG for therapeutic purposes [21], and the information obtained after fragmentation is much more specific than data

generated through the tryptic digestion of whole intact antibodies.

time-of-flight (MALDI-TOF) MS and ESI/MS–MS coupled with RPLC.

been previously studied by fragmentation followed by MS.

of enzymatic fragmentation with papain [9, 10] or new enzymes produced by recombinant methods and available on the market [11]. This type of procedure has not been reported for the fragmentation of porcine IgG, to the authors' knowledge. For instance, procedures have been published for mouse [12, 13], chicken [14], rabbit [15], sheep [16], and human [17, 18] IgGs. The two groups of antibody fragments of primary interest are the antigen-binding fragments (Fab) and

30–40%) can exist in the variable regions of polyclonal IgGs [7, 8].

**66**

Wild-type porcine IgGs were obtained from Université de Nantes (Dr. Jean-Paul Soulillou's Laboratory). Trypsin Ultra™ was purchased from Promega (Wisconsin, USA). The Fabulous™ enzyme was kindly provided by Genovis (Cambridge, MA). Dihydroxybenzoic acid (DHB), sinapinic acid, ammonium bicarbonate, dithiothreitol (DTT), L-cysteine, iodoacetamide (IAA), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). Mini-Protean TGX precast gels (4–15%), Precision Plus™ protein standard, 2-mercaptoethanol, and 4x Laemmli sample buffer were obtained from Bio-Rad (Hercules, CA). Imperial™ protein stain, tris base (2-amino-2-(hydroxymethyl)-1,3-propanediol) and immobilized papain-cross linked and 6% in beaded agarose supplied as 50% glycerol in sodium acetate pH 4.5 were purchased from Thermo Scientific (Rockford, IL). Strata-X C-18 cartridges were obtained from Phenomenex (Torrance, CA). Acetonitrile (ACN) was purchased from EMD-Millipore (Darmstadt, Germany). Sodium phosphate dibasic anhydrous was purchased from McArthur Chemical Co. Ltd. (Montreal, Canada). Hydrochloric acid was purchased from Anachemia (Vancouver, Canada) and deionized water was obtained from an adapted filtration system (Millipore).

#### **2.2 Tryptic digestion of whole porcine IgG**

Porcine IgG (200 μg) was dissolved in 100 mM ammonium bicarbonate (pH~8) and vortexed. A DTT solution (10 mM) was added to the sample, which was then vortexed and left to react at 56°C for 40 min, then cooled to room temperature. After 500 mM IA was added, and the sample was left to react in the dark for 45 min. The excess of IA was quenched with the addition of 100 mM DTT, and the sample was left to react for 10 min in the dark. Trypsin was added and proteolysis took place at 37°C for ~18 h. To deactivate trypsin, the sample was frozen and dried under vacuum. Glycopeptide enrichment was then performed using a ProteoExtract™ glycopeptide enrichment kit (Millipore-Sigma, Etobicoke, ON) according to the manufacturer's procedure [23].

#### **2.3 Papain digestion of porcine IgG**

Just before use, 20 mM sodium phosphate digestion buffer was prepared with a 10 mM cysteine content, and the pH was adjusted to 7 Bead-immobilized papain slurry (20 μL, 50%) was added to an Eppendorf™ tube. The beads were washed twice with 160 μL of digestion buffer and then re-suspended in the buffer. Porcine IgG (200 μg) was dissolved in the digestion buffer. This was added to the tube containing the washed immobilized papain and digestion buffer. The sample was

incubated for ~24 h at 37°C. Constant mixing of the bead slurry was maintained during the whole incubation. The bead-immobilized enzyme was separated from the digest by centrifugation and 20 μL of 10 mM Tris–HCl, pH 7.5 was added before centrifugation. The supernatant, which contained the IgG fragments, was removed.

#### **2.4 Fabulous™ digestion for porcine IgG**

The IgG sample (200 μg) was added to 200 units of Fabulous™ enzyme in 200 μL of 10 mM Tris, 50 mM cysteine buffer. The sample was vortexed and incubated at 37°C for 1 h.

#### **2.5 Fractionation of IgG fragments by HPLC using a SEC column**

The digestion mixtures were injected into a preconditioned SEC-300 4.6 × 300 mm silica-based column (Phenomenex, Torrance, CA). The mixtures were eluted with a mobile phase of 0.1% TFA, 0.1% formic acid in 20% ACN at a flow rate of 0.3 mL/min (manufacturer's recommendation). The HPLC system used was a Waters 1525 binary pump equipped with a Waters 2707 autosampler and a Waters 2998 photodiode array detector (Milford, MA). Fractions were collected, dried, and re-suspended for MALDI-MS analysis.

#### **2.6 Separation of IgG fragments by SDS gel electrophoresis**

Once fractionated by SEC, Fab and Fc components were separated on a Mini-Protean™ Tetra cell system (Bio-Rad). Bio-Rad TGX™ gels used had 10 wells and a density gradient of 4–15%. Wells were washed individually four times with running buffer (10 × tris-glycine-SDS buffer diluted 1 × with water) prior to the loading samples. Each sample fraction containing Fab, Fc, or both Fab and Fc had its own lane on the gel. Each gel was loaded with 15 μL of each fraction (in water) in 11.3 μL of 4 × Laemmli sample buffer, without adding 2-mercaptoethanol. Well 1 was loaded with 10 μL of Precision Plus Protein Kaleidoscope™ standard. Intact-reduced IgG (15 μL, ~14 μg) was loaded into well 2. IgG fragments (~14 μg) were loaded in other lanes. Running buffer was poured in the cell system and the voltage was set at 150 V. Samples were allowed to migrate for 40 min, until the dye front reached the bottom of the gel. The gel was removed from the cell and was rinsed four times with Millipore water, and sufficient Imperial™ protein stain was added. IgG fragments absorbed the stain overnight, and the stain was decanted and replaced with Millipore water until gel bands became visible.

#### **2.7 In-gel tryptic digest of IgG Fab and Fc fragments**

Non-reduced Fab and Fc bands were excised from the gel. Tryptic digestion was performed on each single cut out band. Bands were cut into small pieces and placed into 1.5 mL Eppendorf tubes. The digestion buffer was 50 mM ammonium bicarbonate in water. Each tube contained one lane worth of gel. Gel pieces were washed with wash buffer (1:1 digestion buffer-ethanol) until all protein stain was removed. They were then incubated in absolute ethanol for 10 min. Gel pieces were then rewashed with digestion buffer for 20 min and then incubated again in absolute ethanol for 20 min, which was removed from the gel by vacuum centrifugation. Trypsin solution was added and the tubes were placed on ice where the gel was allowed to swell. Thereafter, excess trypsin solution was discarded. Gel pieces

**69**

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

ACN. Samples were dried down for further analysis.

TFA. Solvent was evaporated from the fractions.

**2.10 MALDI-TOF-MS analysis**

10,000 Da to 160,000 Da.

HPLC-MS/MS analyses.

**2.9 Preparation of samples for MALDI-MS analysis**

**2.11 Preparation of samples for HPLC-ESI-MS analysis**

**2.12 RPLC-MS/MS analysis of Fab and Fc tryptic digests**

sonicated and then ready for HPLC–MS analysis.

were covered with digestion buffer and incubated at 37°C for ~18 h. Glycopeptides and peptides were extracted on C18 cartridges with buffer A, 0.5% acetic acid; extraction buffer B, 5:3 30%; ACN, 0.5% acetic acid; and extraction buffer C, 100%

A solution of PNGase F (4 μL, 10 units/μL) was added to a solution of tryptic glycopeptides. The sample was vortexed and set at 37°C for ~18 h. After the digest, glycans were separated from the de-glycosylated peptides on a C18 cartridge. The cartridge was conditioned with 5 x 1 mL (ACN + 0.1% TFA), then 5 × 1 mL of (H2O + 0.1% TFA). The sample was loaded onto the cartridge and allowed to equilibrate for about 1–2 min. Glycans were eluted with 3 mL H2O + 0.1% TFA and collected in two fractions. Peptides were eluted with 1.5 mL of 50:50 ACN:H2O + 0.1%

Glycopeptide fractions were mixed directly with 5 μL of DHB in 30:70 ACN: water. Samples (1 μL) were then spotted onto the stainless steel target and allowed to dry, for reflector positive mode MALDI-MS. For the Fab and Fc fragments from HPLC fractions, 5 μL of 0.1% TFA and 5 μL of sinapinic acid in 0.1% TFA, 30:70 ACN: water was added, and 1 μL was spotted onto the target already pre-spotted with 0.5 μL of sinapinic acid in ethanol. Spots were then allowed to dry for linear positive mode MALDI-MS.

These analyses were performed on an UltraFleXtreme™ mass spectrometer (Bruker, Billerica, MA) equipped with LID-LIFT™ technology for tandem MS experiments. For positive ionization mode, a nine-peptide calibration mixture with masses ranging from 500 to 5000 Da was used. In linear positive mode, the instrument was calibrated using tryptic peptides of cytochrome C, mass ranging from

For the Fab and Fc tryptic digest fragments, 100 μL of water was added to the pooled C18 cartridge fractions of each Fab and Fc tryptic digests. Samples were

Both digestion mixtures (50 μL) were in turn injected into a preconditioned Acquity BEH C18 (1.7 μm, 2.1 × 50 m) silica-based, reverse phase column, on a Waters Acquity UPLC system (Waters, Mississauga, ON). The flow rate was 0.25 mL/min, and a linear increase from zero to 28% ACN in water with 0.1% formic acid was used. Mass spectrometric detection was performed on a Waters G2 Synapt ESI-MS instrument. Positive ionization mode was used. The analyzer mode was set to high resolution, with a capillary voltage of 3.00 kV and a cone voltage of 25 V. The Progenesis™ software was used to handle and search databases for these

**2.8 Peptide-***N***-glycosidase F (PNGase F) removal of glycans from trypsin** 

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

**digested Fab and Fc fragments**

*Recent Advances in Analytical Chemistry*

**2.4 Fabulous™ digestion for porcine IgG**

re-suspended for MALDI-MS analysis.

incubated at 37°C for 1 h.

incubated for ~24 h at 37°C. Constant mixing of the bead slurry was maintained during the whole incubation. The bead-immobilized enzyme was separated from the digest by centrifugation and 20 μL of 10 mM Tris–HCl, pH 7.5 was added before centrifugation. The supernatant, which contained the IgG fragments, was removed.

The IgG sample (200 μg) was added to 200 units of Fabulous™ enzyme in 200 μL of 10 mM Tris, 50 mM cysteine buffer. The sample was vortexed and

The digestion mixtures were injected into a preconditioned SEC-300 4.6 × 300 mm silica-based column (Phenomenex, Torrance, CA). The mixtures were eluted with a mobile phase of 0.1% TFA, 0.1% formic acid in 20% ACN at a flow rate of 0.3 mL/min (manufacturer's recommendation). The HPLC system used was a Waters 1525 binary pump equipped with a Waters 2707 autosampler and a Waters 2998 photodiode array detector (Milford, MA). Fractions were collected, dried, and

Once fractionated by SEC, Fab and Fc components were separated on a Mini-Protean™ Tetra cell system (Bio-Rad). Bio-Rad TGX™ gels used had 10 wells and a density gradient of 4–15%. Wells were washed individually four times with running buffer (10 × tris-glycine-SDS buffer diluted 1 × with water) prior to the loading samples. Each sample fraction containing Fab, Fc, or both Fab and Fc had its own lane on the gel. Each gel was loaded with 15 μL of each fraction (in water) in 11.3 μL of 4 × Laemmli sample buffer, without adding 2-mercaptoethanol. Well 1 was loaded with 10 μL of Precision Plus Protein Kaleidoscope™ standard. Intact-reduced IgG (15 μL, ~14 μg) was loaded into well 2. IgG fragments (~14 μg) were loaded in other lanes. Running buffer was poured in the cell system and the voltage was set at 150 V. Samples were allowed to migrate for 40 min, until the dye front reached the bottom of the gel. The gel was removed from the cell and was rinsed four times with Millipore water, and sufficient Imperial™ protein stain was added. IgG fragments absorbed the stain overnight, and the stain was decanted and replaced with Millipore water until gel bands

Non-reduced Fab and Fc bands were excised from the gel. Tryptic digestion was performed on each single cut out band. Bands were cut into small pieces and placed into 1.5 mL Eppendorf tubes. The digestion buffer was 50 mM ammonium bicarbonate in water. Each tube contained one lane worth of gel. Gel pieces were washed with wash buffer (1:1 digestion buffer-ethanol) until all protein stain was removed. They were then incubated in absolute ethanol for 10 min. Gel pieces were then rewashed with digestion buffer for 20 min and then incubated again in absolute ethanol for 20 min, which was removed from the gel by vacuum centrifugation. Trypsin solution was added and the tubes were placed on ice where the gel was allowed to swell. Thereafter, excess trypsin solution was discarded. Gel pieces

**2.5 Fractionation of IgG fragments by HPLC using a SEC column**

**2.6 Separation of IgG fragments by SDS gel electrophoresis**

**2.7 In-gel tryptic digest of IgG Fab and Fc fragments**

**68**

became visible.

were covered with digestion buffer and incubated at 37°C for ~18 h. Glycopeptides and peptides were extracted on C18 cartridges with buffer A, 0.5% acetic acid; extraction buffer B, 5:3 30%; ACN, 0.5% acetic acid; and extraction buffer C, 100% ACN. Samples were dried down for further analysis.

#### **2.8 Peptide-***N***-glycosidase F (PNGase F) removal of glycans from trypsin digested Fab and Fc fragments**

A solution of PNGase F (4 μL, 10 units/μL) was added to a solution of tryptic glycopeptides. The sample was vortexed and set at 37°C for ~18 h. After the digest, glycans were separated from the de-glycosylated peptides on a C18 cartridge. The cartridge was conditioned with 5 x 1 mL (ACN + 0.1% TFA), then 5 × 1 mL of (H2O + 0.1% TFA). The sample was loaded onto the cartridge and allowed to equilibrate for about 1–2 min. Glycans were eluted with 3 mL H2O + 0.1% TFA and collected in two fractions. Peptides were eluted with 1.5 mL of 50:50 ACN:H2O + 0.1% TFA. Solvent was evaporated from the fractions.

#### **2.9 Preparation of samples for MALDI-MS analysis**

Glycopeptide fractions were mixed directly with 5 μL of DHB in 30:70 ACN: water. Samples (1 μL) were then spotted onto the stainless steel target and allowed to dry, for reflector positive mode MALDI-MS. For the Fab and Fc fragments from HPLC fractions, 5 μL of 0.1% TFA and 5 μL of sinapinic acid in 0.1% TFA, 30:70 ACN: water was added, and 1 μL was spotted onto the target already pre-spotted with 0.5 μL of sinapinic acid in ethanol. Spots were then allowed to dry for linear positive mode MALDI-MS.

#### **2.10 MALDI-TOF-MS analysis**

These analyses were performed on an UltraFleXtreme™ mass spectrometer (Bruker, Billerica, MA) equipped with LID-LIFT™ technology for tandem MS experiments. For positive ionization mode, a nine-peptide calibration mixture with masses ranging from 500 to 5000 Da was used. In linear positive mode, the instrument was calibrated using tryptic peptides of cytochrome C, mass ranging from 10,000 Da to 160,000 Da.

#### **2.11 Preparation of samples for HPLC-ESI-MS analysis**

For the Fab and Fc tryptic digest fragments, 100 μL of water was added to the pooled C18 cartridge fractions of each Fab and Fc tryptic digests. Samples were sonicated and then ready for HPLC–MS analysis.

#### **2.12 RPLC-MS/MS analysis of Fab and Fc tryptic digests**

Both digestion mixtures (50 μL) were in turn injected into a preconditioned Acquity BEH C18 (1.7 μm, 2.1 × 50 m) silica-based, reverse phase column, on a Waters Acquity UPLC system (Waters, Mississauga, ON). The flow rate was 0.25 mL/min, and a linear increase from zero to 28% ACN in water with 0.1% formic acid was used. Mass spectrometric detection was performed on a Waters G2 Synapt ESI-MS instrument. Positive ionization mode was used. The analyzer mode was set to high resolution, with a capillary voltage of 3.00 kV and a cone voltage of 25 V. The Progenesis™ software was used to handle and search databases for these HPLC-MS/MS analyses.

#### **2.13 Analysis of whole porcine IgG tryptic digest by RPLC-MS/MS**

Separations were conducted on a LC Ultra system (Eksigent, Dublin, CA). A 100 μm × 200 mm analytical column packed with 3 μm Luna C18 (Phenomenex, Torrance, CA) was used, at 500 nL/min flow rate. A 300 μm × 5 mm PepMap 100 trap-column (Thermo Fisher, San Jose, CA) was used to protect the analytical column. The elution gradient was as described above. A Triple TOF 5600 mass spectrometer (ABSciex, Concord, ON) was used in standard MS/MS data-dependent acquisition mode. Mass spectra (250 ms) were collected (*m/z* 370–1250) and followed by up to 20 MS/MS measurements on the most abundant parent ions (400 counts/s threshold, +2 to +5 charge state, *m/z* 100–1500 mass range for MS/MS, 100 ms each). Database search was performed using the Global Proteome Machine (GPM) proteomics data analysis system [24].

#### **3. Results and discussion**

#### **3.1 Mass spectrometric analysis of tryptic digests of whole porcine IgG samples**

In previous studies, wild-type porcine IgG was digested with trypsin, and glycopeptides/peptides were fractionated on a C18 cartridge. Glycopeptide fractions were analyzed by MALDI-MS/MS [2, 3, 30]. In a new separate experiment, the results of which are presented here, all tryptic products were then enriched for glycopeptides using an EMD-Millipore ProteoGlycan™ kit [23]. The glycopeptide-enriched fraction was injected into a RPLC-MS/MS system, using data-dependent MS/MS acquisition. **Figure 1a** shows the MS/MS total ion chromatogram (TIC) obtained from the elution, whereas **Figure 1b** represents the elution of peptides with *m/z* 204

#### **Figure 1.**

*(a) Total HPLC/MS ion chromatogram obtained from the MS/MS spectra of porcine IgG tryptic peptides and glycopeptides after enrichment on EMD-Millipore Proteoglycan™. (b) Selected MS/MS fragment ion chromatogram at m/z 204, indicating the elution times of glycopeptides [25].*

**71**

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

ions as product in their MS/MS spectra, identified as glycopeptides [25]. Although enrichment was performed, there were still non-glycosylated peptides present in the sample. The *m/z* 204 trace shows that glycosylated peptides eluted early in the analysis and that the most abundant compounds eluting in (a) were most probably

A database search using GPM [24] helped to identify some non-glycosylated peptides, while for glycopeptides the extracted *m/z* 204 chromatogram was used to manually identify as many glycopeptides as possible. **Table 1** gives a listing of all peptides detected and sequenced with GPM [24] or manually. For glycopeptide sequencing, MS/MS spectra were treated with CycloBranch [26] after removing abundant glycopeptide ions. As a few sequences are available in UniprotKB [22] for the Fc gamma portion of porcine IgG (K7ZLAZ, L8B180, L8B0S7, L8B0S2, L8B0Z4), and more are available [3] from previously published DNA sequences [5], some peptides show more than one identification source in **Table 1**. Only one entry was found for a porcine IgG Fab portion, P01846, which corresponds to the lambda (λ) constant region. **Figure A1** compares the published porcine IgG Fc heavy chain

Points of interest arising from this table are (i) the presence of a large number of non-glycosylated, even after enrichment, from the Fab and Fc portions of the antibody and (ii) the detection of seven distinct glycosylated peptides, some with complex glycoforms and one with high-mannose glycoforms, thus of the *N*-type. Indeed, all MS/MS spectra of these glycopeptides showed characteristic

and (M + H + 203)<sup>+</sup>

**Figure 2** shows four MS/MS spectra of glycopeptides, starting with two of the complex G0F forms of peptides of (a) constant region EAQFNSTYR [3] and (b) a variable region sequence partially determined as H2N-(300)-QNFSVFR by the CycloBranch software [26]. In these cases, the mass difference between the

and 366 fragment ions are predominant as glycopeptide signature. In **Figure 2c**, a sialylated complex glycoform is featured, the G1FS form of peptide EEQFNSTYR [3]. In previous studies of wild-type pig IgG, the presence of *N*-glycolyl neuraminic acid (NeuGc) is featured exclusively (i.e., no *N*-acetyl neuraminic acid (NeuAc)) has been reported [2, 3]. The fragmentation of NeuGc-containing species produces distinctive *m/z* 308 and 290 fragments, as observed in (c). **Figure 2d** shows the fragmentation of a high-mannose glycoform of a peptide of undetermined

For known peptide sequences such as in **Figure 2a** and **c**, it is possible to find most bare peptide y and b ions, although they appear with very low abundance and are not accounted for by the search engine, due to the domination of all glycopeptide signature ions. There was an attempt by the authors to sequence all unknown glycopeptide sequences, with partial success, as indicated in red in **Table 1**.

Overall results suggest that *N*-glycosylation occurs in the Fc but also in the variable regions of the Fab and/or Fc domains of porcine IgG. Each glycosylated peptide detected indicated patterns linked to *N*-glycosylation, while there was no obvious detection of *O*-glycans. This may be due to the conditions used to enrich the glycopeptides with the EMD-Millipore Kit, which were optimized for *N*-glycosylated peptides [23, 28]. Results obtained with this first workflow will be compared with those generated with a more elaborated procedure involving fragmentation

ions of the bare peptides, next to

bare peptide fragments is 1444. The *m/z* 204

ions appear at *m/z* 1069, with +83 ions (*m/z*

ions [27], with the latter

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

sequences relating to **Table 1** assignments.

fragment ions corresponding to the (M + H)<sup>+</sup>

the characteristic (M + H + 83)<sup>+</sup>

protonated precursors and (M + H)<sup>+</sup>

sequence. The bare peptide (M + H)+

1152) and + 203 ions (*m/z* 1272).

enzymes as discussed below.

being predominant.

non-glycosylated peptides.

#### *Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Recent Advances in Analytical Chemistry*

(GPM) proteomics data analysis system [24].

**3. Results and discussion**

**2.13 Analysis of whole porcine IgG tryptic digest by RPLC-MS/MS**

Separations were conducted on a LC Ultra system (Eksigent, Dublin, CA). A 100 μm × 200 mm analytical column packed with 3 μm Luna C18 (Phenomenex, Torrance, CA) was used, at 500 nL/min flow rate. A 300 μm × 5 mm PepMap 100 trap-column (Thermo Fisher, San Jose, CA) was used to protect the analytical column. The elution gradient was as described above. A Triple TOF 5600 mass spectrometer (ABSciex, Concord, ON) was used in standard MS/MS data-dependent acquisition mode. Mass spectra (250 ms) were collected (*m/z* 370–1250) and followed by up to 20 MS/MS measurements on the most abundant parent ions (400 counts/s threshold, +2 to +5 charge state, *m/z* 100–1500 mass range for MS/MS, 100 ms each). Database search was performed using the Global Proteome Machine

**3.1 Mass spectrometric analysis of tryptic digests of whole porcine IgG samples**

*(a) Total HPLC/MS ion chromatogram obtained from the MS/MS spectra of porcine IgG tryptic peptides and glycopeptides after enrichment on EMD-Millipore Proteoglycan™. (b) Selected MS/MS fragment ion* 

*chromatogram at m/z 204, indicating the elution times of glycopeptides [25].*

In previous studies, wild-type porcine IgG was digested with trypsin, and glycopeptides/peptides were fractionated on a C18 cartridge. Glycopeptide fractions were analyzed by MALDI-MS/MS [2, 3, 30]. In a new separate experiment, the results of which are presented here, all tryptic products were then enriched for glycopeptides using an EMD-Millipore ProteoGlycan™ kit [23]. The glycopeptide-enriched fraction was injected into a RPLC-MS/MS system, using data-dependent MS/MS acquisition. **Figure 1a** shows the MS/MS total ion chromatogram (TIC) obtained from the elution, whereas **Figure 1b** represents the elution of peptides with *m/z* 204

**70**

**Figure 1.**

ions as product in their MS/MS spectra, identified as glycopeptides [25]. Although enrichment was performed, there were still non-glycosylated peptides present in the sample. The *m/z* 204 trace shows that glycosylated peptides eluted early in the analysis and that the most abundant compounds eluting in (a) were most probably non-glycosylated peptides.

A database search using GPM [24] helped to identify some non-glycosylated peptides, while for glycopeptides the extracted *m/z* 204 chromatogram was used to manually identify as many glycopeptides as possible. **Table 1** gives a listing of all peptides detected and sequenced with GPM [24] or manually. For glycopeptide sequencing, MS/MS spectra were treated with CycloBranch [26] after removing abundant glycopeptide ions. As a few sequences are available in UniprotKB [22] for the Fc gamma portion of porcine IgG (K7ZLAZ, L8B180, L8B0S7, L8B0S2, L8B0Z4), and more are available [3] from previously published DNA sequences [5], some peptides show more than one identification source in **Table 1**. Only one entry was found for a porcine IgG Fab portion, P01846, which corresponds to the lambda (λ) constant region. **Figure A1** compares the published porcine IgG Fc heavy chain sequences relating to **Table 1** assignments.

Points of interest arising from this table are (i) the presence of a large number of non-glycosylated, even after enrichment, from the Fab and Fc portions of the antibody and (ii) the detection of seven distinct glycosylated peptides, some with complex glycoforms and one with high-mannose glycoforms, thus of the *N*-type. Indeed, all MS/MS spectra of these glycopeptides showed characteristic fragment ions corresponding to the (M + H)<sup>+</sup> ions of the bare peptides, next to the characteristic (M + H + 83)<sup>+</sup> and (M + H + 203)<sup>+</sup> ions [27], with the latter being predominant.

**Figure 2** shows four MS/MS spectra of glycopeptides, starting with two of the complex G0F forms of peptides of (a) constant region EAQFNSTYR [3] and (b) a variable region sequence partially determined as H2N-(300)-QNFSVFR by the CycloBranch software [26]. In these cases, the mass difference between the protonated precursors and (M + H)<sup>+</sup> bare peptide fragments is 1444. The *m/z* 204 and 366 fragment ions are predominant as glycopeptide signature. In **Figure 2c**, a sialylated complex glycoform is featured, the G1FS form of peptide EEQFNSTYR [3]. In previous studies of wild-type pig IgG, the presence of *N*-glycolyl neuraminic acid (NeuGc) is featured exclusively (i.e., no *N*-acetyl neuraminic acid (NeuAc)) has been reported [2, 3]. The fragmentation of NeuGc-containing species produces distinctive *m/z* 308 and 290 fragments, as observed in (c). **Figure 2d** shows the fragmentation of a high-mannose glycoform of a peptide of undetermined sequence. The bare peptide (M + H)+ ions appear at *m/z* 1069, with +83 ions (*m/z* 1152) and + 203 ions (*m/z* 1272).

For known peptide sequences such as in **Figure 2a** and **c**, it is possible to find most bare peptide y and b ions, although they appear with very low abundance and are not accounted for by the search engine, due to the domination of all glycopeptide signature ions. There was an attempt by the authors to sequence all unknown glycopeptide sequences, with partial success, as indicated in red in **Table 1**.

Overall results suggest that *N*-glycosylation occurs in the Fc but also in the variable regions of the Fab and/or Fc domains of porcine IgG. Each glycosylated peptide detected indicated patterns linked to *N*-glycosylation, while there was no obvious detection of *O*-glycans. This may be due to the conditions used to enrich the glycopeptides with the EMD-Millipore Kit, which were optimized for *N*-glycosylated peptides [23, 28]. Results obtained with this first workflow will be compared with those generated with a more elaborated procedure involving fragmentation enzymes as discussed below.


**73**

**Peptide sequence**

EEQFNSTYR TNNRPTGVPSR

H2N-300-QNFSVFR

SYLALSASDWK

DTNRPSGIPER

STNSRPTGVPSR

FSGSGSGTDFTLK

SSSGFTCQVTHE

TAPSVYPLAPCGR

LLGASVLGVGDIHR

Unknown Unknown Unknown LVESGGGLVQPGGSLR

QSNNKYAASSYLAL

AGGTTVTQVETTKPSK

YAASSYLALSASDWK

VVSVLPIQHQDWLK

QEYREDFVLTVTGK

APASYFVQSVLTVSAK

1667.83

1667.9

12.7

0

1406.81 1415.62 **1467.28** 1495.73 1525.85 1529.76 1604.85 1632.79 1661.92

11 0.39

0.6 0.24 −10.4

2911.26 3073.31

G0F G1F

1

1198.63 **1212.13** 1240.62 1241.62 1258.65 1303.62 1339.56 1388.71

−0.64

−2.6 0.16 2.74 1.27 7.85

*m/z* **(M + H)+**

**1173.5**

**Error (ppm)**

−14.6

*m/z* **glycoform** 2471.01 2618.09 2634.07 2780.13 3087.23 2942.19 3104.23 3249.28 2833.04

G0 G0F G1 G1F G1FS G2F G3F G2FS M9-N

2656.09 2819.14 2981.20

G0F G1F G2F

**Glycoform**

**Identification source\***

IgG1a-b,IgG2a-b,IgG4a-b,IgG5ab,IgG6b, L8B0S7,L8B0S2,K7ZLA7

Unavailable Unavailable

P01846 Unavailable

P01846 Unavailable

P01846 IgG1a-b,IgG4b,IgG5b,IgG6b,L8B180,K7

ZLA7,L8B0Z4

Unavailable

Unavailable

Unavailable

Unavailable

L8B0S2,L8B180,L8B0S7

P01846

P01846

P01846

IgG1ab,IgG3,IgG6b,L8B180,K7ZLA7,L8B0Z4

Unavailable

K7ZJP7

*Characterization of Whole and Fragmented Wild-Type Porcine IgG*

*DOI: http://dx.doi.org/10.5772/intechopen.82792*

#### *Recent Advances in Analytical Chemistry*


#### *Characterization of Whole and Fragmented Wild-Type Porcine IgG DOI: http://dx.doi.org/10.5772/intechopen.82792*

*Recent Advances in Analytical Chemistry*

L8B0Z4

Unavailable

P01846

P01846

IgG2ba-b,IgG4a-b,IgG6ab,L8B180,L8B0S7,L8B0Z4

Unavailable

Unavailable

P01846

IPR 007110 (Ig C1-set)

Unavailable

Unavailable

IgG3

Unavailable

Unavailable

P01846

IgG6a,L8B180,L8B0Z4

**72**

**Peptide sequence**

Unknown

NFSTYR LLVELIR TVTPSECA FSGAISGNK

DLPAPTIR

LLLDLFR LLNGYRR AGGTTVTQVE

LIYQATNR VDPALPLEK NRPTGVPSR TISKATGPSR LSSPATLNSR

Unknown FQQTPGQPPP

EAQFNSTYR

Unknown

855.57 864.38 880.45

882.5 889.55 891.52 962.48 978.54 981.56 983.54 1017.57 1045.56 **1069.55** 1096.54 **1115.49** **1154.53**

−2.09 −19.4

2414.05 2560.12 2722.15

G0 G0F G1F G2F

G1FS G3F

G0F

Unavailable

2884.24 3029.35 3191.50

2599.1

−0.56

−4.6 1.14 3.37 −1.58 −1.9 1.28 −3.44 2933.2

M9

4.44

3.8 2.72 −4.88

*m/z* **(M + H)+**

646.35 **787.37**

0

2231.14 2392.28 2701.08

G0F G1F G1FS G2FS

2863.10

**Error (ppm)**

*m/z* **glycoform**

**Glycoform**

**Identification source\***

Unavailable

IgG1a-b,IgG2a-b,IgG4ab,IgG5a-b, IgG6ab,L8B0S7,L8B0S2,K7ZLA7,L8B180,

