high performance liquid chromatography in phytochemical

High Performance LiquidChromatography inPhytochemical Analysis

CHROMATOGRAPHIC SCIENCE SERIES

A Series of Textbooks and Reference Books

Editor: JACK CAZES

1. Dynamics of Chromatography: Principles and Theory, J. Calvin Giddings2. Gas Chromatographic Analysis of Drugs and Pesticides, Benjamin J. Gudzinowicz3. Principles of Adsorption Chromatography: The Separation of Nonionic Organic

Compounds, Lloyd R. Snyder4. Multicomponent Chromatography: Theory of Interference, Friedrich Helfferich

and Gerhard Klein5. Quantitative Analysis by Gas Chromatography, Josef Novák6. High-Speed Liquid Chromatography, Peter M. Rajcsanyi and Elisabeth Rajcsanyi7. Fundamentals of Integrated GC-MS (in three parts), Benjamin J. Gudzinowicz,

Michael J. Gudzinowicz, and Horace F. Martin8. Liquid Chromatography of Polymers and Related Materials, Jack Cazes9. GLC and HPLC Determination of Therapeutic Agents (in three parts), Part 1 edited by

Kiyoshi Tsuji and Walter Morozowich, Parts 2 and 3 edited by Kiyoshi Tsuji10. Biological/Biomedical Applications of Liquid Chromatography, edited by

Gerald L. Hawk11. Chromatography in Petroleum Analysis, edited by Klaus H. Altgelt and T. H. Gouw12. Biological/Biomedical Applications of Liquid Chromatography II, edited by

Gerald L. Hawk13. Liquid Chromatography of Polymers and Related Materials II, edited by Jack Cazes

and Xavier Delamare14. Introduction to Analytical Gas Chromatography: History, Principles, and Practice,

John A. Perry15. Applications of Glass Capillary Gas Chromatography, edited by Walter G. Jennings16. Steroid Analysis by HPLC: Recent Applications, edited by Marie P. Kautsky17. Thin-Layer Chromatography: Techniques and Applications, Bernard Fried

and Joseph Sherma18. Biological/Biomedical Applications of Liquid Chromatography III, edited by

Gerald L. Hawk19. Liquid Chromatography of Polymers and Related Materials III, edited by Jack Cazes20. Biological/Biomedical Applications of Liquid Chromatography, edited by

Gerald L. Hawk21. Chromatographic Separation and Extraction with Foamed Plastics and Rubbers,

G. J. Moody and J. D. R. Thomas22. Analytical Pyrolysis: A Comprehensive Guide, William J. Irwin23. Liquid Chromatography Detectors, edited by Thomas M. Vickrey24. High-Performance Liquid Chromatography in Forensic Chemistry, edited by

Ira S. Lurie and John D. Wittwer, Jr.25. Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca26. HPLC Analysis of Biological Compounds: A Laboratory Guide, William S. Hancock

and James T. Sparrow27. Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins,

Herbert Schott28. HPLC in Nucleic Acid Research: Methods and Applications, edited by Phyllis R. Brown29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman and E. J. Levy30. Modern Chromatographic Analysis of the Vitamins, edited by André P. De Leenheer,

Willy E. Lambert, and Marcel G. M. De Ruyter

31. Ion-Pair Chromatography, edited by Milton T. W. Hearn32. Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography, edited by

Steven H. Y. Wong33. Affinity Chromatography: Practical and Theoretical Aspects, Peter Mohr

and Klaus Pommerening34. Reaction Detection in Liquid Chromatography, edited by Ira S. Krull35. Thin-Layer Chromatography: Techniques and Applications, Second Edition,

Revised and Expanded, Bernard Fried and Joseph Sherma36. Quantitative Thin-Layer Chromatography and Its Industrial Applications, edited by

Laszlo R. Treiber37. Ion Chromatography, edited by James G. Tarter38. Chromatographic Theory and Basic Principles, edited by Jan Åke Jönsson39. Field-Flow Fractionation: Analysis of Macromolecules and Particles, Josef Janca40. Chromatographic Chiral Separations, edited by Morris Zief and Laura J. Crane41. Quantitative Analysis by Gas Chromatography, Second Edition, Revised

and Expanded, Josef Novák42. Flow Perturbation Gas Chromatography, N. A. Katsanos43. Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto, Kazuhiro Naka-nishi,

and Ryuichi Matsuno44. Countercurrent Chromatography: Theory and Practice, edited by

N. Bhushan Man-dava and Yoichiro Ito45. Microbore Column Chromatography: A Unified Approach to Chromatography,

edited by Frank J. Yang46. Preparative-Scale Chromatography, edited by Eli Grushka47. Packings and Stationary Phases in Chromatographic Techniques, edited by

Klaus K. Unger48. Detection-Oriented Derivatization Techniques in Liquid Chromatography, edited by

Henk Lingeman and Willy J. M. Underberg49. Chromatographic Analysis of Pharmaceuticals, edited by John A. Adamovics50. Multidimensional Chromatography: Techniques and Applications, edited by

Hernan Cortes51. HPLC of Biological Macromolecules: Methods and Applications, edited by

Karen M. Gooding and Fred E. Regnier52. Modern Thin-Layer Chromatography, edited by Nelu Grinberg53. Chromatographic Analysis of Alkaloids, Milan Popl, Jan Fähnrich, and Vlastimil Tatar54. HPLC in Clinical Chemistry, I. N. Papadoyannis55. Handbook of Thin-Layer Chromatography, edited by Joseph Sherma

and Bernard Fried56. Gas–Liquid–Solid Chromatography, V. G. Berezkin57. Complexation Chromatography, edited by D. Cagniant58. Liquid Chromatography–Mass Spectrometry, W. M. A. Niessen and Jan van der Greef59. Trace Analysis with Microcolumn Liquid Chromatography, Milos KrejcI60. Modern Chromatographic Analysis of Vitamins: Second Edition, edited by

André P. De Leenheer, Willy E. Lambert, and Hans J. Nelis61. Preparative and Production Scale Chromatography, edited by G. Ganetsos

and P. E. Barker62. Diode Array Detection in HPLC, edited by Ludwig Huber and Stephan A. George63. Handbook of Affinity Chromatography, edited by Toni Kline64. Capillary Electrophoresis Technology, edited by Norberto A. Guzman65. Lipid Chromatographic Analysis, edited by Takayuki Shibamoto66. Thin-Layer Chromatography: Techniques and Applications: Third Edition, Revised

and Expanded, Bernard Fried and Joseph Sherma67. Liquid Chromatography for the Analyst, Raymond P. W. Scott68. Centrifugal Partition Chromatography, edited by Alain P. Foucault69. Handbook of Size Exclusion Chromatography, edited by Chi-San Wu70. Techniques and Practice of Chromatography, Raymond P. W. Scott71. Handbook of Thin-Layer Chromatography: Second Edition, Revised and Expanded,

edited by Joseph Sherma and Bernard Fried

72. Liquid Chromatography of Oligomers, Constantin V. Uglea73. Chromatographic Detectors: Design, Function, and Operation, Raymond P. W. Scott74. Chromatographic Analysis of Pharmaceuticals: Second Edition, Revised and

Expanded, edited by John A. Adamovics75. Supercritical Fluid Chromatography with Packed Columns: Techniques and

Applications, edited by Klaus Anton and Claire Berger76. Introduction to Analytical Gas Chromatography: Second Edition, Revised and

Expanded, Raymond P. W. Scott77. Chromatographic Analysis of Environmental and Food Toxicants, edited by

Takayuki Shibamoto78. Handbook of HPLC, edited by Elena Katz, Roy Eksteen, Peter Schoenmakers,

and Neil Miller79. Liquid Chromatography–Mass Spectrometry: Second Edition, Revised and Expanded,

Wilfried Niessen80. Capillary Electrophoresis of Proteins, Tim Wehr, Roberto Rodríguez-Díaz,

and Mingde Zhu81. Thin-Layer Chromatography: Fourth Edition, Revised and Expanded, Bernard Fried

and Joseph Sherma82. Countercurrent Chromatography, edited by Jean-Michel Menet and Didier Thiébaut83. Micellar Liquid Chromatography, Alain Berthod and Celia García-Alvarez-Coque84. Modern Chromatographic Analysis of Vitamins: Third Edition, Revised and Expanded,

edited by André P. De Leenheer, Willy E. Lambert, and Jan F. Van Bocxlaer85. Quantitative Chromatographic Analysis, Thomas E. Beesley, Benjamin Buglio,

and Raymond P. W. Scott86. Current Practice of Gas Chromatography–Mass Spectrometry, edited by

W. M. A. Niessen87. HPLC of Biological Macromolecules: Second Edition, Revised and Expanded,

edited by Karen M. Gooding and Fred E. Regnier88. Scale-Up and Optimization in Preparative Chromatography: Principles

and Bio-pharmaceutical Applications, edited by Anurag S. Rathore and Ajoy Velayudhan

89. Handbook of Thin-Layer Chromatography: Third Edition, Revised and Expanded,edited by Joseph Sherma and Bernard Fried

90. Chiral Separations by Liquid Chromatography and Related Technologies, Hassan Y. Aboul-Enein and Imran Ali

91. Handbook of Size Exclusion Chromatography and Related Techniques: Second Edition,edited by Chi-San Wu

92. Handbook of Affinity Chromatography: Second Edition, edited by David S. Hage93. Chromatographic Analysis of the Environment: Third Edition, edited by

Leo M. L. Nollet94. Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis and Discovery,

Paul C.H. Li95. Preparative Layer Chromatography, edited by Teresa Kowalska and Joseph Sherma96. Instrumental Methods in Metal Ion Speciation, Imran Ali and Hassan Y. Aboul-Enein97. Liquid Chromatography–Mass Spectrometry: Third Edition, Wilfried M. A. Niessen98. Thin Layer Chromatography in Chiral Separations and Analysis, edited by

Teresa Kowalska and Joseph Sherma99. Thin Layer Chromatography in Phytochemistry, edited by

Monika Waksmundzka-Hajnos, Joseph Sherma, and Teresa Kowalska 100. Chiral Separations by Capillary Electrophoresis,edited by Ann Van Eeckhaut

and Yvette Michotte101. Handbook of HPLC: Second Edition, edited by Danilo Corradini and consulting editor

Terry M. Phillips102. High Performance Liquid Chromatography in Phytochemical Analysis,

Monika Waksmundzka-Hajnos and Joseph Sherma

Edited byMonika Waksmundzka-Hajnos

Medical University of LublinLublin, Poland

Joseph ShermaLafayette College

Easton, Pennsylvania, U.S.A.

High Performance LiquidChromatography inPhytochemical Analysis

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In Memory ofProfessor Andrzej Waksmundzki

(1910–1998)Pioneer of chromatography in Poland

ix

ContentsPreface............................................................................................................................................ xiiiEditors ..............................................................................................................................................xvContributors ...................................................................................................................................xvii

PART I

1Chapter Overview of the Field of High Performance Liquid Chromatography in Phytochemical Analysis and the Structure of the Book ..............................................3


2Chapter Herbal Drugs and the Role of Chromatographic Methods in Their Analysis ........... 13

Ioanna Chinou

3Chapter Plant Products in Nutrition and Dietary Supplements: Quality Control ....................23

Grazyna Zgórka

4Chapter HPLC in Chemosystematics ....................................................................................... 63

Renate Spitaler and Christian Zidorn

5Chapter Phytochemistry, Phytopharmacology, and the Biological Role of Plant Metabolites .......................................................................................... 89

Krystyna Skalicka-Woźniak, Michał Ł. Hanos, and Kazimierz Głowniak

6Chapter Sample Preparation of Plant Material ...................................................................... 107

Anna Oniszczuk and Anna Hawrył

7Chapter Stationary Phases and Columns in Analysis of Primary and Secondary Metabolites ............................................................................................. 151

Fred Rabel

8Chapter Separation of Nonionic Analytes: Reversed- and Normal-Phase HPLC ................. 185

Mirosław Hawrył and Wojciech Markowski

9Chapter Separation of Ionic Analytes: Reversed-Phase, Ion-Pair, Ion-Exchange, and Ion-Exclusion HPLC.......................................................................................... 195

Monika Waksmundzka-Hajnos and Łukasz Cieśla

x Contents

1Chapter 0 Gradient Elution and Computer-Assisted Method Development ............................. 211

Mirosław Hawrył

1Chapter 1 LC-MS as a Method of Identi�cation and Quanti�cation of Plant Metabolites ...... 257

Günther Stecher, Robert Mayer, Thomas Ringer, Muhammad A. Hashir, Saowapak Kasemsook, Muhammad N. Qureshi, and Günther K. Bonn

1Chapter 2 LC-NMR and Related Techniques for the Rapid Identi�cation of Plant Metabolites ......................................................................................................287

Jean-Luc Wolfender

1Chapter 3 Photodiode Array (PDA) and Other Detection Methods in HPLC of Plant Metabolites ...................................................................................................... 331

Wojciech Markowski and Monika Waksmundzka-Hajnos

1Chapter 4 Quantitative Analysis—Method Validation—Quality Control ............................... 351

Pierre Masson

1Chapter 5 Con�rmation of Chirality of Some Natural Products by the HPLC Method .......... 373

Beata Polak

PART II Primary Metabolites

1Chapter 6 HPLC of Carbohydrates ........................................................................................... 399

Angelika Koch, Simla Basar, and Rita Richter

1Chapter 7 HPLC of Plant Lipids ............................................................................................... 425

Marek Gołębiowski, Monika Paszkiewicz, Łukasz Haliński, and Piotr Stepnowski

1Chapter 8 HPLC Analysis of Amino Acids, Peptides, and Proteins ........................................ 453

Giuseppe Mennella, Antonietta D’Alessandro, and Gianluca Francese

Secondary Metabolites — Shickimic Acid Derivatives

1Chapter 9 Application of HPLC in the Analysis of Phenols, Phenolic Acids, and Tannins ..... 477

Tuulia Hyötyläinen and Maarit Kivilompolo

Contents xi

2Chapter 0 Application of HPLC in Coumarin Analyses .......................................................... 513

Alev Tosun and Petr Tomek

2Chapter 1 HPLC of Flavonoids ................................................................................................. 535

Monika Waksmundzka-Hajnos, Anna Oniszczuk, Mieczysław Hajnos, and Tomasz Oniszczuk

2Chapter 2 HPLC of Lignans ..................................................................................................... 563

Annika I. Smeds

Secondary Metabolites — Isoprenoids

2Chapter 3 HPLC of Mono- and Sesquiterpenes ....................................................................... 579

Angelika Koch, Simla Basar, and Rita Richter

2Chapter 4 HPLC Analysis of Diterpenes ..................................................................................605

Michał Ł. Hajnos

2Chapter 5 High Performance Liquid Chromatography of Triterpenes (Including Saponins) ................................................................................................ 639

Wieslaw Oleszek and Anna Stochmal

2Chapter 6 HPLC of Carotenoids ............................................................................................... 659

Łukasz Ciesla

2Chapter 7 HPLC of Steroids ..................................................................................................... 679

Laurence Dinan, Juraj Harmatha, and René Lafont

2Chapter 8 HPLC of Iridoids ......................................................................................................709

Rilka Taskova, Tetsuo Kokubun, and Kalina Alipieva

Secondary Metabolites — Amino Acid Derivatives

2Chapter 9 HPLC of Indole Alkaloids ....................................................................................... 731

Anna Petruczynik

3Chapter 0 HPLC of Isoquinoline Alkaloids ............................................................................. 769

László Kursinszki, Hajnalka Hank, Ágnes Kéry, and Éva Szőke

xii Contents

3Chapter 1 HPLC of Tropane Alkaloids ....................................................................................803

Tomasz Mroczek

3Chapter 2 HPLC of Alkaloids from the Other Biosynthetic Groups ........................................ 823

Jolanta Flieger

Secondary Metabolites — Compounds Derived from Acetogenine (Acetylocoenzyme A)

33Chapter HPLC Analysis of Polyacetylenes ............................................................................ 887

Lars P. Christensen and Kathrine B. Christensen

3Chapter 4 HPLC of Quinonoid Phytochemicals ....................................................................... 917

Subhalakshmi Ghosh, Madhushree Das Sarma, and Banasri Hazra

Index .............................................................................................................................................. 947

xiii

PrefaceThis is the �rst book to be published that gives a complete description of the techniques, materials, and instrumentation of column high performance liquid chromatography (HPLC) and its appli-cation to essentially all primary and secondary plant metabolites. Together with the companion volume, Thin Layer Chromatography in Phytochemistry that we coedited with Professor Teresa Kowalska in 2008 (Volume 99 in the Chromatographic Science Series edited by Dr. Jack Cazes and published by CRC Press/Taylor & Francis), the two most important analytical approaches used in phytochemical analysis have now been comprehensively covered.

The book is organized into two parts comprising a total of 34 chapters. Part I (15 chapters) begins with a chapter giving an overview of the �eld of phytochemistry and the organization of the book. This is followed by three chapters on the role of HPLC in the analysis of herbal drugs, quality control of plant products in dietary supplements, and chemosystematics, and then a chapter on phytochemistry, pharmacology, and the biological role of plant metabolites. Part I is completed by a series of chapters dedicated to different modes and techniques of HPLC analy-sis: sample preparation; stationary phases and columns; separation of nonionic compounds by normal- and reversed-phase HPLC; separation of ionic compounds by reversed-phase, ion-pair, ion-exchange, and ion-exclusion HPLC; gradient elution and computer-assisted methods; HPLC/mass spectrometry; HPLC/nuclear magnetic resonance spectrometry; HPLC with photodiode array and other detection methods; quantitative analysis and method validation; and HPLC chi-ral analysis.

Part II (19 chapters) contains chapters on the HPLC separation, identi�cation, and quanti�cation of particular classes of compounds in a great variety of sample types (plants, plant extracts, plant-derived products, etc.), starting with chapters on primary metabolites (carbohydrates, lipids, and amino acids, peptides, and proteins) and followed by those on secondary metabolites (shickimic acid derivatives, coumarins, �avonoids, lignans, isoprenoids, diterpenes, tripenes, polyterpenes, steroids, iridoids, amino acid derivatives, isoquinoline alkaloids, tropane alkaloids, other biosyn-thetic alkaloids, polyacetylenes, and quinoids).

Chapters were written by authors with great experience and knowledge in HPLC phytochemi-cal analysis who are working in Austria, Bulgaria, the Czech Republic, Denmark, Finland, France, Greece, Hungary, India, Italy, New Zealand, Poland, Switzerland, Turkey, the United Kingdom, and the United States, which guarantees the book is authoritative and presents an international view of the �eld. The authors were given some latitude to present their material with text, tables, �gures, and references in the manner they believed was best suited to produce the most practical and useful chapters for a wide range of readers with different experience levels and job descriptions.

This book will serve as a laboratory manual, reference book, or source of teaching material for undergraduate and graduate courses in chemistry, biology, and plant science, and it will be an invaluable source of information in research and analytical laboratories associated with universi-ties, dietary supplement production, or governmental regulation. We would be pleased to hear from readers of our book about how it was helpful in their work, as well as noti�cation of any errors and suggestions for changes, additions, and/or deletions if a second edition of the book is published.

We thank Dr. Cazes and Senior Editor Barbara Glunn and Production Coordinator Patricia Roberson of CRC Press/Taylor & Francis for their complete support of this book project from the proposal through publication.

xiv Preface

JS would like to express appreciation to President Daniel H. Weiss and Provost Wendy L. Hill for the continuing support by Lafayette College of his research and publication activities as an emeritus professor.

Monika Waksmundzka-HajnosJoseph Sherma

xv

EditorsMonika Waksmundzka-Hajnos received a PhD in analytical chemistry from the Faculty of Chemistry of Maria Curie-Skłodowska University in Lublin, Poland, in 1980. She is currently pro-fessor of pharmacy and head of the Department of Inorganic Chemistry at the Faculty of Pharmacy of the Medical University of Lublin in Lublin, Poland. Her interests involve the theory and applica-tion of liquid chromatography, taking into consideration the optimization of chromatographic sys-tems for separation of natural mixtures and plant extracts for analytical and preparative purposes. Another scienti�c interest of Dr. Waksmundzka-Hajnos involves the optimization of liquid–solid processes for the extraction of biologically active secondary metabolites from plant material and the optimization of puri�cation processes by liquid–liquid extraction (LLE) and solid-phase extraction (SPE) of crude plant extracts from ballast substances before high performance liquid chromatogra-phy (HPLC) or thin layer chromatography (TLC).

Professor Waksmundzka-Hajnos is author or coauthor of more than 120 papers and approxi-mately 250 conference papers. She has published review articles in journals such as the Journal of Chromatography A, the Journal of Chromatography B, and the Journal of Liquid Chromatography and Research Trends (India). She has also authored a chapter on preparative planar chromatography of plant extracts in the textbook Preparative Layer Chromatography, edited by Teresa Kowalska and Joseph Sherma. Professor Waksmundzka-Hajnos coedited with Professor Kowalska and Professor Sherma the book Thin Layer Chromatography in Phytochemistry (published as Volume 99 in the Chromatographic Science Series by CRC Press/Taylor & Francis), and she coedited with Professor Sherma the current book, HPLC in Phytochemical Analysis. Dr. Waksmundzka-Hajnos has received �ve awards from the Ministry of Health in Poland and two awards from the Polish Pharmaceutical Society for her scienti�c achievements.

Dr. Waksmundzka-Hajnos has taught courses in inorganic chemistry to pharmacy and medical chemistry students for more than 30 years. She has also taught courses in instrumental analysis to students of pharmacy. Over the past 17 years, Dr. Waksmundzka-Hajnos has directed programs for over 40 MSc pharmacy students involved in the theory and practice of different liquid chromato-graphic techniques. She has also supervised four PhD students researching separation science.

Since 2005 Dr. Waksmundzka-Hajnos has been a member of the editorial board of Acta Chromatographica, the annual periodical published by the University of Silesia, Katowice, and by Akademiai Kiado, and since 2008 she has been an editor of that journal. Also since 2008, she has been a member of the editorial board of the Journal of Planar Chromatography-Modern TLC. She has also devoted her time to the development of many chromatographic and hyphenated techniques.

Joseph Sherma received a BS in chemistry from Upsala College in East Orange, New Jersey, in 1955 and a PhD in analytical chemistry from Rutgers, the State University, in New Brunswick, New Jersey, in 1958. He is currently the John D. and Frances H. Larkin Professor Emeritus of Chemistry at Lafayette College, Easton, Pennsylvania. Professor Sherma taught courses in analytical chemis-try for more than 40 years, was head of the Chemistry Department for 12 years, and continues to supervise research students at Lafayette. He has authored, coauthored, edited, or coedited more than 720 publications, including research papers and review articles in more than 50 different analytical chemistry, chromatography, and biological journals; many invited book chapters; and more than 65 books and manuals in the areas of analytical chemistry and chromatography.

In addition to his research in the techniques and applications of thin layer chromatography (TLC), Professor Sherma has a very productive interdisciplinary research program in the use of analytical chemistry to study biological systems with Bernard Fried, Kreider Professor Emeritus of Biology at

xvi Editors

Lafayette College, with whom he has written the book Thin Layer Chromatography (1st–4th edi-tions) and edited the Handbook of Thin Layer Chromatography (1st–3rd editions), both published by Marcel Dekker, Inc., as well as editing Practical Thin Layer Chromatography for CRC Press. Professor Sherma wrote, with Dr. Gunter Zweig, a book on paper chromatography and coedited with him 24 volumes of the Handbook of Chromatography series for CRC Press and 10 volumes of the series Analytical Methods for Pesticides and Plant Growth Regulators for Academic Press. After Dr. Zweig’s death, Professor Sherma edited �ve additional volumes of the chromatography handbook series and two volumes in the pesticide series. The pesticide series was completed under the title Modern Methods of Pesticide Analysis for CRC Press with two volumes coedited with Dr. Thomas Cairns. Three books on quantitative TLC and advances in TLC were edited jointly with the late Professor Joseph C. Touchstone for Wiley-Interscience. For CRC Press/Taylor & Francis, Professor Sherma coedited with Professor Teresa Kowalska Preparative Layer Chromatography and Thin Layer Chromatography in Chiral Separations and Analysis, coedited with Professor Kowalska and Professor Monika Waksmindzka-Hajnos Thin Layer Chromatography in Phytochemistry, and coedited with Professor Waksmundska-Hajnos the current book, HPLC in Phytochemistry.

Professor Sherma served for 23 years as the editor for residues and trace elements of the Journal of AOAC International and is currently acquisitions editor of that journal, and he is now on the editorial advisory boards of the Journal of Liquid Chromatography and Related Technologies; the Journal of Environmental Science and Health (Part B); the Journal of Planar Chromatography-Modern TLC; Acta Chromatographica; Acta Universitatis Cibiniensis, Seria F. Chemia; and Current Pharmaceutical Analysis. Professor Sherma has for 12 years guest-edited with Professor Fried, annual special issues on TLC for the Journal of Liquid Chromatography and Related Technologies, and he regularly guest-edits special sections in the Journal of AOAC International on speci�c subjects in all areas of analytical chemistry. He has also written for 11 years an article on different aspects of modern analytical instrumentation for each issue of the Journal of AOAC International. Professor Sherma has written the biennial review of planar chromatography for the American Chemical Society (ACS) journal Analytical Chemistry continually since 1970. He was the recipient of the 1995 ACS Award for Research at an Undergraduate Institution sponsored by Research Corporation.

xvii

Contributors

Kalina AlipievaInstitute of Organic Chemistry with Centre

of PhytochemistryBulgarian Academy of SciencesSo�a, Bulgaria

Simla BasarDepartment of ToxicologyInstitute for Experimental and Clinical

Pharmacology and ToxicologyUniversity Clinic Hamburg-EppendorfHamburg, Germany

Günther K. BonnInstitute of Analytical Chemistry and

RadiochemistryUniversity of InnsbruckInnsbruck, Austria

Ioanna ChinouDivision of PharmacognosyDepartment of PharmacyUniversity of AthensAthens, Greece

Kathrine B. ChristensenInstitute of Chemical Engineering,

Biotechnology and Environmental Technology

University of Southern DenmarkOdense, Denmark

Lars P. ChristensenInstitute of Chemical Engineering,

Biotechnology and Environmental Technology

University of Southern DenmarkOdense, Denmark

Łukasz CieslaDepartment of Inorganic ChemistryMedical University of LublinLublin, Poland

Laurence DinanLaboratoire Biogenèse des signaux peptidiques-

Equipe de Recherche (BIOSIPE-ER3)Université Pierre et Marie Curie Paris, France

Antonietta D’AlessandroCRA-ORT, Agricultural Research CouncilResearch Center for Vegetable CropsPontecagnano-Faiano, Italy

Jolanta FliegerDepartment of Analytical ChemistryMedical University of LublinLublin, Poland

Gianluca FranceseCRA-ORT, Agricultural Research CouncilResearch Center for Vegetable CropsPontecagnano-Faiano, Italy

Subhalakshmi GhoshDepartment of Pharmaceutical TechnologyJadavpur UniversityCalcutta, India

Kazimierz GłowniakDepartment of PharmacognosyMedical University of LublinLublin, Poland

Marek GołebiowskiLaboratory of Environmental ChemometricsUniversity of GdanskGdansk, Poland

Michał Ł. HajnosDepartment of PharmacognosyMedical University of LublinLublin, Poland

Mieczysław HajnosInstitute of AgrophysicsPolish Academy of SciencesLublin, Poland

xviii Contributors

Łukasz HalinskiDepartment of Environmental AnalysisUniversity of GdanskGdansk, Poland

Hajnalka HankDepartment of PharmacognosySemmelweis UniversityBudapest, Hungary

Juraj HarmathaInstitute of Organic Chemistry and

BiochemistryAcademy of Sciences of the

Czech RepublicPrague, Czech Republic

Muhammad A. HashirInstitute of Analytical Chemistry and

RadiochemistryUniverstiy of InnsbruckInnsbruck, Austria

Anna HawryłDepartment of Inorganic ChemistryMedical University of LublinLublin, Poland

Mirosław HawryłDepartment of Inorganic ChemistryMedical University of LublinLublin, Poland

Banasri HazraDepartment of Pharmaceutical TechnologyJadavapur UniversityCalcutta, India

Tuulia HyötyläinenVTT Technical Research Centre of Finland Espoo, Finland

and

Maj and Thor Nessling FoundationHelsinki, Finland

Saowapak KasemsookInstitute of Analytical Chemistry and

Radiochemistry University of InnsbruckInnsbruck, Austria

Ágnes KéryDepartment of PharmacognosySemmelweis UniversityBudapest, Hungary

Maarit KivilompoloVTT Technical Research Centre of Finland Espoo, Finland

Angelika KochFrohme ApothekeHamburg, Germany

Tetsuo KokubunJodrell LaboratoryRoyal Botanic GardensKew Richmond, Surrey, United Kingdom

László KursinszkiDepartment of PharmacognosySemmelweis UniversityBudapest, Hungary

René LafontLaboratoire BIOSIPE-ER3Université Pierre et Marie Curie Paris, France

Wojciech MarkowskiDepartment of Physical ChemistryMedical University of LublinLublin, Poland

Pierre MassonUnité de Service et de Recherche en Analyses

Végétales et EnvironnementalesInstitut National de la Recherche Agronomique

de BordeauxVillenave d’Ornon, France

Robert MayerInstitute of Analytical Chemistry and


Giuseppe MennellaCRA-ORT, Agricultural Research CouncilResearch Center for Vegetable CropsPontecagnano-Faiano, Italy

Contributors xix

Tomasz MroczekDepartment of PharmacognosyMedical University of LublinLublin, Poland

Wieslaw OleszekInstitute of Soil Science and Plant

CultivationState Research InstitutePulawy, Poland

Anna OniszczukDepartment of Inorganic

ChemistryMedical University of LublinLublin, Poland

Tomasz OniszczukDepartment of Food Process

EngineeringLublin University of Life SciencesLublin, Poland

Monika PaszkiewiczDepartment of Environmental

AnalysisUniversity of GdanskGdansk, Poland

Anna PetruczynikDepartment of Inorganic

ChemistryMedical University of LublinLublin, Poland

Beata PolakDepartment of Physical ChemistryMedical University of LublinLublin, Poland

Muhammad N. QureshiInstitute of Analytical Chemistry and


Fred RabelChromHELP, LLCWoodbury, New Jersey

Rita RichterFrohme ApothekeHamburg, Germany

Thomas RingerInstitute of Analytical Chemistry and


Madhushree Das SarmaDepartment of Pharmaceutical

TechnologyJadavpur UniversityCalcutta, India

Joseph ShermaDepartment of ChemistryLafayette CollegeEaston, Pennsylvania

Krystyna Skalicka-WozniakDepartment of PharmacognosyMedical University of LublinLublin, Poland

Annika I. SmedsProcess Chemistry CentreLaboratory of Wood and Paper ChemistryÅbo Akademi UniversityTurku, Finland

Renate SpitalerInstitute for Food ControlAustrian Agency for Health and Food

Safety (AGES)Innsbruck, Austria

Günther StecherInstitute of Analytical Chemistry and


Piotr StepnowskiDepartment of Environmental

AnalysisUniversity of GdanskGdansk, Poland

xx Contributors

Anna StochmalInstitute of Soil Science and Plant

CultivationState Research InstitutePulawy, Poland

Éva SzokeDepartment of PharmacognosySemmelweis UniversityBudapest, Hungary

Rilka TaskovaSchool of Biological SciencesVictoria University of

WellingtonWellington, New Zealand

Petr TomekInstitute of Microbiology, Academy of

SciencesDepartment of Autotrophic

Microorganisms—Cytotoxicology Division

Trebon, Czech Republic

Alev TosunDepartment of PharmacognosyAnkara UniversityTandogan-Ankara, Turkey

Monika Waksmundzka-HajnosDepartment of Inorganic ChemistryMedical University of LublinLublin, Poland

Jean-Luc WolfenderDepartment of Pharmacognosy and

PhytochemistryUniversity of GenevaGeneva, Switzerland

Grazyna ZgórkaDepartment of PharmacognosyMedical University of LublinLublin, Poland

Christian ZidornInstitute of PharmacyUniversity of InnsbruckInnsbruck, Austria

Part I

3

1 Overview of the Field of High Performance Liquid Chromatography in Phytochemical Analysis and the Structure of the Book


1.1 SURVEY OF PHYTOCHEMISTRY

Phytochemistry, or the chemistry of plants, one of the early subdivisions of organic chemistry, has been of great importance in the identi�cation of plant substances. In a strict sense, phytochemistry is the study of phytochemicals, which are chemicals derived from plants. In a narrower sense, the term is often used to describe the large number of primary and secondary metabolic compounds found in plants. Many of these are synthesized in plant tissues to provide protection against insect attacks, plant diseases, ultraviolet (UV) radiation, environmental contaminants, and so on. Plant metabolites also exhibit a number of protective functions for human consumers. With the devel-opment of new phytochemical methods, additional information has become available for use in conjunction with such research disciplines as plant physiology, plant biochemistry, plant taxonomy, plant biotechnology, and pharmacognosy.

Plant physiology is a discipline concerned with the function, or physiology, of plants. Fundamental processes such as photosynthesis, respiration, plant nutrition, plant hormone functions, tropisms, nastic movements, photoperiodism, photomorphogenesis, circadian rhythms, environmental stress physiology, seed germination, dormancy and stomata function and transpiration, and plant water relationships are studied by plant physiologists. For phytochemical investigations connected with plant physiology, the response of plants to various external factors or environmental conditions is very important and is revealed in the synthesis of secondary metabolites in plant tissues.

Knowledge of the entire plant biochemistry �eld ranges from photosynthesis (the synthesis of natural plant products) to all kinds of genetic engineering with its many commercial applications. Topics include cell structure and function; primary lipid and polysaccharide metabolism; nitrogen �xation; phloem transport; synthesis and function of isoprenoids, phenylpropanoids, and other sec-ondary metabolites; and plant growth regulation and development. Plant biochemistry �eld provides a description of photosynthesis, primary and secondary metabolism, the function of phytohormones, and molecular engineering.

CONTENTS

1.1 Survey of Phytochemistry ........................................................................................................31.2 Procedures of High Performance Liquid Chromatography .....................................................61.3 Organization of the Book .........................................................................................................9

4 High Performance Liquid Chromatography in Phytochemical Analysis

Chemosystematics, also called chemotaxonomy, can be viewed as a hybrid science that com-plements available morphological data to improve plant systematics. Phytochemical constituents (especially the amino acid sequences of common plant proteins) can be used to characterize, describe, and classify species into taxa. Interest in this aspect of systematics has increased with development of rapid, accurate, and precise analytical techniques, and data from as many sources as possible can be employed in plant classi�cation. Evidence from chemical constituents has already led to the reconsideration of many plant taxa. For example, a number of taxonomically dif�cult families have been successfully grouped on the basis of their secondary metabolite pro�les.

The most important application of phytochemical investigation methods is to the �eld of phar-macognosy. Pharmacognosy is the study of medicines derived from natural sources (mainly from plant materials). The history of phytotherapy is almost as long as the history of civilization. The term pharmacognosy was used for the �rst time by the Austrian physician Schmidt in 1811; it is derived from the Greek words pharmakon (“drug”) and gnosis (“knowledge”). Originally—during the nineteenth century and the beginning of the twentieth century—“pharmacognosy” was used to de�ne the branch of medicine or commodity sciences that dealt with drugs in their crude, or unpre-pared, form. Crude drugs are the dried, unprepared material of plant, animal, or mineral origin used for medicine. Although most pharmacognostic studies focus on plants and medicines derived from plants, other types of organisms are also regarded as pharmacognostically interesting, in par-ticular, various types of microbes (bacteria, fungi, etc.) and, recently, various marine organisms. The contemporary study of pharmacognosy can be divided into the �elds of medical ethnobotany, the study of the traditional use of plants for medicinal purposes; ethnopharmacology, the study of the pharmacological qualities of traditional medicinal substances; phytotherapy, the study of the medicinal use of plant extracts; and phytochemistry, the study of chemicals derived from plants (including the identi�cation of new drug candidates derived from plant sources).

Phytotherapy is the study of the use of extracts from natural origin as medicines containing health-promoting agents. In modern science it appeared in the nineteenth century when the �rst biologically active alkaloids (morphine, strychnine, narcotine, caffeine, etc.) were isolated from plants. In the 1930s, chemotherapy began with the appearance and therapeutic usage of synthetic sulfonamides and antibiotics. The lack of studies proving the effects of herbs used in traditional care is especially an issue in the United States, where herbal medicines have fallen out of use since World War II and have been considered suspect since the Flexner Report of 1910 led to the closing of the eclectic medical schools where botanical medicine was exclusively practiced. Furthermore, most herbal studies in the latter part of the twentieth century were published in languages other than English. As it may be more dif�cult to review foreign-language publications, many of these publica-tions have been incorporated into the U.S. Food and Drug Administration (FDA) determinations of drug safety. In 1994, the U.S. Congress passed the Dietary Supplement Health and Education Act (DSHEA) regulating labeling and sales of herbs and other supplements. Most of the 2000 U.S. companies making herbal or natural products choose to market their products as food supplements, which do not require substantial testing. With the development of chemotherapy, it turned out that synthetic drugs exert—along with the positive therapeutic effect—also harmful and often irrevers-ible side effects. Because of this, in recent years a return to phytotherapy has been observed. This return has been further spurred by an appeal of the World Health Organization to screen plant material for biologically active compounds contained therein and their effects, such as pronounced anticancer activity. It is �rmly believed that a great, yet still not fully revealed, therapeutic potential exists in plants, because so far only a few percent out of the estimated 250,000 plant species have been investigated with regard to their usefulness in medicine.

Nowadays, many medicines of natural origin are appreciated for their high effectiveness and low toxicity, and they are widely used commercial products. Plant materials are often obtained from nat-ural sources, although many of the medicinal plants are also cultivated. In view of these facts, there is a high and increasing need for ef�cient purity control of plant material, and further for assessment of their identity and chemical composition, in order to obtain the expected therapeutic effect.

Overview of the Field of HPLC in Phytochemical Analysis 5

In herbal medicine, standardization refers to providing processed plant material that meets a speci-�ed concentration of a speci�c marker constituent. Active constituent concentrations may be mislead-ing measures of potency if cofactors are not present. A further problem is that the important constituent is often unknown. For instance, Saint John’s wort is often standardized with respect to the antiviral con-stituents hypericin or hyperforin or both, although there may be some 24 known possible constituents. Only a minority of chemicals used as standardization markers are known to be active constituents. The process of standardization is not yet carried out consistently: Different companies use different mark-ers, different levels of the same markers, or different methods of testing for marker compounds.

Quality in the use of crude drugs or plant medicines depends on a variety of factors: genetically strong seed; correct species; maturity of the plant at harvest; good soils; air quality; climate; orga-noleptic factors such as intensity of color, �avor, and odor; processing after harvest; and a variety of others. These conditions have been noted in historical herbals, and this was standard pharmacog-nosy curriculum for many years. Storage after collection is also a factor worthy of study. In modern times the foregoing aspects are no less important, but they have been neglected with the advent of laboratory testing, although it generally is true that only certain constituents are identi�ed and mea-sured. Processes like column high performance liquid chromatography (HPLC), gas chromatogra-phy (GC), ultraviolet/visible (UV/VIS) spectrometry, or atomic absorbance spectrometry (AAS) are used to identify species, measure bacteriological contamination, assess potency, and eventually cre-ate certi�cates of analysis for the material. Quality should be overseen by either authorities ensuring good manufacturing practices (GMPs) or regulatory agencies such as the FDA. In the United States, one frequently sees comments that herbal medicines are unregulated, but this is not correct, since the FDA and GMP regulations are in place. In Germany, the Commission E has produced a book of German legal-medical regulations that include quality standards.

The political issues surrounding the safety of crude drugs vary from considering natural rem-edies “safe” regardless of potential dangers to considering them a dangerous unknown. Ephedra has been known to have numerous side effects, including severe skin reactions, irritability, nervous-ness, dizziness, trembling, headache, insomnia, profuse perspiration, dehydration, itchy scalp and skin, vomiting, hyperthermia, irregular heartbeat, seizures, heart attack, stroke, or death. Poisonous plants that have limited medicinal effects are often not sold in material doses in the United States or are available only to trained practitioners. These include Aconite, Arnica, Belladonna, Bryonia, Datura, Gelsemium, Henbane, Male Fern, Phytolacca, Podophyllum, and Veratrum. Secondly are herbs like Lobelia, Ephedra, and Euonymus that cause nausea, sweating, and vomiting, which were traditionally prized for this action. Third are plants such as comfrey and Petasites with speci�c tox-icity due to hepatotoxic pyrrolizidine alkaloids. There are other plant medicines that require caution or can interact with medications, including Saint John’s wort or grapefruit.

Most bioactive compounds of natural origin are secondary metabolites, that is, species-speci�c chemical agents that can be grouped into various categories. A typical protocol to isolate a pure chemical agent of natural origin is bioassay-guided fractionation (BAGF), meaning step-by-step separation of extracted components based on differences in their physicochemical properties, and assessment of the biological activity, followed by another round of separation and assay. Typically, such work is initiated after a given crude drug formulation (normally prepared by solvent extraction of the natural material) is deemed “active” in a particular in vitro assay. If the end goal of the work at hand is to identify which of the scores or hundreds of compounds are responsible for the observed in vitro activity, the path to that end is fairly straightforward: (1) fractionate the crude extract, for example, by solvent partitioning or chromatography; (2) test the fractions thereby gener-ated with in vitro assays; (3) repeat steps 1 and 2 until pure, active compounds are obtained; and (4) determine structure(s) of active compound(s), typically by using spectrometric methods. The most common means for fractionation are solvent–solvent partitioning and chromatographic techniques such as HPLC, medium-pressure liquid chromatography, �ash chromatography, open-column chro-matography, vacuum liquid chromatography (VLC), and thin-layer chromatography (TLC), with each technique being most appropriate for a given amount of starting material. Countercurrent


chromatography (CCC) is particularly well suited for BAGF. After isolation of a pure substance, the task of elucidating its chemical structure can be addressed. For this purpose, the most powerful methodologies available are nuclear magnetic resonance (NMR) spectrometry and mass spectrom-etry (MS). In the case of drug-discovery efforts, structure elucidation of all components that are active in vitro is typically the end goal. In the case of phytotherapy research, the investigator may use in vitro BAGF as a tool to identify pharmacologically interesting or important components of the crude drug. The work does not stop after structural identi�cation of in vitro active substances, however. The task of “dissecting and reassembling” the crude drug one active component at a time, in order to achieve a mechanistic understanding of how it works in phytotherapy, is quite daunting. This is because it is simply too dif�cult, from cost, time, regulatory, and even scienti�c perspec-tives, to study experimental fractions of the crude drug in humans. In vitro assays are, therefore, used to identify chemical components of the crude drug that may reasonably be expected to have a given pharmacological effect in humans and to provide a rational basis for standardization of a crude drug formulation to be tested in humans.

Plant materials, galenic preparations, and isolated compounds proposed for therapy have to meet certain strictly determined standards. With the most important materials, these standards are sim-ply the pharmacopoeia requirements, although a vast number of herbs used in formal and popular medicines are not included in any pharmacopoeia. Standardization of the plant material and of herbal preparations is meant to guarantee their therapeutic value, and it is a result of the investiga-tions on biologically active components.

1.2 PROCEDURES OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Among the chemical methods used for plant examination, chromatographic analysis plays a very important role, and it has been introduced to all modern pharmacopoeias. Due to numerous advantages of the chromatographic methods (such as their speci�city and the possibility to use them for sensitive qualitative and quantitative analysis), they comprise an integral part of medicinal plant analysis.

The following chromatographic methods are most frequently applied in phytochemical analysis: one- and two-dimensional paper chromatography, one- and two-dimensional TLC, HPLC, GC, and CCC. These methods can also be used for isolation of the individual components from the compo-nent mixtures on a preparative and micropreparative scale.

HPLC is a chromatographic technique widely used for qualitative and quantitative analysis of organic compounds present in multicomponent mixtures, such as natural plant extracts. It utilizes a fully automated instrumental system including a column, mobile-phase (eluent) container, mobile-phase pump, injector, and detector. The HPLC system is controlled by a computer program that registers chromatographic pro�les and all data of the individual peaks: retention time, peak height, peak width, surface area of a peak, system ef�ciency, peak symmetry factor, and so on. Because the column providing the separation is connected to the detector, HPLC allows detection and on-line identi�cation of a wide range of organic and inorganic compounds.

The refractive index (RI) detector is an example of a universal detector used in HPLC. However, it is rarely used because all mobile-phase solvents and additives show a signi�cant refractive index response, which makes the use of gradient elution impossible. Other factors, such as the need for temperature control, the effect of dissolved gases in the mobile phase, and low sensitivity, also limit the use of the RI detector for many routine applications. The second type of universal detector is based on evaporative light scattering (ELS). Use of the ELS detector is restricted to nonvolatile analytes and volatile mobile phases.

The detector type most often applied in HPLC is the UV/VIS photodiode array detector (DAD). DAD UV detectors allow simultaneous collection of chromatograms over a range of wavelengths during a single run. Therefore, the DAD provides more information on sample composition than is


provided by use of a single wavelength detector. The UV spectrum of each separated peak is also an important tool for selecting an optimum wavelength to verify peak purity and peak identity. The latter possibility allows comparison of the UV spectra for a standard compound with a sample peak having the same retention time as the standard. The DAD can also be used to examine the chromatograms at different wavelengths, which enables group classi�cation (e.g., taxoids have an absorption maximum at 230 nm, and at 245 nm they absorb poorly). However, UV detection is lim-ited to compounds having chromophore groups (e.g., aromatic rings), and it is not suitable for the compounds that do not absorb in the UV range.

The more selective and sensitive �uorescence (FL) detector is also used in HPLC. Sensitivity of FL detectors is typically three orders of magnitude higher compared to UV detectors, and the detec-tor response is linear over about two or three orders of magnitude for typical conditions. Selectivity is higher because either or both of the excitation and emission wavelengths can be changed. However, only a few analytes possess natural �uorophores. Because of that, derivatization is often necessary prior to use of this detector. Another selective detector used in HPLC is the electrochemical (EC) detector, which operates on the principle of the direct-current amperometry (DCA) or conductivity. These detection methods are applied to compounds that exhibit electrochemical activity. In this case, however, derivatization is also needed to convert the nonelectrochemically active analytes into active ones. Apart from the fact that the analyte must be electrochemically active, the EC detector requires temperature control. This fact and the need for solvents with very high purity, stabilized electrical signals, and so on hampers the use of this detector.

The use of a mass spectrometer as an HPLC detector is becoming commonplace for the qualita-tive and quantitative analysis of mixture components. MS fragmentation patterns can be used to identify each peak. For all MS techniques, the analyte is �rst ionized in the source, since MS can detect only charged species. Ions having discrete mass/charge ratios (m/z) are then separated and focused in the mass analyzer and detected by the detector.

Combination of chromatographic separation techniques with NMR spectrometry is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and mixtures. An on-line HPLC-NMR tandem system has important advantages, espe-cially for structure elucidation of light- and oxygen-sensitive substances, such as hop bitter acids and carotenoid stereoisomers. In such cases, structure elucidation with HPLC-MS is not possi-ble because two isomers often exhibit the same fragmentation pattern. Using the classical method consisting of off-line separation followed by enrichment and transfer to an NMR spectrometry sample tube would result in isomerization of the isolated substances. A closed-loop HPLC-NMR �owthrough system solves this problem. The on-line HPLC-NMR technique also allows continu-ous registration of the time changes as they appear in the chromatographic run. Unequivocal struc-tural assignment of unknown chromatographic peaks is possible in two-dimensional stopped-�ow HPLC-NMR experiments.

Less commonly used in HPLC are polarimetric detectors, used to detect enantiomers, and infra-red and radioactivity detectors.

A main element of HPLC is the column used for the separation processes. Because columns can be used for long periods of time, durable, and well-performing columns are required. For this reason, sorbents used as HPLC packings should reversibly interact with analytes and the mobile-phase components. This means that speci�c interactions (ion–ion, dipole–ion, etc.) should not occur because they cause deactivation of the column packing and a resulting decrease in ef�ciency and separability in the course of usage. Chemically bonded stationary phases with nonpolar alkyl or phenyl ligands or with medium-polarity ligands (containing the CN, NH2, and diol functionalities immobilized on the silica matrix) are mainly used today in the column separations. Application of porous polymers, graphitized carbon, silica, alumina, and the zirconia-based HPLC column pack-ings is also noteworthy.

Reversed-phase (RP) HPLC is one of the most frequently used chromatographic procedures. Today, approximately 80% of all chromatographic separations are performed using nonpolar


chemically bonded phases, containing mainly octadecylsilyl chains (C18). This results from the universality of these phases, relatively low analysis costs, and the ease of the procedure. In such systems, the mobile phase consists of water and organic modi�ers (such as methanol, acetonitrile, tetrahydrofuran, dioxane, etc.), and sometimes extra additives (e.g., buffers of different pH values, ion-pair reagents, ion suppressants, or silanol blockers). These mobile-phase additives are applied for separations of organic electrolytes. RP-HPLC can be performed either by isocratic elution (with a mobile phase having constant composition) or by gradient elution (with changes of the mobile-phase strength resulting from variation in composition in the course of a chromatographic run). Gradient elution is usually obtained by gradual addition of a high-elution-strength solvent to a low-elution-strength solvent (usually water in RP-HPLC). Gradient elution lowers the analysis time and improves separation ef�ciency. The gradient elution mode is applied to mixtures of the components differing considerably in polarity, when the general elution problem occurs. A linear gradient is usually applied, but the gradient pro�le can be programmed to various shapes. Gradient elution suit-able for a given separation can be optimized with the aid of special computer programs simulating separation of a particular mixture (which is characterized with known retention parameters). For separation of organic electrolytes, pH gradients can be applied, or a binary gradient of a mobile-phase modi�er and pH. However, gradient elution limits the use of certain detectors and increases the time needed for equilibration of the column.

The physical properties of the packings and the size of the columns are important parameters in HPLC. Minimization of sorbent particles, increases in the speci�c surface area of the sorbents, and controlled porosity generally cause an increase in column ef�ciency and make separation even of fairly complex mixtures possible. For example, columns with a low particle diameter of 3.5 µm perform bet-ter than those with 5 µm particles. If we compare two columns of the same length, similar resolution on the column with the 3.5 µm particles is available in half the separation time, and the use of a higher mobile-phase �ow rate is also possible without a loss of column ef�ciency. Microbore columns reduce solvent consumption. These columns are especially useful when interfacing an HPLC instrument with mass spectrometers and other instruments requiring small solvent-input volumes. This results in higher mass-detection sensitivity of analytes. In the last decade, monolithic columns have been applied to dif�cult separations. Silica-based monolithic columns contain a novel chromatographic support in which traditional particulate packing is replaced by a single and continuous network of porous silica (known as a monolith). The main advantage of this continuous network is decreased backpressure, due to the presence of macropores (2 µm) throughout the network. This allows high �ow rates and, hence, fast analyses that cannot be obtained with the traditional particulate columns.

Sample preparation is an essential part of HPLC analysis, intended to provide a reproducible and homogeneous solution that is suitable for injection onto the column. The goal of sample preparation is a sample solution that (i) is free of interferences, (ii) will not damage the column, and (iii) is com-patible with an applied HPLC method (i.e., sample solvent is soluble in the mobile phase and does not affect sample retention). Sample pretreatment is usually carried out in a manual off-line mode. However, many sample-preparation techniques have been automated with use of appropriate instru-mentation. Although automation can be expensive and complicated, it is indispensable when large numbers of samples have to be analyzed and the time or labor per sample is excessive. For example, solid-phase extraction (SPE) can be interfaced with a robot to move sample containers and/or the other sample-preparation devices (balances, mixers, dilutors, autosamplers, etc.). Column switch-ing (coupled column chromatography) can also be used as a kind of sample-preparation method. It is used not only for the complete resolution of partly separated fractions from the �rst system but also for the removal of the contaminants (“column killers”) or the late eluters, thus extending the column life and improving resolution of a mixture of components. Column switching can be used as an on-line method of sample pretreatment in the chromatographic run.

Summing up, HPLC is the principal separation and analysis technique in plant chemistry research. It can be used in a search for standardization of plant drugs and for quantitative analy-sis of pharmacologically active compounds. HPLC-DAD enables peak purity control and group


identi�cation (or preliminary identi�cation) of the known extract components. LC-MS or LC-NMR enables identi�cation of the known and unknown plant constituents. These are the bene�ts that make the chromatographic methods widely used in the search for new drugs from the plants that have been used in various ethnomedicines literally for ages. Moreover, such hyphenated techniques enable data collection from numerous plant samples, which proves very useful in chemosystematics. Similar investigations are very helpful for determination of plant drugs or dietary supplements, and of plant food origin and quality.

1.3 ORGANIZATION OF THE BOOK

This book comprises 34 chapters, divided into two parts. Part I consists of 15 chapters and provides general information on those areas of science that are related to phytochemistry and can bene�t from the use of HPLC, such as the instrumentation and chromatographic systems involved. Chapter 2 focuses on herbal drugs and the role of chromatographic methods in the examination of herbs and herbal-product quality by use of �ngerprinting and marker compounds for identi�cation and stand-ardization of botanical drugs by chromatographic methods, especially HPLC.

Chapter 3 is devoted to plant products in nutrition and dietary supplements. It provides quality aspects of botanical products applied and includes a short overview of HPLC analysis of phy-tochemicals (phenolics, carotenoids, phytoestrogens, terpenes, lignans, etc.) in foods (fruit, fruit juices, beverages, wines, tea cultivars, hop resins, beer, etc.) and in dietary supplements (dietary bee products; dietary supplements containing soy, Ginkgo biloba; etc.).

Chapter 4 focuses on the role of HPLC in chemosystematics, also called chemotaxonomy. This chapter starts with a de�nition of this particular branch of phytochemical science, which involves classi�cation of plant organisms based on the qualitative and quantitative differences in their com-position of primary and secondary metabolites. Then the authors highlight the areas of main interest for chemosystematic studies in various branches of science, such as botany, chemical ecology, and food sciences, and the use of HPLC in them. The advantages and limitations of such investigations are also emphasized.

Chapter 5 covers the classi�cation, biosynthesis, and biological importance of plant metabolites, including primary metabolites (carbohydrates, lipids, amino acids, peptides, and proteins) and sec-ondary metabolites from various biochemical pathways. The authors also take into consideration other metabolites with a complicated or mixed biosynthetic origin. The chapter discusses as well the pharmacological activities of primary and secondary metabolites and the therapeutic application of plant materials (natural drugs) in the following cases: nervous system diseases, cardiovascular system disorders, respiratory system diseases, gastrointestinal system disorders, urinary system diseases, gynecological and andrological diseases, rheumatic diseases, and dermatology. Natural compounds having antimitotic, anticancer, and immunomodulatory activities are also presented.

Chapter 6 deals with sample preparation of plant material, taking into account the preliminary processing (collecting, drying, storage, and homogenization of samples) as well as methods of extraction from plant material (traditional methods, Soxhlet extraction, and modern extraction methods such as microwave- or ultrasound-assisted methods or pressurized extraction). The authors also describe procedures applied for sample puri�cation and concentration such as liquid–liquid extraction (LLE), crystallization, SPE, column chromatography, TLC, and membrane separations. Information is provided on methods of hydrolysis of esters, glycosides, and natural polymers and on prechromatographic derivatization.

In Chapter 7, stationary phases and columns that are particularly useful in the analysis of primary and secondary metabolites from plant extracts are described. This chapter covers virtually all col-umns used for this purpose, including different kinds of sorbents (silica, alumina, silica bonded alkyl phases, polymeric supports, polar bonded stationary phases, cellulose, chiral bonded phases, and ion exchange phases), particle size variations, column hardware (stainless steel and polymer), and column sizes. The chapter also presents some methodological problems connected with technical


details of chromatographic columns, such as the effect of column size on separation and ef�ciency, the column life of different chromatographic systems, conditions, kinds of samples, and so on.

The two following chapters deal with the development of methods for separation of plant metabo-lites. Chapter 8 is devoted to optimization of the separation of nonionic analytes by RP and normal-phase (NP) HPLC in isocratic and gradient elution modes. The authors also present applications of particular systems in phytochemical analysis. Chapter 9 deals with the methods of separation of ionic analytes involving RP systems with aqueous buffered mobile phases at various pHs containing ion suppressants or silanol blockers. Ion-pair systems are described, including the effect of mobile-phase pH and buffer type as well as ion-pair type and concentration on the separation of organic electrolytes. The development of methods of ion exchange and ion exclusion chromatography for the separation of ionic analytes is also presented.

Chapter 10 focuses on gradient elution methods and reasons for their application. Multidimensional systems and the use of mixed stationary phases are presented. In this chapter, computer-assisted method development is emphasized.

The next two chapters present methods of identi�cation and/or quanti�cation of plant metabolites by hyphenated techniques such as LC-MS and LC-NMR. Chapter 11 discusses principles of on-line hyphenation of HPLC to MS, including analytical-scale chromatography and miniaturized chro-matographic systems, as well as off-line LC-MS. Applications for structural elucidation of analytes with examples for quanti�cation of selected analytes and in metabolomics are included. Chapter 12 characterizes the principles of LC-NMR by direct and indirect hyphenation as well as problems connected with the compatibility of both techniques. Applications of LC-NMR in plant analysis are presented, as well as limitations of on-line methods.

Chapter 13 deals with the detection methods of analytes applied on-line in the liquid chro-matograph. Detection methods other than MS and NMR are presented for use in quantitative and quantitative analysis. Detector requirements, criteria, and properties of detectors are described, along with selected applications of the UV/VIS DAD, RI, FL, Fourier transform infrared (FT-IR), Ramon Spectroscopy (RS), photothermal, conductivity, ELS, radioactivity, and polarimetry detectors.

Chapter 14 describes quantitative analysis by external standardization, internal standardization, and other methods as well as method validation in HPLC, which includes detection and quanti�ca-tion limits, accuracy, precision, ruggedness/robustness, linearity, speci�city, and so on.

Requirements for chirality con�rmation of some natural plant products by HPLC are the subject of Chapter 15. It describes mechanisms of chiral separations in NP and RP systems and presents applications of these systems in plant analysis.

Part II of the book is divided into subsections that re�ect the types of metabolites that occur in plants. Chapters 16–18 refer to primary metabolites.

Chapter 16 deals with the chemistry of carbohydrates, and their classi�cation and occurrence in the plants as mono-, oligo-, and polysaccharides; glycoconjugates; and aminoglycosides. It provides a historical overview of carbohydrate HPLC and the recommended analytical methods, including sample preparation and the most suitable HPLC systems for the analysis of particular carbohydrate groups.

In Chapter 17, analyses of different classes of plant lipids are presented. The chapter focuses on problems associated with the detection of lipids by HPLC and isolation of lipids from natural sources. Then the authors present chromatographic methods and systems used for separation of nonpolar lipids, polar lipids including phospholipids, glycolipids, and proteolipids.

Chapter 18 focuses on free amino acids, peptides, and proteins, including their occurrence in plants and the use of HPLC to separate the individual groups of these compounds. The authors also present methods for protein HPLC analysis and their importance in taxonomic studies.

The next part of the book deals with the secondary metabolites occurring in plant tissues. It is divided into sections according to the metabolic pathways in which individual substances are synthesized.


Chapter 19 starts with the phenolic compounds that belong to the metabolic pathway of shickimic acid, that is, with phenols, phenolic acids, and tannins. It describes the structure, physicochemical properties, and classi�cation of these compounds; their biological importance; sample-preparation methods; and the various HPLC systems and coupled techniques that are used for their separation and qualitative and quantitative analysis.

Chapter 20 deals with coumarins that belong to the phenol class and are also derived from shickimic acid. Details are provided on a general overview, biological activity of coumarins, and application of HPLC to coumarin analysis by NP, RP, and hyphenated techniques.

Chapter 21 is dedicated to the phenolic compounds originating from a similar pathway as cou-marins, that is, to �avonoids. After a short introduction on the chemistry, biochemistry, and medi-cal signi�cance of �avonoids, the methods for their HPLC analysis are described. The applications of stationary phases and columns, mobile-phase systems and modes, and detectors are discussed. Multidimensional systems and hyphenated techniques for �avonoid analysis are also presented.

The section of the book on secondary metabolites ends with lignans, also originating from shic-kimic acid. Chapter 22 is focused on the chemistry, occurrence in plant material, and pharmaco-logical activity of representatives of this group, followed by procedures for sample preparation and the HPLC analysis. Details about chromatographic systems for NP, RP, and chiral separations of lignans in herbal extracts, preparations, and biological �uids are also reported.

The next section of the book is focused on isoprenoid derivatives, which include several groups of compounds. It starts with Chapter 23 on the volatile compounds (mono- and sesquiterpenes), including their de�nition, classi�cation, occurrence, and importance. Then the following applica-tions of HPLC are discussed: identi�cation and quality control of the volatile fractions in pharma-copoeias and monographs; quality control of traditional Chinese medicines (TCMs); determination of pharmacologic and toxic plant ingredients; improvement of the cultivation of plants; quality control of food, food supplements, and cosmetics; and analysis of active compounds in mosses and fungi.

Chapter 24 covers diterpenoids and presents their structure, physicochemical properties, natu-ral occurrence, pharmacological activity, and chemotaxonomic signi�cance. The details of sample preparation and the analytical HPLC separations of this group of compounds with the aid of dif-ferent chromatographic systems are described, including multidimensional and coupled techniques. The chapter ends with a comparison of the performance of HPLC with that of the other chromato-graphic and related techniques used in diterpenoid analysis.

The next group of compounds that belong to the isoprenoid metabolic pathway are triterpenes, which are described in Chapter 25. After a short introduction on the structure and properties of this group, chromatographic systems and detection methods applied in the analysis of triterpenes (including saponins) are presented.

Chapter 26 is focused on tetra- and polyterpenes; among them, carotenoids represent the most important group of compounds. First, their structure, occurrence, and properties are presented. Then the aspects of the HPLC analysis (such as detection and instability of carotenoids) are empha-sized. The use of polar and nonpolar sorbents and recommended mobile phases are discussed. Practical applications and examples end the chapter.

The next large group of compounds that belong to the isoprenoid pathway are steroids, and they are presented in Chapter 27. In the introductory part of this chapter, the usefulness and validity of HPLC for the separation of steroids are described. Then an overview of the literature is provided, taking into account the classes of phytosterols, steroids (brassinosteroids, bufadienolides, cardeno-lides, ecdysteroids, steroidal saponins, steroidal alkaloids, vertebrate-type steroids, and withano-lides), and the related triterpenoids (cucurbitacins). Structural diversity, separation systems, and the detection and quanti�cation for each class of compounds are presented.

Iridoids are the last group of compounds that belong to the isoprenoid pathway, and they are described in Chapter 28. After an introduction to the structure and physicochemical properties of iridoids, the isolation of this group of compounds from plant material and sample preparation are


emphasized. Finally, HPLC systems and techniques applied to the analysis of iridoids are described, including hyphenated techniques.

The next four consecutive chapters deal with alkaloids synthesized in the plant organisms from amino acids. There are several groups of alkaloids differing in their structure, properties, and bio-logical activity.

Chapter 29 focuses on indole alkaloids. First, the chemical structure, occurrence, and pharma-cological and toxicological importance of this group are discussed. This preliminary information is followed by a detailed description of sample preparation and HPLC separations of indole alkaloids, including chromatographic systems, techniques, and detection methods. Details on the separations of the particular types of indole alkaloids are also presented.

Chapter 30 is devoted to the structure, properties, and biological activity of isoquinoline alka-loids. Details on separation in RP and NP systems, including detection methods for such groups as opium alkaloids and protoberberine alkaloids and derivatives, are presented. The application of LC-MS for these purposes is also described.

Tropane alkaloids are handled in Chapter 31. The chemistry and stereochemistry of tropane and related alkaloids and their biosynthesis and natural occurrence are presented �rst. Various meth-ods of extraction of this group of compounds from plant material are described, followed by the pretreatment of the extracts by liquid–liquid partitioning (LLP), solid-assisted LLP (Extrelut), and SPE. Then information on HPLC of tropane alkaloids including their quanti�cation is provided. The chapter gives detailed information on hyphenated techniques used in tropane alkaloids analysis, and a comparison is made with the results originating from the other separation techniques in use.

Chapter 32 is focused on the remaining groups of alkaloids, including phenylethylamine deriva-tives; quinoline derivatives (Cinchona alkaloids); pyrrolidine, pyrrolizidine, piridine, and piperidine derivatives (Tobacco, Lobelia, Pepper, Pelletierine, Sedum, Senecio alkaloids); quinolizidine alka-loids (Lupine alkaloids); xanthine; imidazole derivatives; and diterpene alkaloids. Preparation of extracts, the most frequently employed HPLC systems, and the detection methods applicable to each individual group are presented.

The last two chapters are devoted to the secondary metabolites derived from acetogenine (acety-locoenzyme A). Chapter 33 deals with polyacetylenes’ distribution in plants and pharmacological activity. Sample-preparation techniques as well HPLC separation, detection, and isolation methods in various systems are described.

Chapter 34 is focused on quinones (antraquinones and naphthoquinones). Their occurrence in plants, pharmacological activity, and HPLC techniques applied to their separation and isolation are discussed.

The authors who agreed to contribute chapters to the book are all recognized international experts in their respective �elds. The book will serve as a comprehensive source of information and training on the state-of-the-art phytochemistry methods performed with the aid of HPLC. It will help in analysts with method development to solve problems connected with practical separations and analyses of plant extract fractions of active metabolites.

A computer-assisted search has found no previous book on HPLC in phytochemical analy-sis. Three editions of the book Phytochemical Methods (1973, 1984, and 1998) by J.B. Harborne (Chapman and Hall, London) had chapters organized by compound type, most of which contained some information on HPLC analysis. However, these information sources are not comprehensive, and the �rst two are now out of date. This book will �ll the void in information in the critical �eld of phytochemical analysis.

This new book is a companion to Thin Layer Chromatography in Phytochemistry, edited by Monika Waksmundzka-Hajnos, Joseph Sherma, and Teresa Kowalska, which was published by CRC Press/Taylor & Francis in their Chromatographic Science Series, edited by the late Jack Cazes. TLC and HPLC have complementary advantages that are of great value in phytochemical analysis, and it is critical to have available the complete information on techniques and applications for both methods provided by these two books.

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2 Chapter 2. Herbal Drugs and the Role ofChromatographic Methods in Their Analysis

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TABLE 2.1

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Gas chromatography Herbal drug analyses, forensicchemistry, forensic toxicology, toxicological analyses,food chemistry Volatiles, secondary metabolites

Thin-layer

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10 Chapter 10. Gradient Elution andComputer-Assisted Method Development

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2. Treiber, L.R., Normal-phase high-performance liquidchromatography with relay gradient elution I. Descriptionof the method, J. Chromatogr. A, 696, 193–199, 1995.

3. Snyder, L.R., and Dolan, J.W., Initial experiments inhigh-performance liquid chromatographic method developmentI. Use of a starting gradient run, J. Chromatogr. A, 721,3–14, 1996.

4. Zhu, P.L., Dolan, J.W., Snyder, L.R., Djordjevic, N.M.,Hill, D.W., Lin, J.-T., Sander, L.C., and Van Heukelem,L., Combined use of temperature and solvent strength inreversed-phase gradient elution IV. Selectivity forneutral (non-ionized) samples as a function of sample typeand other separation conditions, J. Chromatogr. A, 756,63–72, 1996.

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7. Dolan, J.W., Snyder, L.R., and Blanc, T., Selectivitydifferences for C 18 and C 8 reversed-phase columns as afunction of temperature and gradient steepness: II.Minimizing column reproducibility problems, J. Chromatogr.A, 897, 51–63, 2000.

8. Dolan, J.W., Snyder, L.R., Blanc, T., and Van Heukelem,L. Selectivity differences for C 18 and C 8 reversed-phasecolumns as a function of temperature and gradientsteepness: I. Optimizing selectivity and resolution, J.Chromatogr. A, 897, 37–50, 2000 (with erratum, J.Chromatogr. A, 910, 385, 2001).

9. Dolan, J.W., Snyder, L.R., Djordjevic, N.M., Hill, D.W.,

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11. Dolan, J.W., Synder, L.R., Djordjevic, N.M., Hill,D.W., and Waeghe, V., Reversed-phase liquid chromatographicseparation of complex samples by optimizing temperature andgradient time: II. Two-run assay procedures, J.Chromatogr. A, 857, 21–39, 1999.

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14. Dolan, J.W., Snyder, L.R., Saunders, D.L., and VanHeukelem, L., Simultaneous variation of temperature andgradient steepness for reversed-phase high-performanceliquid chromatography method development: II. The use offurther changes in conditions, J. Chromatogr. A, 803,33–50, 1998.

15. Wilson, N.S., Dolan, J.W., Snyder, L.R., Carr, P.W.,and Sander, L.C., Column selectivity in reversedphaseliquid chromatography: III. The physico-chemical basis ofselectivity, J. Chromatogr. A, 961, 217– 236, 2002.

16. Wilson, N.S., Nelson, M.D., Dolan, J.W., Snyder, L.R.,and Carr, P.W., Column selectivity in reversedphase liquidchromatography: II. Effect of a change in conditions, J.Chromatogr. A, 961, 195–215, 2002.

17. Wilson, N.S., Nelson, M.D., Dolan, J.W., Snyder, L.R.,Wolcott, R.G., and Carr, P.W., Column selectivity inreversed-phase liquid chromatography: I. A general

quantitative relationship, J. Chromatogr. A, 961, 171–193,2002.

18. Gilroy, J.J., Dolan, J.W., and Snyder, L.R., Columnselectivity in reversed-phase liquid chromatography: IV.Type-B alkyl-silica columns, J. Chromatogr. A, 1000,757–778, 2003.

19. Gilroy, J.J., Dolan, J.W., Carr, P.W., and Snyder,L.R., Column selectivity in reversed-phase liquidchromatography: V. Higher metal content (type-A)alkyl-silica columns, J. Chromatogr. A, 1026, 77–89, 2004.

20. Wilson, N.S., Gilroy, J., Dolan, J.W., and Snyder,L.R., Column selectivity in reversed-phase liquidchromatography: VI. Columns with embedded or end-cappingpolar groups, J. Chromatogr. A, 1026, 91–100, 2004.

21. Marchand, V., Croes, K., Dolan, J.W., Snyder, L.R.,Henry, R.A., Kallury, K.M.R., Waite, S., and Carr, P.W.,Column selectivity in reversed-phase liquid chromatography:VIII. Phenylalkyl and �uoro-substituted columns, J.Chromatogr. A, 1062, 65–78, 2005.

22. Neue, U.D., Marchand, D.H., and Snyder, L.R., Peakcompression in reversed-phase gradient elution, J.Chromatogr. A, 1111, 32–39, 2006.

23. Kaliszan, R., and Wiczling, P., Theoreticalopportunities and actual limitations of pH gradient HPLC,Anal. Bioanal. Chem., 382, 718–727, 2005.

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33. Concha-Herrera, V., Vivo-Truyols, G., Torres-Lapasio,J.R., and Garcia-Alvarez-Coque, M.C., Limits ofmulti-linear gradient optimisation in reversed-phase liquidchromatography, J. Chromatogr. A, 1063, 79–88, 2005.

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39. Meyer, V.R., Practical High-Performance LiquidChromatography, J. Wiley & Sons, Chichester, UK, 2004.

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80. Bhandari, P., Kumar, N., Singh, B., and Kaul, V.K.,Simultaneous determination of sugars and picrosides inPicrorhiza species using ultrasonic extraction andhigh-performance liquid chromatography with evaporativelight scattering detection, J. Chromatogr. A, 1194,257–261, 2008.

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82. Minocha, R., Thangavel, P., Dhankher, O.P., and Long,S., Separation and quanti�cation of monothiols andphytochelatins from a wide variety of cell cultures andtissues of trees and other plants using high performanceliquid chromatography, J. Chromatogr. A, 1207, 72–83, 2008.

83. Vatinno, R., Aresta, A., Zambonin, C.G., and Palmisano,F., Determination of Ochratoxin A in green coffee beans bysolid-phase microextraction and liquid chromatography withuorescence detection, J. Chromatogr. A, 1187, 145–150,2008.

84. Yuan, J., Liu, Q., Zhu, W., Ding, L., Tang, F., andYao, S., Simultaneous analysis of six aristolochic acidsand �ve aristolactams in herbal plants and theirpreparations by high-performance liquidchromatography–diode array detection–�uorescence detection,J. Chromatogr. A, 1182, 85–92, 2008.

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14 Chapter 14. QuantitativeAnalysis—Method Validation-QualityControl

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3. You, J., Zhao, X., Suo, Y., Li, Y., Wang, H., and Chen,G., Determination of long-chain fatty acids in bryophyteplants extracts by HPLC with �uorescence detection andidenti�cation with MS, J. Chromatogr. B, 848, 283–291,2007.

4. Guignard, C., Jouve, L., Bogéat-Triboulot, M.B., Dreyer,E., Hausman, J.F., and Hoffmann, L., Analysis ofcarbohydrates in plants by high-performance anion-exchangechromatography coupled with electrospray mass spectrometry,J. Chromatogr. A, 1085, 137–142, 2005.

5. Gomez-Serranillos, M.P., El-Naggar, T., Villar, A.M.,and Carretero, M.E., Analysis and retention behavior inhigh-performance liquid chromatography of terpenic plantconstituents with pharmacological interest, J. Chromatogr.B, 812, 379–383, 2004.

6. Huck, C.W., Buchmeiser, M.R., and Bonn, G.K., Fastanalysis of �avonoids in plant extracts by liquidchromatography-ultraviolet absorbance detection onpoly(carboxylic acid)-coated silica and electrosprayionization tandem mass spectrometric detection, J.Chromatogr. A, 943, 33–38, 2001.

7. Tikhomiroff, C., and Jolicoeur, M., Screening ofCatharanthus roseus secondary metabolites byhighperformance liquid chromatography, J. Chromatogr. A,955, 87–93, 2002.

8. Hodisan, T., Culea, M., Cimpoiu, C., and Cot, A.,Separation, identi�cation and quantitative determination offree amino acids from plant extracts, J. Pharm. Biomed.Anal., 18, 319–323, 1998.

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10. Martinez Vidal, J.L., Garrido Frenich, A., Lopez-Lopez,T., Martinez Salvador, I., Hajjaj el Hassani, L., andHassan Benajiba, M., Selection of a representative matrixfor calibration in multianalyte determination of pesticidesin vegetables by liquid chromatography-electrospray tandemmass spectrometry, Chromatographia, 61, 127–131, 2005.

11. Fernandez-Figares, I., Rodriguez, L.C., andGonzalez-Casado, A., Effect of different matrices onphysiological amino acids analysis by liquidchromatography: Evaluation and correction of the matrixeffect, J. Chromatogr. B, 799, 73–79, 2004.

12. Schwartz, H., and Sontag, G., Determination ofsecoisolariciresinol, lariciresinol and isolariciresinol inplant foods by high performance liquid chromatographycoupled with coulometric electrode array detection, J.Chromatogr. B, 838, 78–85, 2006.

13. Lui, H.T., Li, Y.F., Lan, C.Y., and Shu, W.S.,Simultaneous determination of phytohormones in plantextracts using SPME and HPLC, Chromatographia, 66, 515–520,2007.

14. Breinholder, P., Mosca, L., and Linder, W., Concept ofsequential analysis of free and conjugated phytosterols indifferent plant matrices, J. Chromatogr. B, 777, 67–82,2002.

15. Taylor, K.L., Brackenridge, A.E., Vivier, M.A., andOberholster, A., High-performance liquid chromatographypro�ling of the major carotenoids in Arabidopsis thalianaleaf tissue, J. Chromatogr. A, 1121, 83–91, 2006.

16. Krenn, L., Unterrieder, I., and Ruprechter, R.,Quanti�cation of iso�avones in red clover by highperformance liquid chromatography, J. Chromatogr. B, 777,123–128, 2002.


18. Hartmann, N., Erbs, M., Wettstein, F.E., Schwarzenbach,E.P., and Bucheli, T.D., Quanti�cation of estrogenicmycotoxins at the ng/L level in aqueous environmental

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19. Zenkevich, I.G., and Makarov, E.D., Chromatographicquantitation at losses of analyte during samplepreparation: Application of the modi�ed method of doubleinternal standard, J. Chromatogr. A, 1150, 117–123, 2007.

20. Krenn, L., Boros, B., Ohmacht, R., and Jelinek, L.,High separation of opium alkaloids on porous andnon-porous stationary phases, Chromatographia, 51,S175–S178, 2000.

21. El-Shafae, A.M., and El-Domiaty, M.M., Improved LCmethods for the determination of diosmin and/ or hesperidinin plant extracts and pharmaceutical formulations, J.Pharm. Biomed. Anal., 26, 539–545, 2001.

22. Forina, M., Lanteri, S., and Casale, M., Multivariatecalibration, J. Chromatogr. A, 1158, 61–93, 2007.

23. Ortiz, M.C., and Sarabia, L., Quantitativedetermination in chromatographic analysis based on n-waycalibration strategies, J. Chromatogr. A, 1158, 94–110,2007.

24. Martinez-Galera, M., Martinez-Vidal, J.L., GarridoFrenich, A., and Gil Garcia, M.D., Evaluation ofmultiwavelength chromatograms for the quanti�cation ofmixtures of pesticides by high-performance liquidchromatography-diode array detection with multivariatecalibration, J. Chromatogr. A, 778, 139– 149, 1997.

25. Van Nederkassel, A.M., Daszykowski, M., Massart, D.L.,and Vander Heyden, Y., Prediction of total green teaantioxidant capacity from chromatograms by multivariatemodeling, J. Chromatogr. A, 1096, 177–186, 2005.

26. NF EN ISO/CEI 17025, Exigences générales concernant lacompétence des laboratoires d’étalonnages et d’essais,AFNOR, Paris, 2005.

27. Thompson, M., Ellison, S.L.R., and Wood, R., Theinternational harmonized protocol for the pro�ciencytesting of analytical chemistry laboratories, Pure Appl.Chem., 78, 145–196, 2006.

28. Hubert, P., Chiap, P., Crommen, J., Boulanger, B.,Chapuzet, E., Mercier, N., Bervoas-Martin, S., et al., TheSFSTP guide on the validation of chromatographic methods

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29. Gonzalez, A.G., and Herrador, M.A., A practical guideto analytical method validation, including measurementuncertainty and accuracy pro�les, Trends Anal. Chem., 26,227–238, 2007.

30. Van Zoonen, P., Hoogerbrugge, R., Gort, S.M., Van deWiel, H.J., and Van’t Klooster, H.A., Some practicalexamples of method validation in the analytical laboratory,Trends Anal. Chem., 18, 584–593, 1999.

31. Wood, R., How to validate analytical methods, TrendsAnal. Chem., 18, 624–632, 1999.

32. Asnin, L., and Guiochon, G., Calibration of a detectorfor nonlinear responses, J. Chromatogr. A, 1089, 105–110,2005.

33. NF V03-110, Procédure de validation intralaboratoired’une méthode alternative par rapport à une méthode deréférence, AFNOR, Paris, 1998.

34. Huber, L., Validation of analytical methods: Review andstrategy, LC-GC, 11, 96–105, 1998.

35. Den Boef, G., and Hulanicki, A., Recommendations forthe usage of selective, selectivity and related terms inanalytical chemistry, Pure Appl. Chem., 55, 553–556, 1983.

36. Vessman, J., Selectivity or speci�city? Validation ofanalytical methods from the perspective of an analyticalchemist in the pharmaceutical industry, J. Pharm. Biomed.Anal., 14, 867–869, 1996.

37. NF ISO 5725-1, Exactitude (justesse et fidélité) desrésultats et méthodes de mesure – Partie 1: Principesgénéraux et définitions, AFNOR, Paris, 1994.

38. Hibbert, D.B., Systematic errors in analyticalmeasurement results, J. Chromatogr. A, 1158, 25–32, 2007.

39. Thompson, M., Ellison, S.L.R., and Wood, R., Harmonizedguidelines for single-laboratory validation of methods ofanalysis, Pure Appl. Chem., 74, 835–855, 2002.

40. Maier, E.A., Quevauviller, Ph., and Griepink, B.,Interlaboratory studies as a tool for many purposes:Pro�ciency testing, learning exercises, quality control and

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41. NF ISO 5725-2, Exactitude (justesse et fidélité) desrésultats et méthodes de mesure — Partie 2: Méthode debase pour la détermination de la répétabilité et de lareproductibilité d’une méthode de mesure normalisée, AFNOR,Paris, 1994.

42. Wenzl, T., Karasek, L., Rosen, J., Hellenaes, K.E.,Crews, C., Castle, L., and Anklam, E., Collaborative trialvalidation study of two methods, one based on highperformance liquid chromatography-tandem mass spectrometryand on gas chromatography-mass spectrometry for thedetermination of acrylamide in bakery and potato products,J. Chromatogr. A, 1132, 211–218, 2006.

43. Vial, J., and Jardy, A., Interlaboratory studies: Thebest way to estimate the characteristics of dispersion ofan HPLC method and a powerful tool for analyticaltransfers, Chromatographia, 53, S141–S148, 2001.

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45. Maroto, A., Boqué, R., Riu, J., and Rius, F.X.,Evaluating uncertainty in routine analysis, Trends Anal.Chem., 18, 577–584, 1999.

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47. ISO, Guide to the Expression of Uncertainty inMeasurement, ISO, Geneva, 1993.

48. EURACHEM guide, Quantifying Uncertainty in AnalyticalMeasurement, 2nd ed., 2000. Available athttp://www.eurachem.org.

49. Meyer, V.R., Measurement uncertainty, J. Chromatogr. A,1158, 15–24, 2007.

50. Populaire, S., and Campos Gimenez, E., A simpli�edapproach to the estimation of analytical measurementuncertainty, Accred. Qual. Assur., 10, 485–493, 2006.

51. Love, J.L., Chemical metrology, chemistry and the

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52. Hund, E., Massart, D.L., and Smeyers-Verbeke, J.,Comparison of different approaches to estimate theuncertainty of a liquid chromatographic assay, Anal. Chim.Acta, 480, 39–52, 2003.

53. Gonzales, A.G., and Herrador, M.A., Accuracy pro�lesfrom uncertainty measurements, Talanta, 70, 896–901, 2006.

54. Feinberg, M., Validation of analytical methods based onaccuracy pro�les, J. Chromatogr. A, 1158, 174–183, 2007.

55. Dejaegher, B., and Vander Heyden, Y., Ruggedness androbustness testing, J. Chromatogr. A, 1158, 138–157,2007.

56. Nijhuis, A., Van der Knaap, H.C.M., de Song, S., andVandeginste, B.G.M., Strategy for ruggedness tests inchromatographic method validation, Anal. Chim. Acta, 391,187–202, 1999.

57. Ragonese, R., Mulholland, M., and Kalman, J., Full andfractioned experimental designs for robustness testing inthe high-performance liquid chromatographic analysis ofcodeine phosphate, pseudoephedrine hydrochloride andchlorpheniramine maleate in a pharmaceutical preparation,J. Chromatogr. A, 870, 45–51, 2000.

58. Romero, R., Sanchez-Vinas, M., Gazquez, D., Bagur,M.G., and Cuadros-Rodriguez, L., Robustness study for thedetermination of biogenic amines by HPLC, Chromatographia,53, 481–484, 2001.

59. Vander Heyden, Y., Nijhuis, A., Smeyers-Verbeke, J.,Vandeginste, B.G.M., and Massart, D.L., Guidance forrobustness/ruggedness tests in method validation, J. Pharm.Biomed. Anal., 24, 723–753, 2001.

60. Valcarcel, M., and Rios, A., Traceability in chemicalmeasurements for the end users, Trends Anal. Chem., 18,570–576, 1999.

61. Simonet, B.M., Quality control in qualitative analysis,Trends Anal. Chem., 24, 525–531, 2005.

62. Quevauviller, P., Quality Assurance in EnvironmentalMonitoring, VCH, Weinheim, Germany, 1995.

63. Jimenez, C., Ventura, R., de la Torre, X., and Segura,J., Strategies for internal quality control in antidopinganalyses, Anal. Chim. Acta, 460, 289–307, 2002.

64. Masson, P., Quality control techniques for routineanalysis with liquid chromatography in laboratories, J.Chromatogr. A, 1158, 168–173, 2007.

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15 Chapter 15. Confirmation of Chiralityof Some Natural Products by the HPLCMethod

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16 Chapter 16. HPLC of Carbohydrates

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TABLE 16.6

HPLC Conditions of Glucosaminoglycans

Ref. Column/Temp. Mobile Phase Detection A: DerivatizationB: Guard Column/ Cleanup Sample

[39] PGC Hypercarb S 100 × 4.6 mm Gradient: (A) H 2 O–0.05% TFA; (B) AN–0.05% TFA; 0%–100% B Fluorescence:Ex: 330 nm, Em: 420 nm A: Precolumn 2-AB B: Ion exchangefor cleanup Glycoproteins, e.g., fetuin

[40] Anion exchange SS-SAX, 250 × 4.6 mm Linear gradient:0.2–1.5 M NaCl UV 232 nm A: Sample oxymercurationtreatment Tetra-, hexa-, octasaccharide units ofchondroitin sulfate

[40] APS Hypersil 2 (NH 2 ), 5 µm, 200 × 4.6 mm Lineargradient: 100–250 mM NaH 2 PO 4 , 200–400 mM NaH 2 PO 4, 250–450 mM NaH 2 PO 4 UV 206 nm A: Sampleoxymercuration treatment Tri- penta-, heptasaccharide

units of chondroitin sulfate

[41] YMC NH2, 250 × 4 mm, 40°C Linear gradient of 16–800mM NaH 2 PO 4 UV 210 nm, ESI-MS B: Cleanup by labelingwith ANTS followed by PAGE Hyaluronan oligosaccharides

Note: 2-AB, 2-aminobenzamide; ANTS,8-aminonaphthalene-1,3,6-trisulfonic acid; PAGE,polyaclylamide-gel electrophoresis; PGC, porous graphitizedcarbon.

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Secondary Metabolites —

Shickimic Acid Derivatives

19 Chapter 19. Application of HPLC in theAnalysis of Phenols, Phenolic Acids, andTannins

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Isoprenoids

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24 Chapter 24. HPLC Analysis of Diterpenes

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25 Chapter 25. High Performance LiquidChromatography of Triterpenes (IncludingSaponins)

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FIGURE 25.7 (Opposite) Total and selected ion chromatograms

of M. truncatula, cultivar Jemalong A, aer

ial part saponins: (A) total ion chromatogram obtained bynegative-ion liquid chromatography–electrospray

ionization–mass spectrometry (LC-ESI-MS) of plant extractpuri�ed by solid-phase extraction; (B) total ion

chromatogram obtained by negative-ion LC-ESI-MS of amixture of 18 standard saponins; (C) selective ion

chromatogram (m/z 1257 [M-H] – ) representing saponins 2,4, and 7; (D) selective ion chromatogram (m/z

1383 [M-H] – ) representing saponins 3 and 6; (E) selectiveion chromatogram (m/z 1367 [M-H] – ) representing

saponins 12 and 14. Peak areas from the selective ionchromatograms were used to calculate absolute con

centrations based on the standard response curves of 18saponin standards from aerial parts of M. truncatula.

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Amino Acid Derivatives

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31 Chapter 31. HPLC of Tropane Alkaloids

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p p m H 1 0 H 3 H 7 H 1 H 5 H 8 H 6 H 2 , 4 H-10 H-3 H-7H-1 H-5 H-8 H-6 H-2,4

FIGURE 31.7 Correlation spectroscopy (COSY)1 H-nuclearmagnetic resonance (NMR) spectrum of

isomer 1 (3α-senecioyloxy-7β-hydroxytropane) using theHPLC–UV–mass spectrometry–solid-phase

extraction–NMR setup (Taken from Bieri, S., Varesio, E.,Veuthey, J.-L., Munoz, O., Tseng, L.-H.,

Braumann, U., Spraul, M., and Christen, P., Phytochem.Anal., 17, 78, 2006. With permission from Wiley

InterScience.)

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Compounds Derived from

Acetogenine (Acetylocoenzyme A)

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