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Page iii

Modern Derivatization Methods for Separation Sciences
Edited by
Toshimasa Toyo'oka
School of Pharmaceutical Sciences, University of Shizuoka, Japan

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Page iv

Copyright (c) 1999 John Wiley & Sons Ltd,
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Library of Congress Cataloging-in-Publication Data
Modern derivatization methods for separation sciences/edited by Toshimasa Toyo'oka.
p. cm.
Includes bibliographical references and index.
ISBN 0-471-98364-0 (alk. paper)
1. Chromatographic analysis-Methodology. 2. Derivatization.
I. Toyo'oka, Toshimasa.
QD79.C4M63

1999
98-27197
543'.089-dc21
CIP
British Library Cataloging in Publication Data
A catalogue record for this book is available from the British Library
ISBN 0 471 98364 0

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Typeset in 10/12pt Times by Laser Words, Madras, India
Printed and bound in Great Britain by Bookcraft (Bath) Ltd
This book is printed on acid-free paper responsibly manufactured from sustainable forestry, in which at
least two trees are planted for each one used for paper production

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Page v

Contents
List of Contributors
Preface

xi
xiii

1. Pre-treatment for Real Sample Analysis and Choice of Suitable Reagent

1

1.1. Derivatization for Drugs
Akihiko Nakagawa, and Yukinori Kawahara

1

1.1.1. Introduction

2

1.1.2. Application of Derivatization of Drugs

2

1.1.2.1. Determination of Pravastatin Sodium in Plasma by
HPLC with Laser-induced Fluorescence Detection after
Immobilized Antibody Extraction

2

1.1.2.2. Enantiospecific Determination of Ibuprofen in Rat
Plasma Using Chiral Fluorescence Derivatization Reagent, (-)-2[4-(1-aminoethyl)phenyl]6-methoxybenzoxazole

4

1.1.2.4. Specific Fluorogenic Derivatization of Ivermectin Using
Non-fluorescent Reagents

6

1.1.2.4. Other Methods

8

1.1.3. Conclusion

10

References
1.2. Derivatization for Agrochemicals
Hiroyuki Nakazawa and Koichi Saito
1.2.1. Introduction

13

13

1.2.1.1. Sample Preparation

14

1.2.1.2. Derivatization in Food Analysis

15

1.2.2. Analysis of Nutrients

16

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1.2.2.1. Carbohydrates

16

1.2.2.2. Amino Acids and Peptides

17

1.2.2.3. Fatty Acids

22

1.2.2.4. Organic Acids

23

1.2.2.5. Vitamins

24

1.2.2.6. Minerals

26

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1.2.3. Analysis of Food Additives

27

1.2.3.1. Preservatives

27

1.2.3.2. Antioxidants

27

1.2.3.3. Sweeteners

28

1.2.3.4. Emulsifiers

28

1.2.3.5. Other Food Additives

29

1.2.4. Analysis of Veterinary Medicine

29

1.2.4.1. Sulfonamides

30

1.2.4.2. Antibiotics

30

1.2.4.3. Antiparasitic Agents

32

1.2.5. Analysis of Pesticides

34

1.2.6. Analysis of Natural Toxins

35

1.2.6.1. Mycotoxins

35

1.2.6.2. Marine Toxins

39

1.2.6.3. Plant Toxin

42

1.2.7. Analysis of Food Contaminants

42

1.2.7.1. Amines

42

1.2.7.2. Organotin

43

1.2.7.3. Hydroperoxides

45

1.2.7.4. Isocyanate Monomers

45

References

45

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1.3. Derivatization for Environmental Contaminants
Kazuo Iwaki

51

1.3.1. Introduction

51

1.3.2. Environmental Samples

51

1.3.2.1. Gas Sample

51

1.3.2.2. Soil and Various Water Samples

53

1.3.3. Application

55

1.3.3.1. Aldehydes and Ketones in Air

55

1.3.3.2. Aliphatic Amines in Air

57

1.3.3.3. Residual Pesticides and Herbicides in Soil and Water

59

References
2. Reagent for UV-VIS Detection
Kiyoshi Zaitsu, Masaaki Kai and Kenji Hamase

62
63

2.1. Introduction

64

2.2. Label of Amines and Amino Acids (-NH2, NH)

65

2.2.1. Nitrobenzenes

65

2.2.1.1. 1-Fluoro-2,4-dinitrobenzene (FDNB, 2,4-dinitro-1fluorobenzene, Sanger Reagent)

65

2.2.1.2. 4-Fluoro-3-nitrotrifluoromethylbenzene (4-fluoro-3nitrobenzotrifluoride) (FNBT)

66

2.2.1.3. 2,4,6-Trinitrobenzene-1-sulfonic Acid (TNBS)

67

2.2.1.4. Na-(2,4-Dinitro-5-fluorophenyl)-L-alanine amide
(FDAA, 1-Fluoro-2,4-dinitrophenyl-5-L-alanine amide,
Marfey's Reagent)

67

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2.2.2. Acetic Anhydride

67

2.2.3. Isocyanates and Isothiocyanates

67

2.2.3.1. Isocyanates

67

2.2.3.2. Phenyl Isothiocyanate (PITC, Edman Reagent)

68

2.2.3.3. Butylisothiocyanate (BITC)

70

2.2.3.4. 4-N,N-Dimethylaminoazobenzene-4'-isothiocyanate
(DABITC)

70

2.2.4. 1,2-Naphthoquinone-4-sulfonate (NQS)

70

2.2.5. Acyl Chlorides

71

2.2.5.1. Benzoyl Chloride

71

2.2.5.2. Dansyl Chloride (DNS-C1)

72

2.2.5.3. 4-N,N-Dimethylaminoazobenzene-4'-sulfonyl Chloride

(DABS-C1, Dabsyl-Cl)

73

2.2.6. 9-Fluorenylmethyl Chloroformate (9Fluorenylmethoxycarbonyl Chloride, FMOC-CL, Fluorescence and
UV Detection)

73

2.2.7. Ninhydrin

74

2.2.8. Diethylethoxymethylenemalonate (DEMM)

75

2.2.9. 6-aminoquuinolyl-N-hydroxyuccinimidyl Carbamate (AQC)

76

2.2.10. Disuccinimido Carbonate (DSC)

76

2.2.11. Solid-phase Reagent with UV or VIS Light Absorbing
Moiety

76

2.2.11.1. Polymeric 3,5-Dinitrobenzoyl Tagged Derivatization
Reagent
2.2.11.2. Polymeric 6-Aminoquinoline (6-AQ) Tagged

77

78

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Derivatization Reagent
2.2.11.3. Polymeric Benzotriazole Activated Reagent
Containing FMOC Group
2.3. Label of Carboxyl (-COOH)

78

78

2.3.1. Alkyl Halides

78

2.3.2. Aromatic Amines

79

2.3.3. Hydrazines

81

2.3.4. Hydroxylamine

82

2.4. Label of Hydroxyl (-OH)

84

2.4.1. Acyl Halides

84

2.4.2. Acid Anhydrides

86

2.4.3. Isocyanates

87

2.4.4. Other Reagents

88

2.5. Label of Reducing Carbohydrate

89

2.5.1. Reductive Amination

89

2.5.2. l-Phenyl-3-methyl-5-pyrazolone

91

2.5.3. Post-column Derivatizing Reagent

92

2.6. Label of Thiol (-SH)

92

2.6.1. 2-Halopyridinium Salt

92

2.6.2. Disulfide Reagent

93

2.6.3. Other Reagents

94

2.7. Labelling of Other Compounds
2.7.1. l-(2-Pyridyl)-piperazine (PYP)

94
94

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2.7.2. Diethyldithiocarbamate (DDTC)

94

2.7.3. 9-Methylamino-methylanthracene (MAMA)

95

References

95

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3. Reagent for FL Detection
Masatoshi Yamaguchi, and Junichi Ishida

99

Abbreviations

100

3.1. Introduction

102

3.2. Reagents for Amines and Amino Acids

103

3.2.1. General Amino Compounds

103

3.2.1.1. Primary Amines and Amino Acids

103

3.2.1.2. Primary and Secondary Amines and Amino Acids

106

3.2.2. Particular Amines and Amino Acids

116

3.2.2.1. Catecholamines

116

3.2.2.2. Tryptophan and Indolamines

117

3.2.2.3. 5-hydroxyindoleamines (Serotonin Related
Compounds)

117

3.2.2.4. Guanidino Compounds

119

3.2.3. Peptides

121

3.2.3.1. General Peptides

121

3.2.3.2. Arginine-containing Peptides

121

3.2.4. Fluorescence Derivatization for CE

121

3.3. Reagents for Organic Acids

121

3.3.1. Carboxylic Acids

121

3.3.1.1. Fatty Acids

129

3.3.1.2. Prostaglandins

130

3.3.1.3. Steroids

131

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3.3.1.4. Glucuronic Acid Conjugates

132

3.3.2. α-Keto Acids

133

3.3.3. Sialic Acids and Dehydroascorbic Acid

133

3.4. Reagents for Alcohols

135

3.5. Reagents for Phenols

139

3.6. Reagents for Thiols

141

3.7. Reagents for Aldehydes and Ketones

146

3.8. Reagents for Carbohydrates

150

3.9. Reagents for Dienes

152

3.10. Reagents for Nucleic Acid Related Compounds

153

References

158

4. Reagent for CL Detection
Naotaka Kuroda and Kenichiro Nakashima

167

4.1. Introduction

167

4.2. Label of Amines (-NH2, -NH)

171

4.3. Label of Carboxyl (-COOH)

176

4.4. Label of Hydroxyl (-OH) and Thiol (-SH)

178

4.5. Label of Other Functional Groups

179

4.6. Application

183

References

183

5. Reagents for Electrochemical Detection
Kenji Shimada, Tomokazu Matsue and Kazutake Shimada
5.1. Introduction

191

192

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5.1.1. Amperometric Analysis in Flowing Streams

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5.1.2. Electrochemical Detectors

192

5.1.3. Electrode Material

193

5.1.4. Electrode Configuration

194

5.1.5. Mobile Phase

195

5.1.6. Chemical Derivatization

196

5.2. Labeling of Primary and Secondary Amines

198

5.2.1. o-phthalaldehyde

198

5.2.2. Naphthalene-2,3-dicarbaldehyde

199

5.2.3. Ferrocene

200

5.2.4. Isocyanate and Isothiocyanate

202

5.2.5. Salicylic Acid Chloride

203

5.2.6. 2,4-Dinitrofluorobenzene

204

5.2.7. 1,2-Diphenylethylenediamine

204

5.2.8. Bolton and Hunter Type Reagent

204

5.2.9. Others

205

5.3. Labeling of Carboxy Groups

205

5.4. Labeling of Hydroxy Groups

207

5.5. Labeling of Thiol Groups

208

5.6. Labeling of Other Functional Groups

208

5.7. Applications

211

References

213

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6. Derivatization for Resolution of Chiral Compounds
Toshimasa Toyo'oka
6.1. Introduction

217

218

6.1.1. Fundamentals of Stereochemistry

218

6.1.2. Chirality and Biological Activity

220

6.1.3. Chirality Application as Single Isomers

222

6.2. Resolution of Racemates

223

6.2.1. Direct Resolution

223

6.2.2. Indirect Resolution

223

6.3. Reactions of Various Functional Groups

225

6.3.1. Amines

225

6.3.2. Carboxyls

226

6.3.3. Hydroxyls

227

6.3.4. Thiols and Others

227

6.4. Derivatization for LC Analysis

228

6.4.1. Label for Alcohols

228

6.4.2. Label for Carboxylic Acids

230

6.4.3. Label for Aldehydes and Ketones

231

6.4.4. Label for Other Functional Groups

233

6.5. Derivatization for LC Analysis
6.5.1. Label for UV-VIS Detection

233
235

6.5.1.1. Label for Primary and Secondary Amines

235

6.5.1.2. Label for Carboxylic Acids

240

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6.5.1.3. Label for Alcohols

241

6.5.1.4. Label for Other Functional Groups

241

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6.5.2. Label for Fluorescence (FL), Laser-Induced Fluorescence
(LIF) and Chemiluminescence (CL) Detection

243

6.5.2.1. Label for Primary and Secondary Amines Including
Amino Acids 249

249

6.5.2.2. Label for Carboxylic Acids

257

6.5.2.3. Label for Alcohols

262

6.5.2.4. Label for Aldehydes and Ketones

266

6.5.2.5. Label for Thiols

266

6.5.2.6. Label for Other Functional Groups

267

6.5.3. Derivatization for Electrochemical (EC) Detection

267

6.6. Derivatization for Capillary Electrophoresis (CE) Analysis

267

6.7. Conclusion and Further Perspective

284

References

284

Index

291

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Contributors
Kenji Hamase, Ph.D.,
Department of Analytical Chemistry,
Faculty of Pharmaceutical Sciences,
Kyushu University,
3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan,
Phone: +81-92 642-6598,
FAX: +81-92 642-6601,
E-mail: kyushu-u.ac.jp
Junichi Ishida, Ph.D.,
Department of Analytical Chemistry,

Faculty of Pharmaceutical Sciences,
Fukuoka University, Nakakuma,
Johnan-ku, Fukuoka 814-0180, Japan,
Phone: +81-92-871-6631
Kazuo Iwaki, Ph.D.,
Deputy General Manager,
Chemical Analysis Department,
Ebara Research Co. Ltd.,
2-1 Honfujisawa 4-chome,
Fujisawa-shi Kanagawa 251-8502, Japan,
Phone: +81-466-83-8276,
Fax: +81-466-83-3160,
E-mail:
Prof. Masaaki Kai, Ph.D.,
Chemistry of Functional Molecules,
School of Pharmaceutical Sciences,
Nagasaki University,
1-14 Bunkyo-Machi,
Nagasaki 852-8521, Japan,
Phone: +81-95 847-1111,
Fax: +81-95 843-1742,
E-mail:
Yukinori Kawahara, Ph.D.,
Director of Analytical and Metabolic Research
Laboratories,
Sankyo Co. Ltd., Hiromachi 1-2-58,
Shinagawa-ku, Tokyo 140, Japan,
Phone: +81-3-3492-3131
Naotaka Kuroda, Ph.D.,
Department of Hygienic Chemistry,

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School of Pharmaceutical Sciences,
Nagasaki University,
1-14 Bunkyo-Machi, Nagasaki 852, Japan,
Phone: +81-958-47-1111,
Fax: +81-958-48-4219,
E-mail:

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Prof. Tomokazu Matsue, Ph.D.,
Graduate School of Engineering,
Tohoku University,
Aramaki, Aoba, Sendai 980-77,Japan,

Phone:+81-22-217-7209,
Fax:+81-22-217-7293,
E-mail:
Akihiko Nakagawa, Ph.D.,
Analytical and Metabolic Research Laboratories,
Sankyo Co.Ltd.,Hiromachi1-2-58,Shinagawa-ku, Tokyo 140, Japan,
Phone: +81-3-3492-3131,
E-mail:
Prof. Kenichiro Nakashima, Ph.D.,
Department of Hygienic Chemistry,
School of Pharmaceutical Sciences,
Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852, Japan,
Phone: +81-958-47-1111 (Ex. 2526),
Fax: +81-958-42-3549,
E-mail:
Prof. Hiroyuki Nakazawa, Ph.D.,
Department of Analytical Chemistry,
Hoshi University, 2-4-41 Ebara,
Shinagawa-ku, Tokyo 142, Japan,
Phone: +81-3-5498-5763,
Fax: +81-3-5498-5765,
E-mail:
Koichi Saito, Ph.D.,
Senior researcher,
Department of Food Chemistry,
Saitama Institute of Public Health,
639-1, Kamiokubo,
Urawa, Saitama 338, Japan,
Phone: +81-48-853-6121,

Fax: +81-48-840-1041,
E-mail:
Prof. Kazutake Shimada, Ph.D.,
Department of Analytical Chemistry,
Faculty of Pharmaceutical Sciences, Kanazawa University,
Takaramachi 3-1,Kanazawa920-0934,Japan,
Phone: +81-76-234-4459,
Fax: +81-76-234-4459,
E-mail:
Prof. Kenji Shimada, Ph.D.,

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Department of Analytical Chemistry,
Niigata College of Pharmacy,
5-13-2 Kamishinei-cho, Niigata 950-2081, Japan
Phone: +81-25-268-1172,
Fax: +81-25-268-1177,
E-mail:
Prof. Toshimasa Toyo'oka, Ph.D.,
Department of Analytical Chemistry,
School of Pharmaceutical Sciences,
University of Shizuoka,
52-1 Yada, Shizuoka 422-8526, Japan,
Phone: +81-54-264-5656,

Fax: +81-54-264-5593,
E-mail:
Prof. Masatoshi Yamaguchi, Ph.D.,
Department of Analytical Chemistry,
Faculty of Pharmaceutical Sciences,
Fukuoka University, Nakakuma,
Johnan-ku, Fukuoka 814-0180, Japan,
Phone:+81-92-871-6631(Ex.6618),
E-mail:
Prof. Kiyoshi Zaitsu, Ph.D.,
Department of Analytical Chemistry,
Faculty of Pharmaceutical Sciences,
Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan,
Phone: +81-92 642-6596,
FAX: +81-92 642-6501,
E-mail: zaitsu@analysis. phar. kyushu-u.ac.jp

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Preface
Biologically active compounds are ubiquitous in our lives. They are used for medicines, agrochemicals
(e.g. pesticides and harbicides), food additives, biogenic amines and flavors. It is fairly difficult to

determine them with accuracy and precision, because they usually exist in munite amounts, especially
in real biological and environmental samples. The choice of a suitable method that provides good
reproducibility is essential to obtain correct results. Separation analysis represented by various
chromatography is recommended for the quantitation of analytes in complex matrices.
Derivatization was an important technique for analysis using gas chromatography in the early stages.
The main purpose of the derivatization was to add volatility to saccarides and amino acids. In this
derivatization, selectivity and sensitivity were not considered. However, derivatization is the essential
technique in separation sciences using thin-layer chromatography (TLC), liquid chromatography (LC)
and capillary electrophoresis (CE), as well as gas chromatography (GC). For analysis by highperformance liquid chromatography (HPLC), various reagents have been developed to increase
separability, selectivity and sensitivity. This is due to the development of various types of detection
instruments such as ultravioletvisible (UV-VIS), fluorescence (FL), chemiluminescence (CL) and
electrochemical (EC). The use of derivatization in separation sciences is mainly to improve the
chromatographic properties and detection sensitivity.
The major aim of this book is to provide an easyto-read overview of various derivatization methods that
are available for minute analyses of biological importance. Emphasis is placed on practical use, and the
characteristics (merits and demerits) of the various approachs are critically discussed. The derivatization
listed in this book is a reaction which produces covalent binding between the analyte and the reagent.
This book describes recent advances in chemical derivatization for the separation sciences mainly by
GC, LC and CE.
The first chapter presents a general introduction of the pre-treatment of real samples such as biological,
food and environmental. The pretreatment is the clean-up method for derivatization to obtain a trace
amount of analyte without contamination. This part is most important because the accuracy and
precision of the result obtained is dependent on the pre-treatment method, especially in trace analysis.
In Chapters 2 and 3, homogeneous reactions suitable for the derivatization of various functional groups
of trace analytes with UV-VIS and FL labels are described in detail. Preand post-column applications
and typical derivatization procedures are given for each functional group. Chapter 4 deals with the
chemiluminescence (CL) detection

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Page xiv

of fluorophores derived from the fluorogenic reaction and fluorescence labelling reaction. This is one of
the most sensitive detection methods, capable of detecting fmol-amol levels. However, this detection
system is not applicable to all fluorescent materials, a notable disadvantage. In Chapter 5, the theory of
electrochemical reactions and derivatization for eictrochemical detection are described, together with
some examples. Detection is based on a redox reaction of the electrodes and is suitable for compounds
easily oxidized and reduced with low potential. Finally, chiral resolution continues with the
derivatization for effective separation and high-sensitivity detection.
There are some excellent books on derivatization, but they do not address the specifics of the topic.
Each chapter includes sufficient references to the literature to serve as a valuable starting point for more
detailed investigations.
As shown in the bibliography, scientists in Japan are very active in these fields. The contributors and
the authors selected for this book are outstanding research chemists. This book should be useful to
many investigators in various fields, including clinical, pharmaceutical, biological and environmental.
The editor would like to express sincere thanks to the authors for their contributions, as well as to their
colleagues for providing stimulating discussions. Thanks are also due to the entire publishing staff at
John Wiley &
Sons for their continued support and contribution towards the completion of this book.
TOSHIMASA TOYO'OKA
SHIZUOKA, JAPAN

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Page 1

1—
Pre-treatment for Real Sample Analysis and Choice of Suitable Reagent
1.1—
Derivatization for Drugs
Akihiko Nakagawa, and Yukinori Kawahara
Analytical and Metabolic Research Laboratories, Sankyo Co.Ltd., Tokyo, Japan
1.1.1. Introduction

2

1.1.2. Application of Derivatization of Drugs

2

1.1.2.1. Determination of Pravastatin Sodium in Plasma by HPLC
with Laser-induced Fluorescence Detection after Immobilized
Antibody Extraction

2

1.1.2.2. Enantiospecific Determination of Ibuprofen in Rat
Plasma Using Chiral Fluorescence Derivatization Reagent, (-)-2[4-(1-aminoethyl)phenyl]-6-methoxybenzoxazole

4

1.1.2.3. Specific Fluorogenic Derivatization of Ivermectin Using
Non-fluorescent Reagents

6

1.1.2.4. Other Methods

8

1.1.3 Conclusion
References

10
11

Edited by Toshimasa Toyo'oka: Modern Derivatization Methods for Separation Sciences (c) 1999 John Wiley &
Sons Ltd.

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