Modern Methods of Plant Analysis

Modern Methods of Plant Analysis New Series Volume 3 Editors H.E Linskens, Nijmegen J.E Jackson, Adelaide

Volumes Already Published in this Series: Volume 1: Cell Components 1985, ISBN 3-540-15822-7 Volume 2: Nuclear Magnetic Resonance 1986, ISBN 3-540-1591O-X Volume 3: Gas Chromatography/Mass Spectrometry 1986, ISBN 3-540-15911-8

Gas Chromatography/ Mass Spectrometry Edited by H.E Linskens and IE Jackson Contributors R.S. Bandurski G. Combaut A. Ehmann P. Hedden B. Janistyn H. Kameoka H. Kodama D.V. Lynch IK. MacLeod H. Nyberg L.M.S. Palni L. Rivier R.R. Selvendran B.IH. Stevens S.A.B. Tay G.A. Thompson Jr., L. Witte With 98 Figures Springer -Verlag Berlin Heidelberg New York Tokyo

Professor Dr. HANS FERDINAND LINSKENS Botanisch Laboratorium Faculteit der Wiskunde en Natuurwetenschappen Katholieke Universiteit Toernooiveld NL-6525 ED Nijmegen The Netherlands Professor Dr. JOHN F. JACKSON Department of Biochemistry Waite Agricultural Research Institute University of Adelaide Glen Osmond, S.A. 5064 Australia ISBN-13 :978-3-642-82614-6 e-isbn-13 :978-3-642-82612-2 DOl: 10.1 007/978-3-642-82612-2 Library of Congress Cataloging-in-Publication Data. Gas chromatography/mass spectrometry. (Modern methods of plant analysis; new ser., v. 3) 1. Plants - Analysis. 2. Gas chromatography. 3. Mass spectrometry. I. Linskens, H. F. (Hans F.), 1921-. II. Jackson, J. F. (John F.), 1935-. III. Series. QK865.G37 1986 58l.l9'285 86-3875 ISBN-13 :978-3-642-82614-6(U.S.) This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under 54 of the German Copyright Law, where copies are made for other than private use, a fee is payable to "Verwertungsgesellschaft Wort", Munich. by Springer-Verlag Berlin Heidelberg 1986 Softcover reprint of the hardcover 1 st edition 1986 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product Liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. 2131/3130-543210

Introduction Modern Methods of Plant Analysis When the handbook Modern Methods of Plant Analysis was first introduced in 1954 the considerations were: 1. the dependence of scientific progress in biology on the improvement of existing and the introduction of new methods; 2. the difficulty in finding many new analytical methods in specialized journals which are normally not accessible to experimental plant biologists; 3. the fact that in the methods sections of papers the description of methods is frequently so compact, or even sometimes so incomplete that it is difficult to reproduce experiments. These considerations still stand today. The series was highly successful, seven volumes appearing between 1956 and 1964. Since there is still today a demand for the old series, the publisher has decided to resume publication of Modern Methods of Plant Analysis. It is hoped that the New Series will be just as acceptable to those working in plant sciences and related fields as the early volumes undoubtedly were. It is difficult to single out the major reasons for success of any publication, but we believe that the methods published in the first series were up-to-date at the time and presented in a way that made description, as applied to plant material, complete in itself with little need to consult other publications. Contributing authors have attempted to follow these guidelines in this New Series of volumes. Editorial The earlier series Modern Methods of Plant Analysis was initiated by Michel v. Tracey, at that time in Rothamsted, later in Sydney, and by the late Karl Paech (1910-1955), at that time at Tiibingen. The New Series will be edited by Paech's successor H. F. Linskens (Nijmegen, The Netherlands) and John F. Jackson (Adelaide, South Australia). As were the earlier editors, we are convinced "that there is a real need for a collection of reliable up-to-date methods for plant analysis in large areas of applied biology ranging from agriculture and horticultural experiment stations to pharmaceutical and technical institutes concerned with raw material of plant origin". The recent developments in the fields of plant biotechnology and genetic engineering make it even more important for workers in the plant sciences to become acquainted with the more sophisticated methods, which sometimes come from biochemistry and biophysics, but which also have

VI Introduction been developed in commercial firms, space science laboratories, non-university research institutes, and medical establishments. Concept of the New Series Many methods described in the biochemical, biophysical, and medical literature cannot be applied directly to plant material because of the special cell structure, surrounded by a tough cell wall, and the general lack of knowledge of the specific behavior of plant raw material during extraction procedures. Therefore all authors of this New Series have been chosen because of their special experience with handling plant material, resulting in the adaptation of methods to problems of plant metabolism. Nevertheless, each particular material from a plant species may require some modification of described methods and usual techniques. The methods are described critically, with hints as to their limitations. In general it will be possible to adapt the methods described to the specific needs of the users of this series, but nevertheless references have been made to the original papers and authors. While the editors have worked to plan in this New Series and made efforts to ensure that the aims and general layout of the contributions are within the general guidelines indicated above, we have tried not to interfere too much with the personal style of each author. Volume Three - Gas Chromatography/Mass Spectrometry The New Series in Modern Methods of Plant Analysis was initiated in 1985 with a volume on Cell Components, and quickly followed by a second volume on Nuclear Magnetic Resonance (NMR). Both included chapters contributed by world experts on all aspects of the chosen topics. We have now followed the principle adopted in the second volume, that of presenting the application of a relative new and powerful analytical technique, this time devoting the third volume to gas chromatography coupled to mass spectrometry (GCjMS). Here the separation power of GC is combined with the selective detection properties of MS, where compounds are ionized within the mass spectrometer and fragment ions separated and analyzed on the basis of their mass-to-charge ratio. Gas chromatography was one of the earliest techniques used to determine plant hormones. When it is combined with mass spectrometry, a technique capable of functioning as a highly sensitive and selective gas chromatography detector, especially when it is focused on a particular ion, then we have a very sensitive analytical tool indeed. As little as 10 pg may be enough for detection with this technique. Little wonder, then, that GCjMS is often used to determine plant hormones. This is reflected in the make-up of the present volume, where we feature chapters on determination of cytokinins, auxins, gibberellins, cyclic nucleotides, and other biologically active compounds. Other plant materials are not neglected, so that chapters on the determination of tobacco constituents, essential oils, phospholipids, fatty acids, volatile flower compounds, pectic polysaccharides, and terpenoids can also be found in this volume.

Introduction VII As in the previous volume on NMR, we hope that this collection of chapters by world experts on plant analysis by GCjMS will encourage the further use of this highly sensitive and discriminating technique amongst scientists, students, and industrial analysts working with plant materials. Acknowledgements. The editors express their thanks to all contributors for their efforts in keeping to production schedules, and to Dr. Dieter Czeschlik, Ms. K. G6del and Ms. E. Schuhmacher of Springer publishers for their cooperation with this and other volumes in Modern Methods of Plant Analysis. The constant help of Jose Broekmans is gratefully acknowledged. Nijmegen and Adelaide, May 1986 H. F. LINSKENS J. F. JACKSON

Contents The Use of Combined Gas Chromatography-Mass Spectrometry in the Analysis of Plant Growth Substances P. HEDDEN (With 8 Figures) 1 Introduction...... 2 Identification by GC-MS 2.1 Derivatisation... 2.1.1 Methylation.. 2.1.2 Trimethylsilylation. 2.1.3 Permethylation.. 2.2 Gas Chromatography.. 2.3 Qualitative Mass Spectrometry 2.4 Quantitative Mass Spectrometry 3 Gibberellins......... 3.1 Extraction and Purification 3.2 GC-MS.... 4 Abscisic Acid and Related Compounds 4.1 Qualitative GC-MS. 4.2 Quantitative GC-MS 5 Brassinosteroids References........ 1 1 2 2 2 3 3 4 5 9 10 11 14 16 17 18 19 Applications of Mass Spectrometry for the Examination of Pectic Polysaccharides R. R. SELVENDRAN and B. J. H. STEVENS (With 6 Figures) 1 Introduction................... 23 2 Structural Analysis of Pectic Polysaccharides..... 23 3 Determination of the Nature of the Glycosidic Linkages. 24 4 Separation and Identification of Partially Methylated Alditol Acetates. 27 5 Extensions of Methylation Analysis...... 29 5.1 Controlled Partial Acid Hydrolysis Studies........... 29 5.2 fj-eliminative Degradation Studies.............. 30 5.2.1 Neutral Glycosyl Residues Linked to Galacturonosyl Residues 32 5.2.2 Glycosyl Residues Linked to 0-4 of 2,4-Linked Rhamnopyranosyl Residues.. 6 Sequencing of Sugar Residues in Pectins........ 6.1 Partial Acid Hydrolysis............. 6.1.1 Characterisation of Oligo saccharides as Permethylated Derivatives................... 32 33 33 34

Contents IX 6.2 Partial Acetolysis.................... 37 6.3 Enzymatic Hydrolysis.................. 38 7 Sequencing of Pectic Polysaccharides by Partial Depolymerisation of Permethylated Derivatives. 38 8 Experimental 40 References.......... 43 GC-MS Methods for Cyclic Nucleotides in Higher Plants and for Free High Unsaturated Fatty Acids in Oils B. JANISTYN (With 10 Figures) 1 Introduction..................... 47 2 Methods of GC-MS.................. 48 3 Adenosine-3': 5'-Monophosphate (camp) in Maize Seedlings (Zea mays)...................... 48 4 An Isotope Dilution GC-MS Spectrometric Assay for camp in Cultured Tobacco Tissue............. 51 5 Stability of Cyclic Purine Nucleotides in the Presence of Hydrochloric Acid During Extraction........ 55 6 Guanosine-3': 5'-Monophosphate (cgmp) in Maize Seedlings (Zea mays)...................... 56 7 GC-Separation of Synthetic camp and cgmp in a Mixture. 59 8 Cyclic Pyrimidine Nucleotides in Plants?. 60 9 Free High Unsaturated Fatty Acids in Oils 60 10 Conclusions 64 References................ 65 GC-MS Methods for Lower Plant Glycolipid Fatty Acids H. NYBERG (With 4 Figures) 1 Introduction......... 67 2 Extraction of the Plant Material. 67 2.1 Handling and Storage... 67 2.2 Extraction with Organic Solvents 68 2.3 Purification of the Extract... 69 3 Separation of Glycolipids from the Total Lipid Extract. 70 3.1 Column Chromatography..... 70 3.2 Thin-Layer Chromatography (TLC) 72 3.3 Other Applications..... 73 4 Isolation of the Glycolipids...... 74 4.1 Thin-Layer Chromatography... 74 4.2 Localization of the Glycolipids on TLC. 75 4.3 Removing the Spots from the TLC Plates. 76 5 Derivatization of the Glycolipid Fatty Acids for GC-MS 76 5.1 General Features..... 76 5.2 Formation of Methyl Esters. 77 5.3 Silylation and Other Methods 77

x 6 GLC Instrumentation for Fatty Acid Analysis. 6.1 General Features.. 6.2 Carrier Gas System. 6.3 Injection and Injectors 6.4 The Detector.... 7 Column Selection for Fatty Acid GLC Analysis 7.1 Column Types............ 7.2 Supports, Liquid Phases, and Their Characteristics. 8 Interpretation of GC Data and Calculation of Results. 8.1 Identification of Peaks Using Standard Compounds 8.2 Quantitation of Results............ 8.3 External and Internal Standardization..... 9 Fatty Acid Ester Structure Determination by GC-MS 9.1 Equivalent Chain Lengths (ECL)....... 9.2 Semilogarithmic Correlations......... 9.3 Mass Spectrometers and Their Function Principles 9.4 Interpretation of Mass Spectra of Fatty Acid Esters 10 Abbreviations. References...................... Contents 78 78 79 79 82 83 83 84 87 87 88 89 90 90 91 91 92 94 95 Analysis of Phospholipid Molecular Species by Gas Chromatography and Coupled Gas Chromatography-Mass Spectrometry D. V. LYNCH and G. A. THOMPSON, Jr. (With 6 Figures) 1 Introduction.... 100 2 Lipid Preparation....... 101 2.1 Lipid Extraction...... 101 2.2 Purification of Phospholipids. 102 2.2.1 Column Chromatography. 102 2.2.2 Thin Layer Chromatography 103 3 Formation of Derivatives for GC or GC-MS. 104 3.1 Phospholipase C Treatment...... 104 3.2 Conversion of Diacylglycerols to Silyl Derivatives. 106 3.2.1 Formation of Trimethylsilyl Derivatives... 107 3.2.2 Formation of tert-butyldimethylsilyl Derivatives 107 4 Gas Chromatography. 108 5 Mass Spectrometry... 110 5.1 Instrumentation.. III 5.2 Operating Conditions 111 5.3 Identification of Molecular Species 111 5.4 Quantitation of Molecular Species by GC-MS 113 5.5 Quantitation of Molecular Species by GC-MS Following Reduction of Double Bonds Using Deuterium........ 115 5.6 Direct MS Analysis of Underivatized Phospholipids. 117 6 Determination of Positional Distribution of Acyl Chains Using Phospholipase A2. 117 7 Conclusion 118 References........ 119

Contents GC-MS of Plant Sterol Analysis G. COMBAUT (With 2 Figures) 1 Introduction............... 2 Development of GC-MS Plant Sterol Analysis 3 Operations Before GC-MS Sterol Analysis.. 3.1 Extraction and Isolation of Plant Sterols. 3.2 Free Sterols and (or) Sterols from Steryl-Esters 3.3 Purification of Sterolic Fractions 3.4 Derivatization........... 4 Characterization of Sterols....... 4.1 Characterization of Sterols by GC Data 4.2 Characterization of Sterols by MS Data 4.3 Characterization of Sterols by GC and MS Data 4.3.1 A Typical Analysis of 4-Demethyl and 4,4-Dimethyl Sterols from Zea mays................. 4.3.2 Co-Occurrence of LJ5_ and LJ7-Sterols in Tracheophytes 4.3.3 Side Chain-Hydroxylated Sterols from Red Algae 4.3.4 4-Methyl Sterols of Dinoflagellates. 5 Conclusion References.......... GC-MS Methods for Terpenoids L. WITTE Introduction............. 2 Isolation Methods.......... 3 Prefractionation and Ancillary Reactions 4 Gas Chromatography. 5 Retention Data.. 6 Mass Spectrometry. References.... GC-MS of Auxins L. RIVIER (With 22 Figures) 1 Introduction..... 2 The Compounds Involved 3 Reference Compounds. 4 Extraction... 5 Purification..... 6 Columns for GC.. 7 Injection Techniques. 8 Derivatisation 9 Interface Between GC and MS 10 Mass Spectrometer 11 Data Systems. 12 Ionization.... XI 122 123 124 124 124 125 125 125 125 126 127 127 128 129 130 130 131 134 135 136 138 139 140 142 146 147 148 150 152 153 155 157 161 161 163 163

XII 13 GC-MS Strategy for Auxin Analysis. 14 Quantification.... 15 The Internal Standard. 16 Experimental Procedure 17 Conclusions References........ Contents 165 176 178 182 185 185 GC-MS Methods for the Quantitative Determination and Structural Characterization of Esters of Indole-3-Acetic Acid and myo-inositol R. S. BANDURSKI and A. EHMANN (With 4 Figures) Introduction.......... 189 1.1 Discovery of IAA-Inosito1s........ 189 1.2 Occurrence of IAA Conjugates...... 190 1.3 Importance of Measuring and Identifying Hormone Conjugates. 191 2 Quantitative Analysis and Identification of the IAA-Inosito1s. 191 2.1 Analysis After Hydrolysis.......... 191 2.1.1 Methods for Hydrolysis of IAA Conjugates 192 2.1.2 Use ofinterna1 Standards........ 194 2.2 Analysis Before Hydrolysis.......... 195 2.2.1 A Quantitative Estimation ofiaa-inosito1 Using [3H]-IAAmyo-Inosito1 as an Internal Standard. 195 3 Qualitative Analysis of IAA-Inosito1s..... 195 3.1 The Inositol Moiety........... 195 3.2 Derivitization of IAA-Inosito1s for GC-MS. 196 3.3 Mass Spectral Fragmentation Pattern... 197 3.3.1 I-DL-I-0-(Indo1e-3-Acety1)-myo-Inosito1 (6 TMS, MW 769) 201 3.3.2 2-0-(Indo1e-3-Acety1)-myo-Inosito1 (6 TMS MW 769). 201 3.3.3 Di-O-[N-(Trimethy1si1y1) Indo1e-3-Acety1]-O-Tetra-O- Trimethy1si1y1-myo-Inosito1........... 201 3.3.4 Tri-O-[N-(Trimethy1si1y1) Indo1e-3-Acety1]-O-Tri-O- Trimethy1si1y1-myo-Inosito1........... 206 3.3.5 IAA-myo-Inosito1-Arabinoside and IAA-myo-Inosito1- Galactoside.................. 206 3.4 Uses of GC-MS to Identify and Characterize IAA-Esters. 206 4 Conclusions. 210 5 Abbreviations 211 References... 211 GC-MS Methods for Cytokinins and Metabolites L. M. S. PALNI, S. A. B. TAY, and J. K. MACLEOD (With 8 Figures) 1 Introduction....... 214 2 Gas Chromatography (GC)......... 220 2.1 Instrumentation............ 220 2.1.1 Liquid Stationary Phases and Columns 221 2.1.2 Injectors. 222 2.1.3 Detectors............. 224

Contents XIII 2.2 Derivatisation of Cytokinins..... 225 2.2.1 Trimethylsilyl (TMSi) Derivatives 225 2.2.2 Permethyl Derivatives..... 227 2.2.3 tert.-butyldimethylsilyl (t-budmsi) Derivatives 228 2.2.4 Trifluoroacetyl (TF A) Derivatives 229 2.3 Preparative GC. 230 3 Mass Spectrometry..... 230 3.1 Instrumentation.... 230 3.1.1 Sample Introduction 230 3.1.2 Ionisation Methods 231 3.1.3 Analysers..... 231 3.1.4 Data Systems... 233 3.2 Combined Gas Chromatography-Mass Spectrometry (GC-MS) 233 4 Applications of Mass Spectrometry in Cytokinin Analysis 234 4.1 Structural Studies..... 234 4.2 Quantification of Cytokinins......... 236 4.2.1 Internal Standards........... 237 4.2.2 Stable Isotope Dilution Mass Spectrometry 237 4.2.3 Quantification Using GC-MS 240 4.2.4 Probe Analysis.... 241 4.3 Metabolic Profiling..... 244 5 General Remarks and Conclusion 245 References............. 245 GC-MS Method for Volatile Flavor Components of Foods H. KAMEOKA (With 8 Figures) 1 Introduction.............. 2 GC-MS Methods............ 2.1 Preparation Methods of Flavor Samples 2.2 Operational Methods.. 3 Volatile Flavor Components. 3.1 Fruits... 3.2 Vegetables. 3.3 Mushrooms 3.4 Tea... 3.5 Beans and Nuts 3.6 Grains.... 3.7 Jams.... 3.8 Fermentation Products References......... 254 254 255 256 256 256 263 268 270 271 271 272 273. 274 GC-MS Methods for Tobacco Constituents H. KODAMA (With 20 Figures) Introduction................ 2 Cembranoids and Their Degraded Compounds. 277 277

XIV 3 Labdanoids and Their Degraded Compounds 4 Carotenoid-Degraded Compounds 5 Sesquiterpenoids... 6 Terpenoid Glycosides. 7 Linked Scanning References....... Subject Index Contents. 279 280 289 291 294 298 299

List of Contributors BANDURSKI, ROBERT S., Michigan State University, Botany & Plant Pathology Department, East Lansing, MI 48824-1312, USA COMBAUT, GEORGES, Laboratoire de Biologie Vegetale, Universite, Avenue de Villeneuve, F-66025 Perpignan Cedex, France EHMANN, AxEL, Shell Agricultural Chemical Company, P.O. Box 4248, Modesto, CA 95352, USA HEDDEN, PETER, Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS18 9AF, United Kingdom JANISTYN, BORIS, GresserstraBe 8, D-7800 Freiburg, FRG KAMEOKA, HIROMU, Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashiosaka-shi, Osaka 577, Japan KODAMA, HISASHI, Central Research Institute, Japan Tobacco Inc., 6-2 Umegaoka, Midoriku, Yokohama, Kanagawa 227, Japan LYNCH, DANIEL V., Agronomy Department, Cornell University, Ithaca, NY 14853, USA MACLEOD, JOHN KEITH, Research School of Chemistry, Australian National University, G.P.O. Box 1, Canberra, A.C.T. 2601, Australia NYBERG, HARRI, Department of Botany, University of Helsinki, Unioninkatu 44, SF -00170 Helsinki, Finland PALNI, LOK MAN SINGH, Department of Developmental Biology, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra City, A.C.T. 2601, Australia RIVIER, LAURENT, Institut universitaire de Medecine legale, Laboratoire de Toxicologie analytique, CH-I005 Lausanne, Switzerland SELVENDRAN, R.R., Chemistry and Biochemistry Division, AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA, United Kingdom STEVENS, B.J.H., Chemistry and Biochemistry Division, AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, NR4 7UA, United Kingdom

XVI List of Contributors TAY, STEPHEN AlI BOON, Regional Laboratory, Australian Government Analytical Laboratories, P.O. Box 385, Pymble, NSW 2073, Australia THOMPSON, JR., GUY ALLEN, Department of Botany, University of Texas, Austin, TX 78713, USA WITTE, LUDGER, Gesellschaft fur Biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig, FRG