Chromatography of Mycotoxins

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JOURNAL OF CHROMATOGRAPHY LIBRARY- volume 54

chromatography of mycotoxins techniques and applications

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JOURNAL OF CHROMATOGRAPHY LIBRARY- volume 54

chromatography of mycotoxins techniques and applications edited by

Vladimir Betina Department of Microbiology, Biochemistry and Biology, Slovak Technical University, Bratislava, Slovakia

ELSEVIER Amsterdam -London-New York -Tokyo

1993

ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O. Box 21 1,1000AE Amsterdam,The Netherlands

ISBN 0-444-81521-X

0 1993 Elsevier Science Publishers B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science Publishers B.V., Copyright & Permissions Department, P.O. Box 521,1000AM Amsterdam,The Netherlands. Special regulations for readers in the U.S.A. - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the U.S.A. All other copyright questions, including photocopying outside of the U.S.A., should be referred to the copyright owner, Elsevier Science Publishers B.V., unless otherwise specified. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper. Printed in the Netherlands

V

CONTENTS

..................................... ..................................................

List of contributors

xii

Preface

xiii

Part A . Techniques Chapter 1 . Sampling. sample preparation. extraction and clean-up V Betina 1.1 Introduction 1 . 2 Sampling and sample preparation 1 . 3 Sample extraction and clean-up 1 . 4 Illustrative example 1.5 Conclusions References

.

........................................ ............................................ ......................... .......................... .................................... ............................................. ..................................................

Chapter 2 . Techniques of thin layer chromatography R.D. Coker. A.E. John and J.A. Gibbs 2 . 1 Introduction 2.2 Clean-up methods 2 . 3 Normal phase TLC 2 . 3 . 1 Principles 2 . 3 . 2 Practical considerations

3

3 3

4

7 9 9

............. 1 2 ............................................ 12 ........................................ 12 ........................................ 16 ........................................ 16 .......................... 17 2 . 4 Reverse-phase TLC (RPTLC) ............................... 20 2 . 4 . 1 Principles ........................................ 20 20 2 . 4 . 2 Practical considerations .......................... 2 . 5 High performance thin layer chromatography (HPTLC) ...... 2 1 21 2 . 5 . 1 Principles ........................................ 22 2 . 5 . 2 Practical considerations .......................... 2 . 6 Preparative TLC ......................................... 23 2 . 6 . 1 Principles ........................................ 23 Practical considerations .......................... 25 2.6.2 27 2 . 7 Detection ............................................... 27 2 . 7 . 1 Fluorescence detection ............................ 21 2 . 7 . 2 Chemical derivatisation ........................... 2 . 7 . 3 Bioautographic methods ............................ 28 2 . 8 Quantitative and semi-quantitative evaluation ........... 2 8 2 . 9 Illustrative examples ................................... 30 2 . 1 0 Conclusions ............................................ 31 References .................................................. 32

vi

. Techniques of liquid column chromatography P. Kuronen ........................................ Introduction ............................................. Sample pretreatment ...................................... Column chromatography .................................... 3 . 3 . 1 Introduction .......................................

Chapter 3 3.1 3.2 3.3

..........................................

3.3.2

Procedure 3 . 4 Mini-column chromatography 3.4.1 Procedure 3.4.2 Illustrative example 3.5 High-performance liquid chromatography 3 . 5 . 1 Introduction Instrumentation and practice 3.5.2 3.6 Conclusion References

...............................

..........................................

36 36 37 40 40 40 44 45

............................... 46 ................... 46

....................................... ....................... ...............................................

...................................................

Chapter 4 . Techniques of gas chromatography R.W. Beaver 4 . 1 Introduction 4.2 Resolution in gas chromatography 4 . 2 . 1 Definition of resolution 4.2.2 Efficiency 4.2.3 Retention 4.2.4 Selectivity 4.3 Extracolumn resolution 4 . 3 . 1 Resolution through sample clean-up 4.3.2 Chemical derivatization 4.3.3 Resolution through detection 4.4 Conclusions References

....................................... ............................................. ......................... ........................... .........................................

46 48 71 72

78 78 79 79

79

.......................................... 83 ........................................ 84 ................................... 8 6 ................. 8 7 ............................

....................... .............................................. ...................................................

Chapter 5

. Emerging

91 91 96 96

techniques: immunoaffinity chromatography A . A . G . Candlish and W.H. Stimson 99 99 5 . 1 Introduction 101 5 . 2 Immunoaffinity chromatography theory 5 . 3 Practical aspects and instrumentation 103 111 5 . 4 Sample preparation 5.5 Illustrative examples 116 References 122

.................. ............................................. .................... ................... ...................................... ...................................

..................................................

vii

Chapter 6 . Emerging techniques: enzyme-linked immunosorbent assay (ELISA) as alternatives to chromatographic methods C.M. Ward. A.P. Wilkinson and M.R.A. Morgan ..... 6.1 Introduction 6.2 Principles of ELISA 6.2.1 The immune response and polyclonal antibodies 6.2.2 Monoclonal antibodies 6.2.3 Haptens 6.2.4 Specificity of anti-hapten antibodies 6.2.5 Principles of immunoassay 6.2.6 Enzyme immunoassays 6.2.7 Enzyme-linked immunosorbent assays (ELISAS)

........................................... ....................................

............................

....

.......................................... ............ ........................ .............................. ....... preparation .....................................

6.3 Sample 6.3.1 Extraction 6.4 Instrumentation and practice 6.4.1 Instrumentation 6.4.2 Practice 6.5 Illustrative examples 6.5.1 Aflatoxins 6.5.2 Other mycotoxins 6.6 Conclusions References

....................................... ........................... .................................. .........................................

.................................. ....................................... ................................. ............................................ .................................................

124 124 124 124 125 125 125 126 126 127 127 127 128 128 129 132 132 133 134 135

Part B . Applications Chapter 7 . Thin-layer chromatography of mycotoxins V Betina

.

7.1 7.2

7.3

....................................... Introduction ........................................... Aflatoxins ............................................. 7.2.1 Sampling and sample preparation .................. 7.2.2 Extraction and clean-up .......................... 7.2.3 Adsorbents and solvent systems ................... 7.2.4 Detection ........................................ 7.2.5 Selected applications ............................ Sterigmatocystin and related compounds ................. 7.3.1 Extraction and clean-up .......................... 7.3.2 Adsorbents and solvent systems ................... 7.3.3 Detection ........................................

............................ .........................................

1.3.4 Selected applications 7.4 Trichothecenes 7.4.1 Extraction and clean-up

..........................

141 141 143 143 144 149 152 153 162 162 164 166 166 168 169

viii

................... ........................................ ............................ ......................................... .......................................... .................................. ................................ ....................................... .................................... ................................... ...................................... .................................... ............................................ .......................... ................... ........................................ ............................ ............................................ .......................... ................... ........................................

7.4.2 Adsorbents and solvent systems 7.4.3 Detection 7.4.4 Selected applications 7.5 Small lactones 7.5.1 Patulin 7.5.2 Penicillic acid 7.5.3 Mycophenolic acid 7.5.4 Butenolide 7.5.5 Citreoviridin 7.6 Macrocyclic lactones 7.6.1 Zearalenone 7.6.2 Cytochalasans 7.7 Ochratoxins 7.7.1 Extraction and clean-up 7.7.2 Adsorbents and solvent systems 7.7.3 Detection 7.7.4 Selected applications 7.8 Rubratoxins 7.8.1 Extraction and clean-up 7.8.2 Adsorbents and solvent systems 7.8.3 Detection 7.8.4 Selected applications 7.9 Hydroxyanthraquinones .................................. 7.9.1 Extraction 7.9.2 Adsorbents and solvent systems 7.9.3 Detection 7.9.4 Selected applications 7.10 Epipolythiopiperazine-3. 6.diones 7.10.1 Extraction and clean-up 7.10.2 Adsorbents. solvent systems and detection 7.10.3 Selected applications 7.11 Tremorgenic mycotoxins 7.11.1 Adsorbents and solvent systems 7.11.2 Detection 7.11.3 Selected applications 7.12 Alternaria toxins ..................................... 7.12.1 Extraction and clean-up 7.12.2 Adsorbents and solvent systems 7.12.3 Detection 7.12.4 Selected applications

........................................ ............................ ...................... ......................... ....... ........................... ................................ .................. .......................................

169 170 173 178 178 181 183 184 184 186 186 191 196 196 196 197 198 199 199 199 199 200 200 201 201 201 201 203 203 204 204 205 205 205

......................... .................. ....................................... ...........................

207 209 209 209 210 210

............................

....................................... ...................

...........................

ix

............................................. ........................ ................. ...................................... .......................... ................................ ........................ ................. ...................................... ..........................

Citrinin 7 . 1 3 . 1 Extraction and clean-up 7 . 1 3 . 2 Adsorbents and solvent systems 7 . 1 3 . 3 Detection 7 . 1 3 . 4 Selected applications 7.14 a-Cyclopiazonic acid 7 . 1 4 . 1 Extraction and clean-up 7 . 1 4 . 2 Adsorbents and solvent systems 7 . 1 4 . 3 Detection 7 . 1 4 . 4 Selected applications 7 . 1 5 PR toxin and roquefortine 7 . 1 5 . 1 Extraction and clean-up 7 . 1 5 . 2 Adsorbents and solvent systems 7 . 1 5 . 3 Detection 7 . 1 6 Xanthomegnin. viomellein and vioxanthin 7 . 1 6 . 1 Extraction and clean-up 7 . 1 6 . 2 Adsorbents and solvent systems 7.16.3 Detection 7 . 1 6 . 4 Selected applications 7 . 1 7 Naphtho- y -pyrones 7 . 1 8 Secalonic acids 7 . 1 9 TLC of miscellaneous toxins 7 . 2 0 Multi-mycotoxin TLC 7 . 2 1 TLC in chemotaxonomic studies of toxigenic fungi 7 . 2 2 Conclusions References 7.13

............................

210 211 212 212 213 214 214 215 215 216 217

........................

217

...................................

219

................. 2 1 7 ...................................... 2 1 7 .............. 2 1 8 ........................ 218 ................. 2 1 8 ...................................... 218 .......................... 218

...................................... 219 .......................... 220 .................................. 222 ..... 2 3 0 .......................................... 231 ................................................ 233

Chapter 8 . Liquid column chromatography of mycotoxins J.C. Frisvad and U Thrane 8 . 1 Introduction 8 . 2 Column chromatography 8.3 Mini-column chromatography 8 . 4 High performance liquid chromatography 8.4.1 8.4.2 8.4.3 8.4.4 8.4.5 8.4.6 8.4.7 8.4.8

. ..................... .......................................... ................................. ............................ ................ Aflatoxins ...................................... Sterigmatocystin and related compounds .......... Trichothecenes .................................. Small lactones .................................. Macrocyclic lactones ............................ Ochratoxins and related compounds ............... ..................................... .............

Rubratoxins Hydroxyanthraquinones and xanthones

253 253 287 289 290 290 295 296 299 302 303 306 307

X

................

8.4.9 Epipolythiopiperazine.3. 6.diones 8.4.10 Tremorgenic mycotoxins 8.4.11 Alternaria toxins 8.4.12 Toxic peptides 8.4.13 Fusarium toxins other than trichothecenes and zearalenones 8.4.14 Miscellaneous toxins 8.4.15 Multi-mycotoxin analyses by HPLC 8.5 Informative on-line detection methods 8.5.1 Applications of HPLC diode array detection 8.5.2 Applications of HPLC mass spectrometry 8.6 Conclusions References

......................... .............................. .................................

............................... ........................... ............... .................

......

.......... ........................................... ................................................

Chapter 9

.

Gas chromatography of mycotoxins P.M. Scott 9.1 Introduction 9.2 Trichothecenes 9.2.1 Introduction 9.2.2 Derivatization and detection procedures for trichothecenes 9.2.3 Methods for grains. grain foods and feeds 9.2.4 Methods for biological fluids 9.2.5 Methods for animal tissues 9.2.6 Methods for other foods 9.2.1 Additional applications 9.3 Zearalenone 9.3.1 Derivatization and detection procedures for

..................................... .......................................... ........................................ ....................................

308 309 312 314 315 311 319 321 321 354 355 356

313 313 373 313

.............................. 314 ....... 382 ................... 381 ...................... 389

......................... .........................

...........................................

............. ....... .............................. Moniliformin .......................................... Alternaria toxins ..................................... zearalenone and related metabolites 9.3.2 Methods for grains. grain foods and feeds 9.3.3 Other applications

9.4 9.5

9.5.1 Alternariol. alternariol monomethyl ether. altenuene and isoaltenuene 9.5.2 Tenuazonic acids 9.6 Slaframine and swainsonine 9.1 Patulin 9.7.1 Comparison of derivatization and detection procedures for patulin

......................

389 391 392 392 393 395 395 396

............................

...............................................

396 391 398 399

..........................

399

................................

xi

9.7.2 Methods for apple juice and other fruit products 9.7.3 Methods for other foodstuffs 9.8 Penicillic acid 9.8.1 Derivatization and detection procedures for penicillic acid 9.8.2 Methods for agricultural commodities 9.9 Sterigmatocystin 9.9.1 Comparison of detection procedures 9.9.2 Methods for grains 9.9.3 Dihydrosterigmatocystin 9.10 Aflatoxins 9.11 Ergot alkaloids 9.12 Miscellaneous mycotoxins 9.12.1 Sporidesmins 9.12.2 Butenolide 9.12.3 F-Nitropropionic acid 9.12.4 Fumonisins 9.12.5 Fusarin C 9.12.6 Griseofulvin and related compounds 9.12.7 Mycophenolic acid 9.12.8 Kojic acid, terreic acid and terrein 9.12.9 Oxalic acid 9.12.10 "Peptaibol" polypeptide antibiotics 9.12.11 Ochratoxin A 9.12.12 d-Cyclopiazonic acid 9.12.13 Loline alkaloids 9.12.14 Fusarochromanone 9.13 Conclusions References

........................................ .................... .......................................

............................. ............ ...................................... .............. .............................. ......................... ........................................... ...................................... ............................. ................................... ..................................... ......................... ..................................... ...................................... .............

.............................. ........... .................................... ........... .................................. .........................

Subject

.............................. .............................. .......................................... ................................................ index .............................................

400 401 401 401 403 404 404 404 405 405 406 408 408 409 409 409 410 411 411 411 412 412 412 412 413 413 414 414 427

xii

LIST OF CONTRIBUTORS R.W.

BEAVER

V. BETINA

A.A.G.

CANDLISH

R.D. COKER

J.C. FRISVAD

J.A. GIBBS

A.E. JOHN

P. KURONEN

M.R.A. MORGAN

P.M. SCOTT

W.H. STIMSON U. THRANE

C.M. WARD

A.P. WILKINSON

Coastal Plain Station, Department of Plant Pathology, College of Agriculture, The University of Georgia, Tifton, Georgia 31793, USA Department of Microbiology, Biochemistry and Biology, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava, Slovakia RhBne Poulenc Diagnostics Ltd., Montrose House, 187 George Street, Glasgow G1 lYT, Scotland Natural Resources Institute, Central Avenue, Chatham Maritime, Chatham, Kent ME4 4TB, United Kingdom Department of Biotechnology, The Technical University of Denmark, Bygning 221, DK-2800 Lyngby, Denmark Natural Resources Institute, Central Avenue, Chatham Maritime, Chatham, Kent ME4 4TB, United Kingdom Natural Resources Institute, Central Avenue, Chatham Maritime, Chatham, Kent ME4 4TB? United Kingdom Department of Chemistry, University of Helsinki, Vuorikatu 2 0 , SF-00100 Helsinki, Finland AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Sir F.G. Banting Research Centre, Health Protection Branch, Health and Welfare Canada, Tunney-s Pasture, Ottawa, Ontario K1A OL2 Canada Rhane Poulenc Diagnostics Ltd., Montrose House, 187 George Street, Glasgow G1 lYT, Scotland Department of Biotechnology, The Technical University of Denmark, Bygning 221, DK-2800 Lyngby, Denmark AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom

...

Xlll

PREFACE Instruct a wise man and he will be wiser still. Proverbs, 9,9

The idea of this book has gradually developed during my of the Journal of membership of the editorial board Chromatography when I had to referee manuscripts dealing with chromatographic studies of mycotoxins. Another inspiration originated in reflections on a previous book on methods of production, isolation, separation and purification of mycotoxins which I edited in the early 1980s and which has been accepted very positively by many workers in the field. However, without a positive attitude of the Publishers and ethusiastic cooperation of the invited expert scientists the original idea would not have been transformed into reality. The book consists of two parts. In four chapters on Techniques, the most important principles of sample preparation, extraction, clean-up, and of established and prospective chromatographic techniques are discussed in relation to mycotoxins. Two shorter chapters deal with emerging techniques: immunoaffinity chromatography and enzyme-linked immunosorbent assay as alternative to chromatographic methods. In the Applications, the most important data, scattered in the literature, on thin-layer, liquid, and gas chromatography of mycotoxins have been brought together. Mycotoxins are mostly arranged according to families, such as aflatoxins, trichothecenes, lactones etc. Chromatography of individual important mycotoxins and multi-mycotoxin chromatographic analyses are also included. Applications are presented in three chapters devoted to thin-layer, liquid, and gas chromatography. Last but not least, I express my thanks to all the contributors for their excellent cooperation in preparing their manuscripts so that the book might become useful to the researchers who will use it. V. Betina

Editor


PART A

TECHNIQUES


3

Chapter 1 SAMPLING, SAMPLE PREPARATION, EXTRACTION AND CLEAN-UP

V. BETINA 1.1 INTRODUCTION The chromatography of mycotoxins is preceded by a sequence of operations which include sampling, sample preparation, extraction and clean-up. The results of the most sophisticated chromatographic procedure will be determined by the efficacy of these steps. Analytical methods must be extremely sensitive but each natural material which is expected to contain mycotoxins is composed of compounds which may interfere with the analysis, and thus specific methods must be used for a certain class of toxins present in a particular commodity. This chapter will focus upon a description of the sampling, sample preparation, extraction and clean-up procedures associated with chromatography of mycotoxins. 1.2 SAMPLING AND SAMPLE PREPARATION For the analysis of agricultural commodities good sampling techniques are of importance because the contamination of food products such as grain or nuts is most likely to occur in isolated "pockets" of mycotoxins (1). This may be due to mould proliferation and contamination of a few plants suffering from the stress of unfavourable conditions in a small portion of the field. Alternately, "pockets" of toxin may develop during storage of a larger quantity of a commodity because of localized conditions such as isolated areas of high moisture. Sampling, sample preparation, and sampling plans for foodstuffs for mycotoxin analysis have been published by Campbell et al. ( 2 ) , Dickens and Whitaker ( 3 ) and, more recently, by Park and Pohland ( 4 ) . These reviews provide lists of various types of equipment used for sample preparation and sources of supply. Sampling and subsampling procedures recommended for aflatoxins should be adequate for other mycotoxins (5). The problem of sampling is associated with the fact stressed above: there is not a normal distribution of

4

aflatoxins or other toxins within one batch. In general, more heterogeneous samples or food with larger particles require larger sample sizes. Thus, peanuts require a relatively large sample size, whereas progressively smaller sample sizes are needed for corn, wheat, rice, and millet products ( 6 ) . According to Davis et al. (l), a study investigating the presence of mycotoxins in crops in a field requires the geometric division of the field and the acquisition of representative samples from each sector. With a commodity such as corn, the sampling must be coordinated with harvesting so as to obtain kernels from a large number of ears. Sampling of stored crops with probes will only result in representative samples in the lot has been mixed by harvesting or some other mechanical operation. The increments taken from the lot should be mixed and the entire sample ground to reduce particle size and heterogeneity. Chapter 2 6 of the 14th edition of Official Methods of Analysis of the Association of Official Analytical Chemists (7) recommends grinding nuts in a large batch- type cutter to simultaneously mix and reduce particle size to a point at which the ground sample will pass through a No. 14 sieve. The subsample is obtained by systematically dividing the gross sample or utilizing a riffling device. The subsample is then more finely ground so that the particles will pass a No. 2 0 sieve. Analytical samples can then be withdrawn from this more representative subset by randomly dividing the subsample. In order to minimize the formation of mycotoxins during sampling, the analysis of the sample should be performed as rapidly as possible following collection. Warm, moist storage conditions should be avoided to prevent further mycotoxin production. Subsamples should be stored under refrigerated or dried conditions for future analysis. 1.3 SAMPLE EXTRACTION AND CLEAN-UP A variety of extraction and clean-up methods for mycotoxins have been employed. Since mycotoxins occur in a wide variety of commodities and products, the extraction from a sample depends on the physicochemical properties of the sample as well as those of the toxin. In general, the sample or ground sample is subjected to high-speed blending or mechanical shaking in the presence of the extraction solvent system. The slurry is then

5

filtered and is ready for subsequent purification procedures. Diatomaceous earth is sometimes included in the solvent system to speed the filtration step. The most efficient solvents for extracting mycotoxins are the relatively polar solvents such as methanol, acetone, acetonitrile, ethyl acetate, and chloroform. Modern techniques of mycotoxin extraction use water-organic solvent mixtures, e.g., chloroform- water (1O:l) (8). The water wets the substrate and increases penetration of the solvent mixture into the hydrophilic material. The aqueous phase can be an acid solution designed to break interactions between the toxins and sample constituents such as proteins. The small amount of the toxins taken up in the aqueous phase is immediately removed by the organic solvent, giving a rapid isolation procedure. Sodium chloride or other inorganic salts are often included in the aqueous phase to minimize the formation of emulsions during the extraction. The best known extraction and clean-up techniques, as published before 1985, were summarized by the present author (9). Examples of solvent systems utilized in the more recent literature for isolating a variety of mycotoxins are presented in Table 1.1. Extraction procedures employed for extraction of structurally- related families or individual mycotoxins are described in Chapter 7. As a large number of interfering compounds originally present in samples contaminate the primary sample extracts, these components must be removed as completely as possible. For this purpose, a variety of clean-up methods have been used. High levels of additional compounds ca be removed in several ways. For example, high levels of lipids present in certain commodities (cocoa beans, peanuts, peanut butter) would interfere with subsequent analytical procedures. For these foods, nonpolar solvents such as hexane can be included in the original solvent system (25), or they can be added after the homogenization and filtration steps to remove lipid constituents. Primary extracts in mixtures of acetone with water contain proteins that can be precipitated with lead acetate. Sometimes various pigments need to be removed from primary extracts. Scott ( 2 6 ) showed that theobromine could be removed from crude cocoa-bean extracts by treatment with silver nitrate

6

solution. It was also shown by the same author ( 2 7 ) that a coffee-bean extract could be purified by passage through a Florisil column and the unwanted contaminants eluted with tetrahydrofuran.

Aflatoxin B1 Aflatoxin M1 Aflatoxins

Deoxynivalenol Fusarochromanones Gliotoxin Nivalenol and deoxynivalenol Trichothecenes

Compounded feeds Cheese Peanuts, pistachio nuts, soya milk Feeds Maize Moist wheat or rice Agricultural commodities Barley, wheat, fusarium culture on rice Rice culture Cerea1s

Cereals and cereal products Culture filtrate Scirpentriol Sterigmatocystin Mouldy rice Culture filtrate Zearalenone Fermented corn Zearalenone and zearalenols

10 Chloroform, methanolwater, acetonitrile-water Acetone-water (86:14) 11 12

Chloroform Acetone-water ( 8 0 : 2 0 ) Chloroform

13

Acetonitrile-water

16

14 15

(85:15)

Methanol

17

Chloroform Acetonitrile-water

18

19

(85:15)

Organic solvents

20

Ethyl acetate Ethyl acetate Chloroform Acetone

21 22 23

24

In addition to these preliminary clean-up procedures, other clean-up methods include column chromatography, liquid-liquid

7

extraction and commercially available solid-phase extraction (SPE) and chromatography cartridges. The sample extract is usually added to the cartridge in an appropriate solvent. The cartridge is then washed with one or more solvents in which the toxins are insoluble or less soluble than the impurities. The solvent composition is subsequently changed in such a way that the toxins are selectively eluted from the cartridge, and the eluate is collected. SPE techniques are increasingly utilized for the analysis of mycotoxins. Since these types of clean-up methods are sufficiently characterized in Chapter 2, they are not described here. Applications of clean-up procedures were reviewed recently (28) and are also included in Chapter 7 dealing with TLC of mycotoxins. Some more recent examples of clean-up techniques are presented in Table 1.2. The final step prior to analysis of the sample involves concentration of the cleaned-up extract. This is performed using a rotary evaporator operating under reduced pressure and slightly elevated temperature. After concentrating the extract to dryness a small volume of a solvent compatible with the subsequent chromatographic system is used to rinse out the flask and the final volume is adjusted with a gentle stream of nitrogen. The sample can also be placed in a steam bath under a stream of nitrogen for concentration of the sample. 1.4 ILLUSTRATIVE EXAMPLE

Extraction and clean-up of ochratoxin A can be described here as an example. Ochratoxin A present in acidified commomodities is readily soluble in many organic solvents. This charcteristic has been used in several methods. Egan et al., (37) extracted ochratoxin A from ground samples with chloroform, after acidification with aqueous phosphoric acid. Chloroform has also been used to extract the toxin from pig kidney (38), milk (39), and human plasma (40). When extracts of ochratoxin A are purified by immunoaffinity chromatography, methanol (41) or acetonitrile (42) are used. The usual next step is partial purification of the extract to remove lipids and other substances. This step can be sometimes ommited (43). In the method (37) ochratoxins are trapped in a laboratory prepared column containing diatomaceous

8

earth impregnated with sodium bicarbonate solution. Extraneous substances are washed off the column with hexane and chloroform, and the ochratoxins are eluted with benzene-acetic acid ( 9 8 : 2 ) . TABLE 1.2 Examples of clean-up techniques for mycotoxins

Aflatoxin B1

Phenyl non-polar bonded-phase 29 Reversed-phase disposable cartridges 10 Aflatoxins Silica gel column 12 Extract in aqueous methanol defatted 30 with hexane, toxins partitioned into chloroform, placed on silica gel column, eluted with chloroform-acetone Silica gel 60 column eluted with 31 chloroform-methanol ( 8 : 2 ) Sep-Pak Florisil and C18 cartridge 13 CB method 32 Alternaria toxins Liquid-liquid partition and 33 column chromatography Deoxynivalenol 16 Acetonitrile-water extract partially purified on a preparative minicolumn Fusarochromanones Thin-layer or column chromatography 17 18 Precipitation with petroleum ether and Gliotoxin gel permeation chromatography T-2 toxin C18 and silica gel column 34 Trichothecenes Precipitation of proteins with lead 20 acetate, purification of toxins with hexane and chloroform 35 Silica gel minicolumn Zearalenone Zearalenone and Silica gel column and elution with 24 hexane-ethyl acetate ( 8 : 2 ) zearalenols Multimycotoxin Gel permeation chromatography 36 (aflatoxin, ochratoxin and zearalenone)

* CB

=

Contamination Bureau.

9

In the procedure (38) for the determination of ochratoxin A in kidneys of swine, a liquid-liquid partitioning step is used instead. Most recently, first commercial prototypes of immunoaffinity cartridges for ochratoxin A have become available (44). These columns are composed of monoclonal antibodies specific for ochratoxin A and immobilized on Sepharose and packed into small plastic cartridges. The crude extract is forced through the column and ochratoxins are left bound to the immunoglobulin. Extraneous material is washed off the column with water or aqueous buffer, and the ochratoxins are finally eluted with acetunitrile. 1.5 CONCLUSIONS This short chapter was written with the aim to show the necessary operations which usually must precede analytical or preparative chromatography of mycotoxins: sampling, sample preparation, extraction, and clean-up procedures. Examples taken from recent literature concerning extraction were included to show the variety of materials in which the presence of mycotoxins has to be proved or disproved chromatographically. Some recent clean-up techniques were also described.

REFERENCES 1 N.D. Davis, J.W. Dickens, R.L. Freie, P.B. Hamilton, O.L. Shotwell, T.D. Wyllie and J.F. Fulkerson, J. Assoc. Off. Anal. Chem., 70 (1980) 95. 2 A.D. Campbell, T.B. Whitaker, A.E. Pohland, J.W. Dickens and D.L. Park, Pure Appl. Chem., 58 (1986) 305. 3 J.W. Dickens and T.B. Whitaker, in H. Egan, L. Stoloff, P. Scott, M. Castegnaro, I.K. O'Neil and H. Bartsch (Editors), Environmental Carcinogens - Selected Methods of Analysis. Vol. 5 : Some Mycotoxins. ARC, Lyon, 1982, p. 17. 4 D.L. Park and A.E. Pohland, J. Assoc. Off. Anal. Chem., 72 (1989) 399. 5 J.W. Dickens and T.B. Whitaker, in R.J. Cole (Editor), Modern 6 7 8 9 10

Methods in the Analysis and Structural Elucidation of Mycotoxins, Academic Press, New York, 1986, Ch. 2, p. 29. J.E. Smith and M.O. Moss, in Mycotoxins: Formation, Analysis and Significance, Wiley, New York, 1985, p. 104. Official Methods of Analysis of the Association of Official Analytical Chemists, AOAC, Arlington, VA, 14th. ed., 1984, Ch. 26. P.M. Scott, Adv. Thin Layer Chromatogr. (Proc. 2nd Bienn. Symp. 1980), 1982, p. 321. V. Betina, J. Chromatogr., 334 (1985) 211. H.P. van Egmond and P.J. Wagstaffe, Food Addit. Contamin., 7 (1990) 239.

LO 11 J.P. Bijl and C.H. van Peterghem, J. Assoc. Off. Anal. Chem., 70 (1987) 472. 12 R. Biffoli, F. Chiti and G. Modi, Riv. SOC. Ital. Sci. Aliment., 8 (1990) 19. 13 H.P. van Egmond, S.H. Heisterkamp, W.E. Paulsch and H.P. van Egmond, Food Addit. Contamin., 8 (1991) 17. 14 N. Bradburn, R.D. Coker, K. Jewers and K.I. Tomlins, Chromatographia, 29 (1990) 435. 15 M.A. Moreno, A. Olivares and G. Suarez, Mycotoxin Res., 5 (1989) 51. 16 W.C. Gordon and L.J. Gordon, J. Assoc. Off. Anal. Chem., 73 (1990) 266. 17 F.S. Chu, J. Assoc. Off. Anal. Chem., 74 (1991) 655. 18 J.L. Richard, R.L. Lyon, R.E. Fichtner and P.F. ROSS, Mycopathologia, 107 (1989) 145. 19 D.R. Lauren and R. Greenhalgh, J. Assoc. Off. Anal. Chem., 70 (1987) 479. 20 A.N. Kotik and V.A. Trufanova, Gig. Sanit., 1989, No. 9, 53. 21 K.E. Richardson, G.E. Toney, C.A. Haney and P.B. Hamilton, J. Food Protect., 52 (1989) 871. 22 Y. Horie, M. Miyaji, K. Nishimura, H. Toguchi, H. Yamaguchi and S. Udagawa, Proc. Jpn. Assoc. Mycotoxicol., 1989, No. 29, 21. 23 I.A. El-Kady, A.H. Moubasher and S.S.M. El-Maraghy, Egypt. J. Bot., 31 (1988) 99. 24 R. Vesonder and P. Golinski, Mycotoxin Res., 7A (Suppl.), Part I1 (1991) 175. 25 L. Stoloff, J. Assoc. Off. Anal. Chem., 66 (1983) 355. 26 P.M. Scott, J. Assoc. Off. Anal. Chem., 52 (1969) 72. 27 P.M. Scott, J. Assoc. Off. Anal. Chem., 51 (1968) 609. 28 v. Betina, J. Chromatogr., 477 (1989) 187. 29 N. Bradburn, R.D. Coker and R. Jewers, Chromatographia, 29 (1990) 177.. 30 J. Wu, J. Toxicol. Toxin Revs., 9 (1990) 120. 31 0. Sanchey, Food Lab. News, 1989, No. 17, 49. 32 R. W. Beaver, D.M. Wilson and M.W. Trucksess, J. Assoc. Off. Anal. Chem., 73 (1990) 579. 33 M. Kostecki, J. Grabarkiewicz-Szczesna and J. Chelkowski, Mycotoxin Res., 7 (1991) 3. 34 K.A. Koddington, S.P. Swanson, A.S. Hassan and W.B. Buck, Drug Metab. Disposit., 17 (1989) 600. 35 P. Lepom, Arch. Anim. Nutrit., 38 (1988) 799. 36 C. Dunne, M. Meaney and M. Smyth, J. Chromatogr., (In press). 37 H. Egan, L. Stoloff, M. Castegnaro, P. Scott, I.K. O'Neill

38

39 40 41

and €IBartsch . (Editors, Environmental Carcinogens: Selected Methods of Analysis, Vol. 5, Some Mycotoxins, IARC, Lyon, 1982, p. 255. W.E. Paulsch, H.P. van Egmond and P.L. Schuller, in Proceedings, V International IUPAC Symposium on Mycotoxins and Phycotoxins, September 1-3, 1982, Vienna, Austrian Chemical Society, Vienna, 1982, p. 40. M. Gareis, E. Martlbauer, J. Bauer and B. Gedek, Z . Lebens. Unters. Forsch., 186 (1988) 114. A. Breitholtz, M. Olsen, A. Dahlback and K. Hult, Food Addit. Contam., 8 (1991) 183. S.C. Lee and F.S. Chu, J. Assoc. Off. Anal. Chem., 6 7 (1984)

45. 42 N. Ramakrishna, J. Lacey, A.A. Candlish, J.E. Smith and I.A. Goddbrand, J. Assoc. Off. Anal. Chem., 73 (1990) 71. 43 K. Hult, R. Fuchs, M. Peraica, R. PleStina and S . Ceovic, J.

11

Appl: Toxicol., 4 (1984) 326. 44 J. Gilbert, in Proceedings of the International Conference on Fungi and Mycotoxins in Stored Products, Bangkok, Thailand, 23-26 April 1991 (in press).

12

Chapter 2 TECHNIQUES OF THIN LAYER CHROMATOGRAPHY R.D. COKER, A.E. JOHN and J.A. GIBBS

2.1 INTRODUCTION The use of horizontal thin layers as analytical tools was first described, in 1938, by the Russian workers Ismailov and The technique, known as "drop chromatography", Shraiber 1 1 1 . was largely ignored for the following 1 0 years until two American workers, Meinhard and Hall [2l, described the separation of metal ions in aqueous solution using microscope slides coated with an alumina-rich mixture. Of the several separations mentioned, the first and most simple was for aqueous solutions of Fe3+ and Zn2+ ions. The introduction of the thinlayer technique, as a routine analytical method, is generally attributed to the work of Kirchner and his associates in 1951 [3-51. Subsequently, thin layer chromatography (TLC) has been utilised for the separation and quantification of a wide range of compounds, including mycotoxins. The analysis of mycotoxins involves a sequence of discrete operations which includes sampling, sample preparation, extraction, clean-up, quantification and confirmation procedures [6,7]. Needless to say, the validity of the TLC quantification results will be determined by the efficacy of the sampling, sample preparation, extraction and clean-up steps [6-81. This chapter will focus upon a description of the clean-up and quantification procedures associated with thin layer chromatography. 2.2 CLEAN-UP METHODS Since mycotoxins occur in a wide variety of commodities and products, the analyst is faced with the problem of removing a large number of disparate, interfering compounds from the sample extracts. A variety of clean-up methods have been employed [6,81 including column chromatography [9-201, liquid-liquid extraction

and chemical adsorption [241 procedures (see also Chapter 1). Silica gel has been extensively used in column chromatography clean-up. Commodities (and mycotoxins) to which this method has been applied include cereals (aflatoxins) [251, oilseeds (aflatoxins) [ 1 2,15 I, vegetable oils (aflatoxins) [ 10 I, meats (aflatoxins) 1131, spices (aflatoxins) [16,171, dried fruits (aflatoxins) [11,26], wine (aflatoxins) [ l a ] , coffee (aflatoxins) [20], animal feeds (aflatoxins) [9,21 1, milk (aflatoxin M I ) [271, animal viscera (aflatoxins) [19,281, cereals (deoxynivalenol, zearalenone) [291 and porcine kidneys (ochratoxin A ) [301. Similarly, Florisil has been applied to the clean-up of cereals (trichothecenes, moniliformin, butenolide and zearalenone) [31]. Florisil, modified with oxalic acid, has also been used to clean up corn and groundnut meal (aflatoxins) [321 and cellulose columns have been used to clean up animal tissues (aflatoxins, including M i ) [281. Liquid/liquid extraction clean-up procedures, utilising acetonitrile/petroleum ether 1331 and chloroform/aqueous HC1 [34] have been used during the analysis of, for example, maize and barley (zearalenone) [331 and corn (citrinin) [341, whilst chemical adsorbents have been applied to blue cheese (roquefortine) 1351 and black olives (ochratoxin A ) [361. The clean-up methods described above are laborious, time[21-241

consuming and of limited efficiency. Because of these disadvantages, clean-up procedures using commercially available solid phase extraction (SPE) cartridges are increasingly utilised for the analysis of mycotoxins [61. S P E techniques involve the partitioning of analytes and interfering compounds between a mobile and stationary phase. The latter, contained within the cartridge, is composed of a solid adsorbent or an immobilised (bonded) liquid phase [61. Available bonded phases include ethyl (CZ), octyl (C8), octadecyl (C18), cyclohexyl ( C H ) , phenyl ( P H ) , cyanopropyl (CN), diol (20H), aminopropyl (NH2) and a selection of ion exchange phases. S P E clean-up has been applied to the analysis of aflatoxin in groundnuts [371, peanut butter [381, cottonseed [391, and corn [40,41] by utilising silica gel as the solid adsorbent.

14

Florisil has been used for the analysis of aflatoxins in sorghum [42] and green coffee. Bonded phases have been utilised for the analysis of aflatoxin Mi in milk [431, of aflatoxins B1 and M1 in animal tissues [44] and of aflatoxin Bl, ochratoxin A and citrinin in human urine [451. SPE clean-up, utilising the phenyl (PHI bonded-phase, is routinely used in the authors’ laboratory for the analysis of aflatoxins in a range of commodities including maize, cottonseed, peanut butter and palm kernel 146-49,921 (Figure 2.1). The estimation of the aflatoxin content of corn [461, for example, is initiated by extraction with aqueous acetone. A 5 ml aliquot of the filtered extract is diluted with aqueous methanol (6.7% v/v) and acetic acid (1% v/v) mixture (60 ml) and mixed in a reservoir attached to a solvated PH (phenyl) cartridge. Using a vacuum manifold, the sample mixture is drawn through the cartridge at a flow rate of approximately 7 ml rnin-’

Figure 2.1. The elution of solid phase extraction cartridges: aflatoxins are eluted from the phenyl cartridge, through a sodium sulphate drying column, with a volume of chloroform appropriate for the commodity.

15

thus retaining the aflatoxins in the stationary phase. The cartridge containing the aflatoxins is then dried, by pulling air through the cartridge, and, finally, the aflatoxins are eluted with chloroform ( 7 ml). The chloroform solution is drawn through a second cartridge, containing anhydrous sodium sulphate, before removing the solvent by evaporation under nitrogen. The resultant residue may then be stored, in the dark, at -2OOC before quantification. Commodities which produce excessive interfering compounds, such as cottonseed, require additional clean-up steps. Chemical adsorbents such as lead acetate solution may be used, [ 5 0 , 5 1 ] in addition to SPE, in such instances. After extracting with aqueous acetone, lead acetate solution (20% w/v; 2 ml) is added to the aqueous methanolfacetic acid mixture. Diatomaceous earth is also added to act as a filter aid. The SPE clean-up is then performed as previously described. The simplicity of the SPE clean-up makes it ideally suited to the analysis of large numbers of samples. In the authors' laboratory up to 60 samples per day are routinely prepared for quantification using this procedure. The introduction of commercially available liquid handling equipment has facilitated the automation of SPE clean-up procedures. One such application is the analysis of aflatoxin Mi in milk [521. Shepherd et al. [53], using HPLC quantification, compared six clean-up procedures for aflatoxin M1 in milk. Of these six methods, a procedure using a C 1 8 cartridge was the most efficient in terms of cost, analysis time and clean-up efficiency; 0.0005 pg kg-I of aflatoxin M I in whole milk was detected [ 5 4 1 . SPE clean-up procedures are not, of course, universally applicable. The technique, for example, failed to adequately clean-up extracts of sorghum, even when the lead acetate precipitation step was included. However, suitably clean extracts were successfully produced using a Florisil clean-up column [ 4 2 1 .

16 2 . 3 NORMAL PHASE TLC 2 . 3 . 1 Principles

A normal TLC plate consists of a thin, uniform layer of particulate adsorbent, the stationary phase, applied to a flat plate. Silica, alumina and cellulose are frequently used as stationary phases.

The chromatography

is performed

by

the

dropwise application (Figure 2 . 2 ) of microlitre quantities, of a solution of the cleaned-up analyte mixture, to one end of the TLC plate.

The mixture is drawn through the stationary phase,

by capillary action, within the developing solvent (the mobile phase), which is usually contained within a sealed glass tank (Figure

During

2.3).

this

chromatographic

process,

the

components of the analyte mixture are partitioned between the stationary

and

mobile

phases.

The

components

of

greater

polarity will have the greater affinity for the stationary phase and

will

travel

more

slowly

through

the

adsorbent,

thus

effecting the separation of the analyte mixture. In normal phase TLC the stationary phase is more polar than the mobile phase. Typically, the particle distribution will be

Figure 2.2, The application, by micro-pipette, of a sample extract to a two-dimensional TLC plate.

17

Figure 2 . 3 . The development of a two-dimensional TLC plate: the plate is developed in an appropriate solvent in the first direction, air dried and then rotated through 90' before development in the second direction with another solvent

5-80 pm with a mean particle size of approximately 20 pm. 2.3.2. Practical considerations

Normal TLC may take a variety of forms, the chosen method often depending upon the amount of additional sample clean-up required. 2.3.2.1 One and two dimensional TLC

When little additional clean-up is required, one dimensional TLC is often sufficient for the separation and quantification of mycotoxins. Using this method, multiple samples can be simultaneously developed and quantified. However, when the initial clean-up is inadequate, or when there are many sample components of interest, it may be necessary to extend the chromatographic separation into the second dimension. Here, the plate is dried after the first

18

development and is then rotated through 90' and developed in a different solvent, affording better resolution of the components and the removal of interfering compounds. However , one disadvantage of two dimensional (2D) TLC is that only one sample at a time can normally be evaluated (Figure 2 . 4 ) .

Figure 2 . 4 . Two dimensional thin layer chromatography: the plate shown is for a mixture of pure aflatoxins. It has been sprayed with 509 sulphuric acid and dried at 105'C to identify the aflatoxins which change colour from blue-green to light yellow. Both one dimensional and 2D TLC have been applied, with One dimensional great success to the analysis of mycotoxins. TLC, for example, has been applied [55,561 to the analysis of the aflatoxins, the ochratoxins, zearalenone, citrinin, patulin, the trichothecenes, cyclopiazonic acid, the rubratoxins, sterigmatocystin, penicillic acid, butenolide and citreoviridin. Two-dimensional TLC has been applied, for example, to the aflatoxins, ochratoxin A , cyclopiazonic acid and citrinin [ 5 6 1 . 2.3.2.2 Bi-directional development

to samples Bi-directional TLC is also applied [ 4 6 ] requiring substantial, additional clean-up. The first step, in this method involves the application of the samples approximately 3 cm from one long edge of a 1 0 x 20 cm aluminiumbacked plate. A preliminary development is performed to

19

transport interfering compounds into the area between the line of application and the edge of the plate.

This area of the

plate is then removed using a sharp knife.

The TLC plate is

then rotated through 1 8 0 ' phase.

and developed in a suitable mobile

Multiple developments may be employed if necessary.

Bi-directional TLC will be discussed further in Section 2.5. 2.3.2.3

Circular development

Two varieties of this technique are currently in use.

In

circular chromatography, the samples are applied at, or near, the centre of the plate and the development solvent is delivered centrally via a capillary or a wick. developed

in a so-called U

Typically, the plates are

chamber 1571.

separated into diffused arcs.

The analytes are

It is reported that separation

and resolution are better than that normally achieved by linear development 1581.

Figure 2 . 5 . Circular chromatography: the U-Chamber. (by permission of Camag, Switzerland)

In anti-circular chromatography, the samples are spotted around the circumference of the plate and the solvent moves from the edge to the centre of the plate. Theoretically, this technique should result in more compact spots since the area

20

into which the spots can move is restricted. In practice, resulting spots are elongated and some loss of resolution occur. Relatively expensive equipment is required for circular (Figure 2.5). The method has not been widely applied to

the can TLC the

analysis of mycotoxins. 2.3.2.4 Triangular TLC A further variation of TLC is performed using triangular plates as described by Issaq [591 for the separation of dye mixtures. The chromatography is performed on triangular plates, prepared from 5 X 20 cm or 5 X 10 cm plates; the samples are applied along the 5 cm base of the triangle. As in the case of anti-circular chromatography, the area into which the spots migrate is restricted thus reducing spot diffusion. Elongation of the spots occurs after the first development but a second development, in the same solvent system, transforms the spots to a circular shape. The technique has been assessed for the quantification of aflatoxins and cyclopiazonic acid [60]. These preliminary experiments suggested that the resolution was inferior to that obtained with HPTLC and that the geometry of the plates resulted in problems during automated densitometric quantification.

2.4 REVERSE-PHASE TLC (RPTLC) 2.4.1 Principles In RPTLC, the mobile phase is more polar than the stationary phase which is composed of silica to which non-polar groups (typically, C2, C8, C18 or phenyl) have been chemically bonded. Early attempts at RPTLC employed normal phase plates impregnated with paraffin [61I or silanized using a suitable alkyl chlorosilane. Acetylated cellulose plates were also employed [621. 2.4.2 Practical considerations Although RPTLC has not been widely applied to mycotoxins, recent reports include the analysis by RPTLC of ochratoxin A [ 63,641, of a range of toxins using C18 plates (aflatoxins B1 , B2, G I , G2, sterigmatocystin, ochratoxin A, citrinin, penicillic

21

acid, patulin, zearalenone, deacetoxyscirpenol, T-2 toxin, HT-2 toxin, nivalenol, neosolaniol, fusarenone-X, deoxynivalenol and 3-acetyl-deoxynivalenol) [651. High performance RPTLC C8 plates have been used to analyse aflatoxin M1 in milk [661 and, in the authors’ laboratory, C18 plates have been used for estimating zearalenone and alternariol monomethyl ether [671.

2.5 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) 2.5.1 Principles HPTLC has evolved with the development [681 of a) automated sample application and plate interpretation equipment and b) high quality TLC plates. Sample application equipment facilitates the accurate and precise application of nanolitre quantities of cleaned-up sample extracts using automated (or semi-automated) techniques (Figures 2.6a and 2.6b).

Figure 2.6a. The Nanomat 111: a semi-automatic spotting instrument which uses microcaps clamped in precise positions. (by permission of Camag, Switzerland)

High quality (performance) TLC plates are uniformly coated with adsorbents of small particle size, typically within the 2 1 0 pm range, and with a layer thickness of 0.1 - 0.3 mm. Both normal and reversed phase adsorbents are available.

22

Figure 2.6b. The Camag 27210 TLC autosampler: using a fine bore capillary, position on the plate, dosage and dosage rate can be specified for a number of samples and standards, with high precision.

2.5.2 Practical considerations Modern sample application equipment produces a spot of the order of 1 mm diameter which allows up to 30 samples to be

accommodated along the longer side of a 10 x 20 cm plate. The small particle sized adsorbents facilitate the rapid separation of the analyte components, sometimes after only 3 cm of travel. Although a selection of development chambers have been reported [68] for HPTLC, conventional 21 cm (h) x 28 cm (1) x 6 cm (w) glass tanks are successfully used in the authors' laboratory [691. One-dimensional, two-dimensional, circular, anti-circular and bi-directional development methods ( sections 2.3.2.(1-3)) have all been applied to HPTLC plates. In the authors' laboratory bi-directional normal HPTLC is routinely used for the

23

analysis of aflatoxin in a wide range of commodities including edible

nuts,

oilseeds,

[7,8,42,46-491

cereals,

root-crops

and

spices

(Figure 2.7).

Figure 2 . 7 . The analysis of aflatoxins by HPTLC: the plate shown is for samples of cottonseed. There are 25 sample spots and six mixed aflatoxin standards. Some of the contaminants have been removed by a preliminary development in the reverse direction in dry ether and then cutting away the band of impurities before two developments in the normal direction in 6 : 3 : 1 chloroform: xy1ene:acetone. A development tank, for the di-ethyl ether development stage of bi-directional HPTLC and a drying chamber, to remove residual solvent between developments, have been designed and fabricated

[701 in the author's

laboratory

(Figures 2.8a

and

2.8b). Attempts to improve the precision of the development step have

resulted

Chromatography

in

the

technique of Over

(OPTLC) [71,721.

Pressured

In OPTLC,

Thin-Layer

the TLC plate is

covered by a pressurised flexible membrane and the mobile phase is forced through the thin layer with the aid of a pump. A high separation efficiency is reported [71 I 2.9).

for this method

(Figure

TO date OPTLC has not been applied to the analysis of

mycotoxins. 2 . 6 PREPARATIVE TLC

2.6.1 Principles

The

increased

layer

thickness

(1-5

mm)

employed

in

preparative TLC (PTLC) facilitates the application and isolation

of milligram quantities of analyte.

24

Figure 2.8a. A vertical metal tank suitable for ether development. This has the advantages of much quicker development than in a glass tank, better retention of the very volatile solvent, and for aflatoxins, the exclusion of light.

Figure 2.8b.

A

fan-assisted plate dryer.

Figure 2.8. An HPTLC development tank and fan-assisted drying chamber.

25 6

\

1

3

2

10

4 1-

Figure 2 . 9 . The Chompres 1 0 OPLC development chamber. 1 . Bottom support block 7. Solvent inlet valve for 2. Polymethacrylate support plate controlled pumping of the 3 . External frame mobile phase 4 . Position for chromatographic plate 8. Solvent outlet 5. Clamp 9. Water outlet for release 6. Water inlet for supplying of cushion pressure 1 0 . Hydraulic system for pressure to upper side of plate through a plastic foil cushion operating blocks

(by permission of Dr Alfred Huethig Verlag, Heidelberg)

Typically, the sample extract is applied to the plate as a horizontal streak using a manual or automated applicator (Figure 2.10 ) . After development, the separated component "bands" may be located by their UV absorption or fluorescent properties. Alternatively, non-UV absorbing compounds may be visualised by carefully spraying the extremities of the plate with a suitable colour-producing reagent. The individual "bands" can then be carefully scraped from the plate and extracted from the adsorbent using a suitable solvent. 2.6.2 Practical considerations I n PTLC, the increased thickness of the adsorbent layer can lead to vertical band spreading. Tapered-bed plates have been

26

Figure 2.10. The application of sample to a PTLC plate: an even streak of the sample is delivered through a syringe, the plunger being depressed uniformly by the inclined sliding rail.

developed [73] to overcome this problem.

These plates feature a

pre-adsorbent layer of 0.7 mm thickness followed by a tapered separation area which increases in thickness from 0.3-1.7 mm. The tapered bed allows a more uniform mobile phase flow pattern which

reduces

the

vertical

band

spread

and

migration distance of the more polar components.

increases A

the

variety of

mycotoxins have been isolated using PTLC including cytochalasins H and J

[74], chaetoglobosins K and L

[751, proxiphomin and

protophomin [76], citreoviridin [771 and paspalitrem

A

[781, the

trans and cis isomers of zearalenone [791, isotopically labelled ochratoxin

A

zygosporins

[801, the rubratoxins [811, mycophenolic acid [821, [831,

sporidesmins A ,

deoxaphomin

[841,

janthitrems

[851,

C and G [86], PR toxin [87] and the methylated

derivative of territrem C

[88].

PTLC has been used

in the

authors’ laboratory for the partial purification of A l t e r n a r i a mycotoxins [891.

21

2.7 DETECTION After the development of the TLC plate, the separated components are located and quantified. If appropriate, the U V absorbance or fluorescent properties of the analytes may be used for this purpose. Alternatively, chromophores may be introduced into non-UV absorbing compounds by treating (by spraying or dipping) the plates with suitable reagents. Chemical derivatisation i n - s i t u may also be used to confirm the presence of a suspected mycotoxin. 2.7.1 Fluorescence detection The natural fluorescence of mycotoxins under ultra-violet light is widely used in their detection and quantification, allowing the detection, in some cases, of picogram quantities of these compounds. Naturally fluorescent mycotoxins include the aflatoxins (B1, B2, G1 and G2), aflatoxin Mi, zearalenone, ochratoxin A, sterigmatocystin, citrinin, patulin and penicillic acid [go]. The aflatoxins, for example, appear as characteristic blue (B1 and B2) and blue-green (GI and G2) fluorescent spots under long-wave ( 3 6 5 nm) UV light, zearalenone as a blue-green spot, citrinin as a yellow spot, sterigmatocystin as a dull brick-red spot and penicillic acid as a weak, light purple spot. 2.1.2 Chemical derivatisation The formation, in s i t u , of fluorescent derivatives can be used to a ) detect non-fluorescent mycotoxins, b) enhance the fluorescence of naturally fluorescing mycotoxins and c) confirm the presence of presumptive mycotoxins [ 5 6 , 9 0 1 . The non-fluorescent trichothecenes may be detected by chemical derivatisation. T - 2 toxin, for example, appears as a grey-blue fluorescent spot after spraying with 20 per cent concentrated sulphuric acid in methanol and heating at llO°C for 3 to 4 minutes. Alternatively, T-2 toxin will afford a bright blue fluorescence if treated with a mixture of aluminium chloride (in water:ethanol, 1:l) and chromotropic acid (in concentrated sulphuric acid:water, 5 : 3 ) followed by heating at 110OC.

The natural fluorescence of sterigmatocystin may be enhanced, to afford a bright yellow spot, by spraying with a 24 percent solution of aluminium chloride in 9 5 percent aqueous

28

ethanol and heating at 105'C for 10 minutes. The identity of sterigmatocystin may be confirmed by the formation of the acetate [911 or hemiacetal [921 derivatives. Similarly, the long-wave fluorescence of zearalenone and ochratoxin A may be enhanced if the plate is sprayed with aluminium chloride solution. The presence of ochratoxin A may be confirmed by the formation of the ethyl ester derivative. The natural, shortwave (254nm) fluorescence of patulin can be enhanced by treatment with 0.5 percent aqueous 3-methyl-2-benzothiazolinone hydrazone (MBTH) followed by heating at 130'C for 15 minutes. If penicillic acid is treated with MBTH, a visible pale yellow spot is produced. Citrinin tends to streak in many solvent systems and is probably best chromatographed on silica gel TLC plates impregnated with oxalic acid or ethylenediaminetetra-acetic acid (EDTA). The long-wave yellow fluorescence of citrinin may be converted to a green fluorescence by spraying the plate with 14 percent (w/w) boron trifluoride in ethanol. The presence of citrinin may be confirmed by the formation of the acetate derivative. 2.7.3 Bioautographic methods This procedure involves a combination of preparative TLC and biological detection. The trichothecenes T-2 and HT-2, for example, have been isolated by PTLC and detected by their toxicity towards the yeasts Kluyveromyces fragilis and Saccharomyces cerevisiae. The reported limit of detection was 0.2nM per spot [561. Bioautographic detection using Bacillus subtilis has been used in the PTLC of gliotoxin 1941. Similarly, bioautographic methods have also been applied to aflatoxin Bl, kojic acid and sterigmatocystin using Artemia (brine shrimp) larvae as the biological detection salina organism [951.

2.8 QUANTITATIVE AND SEMI-QUANTITATIVE EVALUATION The

quantitative

interpretation

of

developed

(HP))TLC

plates is performed by comparing the fluorescent intensities of standard mycotoxins extracts [90,911.

and

the

mycotoxin

components

of

sample

Using the automated and the semi-automated

29

Figure 2.11a. The Camag TLC I1 Scanner: the plate is secured on a graduated platen using magnetic spacers.

Figure 2.11b. Camag TLC I1 Scanner linked to a SP4270 Integrator and a PC. Figure 2.11. The densitometric quantification of a TLC plate.

30

equipment associated with HPTLC methodology (section 2 . 5 ) picogram quantities of aflatoxin, for example, can be precisely and accurately detected (Figure 2 . 1 1 ) . In the absence of densitometric equipment, the intensities of mycotoxin standard and sample spots may be compared visually. This semiquantitative interpretation is laborious and requires the services of a skilled technician. If mycotoxin standards are not available, the dilution to extinction method may be employed. Decreasing volumes of sample extract (typically 2 5 to 5 pl) are applied to the plate and the smallest volume (V) in which the mycotoxin is visible is then identified. If the minimum quantity of mycotoxin that can be visually detected is known, the concentration of mycotoxin in volume V and, consequently, in the original sample, can be calculated [ g o ] .

ILLUSTRATIVE EXAMPLES HPTLC methods have been developed in the authors' laboratory which facilitate the accurate and precise quantification of aflatoxin in edible nuts, oilseeds and their derivatives, cereals, root crops and spices [ 4 2 , 4 6 - 4 9 1 ; an HPTLC method has also been developed recently for cyclopiazonic acid in groundnut cake, Each newly developed TLC method must be validated by determining the accuracy, precision and limit of detection of that method for any given commodity (including different varieties). The following validation procedure is applied in the authors' laboratory. Mycotoxin-free extracts of the commodity are spiked to afford a range of mycotoxin concentrations, The choice of 2.9

concentration range should depend upon the contamination levels of interest associated with that commodity. Table 2 . 1 shows suggested aflatoxin spiking levels for various commodity types. A second experiment should be performed using the same levels of artificial contamination, but using pure extraction solvent as opposed to the sample matrix. This will eliminate any possible bias in the methodology due to the components of the sample matrix. Six replicate analyses should be carried out Weighted regression at each of the contamination levels. analysis of the data will facilitate the calculation of the accuracy, precision and limit of detection for the method [ 9 2 1 .

31

Table 2.1 Suggested spiking levels for various commodity types. ~

Class of commodity

Typical permitted level (total) (pg/kg)

Suggested range for aflatoxin B1 (pg/kg)*

Foodstuffs

10

0, 2, 4, 8, 10 15, 30, 50, 100

Feedstuffs

50

0, 4, 8, 20, 40 50, 60, 100, 250

Raw materials for feedstuffs

0, 5, 10, 50, 100

200

150, 250, 500

*Concentrations of aflatoxins B1 and GI ; the concentrations of aflatoxins B2 and G 2 are approximately half those shown for B1 and G I .

The proposed method should then be further validated by comparison with an "official method" of analysis.

Naturally-

contaminated samples at two contamination levels are required for this comparison [921.

2.10 CONCLUSIONS

Modern HPTLC is a precise and accurate analytical tool with an efficiency which is comparable to that of high performance liquid assay phenyl

chromatography

(HPLC) and

(ELISA) methods. bonded-phase

enzyme

linked

immunosorbent

The application of a combination of

clean-up

and

HPTLC

to

the

analysis

of

aflatoxin in peanut butter has demonstrated [ 4 9 ] the efficiency of

HPTLC

quantification

as

compared

to

HPLC

and

ELISA

procedures. HPTLC is ideally suited to the analysis of large numbers of accumulated samples. Approximately thirty samples, for example, may be simultaneously chromatographed on a single 10 x 20 cm plate. The ability to perform TLC in a two-dimensional or bidirectional mode enables valuable, additional sample clean-up to be performed during the quantification step. the disposable nature of

TLC

plates

facilitates

Furthermore, the

use

of

32

extreme reaction conditions during the i n - s i t u chemical derivatisation of separated analytes. Undoubtedly, the combination of automated sample clean-up and modern HPTLC methodology can provide the mycotoxicologist with a very powerful analytical tool. Solid phase extraction clean-up combined with bidirectional HPTLC is the method of choice for the analysis of aflatoxins in the authors’ laboratory.

REFERENCES 1 N.A. Izmailov and M.S. Shraiber, Farmatsia, 3 (1938) 1 . 2 J.E. Meienhard and N.F. Hall, Anal. Chem., 21 (1949) 185. 3 J.G. Kirchner, J.M. Miller and K.J. Keller, Anal. Chem. , 23 (1951) 426. 4 J.M. Miller and J.G. Kirchner, Anal. Chem., 24 (1952) 1480. 5 J.M. Miller and J.G. Kirchner, Anal. Chem., 26 (1954) 2002. 6 R.D. Coker and B.D. Jones in R. Macrae (Editor), HPLC in Food Analysis, Academic Press Ltd., London, 1988, p. 335. 7 R.D. Coker in J. Gilbert (Editor), Analysis of Food Contaminants, Elsevier Applied Science Publications, New York, 1984, p. 207. 8 N. Bradburn, R.D. Coker, K. Jewers and K.I. Tomlins, Chromatographia, 29 (1990) 435. 9 G.N. Shannon, O.L. Shotwell and W.F. Kwolek, J. Assoc Off. Anal. Chem., 66(3) (1983) 582. 10 N. Miller, H.E. Pretorius and D.W. Trinder, J. Assoc Off. Anal. Chem., 68(1) (1985) 136. 1 1 D. Boyacioglu and M. Gonul, J. Assoc Off. Anal. Chem., 71(2) (1988) 280. 12 P.M. Scott, J. Assoc Off. Anal. Chem., 52(1) (1969) 72. 13 L.B. Bullerman, P.A. Hartman and J.C. Ayres, J. Assoc Off. Anal. Chem., 52(3) (1969) 638. 14 R.C. Shank, G.N. Wogan, J.B. Gibson and A . Nondastu, Food Cosmet. Toxicol., 10 (1972) 61. 15 J. Karnelic, M. Israel, S. Benado and C. Leon, J. Assoc Off. Anal. Chem., 56(1) (1973) 1452. 16 P.M. Scott and B.P.C. Kennedy, J. Assoc Off. Anal. Chem., 56(6) (1973) 1452. 17 P.M. Scott and B.P.C. Kennedy, Can J. Inst. Food Sci Technol. J., 8(2) (1975) 124. 18 D.M. Takahashi, J. ASSOC Off. Anal. Chem., 57(4) (1974) 875. 19 R.D. Stubblefield and O.L. Shotwell, J. ASSOC Off. Anal. Chem., 64(4) (1981) 964. 20 P.M. Scott, J. Assoc Off. Anal. Chem., 51(3) (1968) 609. 21 S-C. Chen and L. Friedman, J. ASSOC Off. Anal. Chem., 49(1) (1966) 28.

33 2 2 W.A. Pons and L.A. Goldblatt, J. Amer. Oil Chem. SOC., 4 2 ( 6 ) ( 1 9 6 5 ) 471. 2 3 W.A. Pons, A.F. Cucullu and A.O. Franz, J. Assoc Off. Anal. Chem., 5 5 ( 4 ) ( 1 9 7 2 ) 7 6 8 . 2 4 T.R. Romer, J. Assoc Off. Anal. Chem., 6 8 ( 1 9 7 5 ) 5 0 0 . 2 5 G.M. Shannon and O.L. Shotwell, J. Assoc Off. Anal. Chem., 5 8 ( 4 ) ( 1 9 7 5 ) 7 4 3 . 2 6 D. Boyacioglu and M. Gonul, Food Additives and Contaminants, 7 ( 2 ) ( 1 9 9 0 ) 2 3 5 . 2 7 R.D. Stubblefield, J. Amer. Oil Chem. SOC., 5 6 ( 9 ) ( 1 9 7 9 ) 8 0 0 . 2 8 N.L. Brown, S . Nesheim, M.E. Stack and G.M. Ware, J. Assoc Off. Anal. Chem., 5 6 ( 6 ) ( 1 9 7 3 ) 1 4 3 7 . 2 9 G.A. Bennet, S . E . Megalla and O.L. Shotwell, J. Amer. Oil Chem SOC., 6 1 ( 9 ) ( 1 9 8 4 ) 1 4 4 9 . 3 0 C. Wilkein, W. Battes, I. Mehlitz, R. Tiebach and R. Weber, 2 . Lebensm Unters Forsch, 1 8 0 ( 1 9 8 5 ) 4 6 9 . 31 H. Kamimura, M. Nishijima, K. Yasuda, K. Saito, A.

Ilbe, T. Nagayama, H. Ushiyama and Y. Naoi, J. Assoc Off. Anal. Chem., 6 4 ( 5 ) ( 1 9 8 1 ) 1 0 6 7 . 3 2 M.K.L. Bicking, R.N. Knisley and H.J. Svec, J. ASSOC Off. Anal. Chem., 6 6 ( 4 ) ( 1 9 8 3 ) 9 0 5 . 3 3 C.J. Mirocha, B. Schauerhamer and S.V. Pathre, J. Assoc Off. Anal. Chem., 5 7 ( 5 ) ( 1 9 7 4 ) 1 1 0 4 . 3 4 L.K. Jackson and A. Ciegler, Appl. Environ Microbiol., 3 6 ( 3 ) ( 1 9 7 8 ) 408. 3 5 P.M. Scott and B.P.C. Kennedy, J. Agric Food Chem., 2 4 ( 4 ) (1976) 865. 3 6 B. le Tutour, A. Tantoui-Elaraki and A. Aboussalin, J. Assoc Off. Anal. Chem., 6 7 ( 3 ) ( 1 9 8 4 ) 6 1 1 . 3 1 W.J. Hurst, K.P. Snyder and R.A.Martin, Peanut Sci., 11 (1984) 21. 3 8 D. Tosch, A.E. Waltking and J.S. Schlesier, J. ASSOC Off. Anal. Chem., 6 7 ( 1 9 8 4 ) 3 3 7 . 3 9 J.D. McKinney, J. Amer. Oil Chem. SOC., 5 8 ( 1 9 8 1 ) 9 3 5 A . 4 0 J.E. Hutchins and W.M. Hagler, J. Assoc Off. Anal. Chem., 6 6 ( 1 9 8 3 ) 1 4 5 8 . 41 J.E. Thean, D.R. Lorenz, D.M. Wilson, K. Rodgers and R.C. Gueldner, J. Assoc Off. Anal. Chem., 6 3 ( 1 9 8 0 ) 631. 4 2 K. Jewers, A.E. John and G. Blunden, Chromatographia, 27(11/12) (1989) 617. 4 3 N. Takeda, J.Chromat., 2 8 8 ( 1 9 8 4 ) 4 8 4 . 4 4 G. Quan and G.C. Yang, J. Agric Food Chem., 3 2 ( 1 9 8 4 ) 1071. 4 5 D.L. Orti, R.H. Hill, J.A. Liddle and L.L. Neelham, J. Anal Toxicol., 1 0 ( 1 9 8 4 ) 9 7 3 . 4 6 K.I. Tomlins, K. Jewers and R.D. Coker, Chromatographia, 2 7 ( 1 9 8 9 ) 6 7 . 4 7 N. Bradburn, K. Jewers, B.D. Jones and K.I. Tomlins, Chromatographia, 2 8 ( 1 9 8 9 ) 5 4 1 . 4 8 N. Bradburn, R.D. Coker and K. Jewers, Chromatographia, 2 9 ( 1 9 9 0 ) 1 7 7 . 4 9 M.P.K. Dell, S.J. Haswell, O.G. Roch, R.D. Coker, V.F.P. Medlock and K.I. Tomlins, Analyst, 1 1 5 ( 1 9 9 0 ) 1435. 5 0 Official Methods of Analysis, 11th ed., Association of Official Analytical Chemists, Washington D.C., 1 9 7 0 , section 2 6 . 0 3 5 , p . 4 3 2 .

34 5 1 W.A. Pons and L.A. Goldblatt, J. Amer. Oil Chem. SOC., 4 2 ( 6 ) ( 1 9 6 5 ) 4 7 1 . 5 2 L.A. Gifford, C. Wright and J. Gilbert, Food Additives and Contaminants, 7 ( 6 ) ( 1 9 9 0 ) 8 2 9 . 5 3 M.J. Shepherd, M. Holmes and J. Gilbert, J. Chromat., 354 ( 1 9 8 6 ) 305. 5 4 N. Takeda, J. Chromatogr., 2 8 8 ( 1 9 8 4 ) 4 8 4 . 55 V. Betina, J. Chromatogr., 3 3 4 ( 1 9 8 5 ) 2 1 1 . 5 6 V. Betina, J. Chromatogr., 4 7 7 ( 1 9 8 9 ) 1 8 7 . 5 7 R.E. Kaiser, in A. Zlatis and R.E Kaiser (Editors),

HPTLC-High Performance Thin Layer Chromatography, Journal of Chromatography Library Vo1.9, Elsevier Scientific Publishing Company, Amsterdam, 1 9 7 7 , p. 7 3 . 5 8 J. Blome, in A. Zlatis and R.E Kaiser (Editors), HPTLC-High Performance Thin Layer Chromatography, Journal of Chromatography Library Vo1.9, Elsevier Scietific Publishing Company, Amsterdam, 1 9 7 7 , p. 3 9 and 51. 5 9 H.J. Issaq, J. Liq. Chromatogr., 3 ( 6 ) ( 1 9 8 0 ) 7 8 9 . 6 0 N. Bradburn, Natural Resources Institute, personal

communication. 6 1 K. Randerath, Chromatographie sur Couches Minces, Gauthier-Villars, Paris, 2nd ed. ( 1 9 7 1 ) 7 2 . 6 2 R.K. Gilpin and W.R. Sisco, J.Chromatogr., 1 2 4 ( 1 9 7 6 ) 257. 6 3 H.M. Stahr, M. Domoto, Bei Lei Zhu and R.Pfeiffer, Mycotoxin Research, 1 ( 1 9 8 5 ) 3 1 . 6 4 A.A. Frohlich, R.R. Marquardt and A. Bernatsky, J. Assoc Off. Anal. Chem., 7 1 ( 5 ) ( 1 9 8 8 ) 9 4 9 . 6 5 D. Abramson, T. Thorsteinson and D..Forest, Arch. Environ. Contam. Toxicol., 1 8 ( 1 9 8 9 ) 3 2 7 . 6 6 P.A. Biondi, L. Gavazzi, G. Ferrari, G . Maffeo and C. Secchi, J. of High Res. Chrom.& Chrom. Comms., 3 ( 1 9 8 0 ) 92. 6 7 V. Medlock, A.P. Dutta, Natural Resources Institute,

in

preparation. Hara, R.E. Kaiser and A. Zlatis, Instrumental HPTLC, Dr Alfred Huthig Verlag, Heidelberg, 1 9 8 0 . 6 9 R.D. Coker, K. Jewers, K.I. Tomlins and G. Blunden, Chromatographia, 2 5 ( 1 9 8 8 ) 8 7 5 . 7 0 S. Ganguli, Natural Resources Institute, unpublished material. 7 1 H.E. Hauck and W. Jost, J.Chromatogr., 2 6 2 ( 1 9 8 3 ) 1 1 3 . 7 2 S z . Nyiredi, C.A.J. Erdelmeier and 0. Sticher in R.E. Kaiser (Editor), Planar Chromatograhy, Vol. 1, Dr Alfred Huthig Verlag, Heidelberg, 1 9 8 6 , p. 1 1 9 . 7 3 Available from Analtech, Delaware, U S A. 7 4 S.A. Patwardhan, R.C. Sukh Dev Pandey and G.S. Pendse, Phytochemistry, 1 3 ( 1 9 7 4 ) 1 9 8 5 . 7 5 A. Probsl and Ch. Tamm, Helv. Chim. Acta., 6 5 ( 1 9 8 2 ) 6 8 W. Bertsch, S .

1543. 7 6 M. Binder and Ch. Tamm, Helv. Chim. Acta., 5 6 ( 1 9 7 3 ) 2387. 7 7 Y. Ueno in I.F.H. Purchase (Editor), Mycotoxins in Human Health, The Macmillan Press Ltd., London, 1 9 7 1 , p. 115.

35 7 8 R.J. Cole, J.W. Dorner, J.A. Lansden, R.H. Cox, C.

Pape, B. Cunfer, S.S. Nicholson and D.M. Bedel, J. Agric. Food Chem., 25 ( 1 9 7 7 ) 1 1 9 7 . 7 9 S.V. Pathre, C.J. Mirocha and S.W. Fenton, J. Assoc. Off. Anal. Chem., 62 ( 1 9 7 9 ) 1 2 6 8 . 8 0 A.E. de Jesus, P.S. Steyn, R. Vleggaar and P.L. Wessels, J. Chem. SOC., Perkin Trans., 2 ( 1 9 8 0 ) 5 2 . 8 1 C.O. Emeh and E.H. Marth, Arch. Microbiol., 1 1 5 (1977) 157. 8 2 D.F. Jones, R.H. Moore and G.C. Crawley, J. Chem. SOC. C., ( 1 9 7 0 ) 1 5 7 . 8 3 H. Minato, M. Matsumoto and T. Katayama, Annu. Rep. Shionogi. Res. Lab., 23 ( 1 9 7 3 ) 4. 84 M. Binder and Ch. Tamm, Helv. Chim. Acta., 5 6 ( 1 9 3 ) 966. 8 5 R.T. Gallagher, G.C.M. Latch and R.G. Keogh, Appl Environ. Microbiol., 39 ( 1 9 8 0 ) 2 7 2 . 8 6 P.J. Curtis, D. Greatbanks, B. Hesp, A.F. Cameron and A.A. Freen, J. Chem. SOC. Perkin. Trans., 1 ( 1 9 7 7 ) 1 8 0 . 8 7 P.M. Scott, B.P.C. Kennedy, J. Harwig and B.J. Blanchfield, Appl. Environ. Microbiol., 33 ( 1 9 7 7 ) 249. 8 8 K.H. Ling, H.H. Liou, C.M. Yang and C.K. Yang, Appl. Environ. Microbiol., 47 ( 1 9 8 4 ) 9 8 . 8 9 A.E. John, PhD Thesis 1990, CNAA, Portsmouth

Polytechnic. 9 0 R.D. Coker, B.D. Jones, M.J. Nagler, G.A. Gilman, A.J.

Wallbridge and S. Panigrahi, Natural Resources Institute, Mycotoxin Training Manual, 1 9 8 4 . 9 1 Official Methods of Analysis, 1 4 t h ed., Association of Official Analytical Chemists, Washington D.C., 1 9 8 4 , section 2 6 . 1 3 8 . 9 2 S. Nawaz, R.D. Coker and S.J. Haswell, Analyst, 1 1 7 (1992) 67. 9 3 N. Bradburn, Natural Resources Institute, in

preparation. 9 4 V. Betina and Z. Barath, J. Antibiot., 1 7 ( 1 9 6 4 ) 1 2 7 . 9 5 Z . Durackova, V. Bekina and P. Nemec, J. Chromatogr., 116 ( 1 9 7 6 ) 155.

36

Chapter 3 TECHNIQUES OF LIQUID COLUMN CHROMATOGRAPHY PIRJO KURONEN INTRODUCTION Mycotoxins are a heterogeneous class of toxic substances produced by various species of several genera of filamentous fungi. The main producers of mycotoxins belong to the Aspergillus, Fusarium, and Penicillium genera. They play an important role in foodborne diseases of humans and animals, with toxic effects as variable as their composition. There is an increasing concern over food safety. In 1981, Cole and Cox (1) compiled the properties of over 270 mycotoxins from filamentous fungi. It is worth noting that in 1983 altogether about 3000 structurally characterized fungal metabolites were known in the literature ( 2 ) . Watson (3) in 1981 gave a figure of 432 toxic fungal metabolites. Mycotoxins represent a wide range of compound types, various polarities, chemical structures, and acid-base properties. The aflatoxins have been and still are the most important group of mycotoxins because of their frequent contamination of foods and feeds and their extremely severe toxicological effects in animals and man. Therefore they occupy an important position in mycotoxin research, followed in importance by the trichothecenes which now number over sixty (4-6). The commodities with the most mycotoxin problems are maize (corn) and groundnuts (peanuts). This chapter will present the methodology of column liquid chromatography covering sample pretreatment, classical column chromatography, mini-column chromatography, and high-performance liquid chromatography (HPLC), with the main emphasis on HPLC. This is now the most widely used chromatographic method in the analytical laboratory. HPLC allows ultratrace analysis of a wide variety of compounds. Many analytes can be analyzed at a nanogram level, but detection limits of a picogram or even less have been demonstrated in certain cases using special techniques. The 3.1

31

applications of the column liquid chromatographic methods will be presented in Chapter 8. SAMPLE PRETREATMENT The aim of the sampling and sample preparation procedures is to produce as representative a laboratory sample as possible (7). This sample is then analyzed using an analytical procedure which comprises extraction, clean-up, qualitative, quantitative, and confirmatory steps (see Fig. 3.1). 3.2

I

SAMPLE PREPARATION

1

I

I

CHRCNATGRAPHIC

rnYS1S

Fig. 3.1. Flow diagram showing mycotoxin determination.

the

analytical

sequence

for

In the first step of chemical analysis sequence mycotoxins are removed from the food or feed sample by some means of extraction - either by blending or mechanical shaking with solvents such as methanol, acetonitrile, ethyl acetate, acetone, chloroform, dichloromethane, or water, used either singly or as mixtures. This step is illustrated more precisely in Chapter 1. The second step of chemical analysis includes the clean-up of the crude sample extract to remove lipids and other co-extractives which interfere with the detection of mycotoxins at trace-level concentrations and which may contaminate and damage the analytical chromatographic columns. Sample clean-up is normally by far the most time-consuming stage in the analysis sequence of mycotoxins.

38

The choice of sample clean-up method(s) depends critically on the type of mycotoxin(s) and matrix in question, the expected concentration of the mycotoxin(s), and the available final analytical method used for the detection and determination. Generally, analysis of agricultural commodities necessitates extensive clean-up before the final analysis is feasible. The diverse chemical compositions of agricultural commodities have hindered the development of any single method that can be uniformly applied to all products, and therefore a variety of methods is used for this purpose. The techniques used include liquid-liquid partitioning, chemical adsorption, dialysis, and various forms of chromatographic clean-up procedures. Column chromatographic methods are widely used as clean-up procedures using different column packing materials. These methods include classical open column chromatography (Section 3 . 3 ) , chromatography using small disposable pre-packed cartridges (e.g. Sep-Pak, Baker, Supelclean, and Bond Elut), and preparative HPLC (Section 3.5). Solid-phase extraction clean-up. Solid-phase extraction (7-9) has been one of the fastest growing sample (SPE) pretreatment methods. It is a more rapid, efficient, reproducible, and safer method than the traditional liquid-liquid extraction techniques and offers, in addition, a wider range of selectivity. A wide selection of different chromatographic sorbents makes it possible to utilize several extraction mechanisms, thus allowing mycotoxins to be extracted from complex sample matrices. The adsorbents in SPE cartridge columns are chemically similar to the column packing materials used in HPLC (Section 3 . 5 ) , but have a larger particle diameter (40 pm), which facilitates the sample handling process. Silica gel, silica-based non-polar and polar bonded phases , ion-exchange, and size-exclusion phases are available, packed into disposable polyethylene columns. Silica gel-filled cartridges and silica-based bonded-phase packings dominate the market, but polymeric-based cartridges ( 9 ) , which withstand a wider range of solvents and pH values, have become available, too. The standard cartridge sizes are 100 or 400 mg but, in addition, cartridges containing larger amounts of variety of stationary phases (up to 10 g ) are also available. A vacuum manifold is used to facilitate

39

rapid sample handling and solvent elution of the retained compounds. With the 400-mg column, sample sizes as large as 100 ml may be used, but most of the applications reported have used much smaller sample volumes. SPE contains four extraction steps which are illustrated in Fig. 3.2. First, the SPE column is prepared to receive a sample, using a proper solvent. Secondly, the sample extract is applied to the cartridge in a weak solvent, which results in a strong retention of the compounds of interest in the column. Thirdly, the column is washed with a solvent that elutes the less strongly retained sample components: the compounds of interest are finally eluted selectively in a small volume of stronger solvent (ideally about 0.6 ml per 100 mg adsorbent). Step 1

Step 2

Step 3

Step 4

Conditioning

Sample Application

Washing

Elution

Fig. 3.2. Solid-Phase Extraction steps. The SPE process can be used in three different modes: (1) sample clean-up, ( 2 ) sample concentration, and (3) matrix removal. In sample clean-up mode a SPE column retains the mycotoxin and allows impurities to pass through the column. In sample concentration mode large sample volumes are passed through the column and the retained mycotoxin is concentrated by eluting with a small volume of solvent. In the matrix removal mode the SPE column is used to retain interfering impurities and the mycotoxin is allowed to pass through the column. Clean-up and preconcentration procedures for mycotoxin-containing samples, including the use of silica and bonded-phase cartridges, have been reported in numerous references

40

(10-20). Hoke et al. (21) used SPE columns to concentrate mycotoxins (T-2, HT-2, DAS, and DON) so that the assay could respond to low aqueous mycotoxin concentrations after only 24 hours of exposure. Orti (16) has developed a multi-mycotoxin clean-up method which makes it possible to estimate simultaneously aflatoxin B1, ochratoxin A (OCH A), and citrinin (CIT) in human urine, using a sequence of different clean-up cartridges. The SPE technique can nowadays be automated with dedicated instruments available from a number of companies (9). Charcoal/Alumina Clean-up Column. The small charcoal/alumina clean-up columns (No. 213 and No. 215, available from Romer Labs) presented in 1981 by Romer et al. (22) have been effectively used to remove interfering materials from grain, feed, and food extracts prior to the final chromatographic determination of trichothecene mycotoxins (23-25). 3.3

COLUMN CHROMATOGRAPHY 3.3.1 Introduction Classical column chromatographic methods (5,26) have been used widely as clean-up procedures in trace analysis of mycotoxins, although today commercial pre-packed disposable cartridges (Section 3.2) are increasingly replacing these methods. Different modes of chromatography, such as adsorption, partition, ion-exchange, size-exclusion, and affinity Chromatography, have been used. The most widely used of these methods is, however, adsorption chromatography, on which in this section will be mainly focused . 3.3.2 Procedure The sample extract dissolved in a small volume of an appropriate solvent is added slowly and evenly to the top of the column after which the column is washed with one or more solvents in which the mycotoxins are insoluble or at least less soluble than the impurities. Thereafter the solvent composition is The changed so that the mycotoxins are eluted from the column. eluate is collected and concentrated, and the residue is redissolved in a small volume of solvent prior to the final chromatographic analysis or prior to further isolation by preparative HPLC for mass spectrometric confirmation of identity (27,28).

41

3.3.2.1 Stationary phase Silica gel is the most widely used adsorbent for classical column chromatographic clean-up of mycotoxins (29). Other adsorbents used include alumina, charcoal, cellulose powder, magnesium silicate (Florisil), diatomaceous earth (Celite), and macroreticular resin. Reference 29 includes silica gel column chromatographic clean-up methods for the aflatoxins in different commodities. Silica gel clean-up columns have also been used for patulin (PAT) in apple juice, zearalenone (ZEA) in maize, and sterigmatocystin (STE) in wheat and barley. Other column materials have been employed in some mycotoxin methods, but they have not found so widespread use as silica gel. The normal particle size range in traditional gravity column chromatography is 63-200 pm. In flash chromatography (30), where a slight gas (e.g. air, nitrogen) over-pressure is used to increase sample throughput, the optimum particle size is 40-63 pm. Smaller particles increase resistance to solvent flow, resulting in the need for pressurized systems. In addition to average particle size and particle size distribution, the other physical and chemical properties such as specific surface area, average diameter, pore diameter distribution, pore volume, packing density, pH, trace metal content, and activity, are important for adsorption chromatography. The specific surface area is particularly important because sample capacity is proportional to the total surface of the silica gel packed into the column. The activity of the adsorbent is also a very important consideration. It can be controlled by the deliberate addition of a known amount of water to the dried adsorbent, usually in the range of 2-10% water by weight. The bonded-phase silicas (31) are also available for flash chromatography, which has the advantages over traditional column chromatography including moderate resolution, rapid separation times, and reduced band broadening. 3.3.2.2 Solvent system A preliminary screening by TLC is used to establish the optimum mobile phase system for use in adsorption chromatography. A solvent composition is chosen that gives good separation and moves the toxins of interest to an RF value of approximately 0.3 Mixtures of methanol and chloroform (3:97) have to 0.4. frequently been used as mobile phase for aflatoxins and ZEA in

42

several matrices: 5-10% acetone in dichloromethane has also been a good solvent composition for aflatoxins and STE in many matrices. Good results have been obtained on a column for trichothecenes using a solvent mixture of about 5 % methanol in dichloromethane. For acidic mycotoxins such as OCH A, CIT, etc., an acidic mobile phase (e.g. acetic acid-benzene, 1:9) has been used. Mixtures of ethyl acetate or chloroform with hexane have often been used for column chromatographic separations of the less polar mycotoxins. 3 . 3 . 2 . 3 Column packing techniques Classical column chromatography requires only a glass column and a suitable packing material: in addition, flash chromatography requires a flow controller valve. Sometimes a solvent reservoir is added to the top of the column to contain a larger volume of elution solvent. Figure 3.3 presents typical column configurations for classical column chromatography, and flash chromatography.

BleedPort

Needle valve

pressure

Flow controller

8

P Fig. 3.3. Typical all-glass equipment used for classical column chromatography and flash chromatography.

43

Depending on the amount of material to be purified, a column of appropriate diameter (usually 2 0 mm i.d.) is chosen. The adsorbent is supported by a glass frit or preferably a plug of glass wool placed at the bottom of the column (see Fig 3.3). Different methods can be used for column filling. The column can be filled to about 60-70% of its height with the solvent to be used in the separation. Thereafter adsorbent is added to the column in small increments through a filter funnel. The solvent is allowed to run out from the column at a rate not exceeding the addition rate of the adsorbent. This packing method is very time-consuming and may produce difficulties when large columns have to be packed. Alternatively, columns are dry-packed with gentle tapping of the side of the column, or slurry-packed by preparing a slurry of the adsorbent and the required mobile phase which is then poured into the column, and let to settle with the tap open. The column can be vibrated until the stationary phase has completely settled. In each case the column bed should be homogeneous and free of channels. If the column is not properly packed, the channels may result in irregular flow, leading to much band broadening, and the distorted bands are easily observed if coloured substances are chromatographed. In flash chromatography, the column is dry-packed with 15 cm of dry silica gel or bonded-phase silica. The column is then filled with solvent, and gas pressure ( - 2 0 psi) is used to push the air rapidly from the column (31). In many cases it is recommended that a 0.5-1.0 cm layer of anhydrous sodium sulphate is added to the top of the column to protect the adsorbent from water traces in the sample extract. 3.3.2.4 Fractionation and detection The solvent used to pack the column is drained off until it is just over the column bed. Next, the sample, dissolved preferably in a small volume of the mobile phase, is applied slowly and evenly with a pipette on the top of the column bed, and the column is refilled with the solvent. The sample band should be sharp at the top of the column. Some samples may not dissolve, however, in the mobile phase. In this case the sample is usually dissolved in a more polar solvent (solvent of greater elution strength in adsorption chromatography) that may affect column equilibration and may decrease resolution. In this case, the

44

sample dissolved in a more polar solvent is added to a small amount of column packing material and the solvent is removed. This is then packed on the top of the column bed. Alternatively, the sample, dissolved in a mobile phase, may be filtered through an adsorbent cake supported on a Buchner funnel, when the insoluble part of the sample remains on the adsorbent and the components of interest are eluted with the solvent. Excess of solvent is removed and the sample is now applied to the top of the column. If a less-polar sample solvent is used in adsorption chromatography, the sample is concentrated at the head of the column before elution begins. Suitable-sized fractions are collected either manually or with a fraction collector. Elution occurs in classical column chromatography at a flow rate of 0.5-5.0 ml/min depending on column dimensions. In flash chromatography, a single development with about 4 to 5 column volumes of the mobile phase is usually sufficient for simple separations. Elution occurs typically at a flow rate of 5 ml/min which is adjusted with a flow controller valve (see Fig. 3.3). Thus a typical flash chromatographic separation occurs very quickly, in 5-10 min. Elution of the mycotoxins can be monitored by a detuned UV or a refractive index detector. The components can also be determined at the end of the separation by spotting each collected fraction on a TLC plate using the techniques described in Chapter 2.

3.4

MINI-COLUMN CHROMATOGRAPHY One of the most widely used screening methods for certain mycotoxins (usually aflatoxins, OCH A, and ZEA) in contaminated samples before the examination by other analytical techniques has been mini-column chromatography. This technique, using the glass mini-column with an internal diameter of ca. 5 mm, is a special design of a classical column chromatography. Mini-column screening methods (32-43) include the following steps: extraction, purification of the extracts, concentration, and development on a mini-column for detection under UV light. These methods are rapid, simple, and require only little expertise and no sophisticated equipment. The mini-column methods are particularly useful for field analysis. The methods have limitations as well.

45

They are semi-quantitative, having a higher detection limit and less separation power, selectivity, and sensitivity than is obta ned by using HPLC. 3.4.1 Procedure The first mini-column method was introduced by Holaday (32) for detection of aflatoxins in peanuts. The column contained a 45-mm high silica gel layer between glass wool plugs. This column was dipped in the sample extract which drew up by capillary force. The sample components moved upwards in different extents with ascending solvent. After about 10-15 min the column was examined under UV light (365 nm). A blue fluorescent zone characteristic to aflatoxin was visible at the top of the column. Since then a number of refinements and improvements [e.g. by changing solvents (33,35,42) or the composition of the packing material] have been made in the Holaday method. In the method of Romer (36) descending chromatography with a mixture of chloroform and acetone is applied. In this method, packing of a mini-column contains successive zones of alumina, silica gel, and Florisil with calcium sulphate at both ends of the column and the packing materials are held in place by glass wool (Fig. 3.4). Calcium sulphate is a drier and the silica gel and neutral alumina perform clean-up functions.

-

Glass wool Calcium subhate

-

Calcium sulphate Glass wool

Fig. 3.4. Diagram of the Romer mini-column (36). The aflatoxins appear as a tight band at the top of

the

Florisil

46

layer, where they can be detected under UV light. By comparison of a sample column with a reference column containing a known amount of aflatoxin, it is possible to determine whether the sample column contains more or less aflatoxin than the standard column. of Romer was subjected to a The mini-column method collaborative study (36) and approved by the AOAC (44) as an official method for the detection of aflatoxins in the listed commodities (mixed feeds, corn, almonds, peanuts, peanut butter, pistachio nuts, cottonseed). The method does not distinguish between the different aflatoxins. Similar mini-column procedures to those for aflatoxins have been developed for some other fluorescing mycotoxins such as OCH A (37) and ZEA (39) in several commodities. The detection limits vary from 5-20 ng/g. 3.4.2 Illustrative example The mini-column screening method of Romer (36) for the detection of aflatoxins in a wide range of commodities contains the following steps. A 50-9 sample of a blended, ground commodity is extracted in a blender 3 min with acetone-water (85:15, v/v) and filtered. A 0.2 M sodium hydroxide and ferric chloride slurry is added to an aliquot of the filtrate and mixed. Thereafter basic cupric carbonate is added, mixed, and filtered. The acidified filtrate is extracted with chloroform. The chloroform phase is washed with 0.02 M sodium hydroxide. An aliquot of chloroform extract is transferred to a mini-column packed with calcium sulphate, Florisil, silica gel, neutral alumina, and calcium sulphate (see Fig. 3.4). The column is then developed with chloroform-acetone mixture (9:1, v/v) after which the column is viewed under a longwave UV light (365 nm). If aflatoxin is present, it can be seen as a blue or bluish-green fluorescent zone at the top of the Florisil layer. The mini-columns developed with an extract of aflatoxin-free commodity, and of aflatoxin-free commodity containing a known amount of aflatoxin are compared, and the aflatoxin level in the commodity can be estimated. 3.5

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 3.5.1 Introduction HPLC became available for the analysis of foodstuffs nearly twenty years ago. The first published HPLC application f o r

41

mycotoxin research dates from 1973 (45). Since then, the trend has been towards increased use of HPLC for the ultimate separation, detection, and quantification of the mycotoxins in foods, after sufficient clean-up to remove interferences that could give rise to false positives. The important advantages of HPLC are its ability to handle thermally labile, poorly volatile, non-volatile, polar, and ionic compounds. The high resolving power between chemically similar compounds, the speed, increased sensitivity, accuracy and precision of the method, and the variety of detection systems now available make modern HPLC a more suitable technique than other liquid chromatographic techniques. In addition, HPLC is a quantitative technique and is suited for on-line clean-up of crude sample extracts, and finally, it can be automated quite easily. HPLC has limitations as well. The cost of modern HPLC equipment is quite high, and wide experience is necessary for obtaining the best possible benefit from an HPLC system. It must be pointed out that TLC, in its most sophisticated form, is as expensive as a simple HPLC system. There is still no sensitive universal or ideal detector for HPLC. In certain areas sensitivity in HPLC is not of course a problem, but in mycotoxin analysis sensitivity may be a serious limitation. For example, trichothecenes have been analyzed to a limited extent by conventional HPLC; sensitivity is limited, because most of them have weak or only end-absorption in the UV range. The development of efficient and reliable interfaces allowing HPLC to be coupled on-line with mass spectrometry (MS) will do much to overcome a sensitivity problem. The reviews devoted to the HPLC of mycotoxins have been written by Scott (46), Shepherd (47), and Coker and Jones (7). This section will present the instrumentation and practice of the HPLC method in mycotoxin analysis. The basic theory and practical considerations of HPLC will not be covered in detail in this context, because several good books have appeared, and the reader is directed to these. For example, the general principles (48-50), practical HPLC (51), recent progress (52-53), method development (54) and optimization ( 5 5 , 5 6 ) , and troubleshooting ( 5 7 ) in HPLC have been presented in several books mentioned.

48

3.5.2 Instrumentation and practice Requirements for the basic components of HPLC systems suitable for the analysis of mycotoxins are discussed. In addition, the areas where development has recently taken place are pointed out. There is a wide range of modes of chromatography in HPLC that can be employed, making possible the chromatography of many different compound classes. In most mycotoxin analyses, however, it will be profitable to use one of the three primary HPLC methods: reversed-phase (RP), ion-pair (IP), or normal-phase (NP) chromatography. RP-HPLC is nowadays the most commonly used method, and it is a potential technique for multimycotoxin

analysis under gradient elution conditions ( 5 8 - 6 4 ) . 3.5.2.1 Instrumentation Modern liquid chromatographs, which vary widely in sophistication, can be assembled from modular units designed to work independently of each other, or purchased as a single, integrated unit. Each system has its own advantages and drawbacks. Integrated HPLC systems have the advantage of being easier to automate but the drawback of being less flexible. Microcolumn HPLC (micro-HPLC) ( 6 5 , 6 6 ) , which employs columns with internal diameter < 1.0 mm and requires miniaturization of the whole system (pumping system, injector, gradient former, detectors etc.), is left outside this discussion. Micro-HPLC still faces many problems, but promises to be a significant technique in the future because of its low solvent consumption, simpler interfacing with mass spectrometers, lower detection limits when using concentration-dependent detectors (e.g. UV-vis and fluorescence detectors). Solvent reservoir. The solvent reservoir must hold a volume of solvent adequate for repeated analyses and it must be inert with respect to the solvents used, varying from aqueous buffers to hydrocarbons, depending on the mode of chromatography. Air bubbles are the major cause of problems with LC pumps. Problems with dissolved air are usually encountered with protic solvents such as water and alcohols. The best way to avoid bubbles is to thoroughly degas the solvents by heating, application of vacuum or ultrasound, or by sparging with helium. The latter is the most effective and convenient degassing method (65). After initial (a few minutes) vigorous bubbling of helium

49

through the solvents the helium flow is reduced to a trickle during the use of the LC system. Pumping systems. The pump is one of the most important components in HPLC equipment, because its performance directly affects the reproducibility of retention times, quantitative analysis, and detector sensitivity. Various pumps are available, but nowadays reciprocating piston pumps provide the basis f o r most pumping systems. Many pump problems (seal leakage, check valve failure, air bubbles etc.) can be avoided by using appropriate solvents, filtering the solvents and samples, degassing the solvents, and carefully flushing out the buffers. The pump must produce a wide range of flow rates for solvent delivery suitable for the various HPLC modes. F o r analytical and semi-preparative applications, where columns are 10-25 cm long with 1.0 to 10 mm internal diameter (i.d.) and packed with 3-10 pm particles, most modern pumps are able to produce flow rates from as low as 0.01-0.05 up to 5.0-10 ml/min. Although most HPLC pumps operate at pressures of 300-400 bar, the operating pressures should be less than 50% of the maximum capability of the pump (typically < 100-150 bar). Injection devices. Sample introduction is one of the critical steps in HPLC: even the best column will produce a poor separation if injection is not carried out carefully. The most widely used method uses an injection valve (e.g. Rheodyne Model 7010, 7125, or 7410), allowing reproducible volumes to be injected and good quantitative analyses. The six-port valve is the basis of all sample injection valves. The sample is injected with a syringe into a loop (either external or internal) and the solvent flow from the pump is then diverted via the loop to push the sample into the column. Loops can be filled in two ways: complete filling and partial filling. When the complete filling injection technique is used the loop is filled entirely with sample. When the loop is completely filled with the mobile phase, a sample amount equal to the loop volume cannot displace the existing solvent but tends to mix with it. Thus the sample volume must be about five times the volume of the loop (e.g. 100 pl of sample f o r a 2 0 7 1 loop). This is important for quantitative analysis using an external standard method. With the partial loop injection technique, the loop can be partly filled with the sample following

50

the manufacturer's guide. The sample size should not exceed about 50-60% of the loop volume. Although the partial filling method is less precise than the complete filling method, it is necessary in cases where no sample should be lost. Sample size can be adjusted by changing the sample loop volume. For analytical work, the typical sample loop volume is 5, 10, 20, or 50 p l . For very small amounts of sample the special valve (e.g. Rheodyne Model 7413) with an internal loop capacity between 0.5 and 5 p1 can be used. For semi-preparative work sample loops of 1000-2000 pl are appropriate. Automated injection system (autoinjector) is generally an automated version of the six-port injection valve used for manual injections. With the change of precision syringes, the autoinjector offers a wide range of fully programmable injection volumes (from 0.1 to 250 pl). The 25-pl syringe provides the highest accuracy and precision, but even a 1-pl injection is possible from as little as 5 pl of total sample. For large volumes a 250-pl syringe can easily be substituted. The autoinjector can be combined with the autosampler permitting unattended injection of samples into the liquid chromatograph. Column hardware. Columns are available in numerous different configurations and from about a hundred different suppliers. The columns vary in dimensions. They are typically 5-25 cm long when micro-particulate stationary phases of 3-10 pm are used. Longer columns are not worth considering. Columns of internal diameter (i.d.) 1-5 mm are used for analytical purposes in conventional HPLC. Columns of i.d. 1-2 mm (sometimes called microbore columns) increase detectability and reduce solvent consumption. However, while useful for trace analysis when the sample amount is very limited, or for interfacing with detectors such as a mass spectrometer, microbore columns do not give better resolution than regular analytical columns of 4-5 nun i.d. Micro-LC columns (i.d. < 0.5 mm) meet some interest, but most commercial LC instruments are not suitable for the optimum use of such columns. Wider columns of i.d. 10-20 mm are used for semi-preparative work with milligram quantities of sample. Large-scale purifications are performed using preparative liquid chromatography equipment (e.g. Waters Prep LC-500) removing the need for scraping the bands from the preparative plates.

51

Most HPLC column blanks are made of stainless-steel tubes and have compression fittings of various design (e.g. Swagelock, Valco) and steel frits at either end of the column to retain the column packing material. Stainless-steel is resistant to the pressures in HPLC and relatively inert to chemical corrosion. Inert and corrosion resistant glass tubes of various constructions have special uses as column material. Frits made of porous glass, alumina/ceramic, or titanium are also available (66). Recently, less-expensive cartridge columns of metal, glass, or plastic have become popular, since they can be easily replaced, and in many cases only the replacement of the small guard column at the head of the column is necessary. In addition, the cartridge columns can be easily connected into longer lengths for a particular separation. A variation of the cartridge column system is based on radial compression. The radial compression columns, which have been available for many years from Waters Chromatography Division, are loosely packed in a polyethylene sleeve, which in use is placed in a column holder that compresses the column packing material to form a stable bed. Column temperature control. The role of temperature is usually neglected in HPLC, and most HPLC separations are carried out at ambient temperature without the aid of a column oven. Temperature control is nevertheless an important part of separation efficiency. Reproducible retention times and retention indexes (63,67) are possible only if the column temperature is constant. Furthermore, at elevated temperature the viscosity of the mobile phase is lower, which is important with high viscosity mobile phases like the aqueous mixtures used in RP work. In addition, the effect of temperature can be exploited as a means to alter selectivity, since temperature change has a different effect on the retention of compounds (63,67). Further, the column temperature, like the type and composition of the mobile phase, affects the spatial arrangement of the bonded groups of the stationary phase (68,69). The analysis of cyclosporin A with RP-HPLC has required the use of column temperatures as high as 70-80 O C (70), but NP-HPLC using cyano column has allowed reproducible determinations at column temperature of 53 OC (71). The detector signals the presence of sample components and

52

measures their concentration in the mobile phase by producing electric signals. These signals are then conveyed to the recorder and/or display where they are shown as a deviation from the baseline, proportional to the compound concentration. Electronic inteqrators are needed to measure the detector signal. Sample pretreatment equipments. A wide variety of very complex sample clean-up or pretreatment procedures prior to the final HPLC analysis of mycotoxins (see Sections 3.2 and 3.3) are mostly necessary. And these procedures are often laborious, time-consuming, and subject to error and may be a major source of the imprecision of the total assay. Therefore, more and more laboratories use automated robotic arms (e.g. Zymark) or sample pretreatment equipments (e.g. Varian AASP System) to facilitate extraction, clean-up, enrichment, and application of the sample to the HPLC column and to provide for more reliable analysis results (9,71,72). The automated sample pretreatment can be performed also by using column switching technique in which two or more columns in series are connected by a switching valve so that the on-line selective transfer of a fraction or fractions from one column to one or more secondary columns for further separation is possible (72, 73-75). For example, the determination of STE in feed has been performed by HPLC with column switching (76). Smith and Robinson (77) have described a semi-automated HPLC method using column switching for assay of cyclosporin. Computer. Except for instrument control and data acquisition and analysis, the computers may be used for method development (56,78). 3.5.2.2 Normal-phase HPLC When the stationary phase is more polar than the mobile phase, the HPLC mode is called normal-phase (NP) chromatography or often adsorption chromatography. NP-HPLC is carried out with inorganic solids such as silica or alumina and various polar bonded phases (e.g. cyano, amino, diol, nitro) which have been reviewed in many sources (79,80). The polar bonded phases, which are formed from silica particles by binding covalently various polar silanes to the silica surface, are slowly replacing the traditional silica and alumina as packing materials for use in NP-HPLC, although silica and alumina still find widespread use. Silica, for example, has the unique ability to separate isomers,

53 and it is widely used in preparative chromatography. The most commonly used polar bonded phases are aminoalkyl, cyanoalkyl, and 1,2-dihydroxypropyl propyl ether (diol) phases (Fig. 3.5) of which cyanoand amino-derivatized silicas are the most popular. Amino-phases are also weak anion-exchangers, and therefore careful control of pH is important for ionic compounds. The polar bonded phases can be listed in order of increasing polarity: cyano < diol < nitro < amino. In NP-HPLC the compounds are generally eluted in the order of increasing polarity of the compounds. NP-HPLC uses mixtures of organic solvents as mobile phase. Hexane (or pentane, heptane) is generally preferred as the weak solvent and dichloromethane, methyl t-butyl ether, ethyl acetate, or acetonitrile are used as the stronger ones. The strength of the mobile phase in NP-HPLC is increased by raising the proportion of the more polar component in the mixture. Snyder et al. (81) have presented a useful table which shows solvent strength values for some useful organic solvent mixtures for NP-HPLC. The water content of the mobile phase must be carefully controlled to allow to be obtained reproducible results with silica ( 8 2 ) . In NP-HPLC sample-solvent interactions are relatively weak whereas sample-adsorbent or solvent-adsorbent interactions are strong, leading to a different classification of solvent selectivity as compared to RP-HPLC (83,84). The polar interaction between sample molecules and functional groups on the surface of NP packing plays a major role in determining separation selectivity. It must be pointed out that marked differences have been shown in the selectivity between different normal phases (84).

Earlier, NP-HPLC methods, mostly performed on silica columns, were used in mycotoxin analysis [e.g. aflatoxins (85-91), ZEA (92,93), PAT ( 9 4 ) ] , but nowadays RP-HPLC methods are favoured. NP-HPLC methods are desirable for samples dissolved in non-polar organic solvents (e.g. ether or chloroform extracts). If RP separation is used, however, less polar organic solvents should be evaporated to dryness, and the residue redissolved in an appropriate aqueous organic mixture. NP-HPLC (best with silica) is the first choice for preparative scale HPLC, too. 3.5.2.3 Reversed-phase HPLC In the technique of reversed-phase high-performance liquid

54

chromatography (RP-HPLC) the mobile phase is more polar than the stationary phase which is the opposite of NP-HPLC. The RP mode accounts for approximately 70-80% (possibly up to 9 0 % ) of all HPLC separations performed. This popularity depends on the simplicity, versatility, and broad applicability of the method. It has become the method of choice for the analysis of samples ranging from pharmaceutical and drug compounds to environmental pollutants and even large biological molecules. Detailed information on the RP-method may be found in several publications (48,54,55,57,95-97) . The RP techniques have found wide applications in the mycotoxin field, because mycotoxins are a highly miscellaneous set of compounds representing a wide range of polarities and chemical structures and different acid-base properties. RP methods have traditionally employed hydrocarbon-like stationary phases with polar aqueous organic mobile phases. The interaction between solute molecules and the stationary phase depends primarily on dispersion forces (non-specific hydrophobic interactions) and therefore compounds are separated according to their relative hydrophobicity, the most polar compounds being eluted first and the non-polar compounds being retained longer. Although great strides have been made in elucidating the retention mechanism in RP-HPLC, it is still only partially understood, because it is a complex function of the stationary phase, mobile phase, and solute. Stationary phase. The most commonly used stationary phases for RP separations have been and still are C-18 bonded phases, followed by C-8 and shorter n-alkyl, phenyl, or cyanopropyl bonded phases (Fig. 3 . 5 ) . Silica has been the most widely used base material for the aforementioned phases ( 9 8 - 1 0 0 ) . A variety of different procedures have been reported for the synthesis of chemically bonded silica-based packing materials during the last 20 years. Recently, several authors have exhaustively reviewed the preparation and characterization of bonded phases ( 1 0 1 - 1 0 3 ) . One great problem has been, and still is, the difficulty of ensuring reproducibility of the retention properties and selectivities from one commercial RP column to another, and even from one batch to another of the same product. These retention differences occur mainly for polar, particularly basic, compounds.

55

Methyl

Amlno

Hexyl

Q y 3 SI-O-SI-(CH2)nNH2 AH3

Octyl

Q y 3 SI-O-SI-(CH2)7CH3 AH3

+

NIt rl le

'

CH3

d-0-d I-(C

H2) ,C-N

AH3

Octadecyl

Fig. 3.5. The most common silica-bonded stationary phases. Cationic bases can interact quite strongly with silanols by hydrogen-bonding or with ionized silanols by ion-exchange (see The numerous variables involved in the preparation of Fig. 3.6). the RP, starting from the silica itself and ending with the column packing process, explain the great variations between commercial columns from different manufacturers. A standard silica as starting material, standard bonding reaction conditions, a standard procedure to characterize the phase, and standard column packing and testing procedures would be the prerequisites for a high batch-to-batch and column-to-column reproducibility. Another problem of commercially available RP stationary phases is their stability. With silica-based packing it is possible to use mobile phases with a pH between 2 and 8, because silica is soluble at high pH and the Si-C bond binding the hydrocarbon chain to the silica becomes labile at very low pH values. The more pH stable RP stationary phases (from pH 1 to 13) can be made from polymeric resin, but some of these phases may swell or shrink in contact with organic solvents. The pH stability of silica-based RP phase can be enhanced by substituting two bulky sterically protective groups (e.g. isopropyl or t-butyl) for the dimethyl groups on the silicon atom of silane (104).

56

Walters (105) has classified C-18 columns on the basis of two predominant RP retention mechanisms (hydrophobic and silanophilic interactions). This classification scheme will assist in selecting columns with similar performance from among the large number of C-18 brands on the market.

R

I+ I

n

R-N--R

I+

R-N;-R

s

'

,I

7

bH

Fig. 3.6. Interactions of cationic bases with alkyl bonded stationary phase: (a) hydrophobic interactions, (b) ion-exchange, (c) hydrogen bonding. Mobile phase. The properties of some LC solvents are listed in Table 3.1. It is generally accepted that retention in RP-HPLC is mainly controlled by the mobile phase, with the stationary phase playing the secondary role. Optimum selectivity is usually achieved by finding the right composition for the mobile phase. The preferred organic solvents for RP-HPLC are methanol (MeOH), acetonitrile (ACN), and tetrahydrofurane (THF), used in binary, ternary, o r sometimes in quaternary combinations with water. Organic solvents are strong and water is a weak solvent. Solvent strength (= chromatographic elution power) and selectivity are the properties of greatest chromatographic interest. In RP-HPLC solvent strength increases with the decrease in polarity. A change from methanol to acetonitrile or THF can result .in significant selectivity changes for various sample solutes. The Snyder triangle (48,106,107) is a widely accepted aid for characterizing solvent selectivity. Snyder has described a scheme

for classifying common LC solvents according to their polarity or chromatographic strength ( P I values) and according to their relative ability to engage in proton acceptor, proton donor and strong dipole interactions ( = selectivity). Thus solvents having similar functionalities tend to fall within the same selectivity group (see Table 3.1) and should have similar selectivity, while solvents from different groups should exhibit different selectivity for a given separation. However, several discrepancies in the triangle approach have been observed for the experimentally determined selectivities of some solvents (108-110). TABLE 3.1 Selected properties of some LC solvents (48,106,107)

Viscosity

SC

solvent

eoa

-n-Pentane -n-Hexane i-octane

0.00

pfb

0.01 0.01 i-propyl ether 0.28 Ethyl ether 0.38 CNorofonn 0.40 Dichloranethane 0.42 Tetrahydrofuran 0.45 Acetone 0.56 Dioxane 0.56 Mhyl acetate 0.58 Acetonitrile 0.65 n-Propanol 0.82 0.82 i-Prapanol

-

Ethanol Methanol Water

0.88 0.95 Very

( R P ~ )

0.0 0.1 0.1 2.4 2.8 4.1 3.1 4.0 5.1 4.8 4.4 5.8 4.0 3.9 4.3 5.1 10.2

-

-

4.4 3.4 3.5

3.1 -

4.2 3.6 2.6 0.0

Boiling W point Cutoff

(m~a,20 OC) (OC)

(mn)

0.23 0.33 0.50 0.37 0.24 0.57 0.44 0.46 0.32 1.54 0.45 0.37 2.30 2.30 1.20 0.60

195 190 200 220 205 245 230 220 330 220 260 190 210 210 210 205 490

1.00

36 66 99 68 34.5 61 40 66 56 101 77 82 97 82 78 65 100

Selectivity group

-I I VIII V I11

VIa

VIa VIa VIb

I1 I1 I1 I1 VIII

large a Solvent strength parameter for liquid solid chrmtography (LSC) on & l d M (A1 0 )

solvent &?ty parameter calculated fran mhrscimeicierls ciata solvent strength weighting factor in W - H P X ;experimental value A more precise solvent strength parameter ST has been defined for RP systems. ST for any solvent systems can be calculated from equation 1.

s T = I :i si@ i where

ST

is

the

total

solvent

strength

of

the

mixture, Si

58

(Table 3.1) is the solvent weighting factor, and #i is the volume fraction of solvent in the mixture. Approximately equal total solvent strengths will provide equal capacity factors (k') for different solvent mixtures in RP-HPLC. Other factors being equal, ACN has the following advantages over methanol: higher solvent strength, lower viscosity, and lower UV cut-off. Isocratic elution is useful only when toxins with similar retention behavior are to be studied, whereas gradient elution (111-113) is effective for the separation of samples containing compounds with a wide variety range of retention times. Clearly for screening or monitoring of several mycotoxins the only feasible approach is gradient elution (58-64), where great strides have been made in equipment, materials, and a better understanding of the technique. In addition, gradient elution is a valuable technique in concentrating the analyte into a narrow band for more sensitive detection, and gradient elution data can be applied for developing a final isocratic separation. Mobile phase additives. Non-ionic compounds can usually be chromatographed in RP-HPLC in the absence of mobile-phase additives (acids, buffers, ion-pairing reagents, or triethylamine). Ionic or ionizable compounds (e.g. moniliformin, ochratoxins, CIT) are chromatographed by RP-HPLC using one of the two techniques, ion-suppression and ion-pair chromatography ( I P C ) . In the former case the pH of the mobile phase is adjusted to suppress the ionization, which means about 1.0-2.0 pH units below and above the pKa value for an acid and a base, respectively, bearing in mind the pH stability of the stationary phase. It is worth noting that the degree of dissociation of acids and bases is highly solvent dependent. For example, the apparent pKa value of organic acids increases markedly with the organic solvent concentration of the mobile phase (114,115). The pH of the mobile phase also controls the ionization of acidic silanol groups in the RP packing. The pH adjustment usually is performed by using acetic acid (AcOH), phosphoric acid, trifluoroacetic acid (TFA), or different buffers (e.g. sodium or potassium phosphate, ammonium acetate) as mobile phase modifiers. Phosphoric acid is often preferred to acetic acid because of its non-aggressive behavior against the column and liquid chromatographic equipment (116) and a low UV cut-off value of 195 nm. TEA is sometimes

59

added as a silanol blocker to the mobile phase when basic compounds are to be separated. An acidic mobile phase is essential to ensure elutions of the acidic mycotoxins, e.g. OCH A and CIT (63). IPC is frequently, however, a more useful alternative for samples containing ionic or ionizable compounds, particularly if In this technique a the compounds are strong acids or bases. buffer and a so-called ion-pairing reagent is added to an aqueous organic mobile phase. A buffer controls the pH and ion-pairing reagent provides more retention and higher selectivity as compared to the chromatography without these additives. Negatively charged ion-pairing reagents [e.g. alkyl (usually C-5 to C-10) sulphonates] are used for the separation of protonated bases (cations), whereas cationic agents (e.g. tetrabutyl ammonium ion, TBA) are used for the ion-pair separation of carboxylate or other anions (117). For example, moniliformin (118), tenuazonic acid and 3-acetyl 5-substituted pyrrolidine-2,4-diones (119) have been analyzed using ion-pair chromatography. Reporting retention data. There is yet no standard method of reporting retention data in HPLC. The methods most in use today are retention times (t,) , retention volumes (V,) , and capacity factors (k') (Eqn 2), which are all strongly sensitive to variations in the chromatographic parameters. Relative retention expressions such as relative retention times (r) (Eqn 3) and relative capacity factors (r') (Eqn 4) have been used for some HPLC systems (120-122). Capacity factors (k') and relative capacity factors (r') suffer from the need for requiring measurement of the dead time (to), because there is no generally accepted method among numerous suggestions (123-130) for measuring this parameter. In addition, the relative methods involving comparison with an appropriate internal standard, depend on agreement among laboratories which standard to select. As a result the development of retention data libraries for comparison and identification purposes has not proceeded very far. k'

=

(tR

- to)/to

r = tR ( x ) / ~ R ( ~ ~ )

[21 [31

60

An alternative method of reporting retentions relies on the use of an appropriate series of homologous compounds that form a retention index scale. Retention indexes have been widely used in GC but infrequently in HPLC. Some efforts have, however, been made toward establishing retention index scales allowing better reproducibility and documentation of retention data. Baker and Ma (131) made the first proposal for a retention index series suitable for RP-HPLC, studying 2-alkanones as index standard compounds. However, 2-alkanones have only a weak chromophore and they have only limited use as index standard compounds for UV detection. Smith (132,133) and Kuronen (134) later introduced 1-phenyl-1-alkanones (Fig. 3.7a) as retention index standard compounds for RP-HPLC, Smith in an isocratic solvent system and Kuronen in gradient elution conditions. Gradient elution is, however, more applicable in allowing indexes to be determined for compounds with a wide range of polarities in a single chromatographic run (59,63,134-137). Further, a new series of (Fig. homologues 1-[4-(2,3-dihydroxypropoxy)phenyl)]-l-alkanones 3.7b) has been synthesized and evaluated as retention index calibrants in RP-HPLC under gradient elution conditions with UV and DAD (59,63,67,136). This series meets most of the essential requirements for a good reference series and it can serve as index standards for more polar solutes than the 1-phenyl-1-alkanone series. It is worth noting that the cubic spline interpolation (138) is a more precise method than the polygon method in calculating gradient-programmed retention indexes of the solutes because of the non-linearity of the calibration data (67). The gradient-programmed index is a complex function of the experimental conditions. Chromatographic parameters with greatest effect on the reliability of the gradient-programmed indexes are the source of the RP columns, column temperature, the organic modifier of the eluent, the pH of the eluent with ionizable compounds, and the exclusion of those members of the index series strongly determining the shape of the interpolation curve (67,136). The RI system can be used for tentative identifications under specified chromatographic conditions on an interlaboratory basis. The use of retention indexes in RP gradient elution HPLC

61

has been applied for mycotoxins (58,59,62-64)

n=l-ll

n = 1-11

Fig. 3.1. Structures of the homologous series of (a) l-phenyl-lalkanones, (b) l-[4-(2,3-dihydroxypropoxy)phenyl]-l-alkanones. 3.5.2.4 Detection Several sensitive and selective detectors capable of detecting only certain types of compounds have been developed for HPLC, whereas the lack of a sensitive universal detector has been to date one serious limitation of the method. The ideal HPLC detector possessing high sensitivity, low minimum detectability, wide linear dynamic range, good linearity, predictable and fast response, capability of being unaffected by changes in temperature, mobile phase composition and flow rate, capability of detecting all solutes or having predictable specificity, and providing qualitative information on the detected peak will Requirements for perhaps never be developed (95,139-142). detectors naturally vary with a particular separation problem. By far the most commonly used detectors in mycotoxin analysis have been conventional UV-vis and fluorescence detectors. Fortunately, very many mycotoxins (except many trichothecenes) display characteristic and strong UV absorptions at useful wavelengths (1,143,144), In addition, several toxins (e.g. aflatoxins, ZEA, OCH A, CIT) are naturally fluorescent: this property has offered a sensitive alternative to the UV detector. And the powerful combination of chromatography and spectroscopic techniques has become a reality also in HPLC analysis of mycotoxins with the development of the diode array detector (DAD) (59,60,62,63) and many interfacing techniques, especially thermospray (TSP) and dynamic fast atom bombardment (dynamic FAB), allowing HPLC to be coupled on-line with MS (60,64,145,146) .

62

3.5.2.4.1 "Classical" detections Refractive index detector. As a monitor of the refractive index of the eluate, the RI detector is a universal detector responding to all sample types. The pure mobile phase has a specific refractive index which changes when any compound elutes. The detector senses this difference and non-selectively records all peaks. To operate properly the RI detector requires excellent temperature, solvent composition and flow control. It is not amenable to gradient elution. Under favourable conditions the detection limit is about 0.5 pg, and the newer differential refractometers may allow quantitation of as little as 100 ng of most compounds. The RI detector is useful in preparative separation and routine quality control where ultratrace analysis or gradient elution is not required. Earlier, RI detection has been applied to the analysis of T-2, HT-2, and diacetoxyscirpenol (DAS) trichothecenes, with a detection limit of approximately 1 pg (147,148). Conventional UV-vis detector. The UV-vis detector is the most commonly used detector type in HPLC. This is the result of the vast number of UV-absorbing compounds and the great versatility and the excellent convenience and ruggedness of the detector. It can be highly sensitive, has a wide linear range, is unaffected by temperature fluctuation, and is very suitable for gradient elution. UV-vis detectors can be used for quantitation at the low-nanogram level. A primary requirement for successful UV detection is that the mobile phase system has been selected for optical transparency. All compounds absorbing UV or visible light are detected. Molecules absorb at a wavelength above 200 nm provided that they contain one of the following: an aromatic ring, a carbonyl group, a double bond adjacent to an atom with a lone pair of electrons, two conjugated double bonds, bromine, iodine, or sulphur. These groups of compounds do not absorb to the same extent or at the same wavelength. The absorption intensities, measured by molar absorptivity ( E ) , and wavelength maxima are also affected by neighboring groups in the molecule. Absorption increases with increasing conjugated unsaturation. Compounds with higher molar absorptivity produce larger peaks than those with a small molar absorptivity when identical amounts of compounds are injected. It

63

is useful to know the UV spectra of the various sample components (both analytes and interfering compounds) before the analysis, because it is then possible to choose the best detection wavelength. The UV-vis spectra with molar absorptivity values of most known mycotoxins are available from the literature (1). Many mycotoxins display characteristic and strong UV absorptions, allowing the detection of about 1 ppb of toxins with molar absorptivities 1-2.lo4 lmol-lcm-l. The Type A trichothecenes, T-2, HT-2, NEO, and DAS lack conjugated unsaturation and exhibit only end-absorption near 200 nm which means that they have low UV sensitivity at useful wavelength ranges, and can be detected and identified by UV detector only when present in relatively high concentrations. The preparation of p-nitrobenzoate derivatives of the Type A trichothecenes reportedly makes possible their ultratrace analysis in foods with W detection at 254 nm (149). The presence of a conjugated carbonyl in the Type B trichothecenes, DON, NIV, and FUS-X, generates a characteristic UV absorption near 220 nm. The minimum weight wm of a compound (in pg) giving a reasonable absorbance of peak maximum can be calculated from the following equation (150): wm

=

1000 MW(k‘+l) (S/N)(No)L0’5/~LcdcN 2 0.5

where MW is the molecular weight of the compound, k’ is the capacity factor, S/N is the required signal-to-noise ratio (usually > 2), No is the detector baseline noise in absorbance units, L is the column length (cm), E is the molar absorptivity (lmol-lcm-l), Lc is the length of the detector flow cell (cm), dc is the internal diameter of the column, and N is the column plate number. The simplest fixed wavelength UV detectors contain as source a low pressure mercury lamp which emits a sharp line spectrum with a strong line at 253.1 nm (254 nm) . Thus they are limited in their applications allowing only sample molecules absorbing near 254 nm to be detected. UV-vis detectors with a medium pressure mercury lamp as source can offer more fixed wavelengths, including 254, 280, 312, 365, 436, and 546 nm. Variable wavelength UV-vis detectors are the most common

64

absorbance HPLC detectors. Some of them are true recording spectrophotometers which allow a UV-vis spectrum to be generated from an eluting peak trapped in the flow cell (stop-flow of wavelength technique). Others require manual selection (usually 1 9 0 - 3 5 0 nm or 1 9 0 - 7 0 0 nm), allowing selection of a wavelength to maximize sensitivity or remove interfering peaks, thereby improving the accuracy of quantitative determination. Fluorescence detector. Compounds that naturally fluoresce or that can be made to fluoresce through chemical derivatization can be detected with high selectivity and sensitivity by this detector. The fluorescence detector is generally about 1000 times more sensitive than the UV detector. Laser-induced fluorescence (LIF) detector, which is one of the most promising applications of laser-based detection in HPLC, can further improve detectability even to femtogram level. High selectivity means that the compounds of interest can be readily distinguished from a complicated matrix of compounds that do not fluoresce. Both fixed wavelength and scanning fluorescence units are available. Fluorescence detectors can be used with gradient elution. The fluorescent compound being analyzed is excited by W radiation at UV maximum wavelength and the fluorescence energy emitted at a longer wavelength is detected. The intensity of the fluorescence and the position of the excitation/emission wavelength maxima depend on the mobile phase composition, pH, temperature, and dissolved gas content (particularly oxygen) of the mobile phase ( 9 7 ) . For example, halogenated solvents such as chloroform tend to quench or reduce dichloromethane or fluorescence. For example, the aflatoxins, STE, ZEA, ochratoxins, and CIT exhibit significant native fluorescence when subjected to UV irradiation. Under optimum conditions fluorescence detection is about 30-40 times more sensitive than UV detection for aflatoxins. The influence of chromatographic conditions on fluorescence intensity of mycotoxins have been discussed in the literature (7,45-46). Orti et al. (151) have given an excellent examination of chromatographic and spectroscopic properties of hemiacetals of aflatoxins and sterigmatocystins. Picogram quantities of the aflatoxins may be detected by fluorescence detection under appropriate conditions. The sensitivity of the fluorescence

65

detection of the aflatoxins can be further enhanced by several The aflatoxins can be converted to more techniques ( 7 , 4 5 , 4 6 ) . intensely fluorescent derivatives by using pre-column derivatization with TFA (7,45,46,152) or post-column derivatization with iodine or bromine ( 1 5 3 - 1 5 6 ) . A flow cell packed with silica may be used to intensify the fluorescence of the aflatoxins under normal-phase conditions ( 7 , 4 5 , 4 6 ) . In addition, the use of LIF detection enhances the sensitivity. Electrochemical detector. The electrochemical detector (ECD) provides a useful and highly selective and sensitive tool for the detection of readily oxidizing and reducing organic compounds. Examples of compounds that can be detected in oxidation mode are phenols, aromatic and aliphatic amines, thiols, thioketones, and thioethers. Aromatic nitro compounds, amides, oximes, alkyl and aryl halogen compounds, quinones, and amides are suitably detected by electrochemical reduction. Electrochemical reactions occur at the surface of a solid electrode which removes electrons for oxidation and supplies them for reduction. The operating potential of the ECD is set and the current due to oxidation or reduction is measured. The potential applied to the detection can be adjusted to allow discrimination between different electroactive compounds. The majority of applications are in the oxidation mode because dissolved oxygen in the mobile phase and the presence of heavy metals tend to cause problems in the reduction mode. ECD requires the use of a conducting mobile phase, containing aqueous organic (e.g. MeOH, ACN) mixtures and inorganic salts or acids (acetic acid, phosphoric acid), conditions which are compatible with RP-HPLC. These detectors are capable of femtomole sensitivity. ECD has been utilized in the analysis of roquefortine in blue cheese ( 1 5 7 ) and zearalenones in cereals (158) and edible animal tissue ( 1 5 9 ) . 3 . 5 . 2 . 4 . 2 Diode array detection. has The diode array detector (DAD) ( 1 4 0 - 1 4 2 , 1 6 0 - 1 6 4 ) established itself as a powerful LC detector during the last years. DAD uses a photodiode array (e.g. 2 5 6 elements) to detect many wavelengths simultaneously making it possible to provide both multlwavelength chromatographic and spectral information in a single chromatographic run, which makes DAD ideal for the screening and preliminary identification of mycotoxins

66

(59,60,62,63). The newest DAD instruments have competitive sensitivity with other UV detectors. Many graphical and numerical strategies have been developed for the presentation and analysis of the data (161,165,166). The most important capabilities of the DAD are the following: (1) on-the-fly UV-vis spectral scanning; (2) three-dimensional plots; (3) two-dimensional contour plots; (4) UV-spectral overlays; (5) absorbance ratioing (purity parameter) and absorbance ratio plots; (6) derivative spectra; and ( 7 ) recording of chromatograms simultaneously at several wavelengths. The most used function of the DAD is the generation of the on-the-fly UV-vis spectra of separated compounds. Comparison of peak spectra with reference spectra in the library can be used to confirm peak identity. UV-vis spectra can also aid in the identification of unknown peaks, or allow determination of at least the class of the compound. Figure 3.8 presents the on-line UV spectra of some mycotoxins produced with DAD under gradient elution RP-HPLC conditions (63). The spectra are practically identical with those produced off-line with UV-vis spectrophotometers and published in the literature (1,143,144). Insignificantly small shifts (-1-2 nm) are found in some cases. The UV-vis spectrum very often provides little structural information. Therefore qualitative features of the UV-vis spectra can be enhanced by generation of derivatives of the spectra. Verification of the peak homogeneity is provided by the coincidence of UV spectra taken at several points of the eluting peak (usually upslope, apex, and downslope). The three-dimensional presentation of wavelength ( h )-time (t)-absorbance (A) data ( = 3-D plot) is useful in selecting the optimal wavelength for detection sensitivity and selectivity. The two-dimensional contour plot of the absorbance contours in the wavelength-time plane gives symmetrical contours for pure peaks and skewed contours indicate co-elution. Absorbance ratios can also be used for peak homogeneity determinations requiring, however, carefully chosen wavelengths (167), which is easy only for known samples but very difficult if there are unknown impurities. Chromatograms can also be recorded simultaneously at several wavelengths, enabling resolution of co-eluted compounds and making it possible to screen the whole UV-vis region during an analysis, with no UV absorbing compound undetected.

67

Less chromatographic resolution is required with this multichannel detection than with the single-channel UV detector. Qualitative and quantitative analysis is possible f o r moderately overlapping spectra and chromatographic peaks with sophisticated data handling methods. A RP-HPLC gradient elution method has been applied as a multimycotoxin screening method where mycotoxins were characterized using retention indexes based on the 1-phenyl-1-alkanone ( 6 2 ) and l-[4-(2,3-dihydroxypropoxy)phenyl]-lalkanone (63) series and UV-vis spectral data produced with the DAD.

90%LAUl

0.

\

0-

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18

9 0 % - , 7

18

.o

J

289. 0

389.0

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. .

.

,

289.0

J

389.0

W o v e 1e n g t h

C n m l

W a v e l e n g t h

C,im3

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W a v e 1ength

Cnm3

68

90%-

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MU1

STE

-\‘I;

289.0

184.0

389.0

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0-10.0184.0

289.0 W a v e 1 ensth

Cnml

389.0 Cnrnl

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289.0

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Fig. 3.8. The UV spectra of some mycotoxins recorded from 190 to 400 nm with the diode array detector at 50 OC on LiChrosorb Hibar RP-18 column (5 pm, 2 5 0 ~ 4 . 0m m ) . Linear gradient from 20% ACN-HZO (pH=2.5) to 100% ACN in 40 min at the flow rate of 1 ml/min. 3.5.2.4.3 Mass spectrometry detection and identification

The combination of liquid chromatography and spectrometry (LC-MS) is an important technique that offers

mass high

69

sensitivity and selectivity in the analysis of a wide variety of compounds that is difficult or impossible with GC-MS - such as the analysis of many mycotoxins. The most important difference between MS and other LC detectors is its ability to provide structural and molecular weight information. Connecting MS to LC requires an interface device that will convert the liquid phase containing the analyte(s) to a gas phase in the presence of vacuum. The problems associated with interfacing the LC to the MS have been much greater than for the GC. Both quadrupole and magnetic sector MS instruments have been used for LC-MS. Recent developments in LC-MS interfaces have increased the reliability of the technique. Several interfacing techniques have been developed during the last few years including moving belt interface, direct liquid introduction (DLI) interface, thermospray (TSP) interface, particle beam interface, electrospray and atmospheric pressure type interfaces, monodisperse aerosol generation (MAGIC) interface, and dynamic fast atom bombardment (FAB) interfacing (168-171). All these techniques have their own strengths and weaknesses, depending on the LC-MS equipment and the results desired. The two interface techniques, TSP and FAB, offer most potential today and they are already routinely used in several laboratories. The popularity of the TSP interface is largely due to the fact that the total eluent (typically 1-2 ml/min) can be introduced into the ion source, the ionization of the sample may be obtained without the use of a filament, and its ability to operate under RP-HPLC conditions, working best with mobile phases containing a high proportion of water at flow rates between 0.5 and 2 . 0 ml/min (171), when many interfaces begin to fail. In TSP LC-MS, the mobile phase containing the separated analytes is introduced into the MS through a stainless steel capillary tube, which is directly heated by passing a current through it. The mobile phase i s converted by careful temperature regulation into a mist of droplets and carried into the ion source as a supersonic vapour jet. Excess of solvent vapour is removed by an extra vacuum pump, The detectable ions are usually produced by using filament-off ionization ( = buffer ionization). Volatile buffer, generally ammonium acetate, which is added to the mobile phase at a concentration of about 0.05 to 1.0 M, acts as the reagent gas

70

and produces CI-type MS spectra. These spectra usually give pseudomolecular ions [e.g. (M+H)+ and/or (M+NH4)+, in positive ion mode] together with a small degree of fragmentation limiting specificity and giving only little structural information. In the negative ion mode negative ions may be formed by proton abstraction or anion attachment. The amount of each ion species formed depends on the proton affinity of the gaseous analytes (gas-phase acidity). On the other hand little fragmentation may be an advantage when quantitating analytes in the selected ion mode, which produces greater sensitivity than the scanning mode. Furthermore, the pH can be adjusted using either ammonia or acetic acid, and volatile ion-pair reagents can also be used. A buffer ionization mode (pure TSP CI) cannot be used for low-polarity solvents of normal-phase LC-MS. In the cases where the analytes are not readily ionized, or NP-HPLC is used, some TSP devices include an electron filament and discharge ion source to assist in ionization or to make possible the use of normal-phase solvents. In addition, some TSP interfaces may also have adjustable fragmentor electrodes, which produce molecular fragments by increasing the rate of intermolecular collisions, therefore being useful for structure elucidation. Further, the combination of HPLC with MS-MS instrumentation, which is capable of fragmenting molecular ions into structurally significant daughter fragments under collision with an inert gas, can be used for structural studies. The response and sensitivity of TSP LC-MS using filament-off, filament-on, or discharge-on CI is very compound-dependent, and can be affected by several physical ionization factors. FAB has been an alternative ionization technique for several years. Nowadays dynamic FAB systems are available for use as interfaces in LC-MS. The flow rates in this system are in the order of 1-5 pl/min, and therefore use of microbore columns or post-column splitting before MS are necessary. Furthermore, the system requires the matrix (4-10% glycerol) in the mobile phase. The matrix can be added either to the mobile phase or using a post-column addition. The latter method has been found to have no significant effect on the retention time but may cause peak broadening (64). An argon or xenon molecular beam is used in the bombardment.

71

TSP LC-MS (60,64,145,146) have proven very useful for the

and also dynamic FAB LC-MS (64) analysis of a wide range of

mycotoxins; both being applicable as multimycotoxin methods. CONCLUSION HPLC plays an important role in the analysis of mycotoxins. It is a powerful analytical technique being able to separate a wide range of mycotoxins, being quantitatively precise, and in many cases a very sensitive technique. It demands, however, a good understanding of the problems involved in the application of this technique to the food analysis in order to produce reliable and accurate data. Mini-column chromatographic methods, especially developed for aflatoxins, are particularly useful for field analysis and as screening tests for agricultural commodities when rapid decisions have to be made for accepting or rejecting a lot. Efficient extraction and clean-up of the samples are very critical to successful HPLC. The purity of the residue obtained from the sample pretreatment will have a major influence on both detection sensitivity and degree of confidence in the result. Earlier, classical open column chromatographic methods have been widely used as a preliminary clean-up for trace analysis of mycotoxins by HPLC, but nowadays the replacement of classical laboratory-packed glass columns by commercially available cartridge clean-up columns has greatly simplified sample purification, making possible higher reproducibility between different laboratories. The dedicated TSP and FAB LC-MS instruments can now provide a powerful technique to the analysis of mycotoxins. In addition, the reliability of the detection can be greatly improved by the use of retention indexes, which offer an independent identification, additional to the data produced by DAD or the MS-data. In the near future the completely automated, unattended HPLC assay of mycotoxins, starting from the sample extraction and ending with the identification of the toxins and the calculation of the quantitative results, in one operation, will become a reality. 3.6

72

REFERENCES 1 R.J. Cole and R.H. Cox, Handbook of Toxic Fungal Metabolites, Academic Press, New York, 1981. W.B. Turner and D.C. Aldridge, Fungal Metabolites 11, 2 Academic Press, London, 1983. D.H. Watson, CRC Crit. Rev. Food Sci. Nutr., 22 (1985) 117. 3 C.W. Hesseltine, in: P.S. Steyn and R. Vleggaar (Eds.), My4 cotoxins and Phycotoxins, Elsevier, Amsterdam, 1986, p. 1. P.M. Scott, in: K. Robinson (Ed.), Developments in Food 5 Microbiology, Vol. 4, Elsevier, Amsterdam, 1988, p. 47. Y. Ueno, CRC Crit. Rev. Toxicol., 14 (1985) 99. 6 7 R.D. Coker and B.D. Jones, in: R. Macrae (Ed.), HPLC in Food Analysis, Academic Press, London, 1988, p. 335. 8 R.E. Majors, LC-GC, 4 (1986) 972. R.E. Majors, LC-GC Int., 2(2) (1989) 12. 9 J.E. Thean, D.R. Lorenz, D.M. Wilson, K. Rodgers, and 10 R.C. Gueldner, J. Assoc. Off. Anal. Chem., 63 (1980) 631. 11 J.D. McKinney, J. Am. Oil Chem. SOC., 58 (1981) 935A. J.E. Hutchins and W.M. Hagler, J. Assoc. O f f . Anal. Chem., 12 66 (1983) 1458. W.J. Hurst, F.P. Snyder, and R.A. Martin, Peanut Sci., 11 13 (1984) 21. D. Tosch, A.E. Waltking, and J.F. Schlesier, J. Assoc. Off. 14 Anal. Chem., 67 (1984) 337. G.-S. Gian and G.C. Yang, J. Agric. Food Chem., 32 (1984) 15 1071. D.L. Orti, R.H. Hill, Jr., J.A. Liddle, L.L. Needham, and 16 L. Vickers, J. Anal. Toxicol., 10 (1986) 41. 17 N. Takeda, J. Chromatogr., 288 (1984) 484. H. Cohen and M. Lapointe, J. Assoc. Off. Anal. Chem., 69 18 (1986) 957. 19 H. Terada, H. Tsubouchi, K. Yamamoto, K. Hisada, and Y. Sakabe, J. Assoc. Off. Anal. Chem., 69 (1986) 960. K.C. Ehrlich and L.S. Lee, J. ASSOC. O f f . Anal. Chem., 67 20 (1984) 963. 21 S.H. Hoke, C.M. Carley, E.T. Johnson, and F.H. Broski, J. Assoc. Off. Anal. Chem., 70 (1987) 661. 22 T.R. Romer, D.E. Greaves, and G.E. Gibson, presented at 6th Annual Spring Workshop of the AOAC, Ottawa, Ontario, Canada, May 12-14, 1981. 23 R.E. Eppley, M.W. Trucksess, S. Nesheim, C.W. Thorpe, and A.E. Pohland, J. Assoc. Off. Anal. Chem., 67 (1984) 43. H.L. Chang, J.W. DeVries, P.A. Larson, and H.H. Patel, 24 J. ASSOC. Off. Anal. chem., 67 (1984) 52. T.R. Romer, J. ASSOC. Off. Anal. Chem., 69 (1986) 699. 25 C.P. Gorst-Allman and P.S. Steyn, in: V. Betina (Ed.), 26 Mycotoxins - Production, Isolation, Separation, and Purification. Developments in Food Science, Vol. 8, Elsevier, Amsterdam, 1984, p. 59. 27 W.C. Brumley, S. Nesheim, M.W. Trucksess, E.W. Trucksess, P.A. Dreifuss, J.A.G. Roach, D. Andrzejewski, R.M. Eppley, A.E. Pohland, C.W. Thorpe, and J.A. Sphon, Anal. Chern., 53 (1981) 2003. W.C. Brumley, M.W. Trucksess, S.H. Adler, C.K. Cohen, 28 K.D. White, and J.A. Sphon, J. Agric. Food Chem., 33 (1985) 326. L. Stoloff and P.M. Scott, in: S. Williams (Ed.), Official 29 Methods of Analysis, 14th ed., Association of Official Ana-

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59

lytical Chemists, Washington, D.C., 1984, p. 477. W.C. Still, M. Kahn, and A. Mitra, J. Org. Chem., 43 (1978) 2923. R.E. Majors and T. Enzweiler, LC-GC Int., 2(3) (1989) 10. C.E. Holaday, J. Am. Oil Chem. SOC., 45 (1968) 680. J. Velasco, J. Am. Oil Chem. SOC., 49 (1972) 141. P. Hald and P. Krogh, J. Assoc. Off. Anal. Chem., 58 (1975) 156. C.E. Holaday and J. Lansden, J. Agric. Food Chem., 23 (1975) 1134. T.R. Romer, J. Assoc. Off. Anal. Chem., 58 (1975) 500. T.R. Romer and A.D. Campbell, J. Assoc. Off. Anal. Chem., 59 (1976) 110. C.E. Holaday, J. Am. Oil Chem. SOC., 53 (1976) 603. T.R. Romer, N. Ghouri, and T.M. Boling, J. Am. Oil Chem. SOC., 56 (1979) 795. C.E. Holaday, J. Am. Oil Chem. SOC., 57 (1980) 491A. C.E. Holaday, J. Am. Oil Chem. SOC., 58 (1981) 931A. O.L. Shotwell and C.E. Holaday, J. Assoc. Off. Anal Chem., 64 (1981) 674. R.B. Sashidhar, R.B. Bhat, and S. Vasanthi, Curr. Sci., 58 (1989) 882. S . Williams (Ed.), Official Methods of Analysis, Association of Official Analytical Chemists, Washington, D.C., 1984. J.W. Seiber and D.P.H. Hsieh, J. ASSOC. Off. Anal. Chem., 56 (1973) 827. P.M. Scott, in: J.F. Lawrence (Ed.), Trace Analysis, Vol. 1, Academic Press, New York, 1981, p. 193. M.J. Shepherd, in: R.J. Cole (Ed.), Modern Methods in the Analysis and Structure Elucidation of Mycotoxins, Academic Press, New York, London, 1986, p. 293. L.R. Snyder and J.J. Kirkland, Introduction to Modern Liquid Chromatography, 2nd ed., John Wiley and Sons, Inc., New York, 1979. M.T. Gilbert, High Performance Liquid Chromatography, Wright, Bristol, 1987. P.R. Brown and R.M. Hartwick (Eds.), High Performance Liquid Chromatography, Wiley, New York, 1989. V.R. Meyer, Practical High Performance Liquid Chromatography, Wiley, Chichester, 1988. C. Horvath (Ed.), High Performance Liquid Chromatography, Advances and Perspectives, Vol. 5., Academic Press, San Diego, 1988. J.C. Giddings, E. Grushka, P. Brown (Eds.) , Advances in Chromatography, Vol. 28, Marcel Dekker, New York, 1989. L.R. Snyder, J.L. Glajch, J.J. Kirkland, Practical HPLC Method Development, Wiley, New York, 1988. S. Ahuja, Selectivity and Detectability Optimization in HPLC, Wiley, New York, 1989. J.C. Berridge, Techniques for the Automated Optimization of HPLC Separations, Wiley, New York, 1985. J.W. Dolan and L.R. Snyder, Troubleshooting LC Systems, Humana Press, Clifton, 1989. D.W. Hill, T.R. Kelley, K.J. Langner, and K.W. Miller, Anal. Chem., 56 (1984) 2576. P. Kuronen, in: M. Rautio (Ed.), Systematic Identification of Mycotoxins, B . 5 . Identification of Selected Trichothe-

14

60 61 62 63 64 65 66 67 68 69 70 71 72 13 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

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97 98 99 100 101

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C.F. Poole and S.A. Schuette, Contemporary Practice of Chromatography , El sevier , Amsterdam , 1984 . K.K. Unger, Porous Silica, Its Properties and Use as Support in Column Liquid Chromatography, J. Chromatogr. Library, Vol. 16, Elsevier, Amsterdam, 1979. H. Engelhardt and H. Elgass, in: C. Horvdth (Ed.), High-Performance Liquid Chromatography, Advances and Perspectives, Vol 2., Academic Press, New York 1980, p. 57. K.K. Unger and B. Anspach, Trends Anal. Chem., 6 (1987) 121. L.C. Sander and S.A. Wise, CRC Crit. Rev. Anal. Chem., 18 (1987) 299. P.J. Van den Driess, H.J. Ritchie, and S. Rose, LC-GC, 6 (1988) 124. J. Nawrocki and B. Buszewski, J. Chromatogr., 449 (1988) 1. J.J. Kirkland, J.L. Glajch, R.D. Farlee, Anal. Chem. , 61 (1989) 2. M.J. Walters, J. Assoc. Off. Anal. Chem., 70 (1987) 465. L.R. Snyder, J. Chromatogr., 92 (1974) 223. L.R. Snyder, J. Chromatogr. Sci., 16 (1978) 223. J.J. Lewis, L.B. Rogers, and R.E. Pauls, J. Chromatogr. , 264 (1983) 339. S.D. West, J. Chromatogr. Sci., 25 (1987) 122. S.D. West, J. Chromatogr. Sci., 27 (1989) 2. Ref.48, Chapter 16. P . Jandera and J. Churbcek, Gradient Elution in Column Liquid Chromatography, J. Chromatogr. Library, Vol. 31, Elsevier, Amsterdam, 1985. L.R. Snyder and M.A. Stadalius, in: C. Horvdth (Ed.), High Performance Liquid Chromatography, Advances and Perspectives, Vol. 4., Academic Press, New York, 1986, p. 195. G. Vigh, 2 . Varga-Puchony, A. Bartha, and S. Balogh, J. Chromatogr., 241 (1982) 169. D. Palalikit and J. H. Block, Anal. Chem., 52 (1980) 624. R. Schwarzenbach, J. Chromatogr. , 251 (1982) 339. A.P. Goldberg, E. Nowakowska, P.E. Antle, and L.R. Snyder, J. Chromatogr., 316 (1984) 241. M.J. Shepherd and J. Gilbert, J. Chromatogr., 358 (1986) 415. M.H. Lebrun, F. Gaudemer, M. Boutar, L. Nicolas, and A. Gaudemer, J. Chromatogr. , 464 (1989) 307. R. Gill, A.C. Moffat, R.M. Smith, and T.G Hurdley, J. Chromatogr. Sci., 24 (1986) 153. R. Gill, M.D. Osselton, R.M. Smith, and T.G. Hurdley, J. Chromatogr., 386 (1987) 65. R.M. Smith, T.G. Hurdley, R. Gill, and M.D. Osselton, J. Chromatogr., 398 (1987) 73. A.M. Krstulovic, H. Colin, and G. Guiochon, Anal. Chem., 54 (1982) 2438. H. Engelhardt, H. Muller, and B. Dreyer, Chromatographia, 19 (1984) 240. G.E. Berendsen, P.J. Schoenmakers, L. de Galan, E. Vigh, 2 . Varga-Puchony, and J. Inczedy, J. Liq. Chromatogr., 3 (1980) 1669. M.J.M. Wells, and C.R. Clark, Anal. Chem., 53 (1981) 1341. O.A.G.J. Van der Houwen, J.A.A. van der Linden, and A.W.M. Indemans, J. Liq. Chromatogr., 5 (1982) 232. K. Jinno, Chromatographia, 17 (1983) 367. R.M. McCormick and B.L. Karger, Anal. Chem., 52 (1980) 2249.

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130 131 132 133 134

135

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137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156

P.J.M. Van Tulder, J . P . Franke, and R.A. de Zeeuw, J. High Resolut. Chromatogr. Chromatogr. Comm., 10 (1987) 191. J.K. Baker and C.-Y. Ma, J. Chromatogr., 169 (1979) 107. R.M. Smith, J. Chromatogr., 236 (1982) 313. R.M. Smith, in: J.C. Giddings, E. Grushka, and P.R. Brown (Eds.), Advances in Chromatography, Vol. 26, Marcel Dekker, New York, 1987, p. 277. P. Kuronen, in: J. Enqvist and A. Manninen (Eds.), Systematic Identification of Chemical Warfare Agents, B.3. Identification of Non-PhosphOrus Warfare Agents, the Ministry for Foreign Affairs of Finland, Helsinki, 1982, p. 43. P. Kuronen, in: J. Enqvist and A. Manninen (Eds.), Systematic Identification of Chemical Warfare Agents, B.4. Identification of Precursors of Warfare Agents, Degradation Products of Non-Phosphorus Agents, and Some Potential Agents, the Ministry for Foreign Affairs of Finland, Helsinki, 1983, p. 51. P. Kuronen, in: M. Rautio (Ed.), Air Monitoring as a Means for Verification of Chemical Disarmamemnt, C.2. Development and Evaluation of Basic Techniques, Part I, the Ministry for Foreign Affairs of Finland, Helsinki, 1985, p. 162. P. Kuronen, Proc. 2nd Int. Symp. Protection Against Chemical Warfare Agents, National Defence Research Institute, NBS Research Dept., Umea, 1986, p. 261. W.A. Halang, R. Langlais, and E. Kugler, Anal. Chem., 50 (1978) 1829. Ref. 48, p . 125. R.P.W. Scott, Liquid Chromatography Detectors, J. Chromatogr. Library, Vol. 33, 2nd ed., Elsevier, Amsterdam, 1986. Ref. 55, p. 505. P.C. White, Analyst, 109 (1984) 667; 973. A.E. Pohland, P.L. Schuller, and P.S. Steyn, Pure Appl. Chem., 54 (1982) 2219. V. Betina (Ed.), Mycotoxins - Production, Isolation, Separation, and Purification. Developments in Food Science, Vol. 8, Elsevier, Amsterdam, 1984, pp. 87-485. R.D. Voyksner, W.M. Hagler, J r . , K. Tyczkowska, and C.A. Haney, J . High Resolut. Chromatogr. Chromatogr. Commun., 8 (1985) 119. R.D. Voyksner, W.M. Hagler, Jr., and S.W. Swanson, J. Chromatogr., 394 (1987) 183. R. Schmidt, E. Ziegenhagen, and K. Dose, J. Chromatogr., 212 (1981) 370. R. Schmidt and K. Dose, J. Anal. Toxicol., 8 (1984) 43. R. Maycock, and D. Utley, J. Chromatogr., 347 (1985) 429. Ref. 54, p. 87. D.L. Orti, J. Grainger, D.L. Ashley, and R.H. Hill, Jr., J . Chromatogr., 460 (1989) 269. D.L. Park, S. Nesheim, M.W. Trucksess, M.E. Stack, and R.F. Newell, J. Assoc. Off. Anal. Chem., 73 (1990) 260. H. Jansen, R. Jansen, U.A.T. Brinkman, and W. Fsei, Chromatographia, 24 (1987) 555. W.J. Hurst, F.P. Snyder, and R.A. Martin, J. Chromatogr., 409 (1987) 413. J.W. Dorner and R.J. Cole, J. Assoc. Off. Anal. Chem., I1 (1988) 43. W.T. Kok, T.C.H. van Neer, W.A. Traag, and

157 158

L.G.M.T. Tuinstra, J. Chromatogr., 3 6 3 ( 1 9 8 6 ) 2 3 1 . G.M. Ware, C.W. Thorpe, and A.E Pohland, J. Assoc. Off. Anal. Chem., 6 3 ( 1 9 8 0 ) 6 3 7 . M.R. Smyth and C.G.B. Frischkorn, Anal. Chim. Acta, 1 1 5 (1980) 293.

159 160 161 162

J.G. Roybal, R.K. Munns, W.J. Morris, J.A. Hurlbut, and W. Shimoda, J. Assoc. Off. Anal. Chem., 7 1 ( 1 9 8 8 ) 2 6 3 . D.G. Jones, Anal. Chem., 57 ( 1 9 8 5 ) 1 0 5 7 A . D.G. Jones, Anal. Chem., 57 ( 1 9 8 5 ) 1 2 0 7 A . T. Alfredson and T. Sheehan, J. Chromatogr. Sci., 2 4 ( 1 9 8 6 ) 473.

163 164 165 166 167 168 169 170 171

G.W. Schieffer, J. Chromatogr., 3 1 9 ( 1 9 8 5 ) 3 8 7 . Ref. 57, p . 3 2 1 . T. Alfredson, T. Sheehan, T. Lenert, S. Aamodt, and L. Correia, J. Chromatogr., 3 8 5 ( 1 9 8 7 ) 2 1 3 . S . Ebel and W. Mueck, Chromatographia, 2 5 ( 1 9 8 8 ) 1 0 3 9 . H. Cheng and R.R. Gadde, J. Chromatogr. Sci., 2 3 ( 1 9 8 5 ) 2 2 7 P.A. Ireland, in: R. Macrae (Ed.), HPLC in Food Analysis, Academic Press, London, 1 9 8 8 , p. 4 7 1 . P. Newton, LC-GC Int., 3 ( 9 ) ( 1 9 9 0 ) 2 8 . W.J.B. Lanchflower, Spectroscopy Int., 2 ( 4 ) ( 1 9 9 0 ) 3 7 . R.D. Voyksner and C.A. Haney, Anal. Chem., 5 7 ( 1 9 8 5 ) 9 9 1 .

78

Chapter 4 TECHNIQUES OF GAS CHROMATOGRAPHY R. W. BEAVER

4.1.INTRODUCTION The mycotoxins are of concern when they occur in animal or human feedstuffs. The extreme toxicity and/or carcinogenicity of many of the mycotoxins necessitates their detection and determination at very low levels. The combination of chemically complex matrices such as foodstuffs with the need to detect a single analyte at low levels presents a formidable challenge to the analytical chemist. Gas chromatography (GC) is an analytical technique which is, in many cases, capable of meeting this challenge. The utility of GC in the analysis of mycotoxins is dependent on the ability of the technique to resolve the mycotoxin of interest from other constituents in the matrix. GC, and capillary GC in particular, is often referred to as a high resolution chromatographic technique. While this is certainly true, contributions to resolution which occur prior to the GC column (such as clean-up and extraction schemes which serve to isolate the mycotoxins from interferences) and after the GC column (such as detectors which exhibit a high degree of specificity or selectivity for the mycotoxin of interest) are often overlooked in the search for high resolution. Since a broad view of resolution would refer to whatever means are employed to separate the analyte from interferences, both chromatographic resolution (which occurs on the GC column) and extracolumn resolution (such as through clean-up and detection) are important in analyses. In this chapter, the basic theory of GC, which leads to ways of controlling resolution on the GC column, will be discussed. Various techniques for achieving extracolumn resolution will also be examined. Where applicable, specific examples of mycotoxin determinations will be used to illustrate the discussion. The examples will be chosen for their pertinence to the discussion and are not intended to present a comprehensive review of the determination of mycotoxins by GC. The reader is referred to Chapter 9 in this book for such a review.

79

4.2.RESOLUTION IN GAS CHROMATOGRAPHY 4.2.1 Definition of Resolution In order to accurately identify and quantitate a chromatographic peak, the peak of interest must be adequately resolved, or separated, from all adjacent peaks. Resolution can be measured directly from the chromatogram (1) according to equation 111

where tR1and tR2are the retention times of the peaks of interest and W,, and W,, are the peak widths (same units as t,, and tR2) at the base line. Snyder and Kirkland (2) provided a set of fiqures which illustrate the effect that different R, values have on the appearance of chromatographic peaks. An R, value of 1.25 corresponds to essentially complete, or base line, resolution of peaks of approximately the same size. While equation [l] provides a means of measuring the resolution for two peaks in a chromatogram, it provides no insight into the physical and chemical parameters in the chromatographic process which affect resolution. An alternative expression for R, (3) is given in equation [2]

where N is the number of theoretical plates generated by the column (or, synonymously, the efficiency of the column), k is the capacity factor, and cx is the selectivity or separation factor. Equation [2] couches resolution in terms of the three fundamental factors over which the chromatographer has control: 1) efficiency (N); 2) retention (k); and 3) chemical interaction between analytes and the column stationary phase ( cx 1 . 4.2.2 Efficiency Efficiency in GC refers to the ability of the GC system to generate narrow peaks. Obviously, the narrower the peaks, the less likelihood that adjacent peaks will overlap. Efficiency is measured as a quantity known as a theoretical plate (N) and can be measured from the chromatogram as in equation [31 N

=

16

( 2)'

I31

80

where t, is the peak retention time and W, is the peak width at the base line (1). Column efficiencies are usually reported as plates per meter so that columns of different lengths can be compared. Alternatively, column efficiency can be expressed as height equivalent to a theoretical plate, or H: H = L/N

t41

where L is the column length. Thus, the more efficient the column the smaller the value of H. While equation [4] provides a method by which to measure H for a given column, it provides no insight into the physical parameters which affect H. The discussion which follows presents the factors which affect H in simple, intuitive terms which are easily visualized and which are sufficient for understanding GC efficiency in a broad sense. However, for the rigorous derivation of the van Deemter equation and for a theoretical discussion of the Golay equation the reader is referred to the text by Perry (4) and to the work of Ogan and Scott (5) and of Sandra ( 6 ) . The van Deemter equation can be written as follows:

where A , B, and C will be discussed below and 1 ~ .is the average linear velocity of the carrier gas (the terms carrier gas and mobile phase will be used interchangeably) through the column. For packed columns, A describes the contribution to peak broadening which results from the various paths which different analyte molecules take as they migrate through and around the particles comprising the column packing. It should be noted that the A term is independent of the carrier gas velocity. In packed columns, the A term of equation 151 is minimized by using small particles of the narrowest possible size distribution. In capillary columns the A term of equation [ 5 ] vanishes, and, with slight modifications, equation 1 5 1 becomes the Golay equation ( 5 , 6 ) . For both packed and capillary columns, the B and C terms of equation [5] describe the variation of plate height (H) with mobile phase linear velocity. Although, as previously noted, the true situation is complex, to a first approximation the B and C terms can be considered to be identical in both packed and capillary columns.

81

Examination of equation [ 5 1 shows that the B term contribution to peak height decreases with increasing mobile phase linear velocity. Thus, B contains the factors which relate to analyte longitudinal diffusivity in the mobile phase. A s a group of analyte molecules traverses the column, they will tend to diffuse longitudinally through the mobile phase. This results in an increase in H since the analyte molecules are dispersed over a greater length of the column. Thus, the faster the mobile phase flows through the column the less time analyte molecules have to diffuse. Since dispersion due to the B term is related to diffusivity in the mobile phase, peaks spread less due to the B term when nitrogen is used as a carrier gas and the most when hydrogen is used as the mobile phase (noting that nitrogen, helium, and hydrogen are the most commonly used GC carrier gases). In order for any separation to take place on a GC column, the analytes must be absorbed into the stationary phase have different affinities for the stationary phase so that different migration rates occur. The C term of equation 1 5 1 , which increases with increasing carrier gas velocity, accounts for non-equilibrium between the stationary and mobile phases. This term behaves conversely to the B term in that increased diffusivity of the analyte in the mobile phase results in a smaller value for C. Thus, hydrogen carrier gas minimizes C. The C term also contains contributions due to the thickness of the stationary phase (either the layer coated on the particles in packed columns or on the wall of capillary columns), contributions due to the diameter of capillary columns, and contributions due to analyte diffusivity in the stationary phase. Plots of H vs p (see for example reference 6) describe a flattened hyperbola. For a given column, i. e. fixed stationary phase film thickness and column diameter, minimum H values are obtained with hydrogen as the carrier gas. However, due to safety considerations, a small sacrifice in ultimate achievable plates is usually made and helium is most often the carrier gas of choice. For capillary columns, the choice of column diameter and stationary phase film thickness is a matter of compromise. Ettre and co-workers ( 7 , 8 ) and Leclercq &. (9) have investigated the effects of film thickness and column diameter on efficiency. In general, thin films (i.e. 0.1 pm) and small column diameters lead to higher efficiencies. However, columns of larger diameter and with

82

thicker stationary phase films (or packed columns) have higher sample capacities. It is often falsely assumed that capillary columns are inherently more efficient than packed columns. However, procedures for producingpacked columns with greater than 3000 platesjmeter have been published (10). Well made capillary columns also provide on the order of 3000 plates/meter. However, due to pressure constraints, the length of packed columns is limited to approximately 3 - 4 meters. Capillary columns of 5 0 or 100 meters are routinely used. Thus, capillary columns are capable, if required, of providing 20-30 times the theoretical plates of packed columns. It is well to examine, in practical terms, what effects all of the above discussed parameters have on actual GC separations. Despite the considerable theoretical interest in efficiency, most laboratories will perform GC analyses on commercially obtained columns, either packed or capillary, operated at close to the optimum carrier gas velocity and the carrier gas will be helium. Therefore, the only way to gain significant numbers of theoretical plates will be to increase the column length. With capillary columns especially, this is easily done. However, it must be remembered that doubling column length will double the analysis time. It is also important to remember that R, varies as the square root of N so that doubling column length (and analysis time) results in only about a 40% increase in R,. Despite the time penalty, difficult separations can often be achieved only by increasing N (other methods of controlling R, are discussed later) and the wide use and availability of capillary columns has made many previously difficult to achieve separations routine. Two reports by Bata and co-workers (11,121 provide excellent examples of the use of increased efficiency to enhance a mycotoxin determination. In the first report (ll), deoxynivalenol (DON) was extracted from wheat and, after clean-up and derivatization, the extract was separated on a 1 2 m x 0 . 2 5 nun i.d. capillary column. The method could reliably determine DON to approximately 100 ppb. Although no interferences were noted under the analysis conditions (i.e. the DON peak was sufficiently resolved from all other components), Bata and associates were able to reduce the quantifiable level of DON to 5 0 ppb by carrying out the analysis on a 0.13 mm i.d. column ( 12) . By reducing the band width of the DON peak ( increasing

83

efficiency) the height of the peak was enhanced and the mass of DON required to produce a recognizable signal was reduced. 4.2.3 Retention The second variable in equation 121 which affects R, is the capacity factor, symbolized by k and defined k

=-R+t

- t

where tR is the retention time of the peak of interest and to is the retention time of an unretained compound. (At reasonably high temperatures the retention time of methane gives a sufficiently accurate estimation of to). Upon examination of equation [2], it is apparent that only one value of k is used to calculate R,, while R, refers to the separation between two peaks. For closely spaced peaks (the value of R, becomes irrelevant for widely separated peaks), an average value of k is used in equation [21. The value of k has a profound effect on R,. Equation [ 2 ] shows that, for k = 0, R, = 0. In other words, without retention no resolution is possible. A l s o , for values of k < 10, small increases in retention have large effects on R,. If k is increased from 0.5 to 5, R, increases 250%. However, a further increase in k from 5 to 10 results in only an additional 9% increase in R,. Thus, the standard recommendation is that k be adjusted so that peaks elute with values of k ranging from G. 1 - 1 0 (2). Values of k greater than about 10 result in much longer analysis times with minimal improvements in R,. If k is expressed as in equation [71, ( 1 3 )

171 where C, is the concentration of analyte in the stationary phase, C, is the concentration of the analyte in the mobile phase, d, is the stationary phase film thickness, and r is the column radius, the factors which affect k are evident. As the temperature is increased, C, decreases and C, increases so that the ratio CJC, becomes smaller and k decreases. Thus, retention (k) is inversely proportional to temperature and k can be controlled by varying the separation temperature. Alternatively, equation [ 7 ] suggests that the ratio of stationary phase film thickness to column diameter affects k.

84

Since most mycotoxins are large molecules with numerous polar functional groups (14), the usual problem in GC separations of these compounds is one of too much retention ( k > 10) rather than too little. The first step in achieving a value of k B > C (see Table 7.21). TABLE 7.21 RF x 100 values of cytochalasins on Silufol sheets Adapted from ref. 130. cytochalasin A B C

silica gel

SorbentX RF x looxx

Sil G Fo 1 Sil G Fo 1 Sil G Fol

G plates and

DetectionXXX

A

B

C

D

E

F

G

H

1

41 34

58

25

85

17

33

42 46 34 24 27

23

83

13 13 10 10

78 74 75 74

20 08

31 16

46 44 24 26 14 20

99 95 98 94

Pale Blue Beige Violet Blue

90

Beige

28

34 19 32

07

14

0

08

04

09

2

Orange

85

Sorbents: Sil G, silica gel G; Fol, Silufol sheets. xx Solvent systems: A, benzene-methanol-acetic acid (24:2:1); B, toluene-ethyl acetate-90% formic acid (6:3:1); C, chloroform-methanol (4:l): El benzene-ethanol (95:5); D, chloroform-methyl isobutyl ketone (4:l); F, chloroform-acetone (9:l): G, chloroform-acetic acid-diethyl ether (17:1:3); H I n-butanol-acetic acid-water (4:1:4, upper layer). xxx Detection: 1, panisaldehyde; 2, W at 366 nm after panisaldehyde. TLC on silica gel G plates of several derivatives of cytochalasin B was carried out with chloroform containing 0.5-20% as the solvent system (333). Lees and Lin (335) used TLC to purify 7,20-diacetylcytochalasin B on silica gel GF plates. In the system chloroform-ethyl acetate (l:l), the RF values of cytochalasin B, dihydrocytochalasin B and diacetylcytochalasin B were 0.4, 0.5 and 0.6, respectively. Aldridge and Turner (336) separated cytochalasins C and D on silica gel G plates using chloroform-methanol-formic acid (90:5:5) as the solvent system. The plates were then s rayed with 5% ethanolic sulphuric acid and heated at ca. 110 C for a few minutes. Cytochalasin C gave an orange and cytochalasin D a yellow fluorescence under W light. Hayakawa et a l . (337) carried out TLC of cytochalasin D on silica gel GF with (or benzene-ethyl acetate (7:3) or chloroform-methanol both) and the spots were rendered visible by spraying with concentrated sulphuric acid followed by heating at ca. 180 C.

(854)

193

A variety of solvent systems have been used by Chappuis and T a m (338) in the analytical and preparative TLC of derivatives and degradation products of cytochalasin D. In PLC, Kieselgel 60 PF25 was the sorbent, and analytical TLC was carried out on Fertigpfatten 60 F2 4. In the course isolation of cytochalasins H and J, TLC and PLC were carried out on silica gel layers containing 15% of gypsum (339). Chromatography of crude diethyl ether extracts from Phomopsis p a s p a l i with chloroform-methanol (9:l) as the solvent system and spraying with concentrated sulphuric acid revealed four spots with RF values 0.56 (yellow, minor), 0.49 (red, major), 0.36 (yellow, minor) and 0.32 (red, major). The red spots corresponded to cytochalasin H (kodo-cytochalasin-1) (kodo-cytochalasin-2). In a typical and cytochalasin J experiment, 337 mg of the ether extract on PLC gave 132 mg of cytochalasin H and 28 mg of cytochalasin J. A TLC method for the determination of cytochalasin H production was reported by Mujumdar e t a l . (340). Capasso e t a l . (341) showed that the phytopathogenic fungus, Ascochyta heteromorpha, produces cytochalasins A and B both in v i t r o and in v i v o . Elegant HPLC, TLC and HPTLC techniques were used in their work. In TLC and HPTLC, 7 cytochalasins were compared. Data for HPTLC are shown in Table 7.22.

02

TABLE 7.22 HPTLC data for cytochalasins Adapted from ref. 341. cytochalasin

RF x 100 in systemX

Detection limit (ng)

A

B

C

0.70 0.55 0.59 0.54

0.60 0.48 0.40

0.73 0.55 0.58

0.23

0.56

0.62

0.55

0.60

0.23 0.45

0.59

160 40 40 80 120

0.37

0.23

0.42

230

320

Solvent systems on Kieselgel 60 F254: A, chloroform-methanol ethyl acetate-n-hexane (70:30); c , D, chloroform-2propanol (90:lO). (92:8); B,

From

Phomopsis

sp.

six

new

cytochalasans,

named

194

cytochalasins N, 0, PI Q, R and S were isolated, together with the four known compounds, epoxycytochalasins H and J and cytochalasins H and J (342). The dichloromethane extract of the culture on wheat was separated by silica gel CC and HPLC. The fractions containing cytochalasins were detected as fluorescent spots under an W light on TLC plates after spraying with 50% sulphuric acid and heating. Kieselgel 60 F 54 precoated plates were used for TLC. The identities 0 % the four known cytochalasins were confirmed by the direct TLC and IR comparisons. 7.6.2.2 Zygosporins. PLC was used in the isolation and purification of zygosporins from a culture of Zygosporium masonii (343). The culture filtrate was extracted with ethyl acetate, the washed and dried extract was evaporated to about one-third of its volume and the separated product was filtered off. The filtrate was evaporated in vacuo to give crude cytochalasin D (zygosporin A ) and a paste ( A ) . Recrystallization of the crude cytochalasin D from acetone gave the pure compound and a residue (B). From the combined residues A and B zygosporins were isolated using CC and PLC. Fraction 2 , eluted from the silica gel column with chloroform, was crystallized from ethyl acetate to give a crystalline product (C) and a paste (D). The latter was chromatographed on alumina to give an oil and a paste (E), eluted with chloroform-methanol. Fraction 3, eluted from the silica gel column with chloroform-methanol (9:1), was dissolved in light petroleum and the precipitate (F) was collected. The crystalline product C was separated into cytochalasin D (R 0.40) and zygosporin E (RF 0.48) by PLC with ethyl acetate as tie solvent. The paste E was re-chromatographed on silica gel to give an amorphous powder, which was separated into zygosporin G (R 0.35) and zygosporin F (RF 0.28) by PLC using tofuene-methanol (10:1) as the solvent system. The precipitate F was separated into cytochalasin D (RF 0.50) and zygosporin D (RF 0.40) by PLC using chloroform-methanol (1O:l) as the solvent system. In addition to the use of PLC in the isolation of zygosporins, TLC has been used to characterize degradation products and derivatives of the four zygosporins (343, 344). RF values of zygosporins on silica gel plates developed with chloroform-methanol (9:l) were reported (345): 0.50 for zygosporin A (cytochalasin D), 0.40 for zygosporin D, 0.55 for zygosporin E and 0.57 for zygosporin F and G. 7.6.2.3 Aspochalasins. Keller-Schierlein and Kupfer (346) isolated aspochalasins A, B, C and D from Aspergillus parasiticus. TLC of the aspochalasins was performed on Kieselgel 60-Fertigplatten F254 and the spots were rendered visible by spraying with 50% sulphuric acid and heating at 200° C, with iodine vapour or fluorescence under W light. RF values of 0.35, 0.27 and 0.54 were obtained for aspochalasins C, D and B,

195

respectively (in ethyl acetate, blue fluorescence). In chloroform-methyl acetate (4:1), aspochalasins A and B had 0.26, respectively. TLC data for RF . values of 0.53 and derivatives and degradation products of aspochalasins were also given by these workers. 7.6.2.4 Deoxaphomin, proxiphomin and protophomin. Binder and Tamm (347) isolated deoxaphomin by PLC from mother liquors after crystallization of phomin (cytochalasin B ) as follows. The mother liquors were combined and deoxaphomin was separated on preparative plates with chloroform-acetone (3:l). The crude product was further purified using four preparative separations (chloroform-acetone, 3:l; twice with chloroform-acetone-formic acid, 90:5:5; chloroform-acetone, 3:l). The compound was extracted with chloroform-acetone (1:l) and the extracts were checked for their purity by means of TLC (chloroform-acetone, 3:l; chloroform-acetone-formic acid, 90:5:5). Proxiphomin and protophomin were isolated by Binder and Tamm as follows (3481.The residue after isolation of phomin and deoxaphomin was chromatographed on a Kieselgel column. The fractions, eluted with methylene chloride-methanol (9:l) and containing several non-polar components, were combined and chromatographed again on Kieselgel. From the eluate in methylene chloride crude proxiphomin was obtained and the methylene chloride-methanol (98:2) fractions contained protophomin. The crude preparation of proxiphomin was purified using PLC in methylene chloride-methanol. Extraction of the main zone with chloroform-acetone (4:l) resulted in 55 mg of chromatographically pure proxiphomin. TLC was carried out with methylene chloride-methanol (98:2) and methylene chloride-ethyl acetate (9:l). The protophomin-containing fractions were chromatographed on PLC layers, yielding crude protophomin, which was submitted to further purification by PLC : twice with chloroform-acetone-formic acid (96:2:2) and once with methylene chloride-methanol (98:2). Extraction of the zones with chloroform-acetone (3:l) yielded almost pure protophomin. 7.6.2.5 Chaetoglobosins. With extracts from Diplodia macrospora cultures, Probst and Tamm (349) used TLC to check the presence of compounds with a positive reaction to phenols and indoles - spraying with 5% solutions of ammonium cerium(1V) nitrate in acetone and of hydroxylammonium chloride in 80% aqueous acetone. The extracts with positive reactions were cleaned up on a silica gel column. The fractions containing chaetoglobosins K and L were purified by PLC on silica gel plates using toluene-ethyl acetate-formic acid (5:4:1) as the solvent system. Chaetoglobosins A , B , C, D and E were analysed on silica gel using benzene-ethyl acetate (1:1) and F254 benzene-chloroform-methanol (10:10:3) as the solvent systems.

196

Metabolites were detected by W irradiation at 254 and 365 nm and by spraying with Ehrlich's reagent and coloration after heating (350). The RF value of chaetoglobosin K was 0.53-0.56 on silica gel 6 0 plates developed with toluene-ethyl acetate-formic acid (5:4:1) and it was observed by Cutler e t a l . (351) as a dark spot under short-wave W light. Probst and Tamm (352) reported decreasing RF values of five chaetoglobosins on the same sorbent and with methylene chloride-methanol (95:5), showing increasing polarity from left to right, as follows: 19-0-acetylchaetoglobosin A > chaetoglobosin C > 19-0-acetylchaetoglobosin B > 19-0-acetyl chaetoglobosin D > chaetoglobosin A. Sekita e t a l . (353) used TLC in their work on chaetoglobosins A-J. TLC was also used by Cole e t a l . (354) in the isolation and identification of two new cytochalasans from Phomopsis s o j a e . 7.7 OCHRATOXINS The ochratoxin group consists of ochratoxin A and its methyl and ethyl (ochratoxin C) esters, ochratoxin B, its methyl and ethyl esters, and 4-hydroxyochratoxin A. Ochratoxin A and its esters are the toxic members of the group. 7.7.1 Extraction and clean-up Extraction and clean-up procedures for ochratoxins were reviewed by Steyn (355). Mouldy material can be extracted with various solvents and their combinations, such as methanol-water, acetonitrile-aqueous KC1, chloroform-methanol, or mixtures of organic solvents with diluted phosphoric acid. Ochratoxin A has to be determined in various materials. According to a Steyn's review, the problem of ochratoxin A contamination has been brought closer to home by reports of its occurence in barley, corn, swine tissue, pig serum, pig kidneys, sausages, commercial roast coffee, and human serum, kidneys or milk. A significantly higher incidence of ochratoxin A has been found in blood serum of patients with urinary systemic tumours and/or endemic nephropathy living in an endemic area of Bulgaria than in people from a non-endemic area. Clean-up procedures for ochratoxins include CC, gel filtration chromatography, solvent partition or dialysis. One of the recent methods, published by Cohen and Lapointe (356), employs a new extraction solvent (ethanol-chloroform-5% aqueous acetic acid) and clean-up using a Sep-Pak silica cartridge followed by a cyan0 cartridge. Another method (357) includes the use of a C Sep-Pak cartridge. 7.7.2 hisorbents and solvent systems. Silica gel, oxalic acid-treated silica gel, and rice starch have been reported as adsorbents for TLC of ochratoxins. Solvent systems and other TLC data are given in Table 7.23.

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TABLE 7.23 TLC data for ochratoxins Adsorbent

Silica gel Silica gel

Rice starch Silica gel G

Oxalic acidtreated silica gel Silica gel

RF X 100

Solvent systemX

Ref.

AX

BX

C6H6-HOAC (3:l) Tol-EtOAc-HOAc (5:4:1) C6Hs-HOAC ( 4 ~ 1 ) Tol-TCE--OH-HOAc (80:15:4:1) Tol-HOAc (20:0.15) C Hs-MeOH-HOAC (24:2:l) Tol-EtOAc-FA (6:3:1) C6H -EtOH (95:5) CHCP3-MIBK (4:l) CHC13-Me CO (9:l) CHC 1 -HOlc-E t 0 nBuOH-HOAc-H26 (4:1:4, upper layer) CHC13-MeOH (98:2) CHC13-Me2C0 (9:l)

50 70 40 60

35

43 52

30 41

59 34xx llXXX 23xxx 56 95

46 72 12xx 75 0 53xx 02 73 33 86 79 87

32 34

197

CsH6-HOAC (3:l)

50

358 , 360 358, 361 362

CHC13-HOAc (4:l) C6H6-HOAC (25:l)

Cx

HAx 358 359

80 130

80

35

25 55

Abbreviations: A, ochratoxin A: B, ochratoxin B: C, ochratoxin C; HA, 4-hydroxy-ochratoxin A: C6H6, benzene: HOAc, acetic acid: Toll toluene: EtOAc, ethyl acetate; TCE, trichloroethylene: AmOH, amyl alcohol: MeOH, methanol: FA, 90% formic acid; EtOH, ethanol: CHCl chloroform; MIBK, methyl isobutyl ketone: Me2C0, acetone: EZiO, diethyl ether: nBuOH, n-butanol. xx Tailing. xxx Elongated spot. 7.7.3 Detection A generally used technique is to view the plate under long-wave (366 nm) W light; ochratoxin A appears as a green fluorescent spot (blu-green on acidic plates) and ochratoxin B has blue fluorescence. The fluorescence of the ochratoxins changes to purple blue on exposure to ammonia fumes or Spraying with aqueous sodium hydrogen carbonate or sodium hydroxide (358, 359). The presence of ochratoxin A on chromatograms can also be confirmed by boron trifluoride derivatization (360).

I98

7.7.4 Selected applications In a report on TLC systematic analysis of 37 fungal metabolites in eight solvent systems, data for ochratoxins A, B and C were included (130). A very efficient separation of ochratoxins A and B was achieved by impregnation of the silica gel with oxalic acid (197). The TLC plates were then developed with the neutral solvent systems: chloroform-methyl isobutyl ketone (4:1), chloroform-methanol (98:2) or chloroform-acetone (9:l). Semi-quantitative and quantitative methods for the determination of low levels of ochratoxin A have been developed ( e . g . , refs. 363-365) and have been reviewed (355, 366-374). Patterson and Roberts (375) applied two-dimensional TLC to the analysis of feedstuffs. The chromatograms were developed with toluene-ethyl acetate-90% formic acid (6:3:1) (first direction) and chloroform-acetone (9:l) (second direction) and then examined at 366 nm. Quantitation of ochratoxin A was described by Johann and Dose (376). In a study on postharvest production of ochratoxin A inbarley, Haggblom and Ghosh (377) used DC Alufolien Kieselgel 60 with benzene-acetic acid (9:l) as the solvent system. Quantitation at 365 nm was carried out by fluorodensitometry. TLC has been applied in the quantitative determination of ochratoxin in vegetable foods by Asensio e t a l . (378). TLC remains one of the chief methods for the detection, identification and quantitation of ochratoxin A. Stahr e t a l . (379) included TLC among methods of chemical analysis for ochratoxin poisoning. Problems of streaking of ochratoxin A and B spots in neutral solvent systems accompanied by increasing RF values with increasing amount applied and the effects of acidic modifiers on these values have been discussed by Nesheim and Trucksess (21). Tsubouchi e t a l . (380) tested the heat stability of ochratoxin A in contaminated coffeee beans. The method developed by Nesheim e t a l . (371) for the determination of ochratoxins A and B in barley is very sensitive and specific for ochratoxin A. The method was adopted by the Association of Official Analytical Chemists as an official, first action method (369). It was also used by PleStina e t a l . (370) in the analysis of food samples from areas in Yugoslavia where Balkan endemic nephropathy is a major problem. The fluorescence intensity can change when ochratoxin A is exposed to ammonia-methanol vapour and the magnitude of the change is influenced by the residual mobile phase. This observation was exploied by Nesheim e t a l . (371). Samples are spotted on TLC plates in benzene-acetic acid (9:l) and benzene-acetic acid-methanol (90:5:5) is used as the mobile phase. The developed plate is exposed to ammonia-methanol vapour and then was covered with another glass plate to prevent evaporation of the ammonia-methanol. If the ammonia-methanol

199

does escape and the fluorescence intensity drops, it can be restored by re-exposure to fresh ammonia-methanol. The fluorescent spots under these conditions are stable for several days, whereas they occasionally fade in a few minutes on acidic plates. The method is recommended for most commonly contaminated commodities such as corn, barley and pig tissue. The method includes a confirmatory step. Methyl esters are prepared with boron trifluoride as a catalyst. The esters are identified by comparing the RF values of standard and analyte derivatives. PLC with benzene-acetic acid (4:l) as the solvent system was used for the purification of isotopically labelled ochratoxin A (372). Conversion of ochratoxin C into ochratoxin A in rats was studied by Fuchs et a l . (373) and ochratoxin A-containing fractions from a silica gel column were purified by PLC in toluene-dioxane-acetic acid (95:35:4). Ochratoxin A has been included in multi-mycotoxin analytical methodology (374). Other multi-mycotoxin analyses, in which ochratoxins have been included, are described in Section 7.15. RUBRATOXINS Rubratoxins A and B are structurally related toxins. Their production, physical, chemical and biological properties were summarized by Davis and Richard (381). 7.8.1 Extraction and clean-up The more toxic rubratoxin B can be extracted after concentrating the culture filtrate and mycelial washing, the concentrate being acidified with HC1 and extracted with diethyl ether. The ether extract is evaporated and the residue is dissolved in acetone and analysed by TLC (382). For corn, extraction with ethanol, acetone and ethyl acetate yields the maximum amount of rubratoxin A, whereas refluxing with diethyl ether yields the maximum amount of rubratoxin B. For rice, extraction with ethyl acetate in benzene yields the maximum amount of rubratoxin A, whereas extraction with ethyl acetate-benzene and diethyl ether yields the maximum amount of rubratoxin B (381). Hayes and McCain (383) reported that acetonitrile was satisfactory for extracting rubratoxin B from corn. 7.8.2 Adsorbents and solvent systems TLC of rubratoxin can be accomplished according to Cottral ( 3 8 4 ) as follows. Spotting of the silica gel plates should be carried out under nitrogen to prevent oxidation and internal and external standards should be included on the plates. The solvent system is chloroform-methanol-glacial acetic acid-water

7.8

(80:20:1:1). 7.8.3 Detection

Rubratoxin adopts a greenish fluorescence after heating the

200

plate at 2OO0C for 10 min. The intensity of the fluorescence can be increased by subsequently spraying the plate with 21,71-dichlorofluorescein; however, the background will also have a yellow-green fluorescence (383). Whidden et al. (64) quantitated rubratoxin B according to Hayes and McCain (383) and described the following confirmatory tests. The fluorescent derivatives, which were formed from rubratoxin B on a TLC plate after heating at 2OO0C for 10 min, were exposed to ammonia vapour for 10 min. Examination under long-wave W light revealed a change in the intensity and colour of the fluorescence. Rubratoxin was then more easily observed as a light blue spot, although the detection limit remained the same. Further, the fluorescence intensity of fluorescent greatly reduced, which compounds near rubratoxin B was considerably improved the contrast and thereby the ease of detecting rubratoxin. Also, after prolonged heating of the TLC plates at 100°C for 2-10 h with ammonium hydrogen carbonate, rubratoxin became visible under W light. The reactions of ammonia and ammonium hydrogen carbonate with rubratoxin B both produced very similar fluorescent derivatives on the TLC plates. The ammonium ion apparently combined with the anhydride derivative of rubratoxin to produce an amide or imide, which reacted with chlorine fumes and a spray reagent to produce a colour reaction. The spray reagent was prepared by mixing equal volumes of a 0.2 M pyridine solution of l-phenyl-3-methyl-2-pyrazolin-5-one and 1 M aqueous potassium cyanide. Subsequently, rubratoxin first turned pink under visible light, then quickly changed to blue and subsequently brown. The detection limit was 10 pg. 7.8.4 Selected applications TLC data for rubratoxins reported by Hayes and Wilson (385) were as follows: on silica gel HF254 plates with chloroform-methanol-glacial acetic acid (80:20:2) the R values for rubratoxin A and B were 70 and 56, respectively. With six of the eight solvent systems used by buraekova et a l . (130) no migration of rubratoxin B was observed on Silufol plates. With chloroform-methanol (4:l) and n-butanol-acetic acid-water (4:1:5, upper layer) its RF values were 0.28 and 0.88, respectively. Emeh and Marth (382) used PLC on freshly activated plates prepared with silica gel HF 4+ and developed the plates with 137. ethyl acetate-acetic acid

(82:

7.9 HYDROXYANTHRAQUINONES

The most important hydroxyanthraquinone mycotoxins are emodin, luteoskyrin and rugulosin. TLC of these and related mycotoxins has been reviewed (5, 30, 386, 387).

20 I

7.9.1 Extraction Anke et al. (388) extracted the mycelia of aspergilli with acetone (50 mL/g mycelium) and the culture broth with ethyl acetate (1:l). The extracts were concentrated to 5% of their volumes and aliquots were used directly for TLC. Ethyl acetate was also used to extract culture filtrates of Trichoderma viride (389). After drying with anhydrous sodium sulphate, the solvent was evaporated under reduced pressure and the residue was dissolved in acetone prior to TLC. 7.9.2 Adsorbents and solvent systems Silica gel is usually used as the adsorbent, sometimes impregnated with oxalic acid. For PLC, 58 g of silica gel PF 45 (Machery, Nagel and Co.) were mixed with 120 mL 0.2 M oxaiic acid and poured on glass plates (20 x 40 cm); after drying the plates were activated for 3 h at 13OoC (388). The following solvent systems were used by Anke et al. (388): (a) chloroform-methanol (97:3); (b) carbon (90:10) : (C) benzene-ethyl tetrachloride-chloroform acetate-acetic acid (45:55:1); ( d ) light petroleum (b.p. 40-60°C)-ethyl formate-formic acid (90:4:1). 7.9.3 Detection The hydroxyanthraquinones give yellow, orange or red spots on TLC plates. They are also detected by spraying the plates with a saturated solution of magnesium acetate in methanol or 5% potassium hydroxide in methanol (386). Varna et al. (387) compared detection with methanolic solutions of magnesium acetate and copper acetate. The colour obtained with 0.2% copper acetate was more stable than that with magnesium acetate. The colour obtained with copper acetate increased for 2 h and then remained stable for 24 h. buraekova et al. (130) detected luteoskyrin and rugulosin with panisaldehyde reagent. Spots of two hydroxyanthraquinones from Trichoderma viride on Silufol plates became intensely orange and violet, respectively, when the plate was exposed to ammonia fumes (389). 7.9.4 Selected applications Analytical TLC was used to characterize emodin on silica-7GF plates developed with (a) toluene-ethyl acetate-formic acid (5:4:1) and (b) chloroform-acetone (83:7). Orange-red spots in visible light had R values of 0.80 in the former system and 0.45 in the latter (380). On silica G plates impregnated with 0.5 M oxalic acid and developed with benzene-hexane (l:l), rugulosin gave an RF value of 0.25 (391). An RF value of 0.40 was reported (392) for luteoskyrin chromatographed on silica gel G plates impregnated with 0.5 M oxalic acid using acetone-n-hexane-water (6:3:1.5) as the solvent system. for hydroxyanthraquinones from Penicillium TLC data islandicum are given in Table 7.24. The separation of skyrin, (rugulin), rugulosin and 2,2-dimethoxy-4a,4a-dehydrorugulosin

202

a minor metabolite from Penicillium rugulosum, obtained by CC was monitored by TLC on Silufol plates developed with chloroform-ethyl acetate (2:l). Detection was carried out at 366 nm and by bioautography using Bacillus subtilis (393). Two main anthraquinones from a colour mutant of Trichoderma viride, 1,3,6,8-tetrahydroxyanthraquinone and l-acetyl-2,4,5,7-tetrahydroxy-9,10-anthracenedioneI were purified by PLC on Silufol plates using benzene-acetone (75:25) for repeated development (394). Quantitation of emodin and its major hepatic metabolite, w-hydroxyemodin, was performed by TLC as described by Murakami et a l . (395). TABLE 7.24 TLC data for hydroxyanthraquinones from Penicillium islandicum Adapted from ref. 386. Compound

RF X 100

C

D

82

85

95

65(Y)"

84

A Islandicin Chrysophanol Iridoskyrin Roseoskyrin Dianhydrorugulosin Catenarin Punicoskyrin Rhodoislandin A Rhodoislandin B Auroskyrin Emodin Skyrin Aurantioskyrin Dicatenarin Luteoskyrin Deoxyluteoskyrin 4a-Oxyluteoskyrin Rubroskyrin Deoxyrubroskyrin

B

90 70

Solvent systems: A, benzene-hexane (1:1); B, benzene-acetone (20:l); C , benzene-acetone (4:l); D, acetone-n-hexane-water (5:5:3.5, upper layer). xx Y, yellow on spraying with magnesium acetate reagent. The remaining pigments red or purple using the same detection.

203

7.10 EPIPOLYTHIOPIPERAZINE-3,6-DIONES This class of fungal secondary metabolites includes compounds with various biological activities such as mycotoxins ( e . g . , sporidesmins) or antibiotics ( e . g . , gliotoxin). The isolation, separation, purification and chemical and biological properties have been summarized by Nagarajan (396). TLC of these metabolites has been reviewed by the present author (30). 7.10.1 Extraction and clean-up Most data on extraction and clean-up procedures included in this section are taken from the above review (396) where references to the original literature may be found. Hyalodendrins are extracted from the filtered culture broth with chloroform and the extract is evaporated to dryness. The residue is chromatographed on silica gel. Benzene-chloroform (65:35) eluates afford hyalodendrin. Later fractions give bisdethiodi(methy1thio)hyalodendrin. Gliotoxins can be extracted from filtrates with chloroform or benzene. CC on silica gel or crystallization without chromatography have been described. Most recently, screening techniques of A s p e r g i l l u s f u m i g a t u s isolates for gliotoxin production were described by Richard e t a l . (397) as follows. The liquid cultures were harvested by filtering and the filtrate was extracted three times with chloroform. The extracts were combined and placed at 4OC overnight. The chloroform layer was absorbed on a hydrophilic matrix column (Chem Tube CT-2050) and gliotoxin was eluted with chloroform. The eluate was evaporated to dryness and redissolved in 2 m L of methylene chloride. The 2 mL of extract from each isolate was placed on a silica gel Sep-Pak primed with 5 mL of methylene chloride. Each Sep-Pak was eluted with 2 mL of each of the following solvents and saved separately: hexane, ether, ethyl acetate, chloroform, and methanol. Each eluate was evaporated to dryness and redissolved in 100 pL methylene chloride for use in TLC analysis. The latter workers described the extraction and clean-up procedure for gliotoxin from rice culture as follows: (1) Extract rice in flask with 250 mL chloroform (overnight, static). ( 2 ) Filter extract into evaporating flask. (3) Extract rice a second time with 200 mL chloroform (8-12 h, static). (4) Filter and combine with extract above. (5) Evaporate combined extracts to dryness and redissolve in 10 mL chloroform. (6) Add 250 mL light petroleum (b.p. 3O-6O0C) and place at 4OC for 12-24 h. (7) Filter solvent, save and evaporate to dryness. Discard precipitate. (8) Redissolve residue in 5 mL of methylene chloride-cyclohexane ( 5 0 : 5 0 ) and inject onto gel permeation column. (9) Elute with methylene chloride-cyclohexane ( 5 0 : 5 0 ) at 5 mL/min discarding first 100 mL fraction and collecting four 20-mL fractions. (10) Combine fractions 2-4, evaporate to dryness. Redissolve for TLC or HPLC analysis.

204

Aranotin and related compounds are extracted from filtered broth with ethyl acetate and the extract dried over anhydrous sodium sulphate. Evaporation of the solvent under reduced pressure affords the crude antibiotic complex. The individual metabolites are separated by CC and the separation is monitored by TLC. Sporidesmins which are responsible for facial eczema in grazing animals in New Zealand were isolated from cultures of the fungus P i t h o m y c e s c h a r t a r u m . A mixture of the culture and water-methanol (2:3) was stirred for 24 h and filtered. The residue was extracted again with water-methanol. The aqueous methanol extracts were combined and concentrated. The concentrate was diluted with water, extracted with diisopropyl ether and the extract evaporated to dryness. The residue was washed with light petroleum. The lipid-free residue was dissolved in benzene and separated on a silica gel column using a benzene-ethyl acetate gradient. Sporidesmin B eluted first, followed by sporidesmin and sporidesmin E. The next fractions contained sporidesmin G and D. Sporidesmins H and J were isolated by PLC from the next eluates. 7.10.2 Adsorbents, solvent systems and detection Silica gel is usually used as the adsorbent. Some solvent e t a l . (398) detected systems are given below. Hodges sporidesmins by spraying with 5% aqueous silver nitrate or viewing under reflected short-wave W light. In TLC of melinacidin, bioautography with B a c i l l u s s u b t i l i s was employed (399). Sirodesmins were detected by spraying with chromic acid and heating ( 4 0 0 ) . Gliotoxin was visualized with a spray reagent of 5% silver nitrate in 90% ethanol. Other detections are given below. 7.10.3 Selected applications PLC has been used in the preparation of sporidesmin H and J (396). Hodges e t a l . (398) characterized sporidesmin A on silica gel F plates with benzene-ethyl acetate (4:l) and chloroform-me%nol ( 19: 1 ) as the solvent system, resulting in RF values of 0.38 and 0.57. respectively. The melinacidin factors were differentiated from each other (399) on silica gel G plates using the solvent systems toluene-ethyl acetate (1:l or 3:2) and methylene chloride-ethyl acetate (7:3). Gliotoxin was analysed by Richard e t a l . ( 3 9 7 ) on silica gel 60 plates including internal and external standards of gliotoxin (at least 500 ng of gliotoxin per spot). The plates were developed 10 cm in an unlined tank with methylene chloride-methanol (97:3). Repeated PLC of fractions from a silica gel column afforded sirodesmins A, C and G (400). Analytical TLC was performed on (1:2), silica gel GF with toluene-ethyl acetate chloroform-methanof5formic acid (95: 4 :1 ) and chloroform-methanol

205

(95:5) as the solvent systems. Elution of hyalodendrin from a silica gel column was monitored by TLC on silica gel GF254 and detection under W light, giving an RF value of 0.60. Hyalodendron tetrasulphide was obtained from an enriched CC fraction by PLC (401). The latter compound also gave an RF value of 0.5 on Kieselgel plates developed in benzene-acetone (9:l) (402). TLC followed by bioautography has been used for the antibiotic A30641 (403), antibiotics of the A26771 series (404), aranotin and its derivatives (405). Epicorazines A and B were purified by means of PLC (406). 7.11 TREMORGENIC MYCOTOXINS Except for the territrems, the known tremorgenic mycotoxins have in common an indole moiety and can be placed into the following groups : the paspalitrem group, the fumitremorgin-verruculogen group, the penitrem group, the janthitrems and the tryptoquivaline group. TLC has been used in monitoring the CC separation and purification of most of the tremorgens, and also in preparative and qualitative separations. 7.11.1 Adsorbents and solvent systems Silica gel has been used in most TLC studies of the tremorgens. Solvent systems are mentioned in the applications. Penitrems A-F are unstable in chloroform when exposed directly to light, presumably as a result of acid formation in the solvent. Hence, its use must be avoided in work with these toxins (407). 7.11.2 Detection Detection methods used in TLC of indole-derived tremorgens include short- and long-wave W light and the following spray reagents: 50% sulphuric acid in ethanol without and with heating, cerium(1V) sulphate in sulphuric or phosphoric acid, phosphomolybdic acid, iron(II1) chloride, aluminium chloride, m-dinitrobenzene, 2,4-dinitrophenylhydrazine and Van Urk reagent. The following results have been obtained with these detections. 7.11.2.1 Paspalitrem group. Aflatrem appeared as a dark spot under long-wave W light; spraying with m-dinitrobenzene caused the spots to turn a non-specific brown colour, but spraying with phosphomolybdic acid with applied heat turned the spots an orchid to violet colour (408, 409). Paspaline and paspalicine were detected as pale green spots with Van Urk reagent (410). Paspaline and paspalitrem A were revealed as grey-blue spots in visible light after spraying with 50% ethanolic sulphuric acid and heating for 5 min at 150 C and were fluorescent under long- and short-wave W light. Under the same conditions, paspalitrem B was visible as a green spot immediately after spraying (411).

206

Paxilline was detected after spraying TLC plates with 50% ethanolic sulphuric acid or 3 % phosphomolybdic acid and heating for 5 min at 100°C. With the latter treatment paxilline gave a dark blue spot and with the former a greenish grey spot. It was also revealed under long-wave W light as a blue-grey fluorescent spot after the former but not latter treatment (412). Cockrum et a l . (413) detected paxilline as spots showing a characteristic colour (purple-blue fading through yellow with a blue border to salmon pink) when sprayed with a 10% solution of cerium(1V) sulphate in concentrated phosphoric acid, diluted immediately before use with acetone (1:4). 7.11.2.2 Fumitremorgin-verruculogen group. Fumitremorgin A develops a slate grey-blue spot under visible light or immediately after a mustard-coloured spot under W light spraying with 50% ethanolic sulphuric acid (414). Fumitremorgin C develops a bright orange spot immediately after spraying with the same reagent and minimal heating (415). Fumitremorgin B was detected under W light and with the following spray reagents: (a) cerium(1V) sulphate (1% solution in 3 M sulphuric acid): (b) 2,4-dinitrophenylhydrazine (1 g), concentrated sulphuric acid (7.5 mL), ethanol (75 mL) and water (170 mL); (c) iron(II1) chloride ( 3 % solution in ethanol). Characteristic colours of fumitremorgin B were light purple at 254 nm, yellow-brown with rea ent (a) immediately and also after heating for 10 min at 1108C, light orange with reagent (b) after heating and orange with reagent (c) after heating. The most sensitive detection was at 254 nm with reagent (a). The lowest detectable amount of fumitremorgin B was 1 pg (197). Verruculogen (416) and 15-acetoxyverruculogen (414) become visible immediately after spraying with 5 0 % ethanolic sulphuric acid as slate-grey spots under visible light. When sprayed with a 10% solution of cerium(1V) sulphate in concentrated phosphoric acid, diluted immediately before use with acetone (1:4), verruculogen produced pinkish blue spots, fading to yellow-green (413). Mycotoxin TR-2 produced a light-brown fluorescent spot after spraying with 50% ethanolic sulphuric acid and heating for 5 min at 100°C (414). 7.11.2.3 Penitrem group. Penitrem A was revealed as a blue spot after spraying with 50% ethanolic sulphuric acid and heating (417). Penitrems A and B produce stable green spots after spraying with 1-2% iron(II1) chloride in butanol and gentle heating (418). Penitrems A-F give blue spots immediately after spraying with cerium(1V) sulphate, which become stable dark purple after heating (407). 7.11.2.4 Janthitrems. Unlike all previously discovered Penicillium tremorgens, the janthitrems are highly fluorescent under long-wave W light. The intense blue fluorescence is reminiscent of that of the aflatoxins. They can be also detected by spraying the TLC plates with Ehrlich reagent and exposure to HC1 vapour for 5-10 min, resulting in grey-green spots (419).

201

7.11.3 Selected applications Aflatrem on silica gel G plates developed in chloroform-methanol ( 9 5 : 5 ) was characterized by an RF value of about 0.8 (408). TLC was applied in monitoring the CC purification of paspalicine (410) and paxilline (412), TLC of paspaline and paspalicine carried out on Kieselgel HF plates using chloroform as the solvent gave RF values of 0.35 and 0.7, respectively. PLC was used to isolate and to purify paspalinine, paspalitrem A and paspalitrem B. The three tremorgens appeared on silica gel GH-R plates, developed in chloroform-acetone (93:7), at RF 0.60 (paspalitrem A), 0.52 (paspalinine) and 0.20 (paspalitrem B) (411). The RF values of paxilline on silica gel GH-R and on silica gel 60 F254 were 0.75 and 0.52, respectively (412, 413), when developed in toluene-ethyl acetate-formic acid (5:4:1). TLC was used to check paxilline in fractions from CC during purification of the toxin. Spraying with Ehrlich's reagent followed by heating revealed paxilline by its colour, yellow becoming green. Complementary detection involved spraying with 50% ethanolic sulphuric acid and heating at 100°C for 5 min (420 ) . Analytical or preparative TLC has been applied in studies on the role of paxilline in the biosynthesis of lolitrem B (421) and penitrems A and E (422, 423). The RF values of fumitremorgin A on silica gel GH-R plates in chloroform-acetone (97:3) and toluene-ethyl acetate-formic acid (5:4:1) were 0.30 and 0.65, respectively (414). In the latter system, the RF value of fumitremorgin C was 0.55 (415). Using the same adsorbent, fumitremorgin B had an RF value of 0.67 in diethyl ether and 0.38 in acetone-methylene chloride (5:95) as the solvent system (424). Mean RF values of fumitremorgin B on Merck pre-coated silica gel F 54 plates in six solvent systems were reported (197) as folfows: 0.51 in chloroform-methanol (97:3); 0.36 in chloroform-acetone-n-hexane (7:2:1); 0.28 in chloroform-acetone (9:l); 0.14 in ethyl acetate-n-hexane (1:l); 0.71 in chloroform-acetone-2-propanol (85:15:20); and 0.30 in benzene-chloroform-acetone (45:40:15). For verruculogen chromatographed on either MN-Kieselgel GH-R (416) and silica gel (413) plates developed in toluene-ethyl acetate-formic :zidF2?g:4: 1) , RF values of 0.65 and 0.48, respectively, have been reported. Recently, effects of temperature , light , and water activity on growth of a heat-resistant mould, Neosartorya f i s c h e r i , and production of fumitremorgins A and C and verruculogen were investigated by Nielsen and co-workers (425). Mycotoxins were analyzed by TLC on silica gel plates developed in chloroform-acetone (93:7). Fractions having the same secondary metabolite profile on thin-layer chromatograms were combined, concentrated under vacuum, and analyzed by HPLC. PLC has been used as a purification step for penitrem A (417). TLC data for the toxin have been reported by Gorst-Allman and Steyn (197), Ciegler (417) and Wilson et al.

208

procedure for the quantitative detection of penitrems (then called tremortins) in agricultural products involved extraction with chloroform-methanol (2:l) followed by TLC and Richard and Arp (4281, using colorimetric assay (427). extraction and TLC analysis, reported on the occurrence of penitrem A in mouldy cream cheese. Simple HPLC and TLC systems for the separation, identification and quantitation of the various penitrems in culture extracts were devised by Maes et a l . (407). As the penitrems are unstable in chloroform when exposed directly to light, all contact of the penitrems with this solvent was avoided. The most efficient solvent systems for the TLC separation of the penitrems were found to be (a) n-hexane-ethyl acetate (70:30), (b) dichloromethane-acetone (85:15) and (c) benzene-acetone (85:15). In solvent system (a) penitrems B and F and penitrems C and D still overlapped, whereas penitrems C and E overlapped in system (b). The only system that gave a complete separation of all the penitrems was (c) when the chromatogram was developed twice. The order of decreasing RF values for the penitrems was F, B, A, E l C and D (see Table (426). A

7.25).

TABLE 7.25 TLC of penitrems Data from refs. 407 and 429 Penitrem

A B C D E F

R, x loox A

B

C

D

16 18 9 9 13 18

49 53 39 37 46 55

37 39 28 26 33 42

32 36 22 22 28 36

E 46 32 29 50

Solvent systems: A, n-hexane-ethyl acetate (70:30); 8, dichloromethane-acetone (85:15); C, benzene-acetone (85:15); D, n-hexane-ethyl acetate (6:4); E, methylene chloride-ethyl acetate (9:l). PLC has been used in the purification of the janthitrems but CC on Mallincrodt Silica ARCC-7 silica gel was more succesful (419 ) . The three major janthitrems have the following RF values on silica gel 60 F254 pre-coated plates, developed in toluene-ethyl acetate-acetone (3:2:1): janthitrem A 0.61, janthitrem B 0.54 and janthitrem C 0.74.

209

Territrems are metabolites of Aspergillus terreus (430, 431). Territrems A and B were separated by TLC in the following solvent systems (431): (a) benzene-ethyl acetate (1:l); (b) toluene-ethyl acetate-658 formic acid (5:4:1); and (c) benzene-ethyl acetate-acetic acid (55:40:5). Detection is based on blue fluorescence of the territrems (430). Territrem C exhibited light-blue fluorescence on silica gel 60 F254 pre-coated plates at RF values of 0.25 in system (a), 0.43 in system (b) and 0.42 in system (c). The intensity of fluorescence was quenched when the concentration was higher than 20 pg per spot. The fluorescence intensity also gradually faded after development in system (a), but was enhanced and turned greenish in acidic solvent systems. PLC was used to isolate the methylation product of territrem C and its identity with territrem B was proved (431). More recently, Peng et al. (432) succeded in isolating another related metabolite. As its RF values in TLC were between those of territrems B and C , the compound was designated territrem B'. 7.12 ALTERVARIA TOXINS Alternaria mycotoxins and phycotoxins have received much interest in recent years. Production, isolation, clean-up procedures and chromatographic techniques (TLC, GC and HPLC) for the determination of alternariols, altenuene and tenuazonic acid were reviewed (433). TLC is the most widely used technique for the detection of these mycotoxins (for a review, see ref. 434). 7.12.1 Extraction and clean-up Palmisano et al. (435) extracted undried cultures of rice, maize and tomatoes or naturally contaminated samples (50 g ) in a blender with 75 mL of methanol and filtered. A 40-mL portion of the filtrate was clarified by addition of 80 mL of 5% aqueous ammonium sulphate and filtered. A 90-mL volume of the filtrate (corresponding to 20 g of the original substrate) was extracted twice with 5 mL of methylene chloride. For oleaginous samples (sunflower seeds or corn kernels) a defatting step, with 30 mL of phexane, preceded the methylene chloride extraction. The combined extracts containing the dibenzo-a-pyrone and perylene derivatives were evaporated to dryness and reconstituted with 1 mL of methanol. According to another technique (436), ground samples or kernels or chaffs (4-10 g) were extracted with 50 mL of methanol, filtered, evaporated to small volume, and, if needed, purified on Celite 545 column. 7.12.2 Adsorbents and solvent systems With Alufolien Kieselgel 60 F254 (Merck), the solvent systems used (436) were: (a) toluene-ethyl acetate-formic acid (6:3:1) and/or (b) chloroform-ethanol-ethyl acetate (90:5:5). The obtained results are given in Table 7.26.

210

TABLE 7.26 TLC data for Alternaria toxins Adapted from ref. 436. Toxin

Alternariol Alternariol methyl ether A1tenuene Altertoxin Tenuazonic acid

R,

x 100x

A

B

44

32 78

59 20 34 29

15 28 10

Solvent systems: A, toluene-ethyl acetate-formic B, chloroform-ethanol-ethyl acetate (90:5:5).

acid

(6:3:1):

7.12.3 Detection Alternaria toxins can be detected by quenching of fluorescence under UV light at 254 nm (tenuazonic acid) or by their fluorescence at 365 nm after spraying with a 20% aluminium chloride in ethanol. Yellow-orange fluorescence is characteristic for altertoxin and violet-blue for alternariol, alternariol methyl ether, and altenuene (436). 7.12.4 Selected applications TLC data for alternariol, alternariol monomethyl ether, ltertoxin I and I1 and tenuazonic acid were published (437). %-labelled alternariol and alternariol monomethyl ether were isolated from ethyl acetate extracts of conidia of A. alternata by PLC. Two solvent systems were used (438): (a) toluene-dioxane-acetic acid (95:25:4) and (b) methanol-2 M HC1 (5:l). Visconti et al. (439) used TLC to determine alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in olives. Altenuene, alternariol, alternariol methyl ether (dibenzo-a-pyrone derivatives), altertoxin-I and altertoxin-11 (perylene derivatives) were found in extracts of artificially infected maize, rice and tomato samples and naturally contaminated sunflower sedds (435). Natural occurrence of Alternaria toxins (alternariol and alternariol methyl ether) in the grain and chaff of cereals was detected (436). 7.13 CITRININ TLC has been used by many workers to characterize, identify and quantitate citrinin in various commodities and also in preparative work. Chromatographic methods, including TLC, were reviewed (11, 30, 440).

21 1

7.13.1 Extraction and clean-up Extraction solvents and clean-up techniques for citrinin are given in Table 7.27. Chloroform, ethyl or butyl acetate and methanol are the most commonly used solvents for extraction. Originally, precipitation from culture filtrates with concentrated hydrochloric acid was applied (441). In clean-up procedures, silica gel CC, Extrelut columns or partition at different pH values between aqueous and organic phases have been used. TABLE 7.27 Extraction and clean-up of citrinin Materia1

Extraction solvent( s

Clean-upx

Culture filtrate

Precipitation with conc. HC1 (12.5 mL/L)

Culture filtrate

EtOAc at pH 2.5

Culture filtrate

CHC13 followed by EtOAc from conc. filtrate

Culture filtrate

BuOAc at pH 2.5

Corn

CHC13

Culture filtrate Culture filtrate

CHCl3

Crude CIT dissolved in 441 CHC13 crystallization from h O H Partition into buffer pH 442 8.5, re-extraction with CHC13 at pH 2.5, evaporation, partition between CC14 and (CH20H)2, CC14 phase evaporated, crystallization from Me CO cc silica gel, efution 443 with CHC13, partition into 0.2 M NaHCO , acidification , crysta? 1ization of precipitate from EtOH Evaporation, dissolution 444 in C6H6, partition into sat. aq. KHC03, re-extraction with c H at pH 3.8, evaporation ,6dfssolution in EtOH Extract rinsed with dil. 445 HC1 and H20, partition into 0.1 M NaHC03, reextraction with CHCl at pH 2.5 and concentragion, partition into 0.1 M NaHC03, precipitation (pH 2.5) Concentration and TLC 446

Static culture Culture filtrate and mycelia

EtOAc

CHC13 at pH 1.5

EtOAc (filtrate) Hot EtOAc (mycelium)

Ref.

Evaporation, dissolution 447 in CHC13 or 0.1 M buffer (PH 10) Concentration 443 Extract passed through Na SO4 and concentrated u d e r N2

448

212

TABLE 7.27 Conth u e d ~

~~

~

Ref.

Materia1

Extraction solvent( s

Clean-upx

Tomatoes

MeOH and Hex

Maize

MeOH-CHC13 (1:l)

Centrifugation, 5 M 448 H2S04 added, partition into CHC13, evaporation, dissolution in CHCl Filtration , evaporazion , 197 partition Hex-90% MeOH (l:l), MeOH layer evaporated, partition CHC13H20 (l:l), CHC13 layer extracted with sat. NaHC03, re-extraction with CHC13 at pH 2, concentration CHC13 layer passed 449 through Extrelut column

Cereal grains CHC13-0.1 M H3P04 (15:2)

Abbreviations: CIT, citrinin: EtOAc, ethyl acetate: CHC13, chloroform: CC14, carbon tetrachloride: (CH20H)2, ethylene glycol: Me2C0, acetone: BuOAc, butyl acetate: C6H6, benzene: EtOH, ethanol: MeOH, methanol: Hex, n-hexane. 7.13.2 Adsorbents and solvent systems Silica gel is the most often used adsorbent in the TLC of citrinin. Better results were obtained on oxalic acid pre-treated plates. Silufol plates were impregnated with 0.25 M oxalic acid in methanol by developing the plates in the solution. The plates were then dried in air and spotted (450). Marti et a l . (451) dipped inactivated silica gel 60 in 10% oxalic acid. Gorst-Allman and Steyn (197) immersed the plates in a 10% solution of oxalic acid in methanol for 2 min. After heating at llO°C for 2 min and cooling, the plates were immediately spotted and developed. Gimeno (452) found glycolic acid to be better then oxalic acid because of reduced diffusion of the citrinin spots and hence enhanced detectability. A variety of solvent systems have been used by various workers: some of them are mentioned in 7.13.4. 7.13.3 Detection Citrinin can be observed on chromatograms under UV light owing to its yellow fluorescence. In addition, several spray reagents have been employed. Curtis et a l . (453) used a freshly prepared solution of a stabilized diazonium salt of o-dianisidine (0.05 g in 4 0 mL of methanol-water, l:l), followed by methanol-aqueous ammonia (1:l) to promote the coupling reaction. Citrinin produced a pale pink colour. Improved colour

213

resolution was obtained if the TLC plates were allowed to dry overnight before spraying. After spraying with a 3% solution of iron(II1) chloride in methanol, citrinin is detected as a brown spot (450). Citrinin was also detected with p-anisaldehyde spray reagent (130). Gorst-Allman and Steyn (197) detected citrinin and other acidic mycotoxins under short- and long-wave UV light or by spraying with cerium(1V) sulphate, 2,4-dinitrophenylhydrazine and iron(II1) chloride reagents. Marti et a l . (451) obtained a detection limit of 20 ng per spot of citrinin by measuring the yellow-green fluorescence under UV light. 7.13.4 Selected applications Curtis et a l . (453) examined phenolic metabolites including citrinin using Kieselgel G plates and benzene-methanol-acetic acid (10:2:1) as the solvent system. Betina and Binovska (444) monitored the production of citrinin in the course of a submerged fermentation. The cleaned-up samples (see Table 7.27, ref. 444) were spotted on oxalic acid-impregnated Silufol plates. The most suitable solvent systems were benzene-methanol-acetic acid (10:2:1) and benzene-methanol (95:5). In producing 14C-citrinin by P. citrinum, Phillips et a l . (454) isolated and purified the compound by the method of Davis et a l . (441). The identity and purity of citrinin were established by TLC using diethyl ether-hexane-formic acid (75:25:1) and ethyl acetate-acetone-0.1 M (40:40:20) as the solvent systems. A single peak of radioactivity appeared, which co-chromatographed with authentic, chemically pure citrinin. The production of citrinin in corn was monitored by TLC on silica gel F2 using the solvent system chloroform-methanol (75:25) and dezection under 366 nm UV light (445). Harwig et al. (448) detected citrinin in extracts from Penicillium spp. cultures isolated from decaying tomato fruit, and also in tomato extracts, using silica gel plates and the solvent systems toluene-ethyl acetate-formic acid (5:4:1) and ethyl acetate-acetone-water (5:5:2). TLC analysis and chemical confirmation of citrinin in barley were reported by Hald and Krogh (455). TLC quantitations of citrinin have been published. Wu et a l . (446) separated citrinin-containing extracts by TLC on Adsorbosil-1 using toluene-ethyl acetate-formic acid (6:3:1) as the developing solvent and fluorodensitometry. Damodaran et a l . (447) reported a procedure for the isolation and quantitation of citrinin in culture filtrates. Cleaned-up samples were spotted on to silica gel plates and developed in toluene-ethyl acetate-formic acid (5:4:1). The fluorescent portions were citrinin was extracted with scraped off , carbonate-hydrogencarbonate buffer pH 10 and the determinations were carried out using Folin's reagent. Further quantitations have been reported by Ciegler et a l . (443) and Damoglou et a l .

2 14

(456). The latter procedures were shown to be of importance in the separation and identification of dihydr itrinone and ochratoxin A as products of conversion of “C-citrinin by Penicillium viridicatum (457). The presence of radiolabelled products on TLC plates formed by the breakdown of citrinin was assessed by autoradiography. 7.14 a-CYCLOPIAZONIC ACID Of the known tetramic acids, a-cyclopiazonic acid is the most studied. Data concerning the production, isolation, separation and purification of this and related toxins were reviewed by Cole (458). 7.14.1 Extraction and clean-up Gorst-Allman and Steyn (197) carried out extraction of Penicillium cyclopiumcontaminated maize with methanolchloroform (1:1), the mixture was filtered and the filtrate evaporated to dryness. The residue was partitioned between n-hexane and 90% methanol (1:l) and the methanol layer was evaporated to dryness. The solid was partitoned between chloroform and water (1:l) and the chloroform layer was extracted with saturated sodium hydrogencarbonate solution. The aqueous layer was acidified to pH 2 and extracted with chloroform. The chloroform extract was concentrated and contained a-cyclopiazonic acid. LeBars (459) extracted cheese samples with azeotropic chloroform-methanol. The filtered and evaporated extract was dissolved in acetone-water-lead acetate solution. A saturated solution of sodium sulphate and Celite were added and the suspension was filtered. The filtrate was defatted by partition against hexane, acidified to pH 3 and extracted with chloroform. The centrifuged and dried extract was evaporated to dryness and dissolved in the minimum volume of chloroform for TLC. Benkhemmar et al. (460 ) extracted cyclopiazonic acid from culture filtrates a modified Le Bars technique. A 40-mL portion of culture FXltrate, adjusted to pH 3 with aqueous HC1 ( 5 0 : 5 0 ) , was extracted with four volumes of methanol-chloroform (1:4). The methanol-chloroform layer was decanted and retained, and then it was dried with Na2S04, filtered, and vacuum concentrated to dryness. The crude extract was taken up in chloroform for characterization by TLC to discrimiate cyclopiazonic acid-producing from non-producing strains of Aspergillus oryzae. According to Lansden (461), samples of peanuts or corn were extracted with methanol-chloroform (20:80); the extract was stripped of most interferences by partitioning with aqueous sodium hydrogencarbonate followed by acidification and repartitioning with chloroform. Rao and Husain (462) extracted the toxin from culture filtrates and solid substrates as follows. Twenty five mL of the

215

filtrate was extracted twice with equal amounts of chloroform. The pooled extract was dried over anhydrous Na2S04 and evaporated to dryness. The residue was dissolved in one mL chloroform and used for TLC. The solid substrates were first defatted with petroleum ether by thorough extraction using a mechanical shaker. The defatted substrate was extracted with 200 mL of chloroform twice for 24 h on a mechanical shaker. The extracts were pooled, filtered, dried over anhydrous Na2S04 and evaporated to dryness. The residue was dissolved in one mL of chloroform and used for TLC. Extraction procedures of Hermansen e t al. (463 ) started by homogenization of culture broths. The homogenate was acidified with 2 M HC1 to pH 2 and extracted with chloroform-methanol (4:l) by shaking for 14 h. After filtration the phases were separated, and the organic phase was dried (Na2S04) 2a;; evaporated to dryness. The residue was dissolved in chloroform. Isolates of Aspergillus and Penicillium species from dried beans, corn meal, macaroni and pecans were examined for their ability to produce cyclopiazonic acid. From static fungal cultures in 100 mL volumes of a culture medium, the culture broth and the mycelial mat were extracted by adding 100 mL chloroformin the flask and soaking with occasionally shaking for 24 h (464). The mixture was heated in a steam bath until it boiled. After cooling, 10 mL of the chloroform layer was withdrawn and filtered through 5 g anhydrous Na2S04. The filtrate was collected in a vial. The sodium sulphate was washed with 2 mL chloroform and was collected in the same vial. The extract was evaporated to dryness on a steam bath under a stream of nitrogen. 7.14.2 Adsorbents and solvent systems Silica gel TLC plates have been impregnated with oxalic or tartaric acid (197, 459). A variety of solvent systems have been used, e . g . , (a) chloroform-methyl isobutyl ketone (4:1), (b) chloroformmethanol (98:2), (c) chloroform-acetone (9:1), (d) ethyl acetate-2-propanol-ammonia solution (20:15:10), (el chloroform-acetone (95:5), or (f) toluene-ethyl acetate-formic acid (5:4:1). Systems (b) and (c) are recommended for acidic mycotoxins. Systems (a), (d) and (f) were used by Le Bars (459) for the quantitation of cyclopiazonic acid from commercial cheese samples. 7.14.3 Detection Cyclopiazonic acid can be detected by derivatization with Ehrlich's reagent. Lansden (461) recommended the following preparation of this spray reagent: 1 g 4-dimethylaminobenzaldehyde is dissolved in 75 mL ethanol and 25 mL concentrated HC1 are added. The dried plates are sprayed with the reagent until first appearance of blue spot among cyclopiazonic acid standards. Colour is developed within 10 min, without heating.

2 16

In addition to Ehrlich's reagent, cyclopiazonic acid can be detected with either iron(II1) chloride or concentrated sulphuric acid and heating (465). Other detection methods were reported by Gorst-Allman and Steyn (197). 7.14.4 Selected applications A semi-quantitative TLC in the presence of appropriate internal and external standards was published by Hermansen et al. (463). The analyses were performed on silica gel 60 precoated on glass. Before use the TLC glates were dipped in 0.3 M aqueous oxalic acid and dried at 110 C for 2 h. Standards and samples (2 pL) were applied on the plate and developed in toluene-ethyl acetate-formic acid (5:4:1) followed by drying at room temperature. Cyclopiazonic acid was detected with Ehrlich's reagent and showed as a spot with RF 0.70. Using Lansden's procedure (461) and detection with Ehrlich's reagent, the toxin was quantitated by reflection densitometry at 540 nm. The detection limit was 25 ng per spot. A simple determination of cyclopiazonic acid in contaminated food and feeds was described by Rathinevalu et al. (466). Semi-quantitative TLC of the toxin in extracts from culture media has been reported by Trucksess et al. (464) and Hermansen et al. (463). al. (460) applied TLC to discriminate Benkhemmar et cyclopiazonic acid-producing (CPA ) from non-producing (CPA-) strains of Aspergillus oryzae. TLC was performed on oxalic acid-impregnated silica gel plates and chloroform-methyl isobutyl ketone (4:l) as the solvent system. After detection with Ehrlich's reagent, the toxin from CPA+ strains revealed a blue-violet spot at R 0.75. TLC has been appfied in studies on the production of cyclopiazonic acid by Penicillium verrucosum var. cyclopium (467). TLC was performed on silica gel G-1500 LS 254 with ethyl acetate-2-propanol-25% ammonia solution (20:15:10). The toxin was measured quantitatively with a spectrodensitometer with a digital counter and integrator at 282 nm. It was detected as a violet spot under visible light after spraying with Ehrlich's reagent. In screeing the toxin in agricultural commodities, Rao and Husain (462) applied PLC to chloroform extracts from culture filtrates. The standard was spotted at one end of the plate. After development (the same system as in the latter paper), the standard was detected with Ehrlich's reagent (the remainder of the plate being covered with a glass plate). When the standard was detected, the covering plate was removed and the TLC plate exposed to iodine vapour. The area with an R value corresponding to the standard spot and coloured witK iodine vapour was scraped off, eluted with methanol, and used for colorimetric determination of the toxin using a modification of Ehrlich's reagent. In addition to TLC methods, cyclopiazonic acid in agricultural products and foods can be successfully determined by HPLC (see Chapter 8).

217

7.15 PR TOXIN AND ROQUEFORTINE PR toxin and roquefortine are secondary metabolites of strains of Penicillium rogueforti and have been isolated from fungal isolates from blue cheese and other sources. The production, isolation and chromatographic techniques were reviewed (30, 468). 7.15.1 Extraction and clean-up Still (469) extracted PR toxin from culture filtrates with chloroform and Scott et al. (470) used ethyl acetate. Two basic procedures for extraction and clean-up from blue cheese were published by Scott and Kanhere (471). In the first procedure, the sample was extracted with a mixture of methanol-water and hexane and centrifuged. After filtration, the methanol-water layer was extracted with chloroform, the extract was evaporated, the residue was dissolved in chloroform and immediately analysed by TLC for PR toxin and/or PR imine. In the second procedure, cheese was blended with ethyl acetate and centrifuged. The extract was evaporated and partitioned between hexane and acetonitrile. The acetonitrile layer was evaporated and the residue was dissolved in chloroform for immediate TLC analysis. Roquefortine is present mainly in the mycelium of P. roqueforti. Extraction and clean-up procedures were summarized by Scott (472). CC procedures for the separation of roquefortine from other alkaloids isolated from P. rogueforti or other penicillia have also been described. It has been found that roquefortine could be eluted with chloroform-methanol-25% ammonia solution (70:10:0.5) from silica gels (473) and with chloroform-ethanol (95:5) from basic alumina (474). Fractions from CC columns were monitored by TLC. 7.15.2 Adsorbents and solvent systems Solvent systems for the TLC of PR toxin on silica gel include chloroform-methanol (96:4), chloroform-2-propanol (1O:l or 4:1), toluene-ethyl acetate-formic acid (5:4:1 or 6:3:1) and toluene-ethyl acetate (30:70) saturated with water (471,475, 476). Solvent systems for roquefortine that have been used with silica gel TLC plates (477, 478) include chloroform-methanol-28% ammonia solution (90:10:1), chloroform-methanol (9:1), chloroform-re-distilled diethylamine (8:2), chloroform-ethanol (10:1.5), acetone-chloroform (3:2) and benzene-methanol (93:7). 7.15.3 Detection PR toxin can be detected by its green fluorescence under long-wave UV light following exposure of the chromatograms to short-wave UV light for about 0.5 min (471, 475). After spraying the chromatograms with 50% sulphuric acid, the toxin appears as a yellow spot (475). The toxin was quantitated in situ by

218

fluorodensitometry

after

spraying the plates with 1% in concentrated HC1-acetone (1:lO) or in ethanol with subsequent exposure to HC1 fumes for 10 mini the latter is the preferred method (476). Roquefortine on TLC plates can be detected as a blue-grey spot after spraying with 5 0 % sulphuric acid and heating at llO°C for 10 min (479). Other spray reagents are Pauli reagent (480), Van Urk reagent (473) and Ehrlich's reagent (474, 478). pdimethylaminobenzaldehyde

7.16 XANTHOMEGNIN, VIOMELLEIN AND VIOXANTHIN

Xanthomegnin, viomellein and vioxanthin are toxic metabolites of a number of fungi including Aspergillus and Penicillium species: these micromycetes are of particular interest because they are routinely implicated in toxin contamination of foods and feeds. 7.16.1 Extraction and clean-up Wall and Lillehoj (481) used the following extraction and clean-up procedure. A strain of A. ochraceus was cultivated on rice for 10 days. The mouldy rice was extracted by suspension in methylene chloride and grinding. The extract was filtered and the solvent removed by vacuum evaporation. The crude oil was subsequently extracted three times with acetonitrile and the acetonitrile solutions were used for chromatography. 7.16.2 Adsorbents and solvent systems TLC methods for the detection of xanthomegnin and viomellein utilize silica gel plates and benzene-methanol-acetic acid (18:l:l) or toluene-ethyl acetate-formic acid (6:3:1) as the solvent systems (482). 7.16.3 Detection After standing for 6 h, the spots of xanthomegnin turn from yellow to orange and those of viomellein turn from yellowish green to yellowish brown. Exposure to ammonia fumes turns the compounds from yellow to purple (482). The detection limits were 0.1 pg for xanthomegnin and 0.3 kg for viomellein. 7.16.4 Selected applications Standards of xanthomegnin and viomellein were prepared by Wall and Lillehoj (481) by PLC on silica gel plates that were developed in benzene-methanol-acetic acid (18:l:l). Appropriate bands were scraped off the plates and the compounds were eluted with methylene chloride. The solvent was removed under a stream of nitrogen and standards were stored as dry films in a freezer. Purity was determined by TLC and HPLC comparisons with reference compounds. In a screening for toxigenic isolates of Aspergillus ochraceus from green coffee beans, Stack et al. (483) applied TLC in detecting xanthomegnin, viomellein and vioxanthin in addition to ochratoxins.

219

7.17

NAPHTHO-r-PYRONES

Monomeric and dimeric naphtho-r-pyrones have been isolated from the mycelium of Aspergillus niger by several groups of workers. Ehrlich et al. (484) subcultured an A. niger isolate on rice, corn, cottonseed and two liquid media. After incubation, the culture (in the case of culture on liquid media, the mycelial mat) was extracted with methylene chloride. The solvent was evaporated and the residual red paste was treated with 9 volumes of cold hexane and kept at 5OC overnight. The red precipitate was collected, dissolved in methylene chloride and filtered. Samples were examined by HPTLC. HPTLC was carried out on LHP-KF plates (Whatman) and developed with benzene-ethyl acetate-formic acid (10:4:1). Components were identified by their colour, fluorescence under long-wave UV light and colour after spraying with Gibbs reagent. HPTLC showed that the mixture contained more than 18 components, contained but only the material migrating at RF 0.5-0.8 naphtho-r-pyrones. The results are given in Table 7.28. TABLE 7.28 HPTLC data for naphtho-r-pyrones Adapted from ref. 484. Compound Flavasperone Fonsecin monomethyl ether Rubrofusarin Aurasperone A Isoaurasperone A Aurasperone B Aurasperone D Aurasperone C

RF x loox 81 76 72 67 61 56 53 49

Gibbs test

Fluorescence

Blue Brown Blue-green Violet Red-violet Brown Violet Brown

Violet Violet Orange Yellow Yellow Yellow Yellow Yellow

With benzene-ethyl acetate-formic acid (100:40:10) on Whatman LHP-KF 7.18 SECALONIC ACIDS

The secalonic acids are xanthone dimers with identical molecular masses and molecular formulae, differing in their stereochemistry. Secalonic acid D is the most studied member of this group (485). Methods used for the production, isolation, separation, purification and detection of secalonic acid D have been summarized (486). TLC and HPLC techniques were also included. On TLC plates, secalonic acids can be detected by quenching fluorescence (487) or by spraying with cerium(1V) sulphate

220

reagent (488), iron(II1) chloride, or panisaldehyde reagent (130). RF values of secalonic acids in a variety of solvent systems are given in Table 7.29. Ciegler et al. (487) quantitated secalonic acid D on pre-coated silica gel F254 plates using benzene-ethyl acetate-formic acid (100:40:10) as the solvent system. TABLE 7.29 RF x 100 values for secalonic acids SorbentX

OA-treated silica gel TA-treated silica gel Silufol

Solvent systemX

CHC13-MP (9:l)

Secalonic acid A

B

D

23

46

23

CHC13-Pen

17

C6H6-MeOH-HOAC (24:2:1) Tol-EtOAc-FA (6:3:1) CHC1,-MeOH (4:l)

28 32 68

Ref. F 488, 489 29

490

130

Abbreviations: OA, oxalic acid; TA, tartaric acid; CHC13, chloroform; MP, 4-methyl-2-pentanone; CgHg, benzene; MeOH, methanol; HOAc, acetic acid; Toll toluene; EtOAc, ethyl acetate; FA, 90% formic acid; Pen, 2-pentanone. 7.19 TLC OF MISCELLANEOUS TOXINS In this section, TLC data for the following compounds are included: moniliformin, wortmanin, echinulin, fusaric acid analogues, fusarin C, viridin and toxic peptides. Jansen and Dose (491) described a quantitative TLC determination of moniliformin in vegetable foods and feeds. Crude acetonitrile extracts of Fusarium moniliforme cultures were checked for moniliformin (492, 493) by spotting, together with a standard, on pre-coated thin layers of silica gel 60 and developing in chloroform-methanol-formic acid (70:30:0.16). The toxin was detected by spraying and heating with 0.5% aqueous 3-methyl-2-benzothiazolinone hydrazone hydrochloride. The limit of detection was approximately 8 wg/g in corn culture. Most recently, Chelkowski et al. (494) published a simple TLC method for moniliformin detection. Rice cultures were dried and powdered. Moniliformin was extracted from 3 g of powdered culture with 6 mL of water to form a slurry, diluted after 15 min with 34 mL of ethanol. This suspension was kept overnight in a refrigerator and filterd the next day. The filtrate (1-20 pL) was spotted on to Merck 5553 TLC plates. A moniliformin standard in methanol (100 pg/mL) in amounts of 0.2, 0.5, and 1 pg in

22 1

a spot was placed on the same plate. Plates were developed in chloroform-methanol (6:4) as the solvent system and moniliformin 0.5% water solution of MBTH Aldrich visualized with (3-methyl-2-benzothiazoline-hydrazone hydrochloride, freshly prepared) after heating for 10 min at 14OoC. The spots which appeared were red-violet, with detection limit 0.1 pg in each spot. In acidic atmosphere the colour of spots turns into brown-grey and even green, so it is necessary to avoid contact of chromatograms with vapours of volatile acids (HC1 and others). Ammonia vapours intensify formation of the carmine-red colour. The authors recommended placing developed plates for 5 min into a tank with ammonia vapours before they are sprayed with MBTH. Shepherd and Gilbert (495) developed an effective HPLC method for moniliformin. PLC on silica gel plates developed with chloroform-methanol (97:3) was used to purify a haemorrhagic factor from Fusarium oxysporum identical with the antibiotic wortmannin (496). Echinulin was isolated by means of PLC from acetone extracts of feed refused by swine. The solvent system was ethyl acetate-hexane (8:2) and the toxin turned blue in the presence of panisaldehyde reagent at llO°C. The anisaldehyde-reactive material from the PLC was identified with echinulin by its UV and IR spectra (497). Viridin, a steroid-like antibiotic, is converted by viridin-producing fungi intc its dihydro derivative, viridiol, which is ineffective as an antibiotic but is a potent phytotoxin. Both metabolites were isolated from culture extracts by means of PLC (498). TLC was used to characterize two new fusaric acid analogues from Fusarium moniliforme (499). Fusarin C is a mutagenic mycotoxin produced by Fusarium moniliforme. Its natural occurrence in corn was reported by Gelderblom et al. (500). Corn samples were extracted with water and methylene chloride-2-propanol (1:l). After filtering, drying the extract and evaporating to dr ness, the residue was extracted with petroleum ether (60-808C) and chloroform. The petroleum ether was re-extracted with acetonitrile and the residues from the chloroform and acetonitrile extracts were chromatographed on a column of silica gel with methylene chloride-methanol (19:l) as the eluent. Scott et a l . (493) used acetonitrile to extract ground corn, corn meal, or wheat flour. After filtration and evaporation of the solvent, clean-up was carried out on small disposable amino bonded phase or silica gel columns with methylene chloride-methanol (9:l) as eluting solvent. TLC on silica gel has been used by Farber and Sanders (501) or by Wiebe and Bjeldanes ( 5 0 2 ) using chloroform-methanol (9:l) or chloroform-2-propanol (9:l) as the solvent

222

systems.Standards and positive samples were identified by the presence of bright yellow spots under visible light ( R F in the former system, 0.32 to 0.35). Jackson e t al. (503) assessed fusarin C standard purity by TLC, nuclear magnetic resonance, and mass spectral analysis. TLC has also been used to characterize cyclosporin A extracted from rice (504). Silica gel plates were developed in 3 solvent systems: (1) n-butanol-acetic acid-water (4:1:1), (2) chloroform-acetic acid-methanol (85:10:5), and (3) ethyl acetate-hexane-acetone (2:l:l). TLC plates were dried with a hot-air blower gun placed in an iodine chamber for 15 min to detect iodine-reactive substances. Iodine was sublimed from the plates by placing them in an oven at llO°C for 15 min. The plates were sprayed with 6 M HC1 and oven-dried at llO°C for 30 min. Dried plates were sprayed with 0.1% ninhydrin solution in n-butanol. Orange to brown spots had RF values: 0.83 in system 1: 0.92 in system 2: 0.81 in system 3. 7.20 MULTI-MYCOTOXIN TLC Various multi-mycotoxin methods have been published for the simultaneous detection of a number of mycotoxins, which differ in the extraction solvents, clean-up procedure and final detection TLC procedure. In clean-up techniques, mini-column chromatography has been used by several workers ( e . g . , refs. 60, 61, 505-507). Patterson et al. (508) used a dialysis clean-up procedure. A final TLC analysis has been adopted in the following selected instances. Originally, Eppley (61) described a screening method for zearalenone, aflatoxin and ochratoxin. His techniques were subsequently used or adapted by various workers. Steyn (465) reported a TLC system for the simultaneous separation and detection of eleven mycotoxins, in which extensive purification of acidic mycotoxins was achieved by removal of the neutral material. The procedure used silica gel G TLC plates impregnated with oxalic acid, with development in chloroform-methyl isobutyl ketone (4:l). The mobility of the neutral mycotoxins was essentially unaffected when oxalic acid was omitted, whereas the acidic mycotoxins, e . g . , cyclopiazonic acid and secalonic acid, and also ochratoxins remained at the origin. The mycotoxins were detected by examination of TLC plates under long-wave UV light and spraying with 1% cerium(1V) sulphate in concentrated sulphuric acid or 1% ethanolic iron(II1) chloride. Later, Gorst-Allman and Steyn (197) used the following spray reagents: (a) 2,4-dinitrophenylhydrazine (1 9)-concentrated sulphuric acid (7.5 mL)-ethanol (75 mL)-water (170 mL); (b) hydrazono-2,3-methylbenzothiazole hydrochloride (0.5% aqueous solution); (c) iron(II1) chloride ( 3 % solution in ethanol): (d) aluminium chloride (1% solution in chloroform): (e) Ehrlich reagent: (f) cerium(1V) sulphate (1% solution in 3 M sulphuric acid): (9) vanillin (1% in 50%

223

phosphoric acid). The plates were sprayed, the immediate effects noted, and they were then heated at llO°C for 10 min. Iodine and ammonia fumes were also used for some plates. Characteristic colours were reported. Whidden et al. (507) developed a method for simultaneous extraction, separation and qualitative analysis of rubratoxin B, aflatoxin B1! diacetoxyscirpenol, ochratoxin A, patulin, penicillic acid, sterigmatocystin and zearalenone in corn. Mycotoxins were extracted with acetonitrile, sequentially eluted from a silica gel mini-column and rendered visible by TLC. A flow chart for the extraction and separation of the eight mycotoxins is presented in Fig. 7.1. Fractions 11-IV were analysed on the same TLC plate using external and internal standards and the solvent system toluene- ethyl acetate-formic acid (6:3:1). Fraction V (containing rubratoxin B) was applied to a separate TLC plate together with external standards (five concentrations of the toxin) and developed in acetonitrile-acetic acid (100:2).

Ground sample Acetonitrile Residue

Filtrate Wash with

II

isooctane

I

I

Isooctane

Acetonitrile

(g5:r

acetone

ether

methanol

Fig. 7.1. Flow chart for the extraction andseparation of mycotoxins Adapted from Whidden et al. (ref. 507).

224

A multi-mycotoxin method involving a membrane clean-up step two-dimensional TLC was published by Patterson et a l . (508). Fishbein and Falk (509) developed TLC procedures for five types of mycotoxins (aflatoxins, ochratoxins, aspertoxin, 0-methylsterigmatocystin and sterigmatocystin) and some other fungal metabolites. Stoloff et a l . (510) described a multi-mycotoxin TLC method for aflatoxins, ochratoxins, zearalenone, sterigmatocystin and patulin in a number of agricultural products. They used silica fluorophores and 9e1 plates with internal benzene-methanol-acetic acid (18:l:l) or hexane-acetone-acetic acid (18:2:1) as the solvent system. The developed plates were viewed under both short- and long-wave UV light. The limits of detection ranged from 20 (aflatoxin) to 450 wg/kg (patulin). Joseffson and Moller (505) reported detection limits of aflatoxin 5, ochratoxin 10, patulin 50, sterigmatocystin 10 and zearalenone 35 pg/kg by using gel filtration on Sephadex LH-20 as a clean-up procedure prior to TLC. Wilson et a l . (290) published a method for the detection of aflatoxins, ochratoxins, zearalenone, citrinin and penicillic acid. Mycotoxins in chloroform extracts were isolated by CC and then separated by TLC on Adsorbosil-1 pre-coated plates. Moubasher et al. (511) evaluated the toxin-producing potential of fungi isolated from blue-veined cheese. The toxins tested for were aflatoxins, patulin, versicolorin, sterigmatocystin, ochratoxin A, kojic acid and penicillic acid. Coman et a l . (512) reported a TLC analysis of feed samples in which four aflatoxins, ochratoxin A, zearalenone, sterigmatocystin and T-2 toxin were detected. Zearalenone, T-2 toxin, neosolaniol and HT-2 toxin were detected in grains of barley, wheat and oats by Ilus et a l . (513) as follows. Toxins were extracted with ethyl acetate, purified on a Kieselgel TLC plate and analysed by TLC using acetone-hexane as the solvent with detection at 3 6 6 nm or with panisaldehyde reagent. Nowotny et a l . (514) detected citrinin, ochratoxin A and sterigmatocystin in samples of commercial cheese using TLC and HPLC. Gimeno and Martins (515) described a rapid TLC determination of mycotoxins which can often be found in fruits and fruit products. The method was tested for patulin, citrinin and aflatoxin in apples and pears and their juices and jams. The mycotoxins were extracted with a mixture of acetonitrile and 4% aqueous KC1 (9:l). The extract was cleaned up with water and then acidified, and the toxins were recovered with chloroform and separated by TLC. Toxin identity was confirmed with various developing solvents, spray reagents and chemical reactions and then quantitated by the limit of detection method. The minimal detectable concentrations were: patulin 120-130, citrinin 30-40, aflatoxin B1 and G1 2-2.8 and aflatoxin B2 and G2 2 wg/kg * and

22 5

A method for the routine examination of mouldy rice, wheat bread and other vegetable foodstuffs was published by Johann and Dose (376). The mycotoxins are first extracted with acetonitrile-4% KC1 and cyclohexane and then transferred from acetonitrile into a methylene chloride phase and separated by two-dimensional TLC. Aflatoxins are determined fluorimetrically after development in chloroform-acetone (9:l) and methylene chloride-acetonitrile (8:2). Other mycotoxins (ochratoxin A, patulin, penicillic acid, and sterigmatocystin) are analysed on separate plates with toluene-ethyl acetate-acetic acid (6:3:1) and benzene-acetic acid (8:2). Citrinin is chromatographed on a plate pre-treated with oxalic acid. Citrinin and ochratoxin A, like the aflatoxins, can be immediately determined by fluorimetry, whereas the other toxins have to be converted into fluorescent derivatives using spray reagents (penicillic acid using diphenylboric acid-2-ethanolamineI patulin using N-methylbenzthiazolone-2-hydrazone and sterigmatocystin using aluminium chloride) for quantitative determination. Gorst-Allman and Steyn (197) separated 13 mycotoxins as neutral (aflatoxin B1, sterigmatocystin, zearalenone, patulin, T-2 toxin, roquefortine, penitrem A , fumitremorgin B and roridin A) and acidic (citrinin, ochratoxin A , a-cyclopiazonic acid and penicillic acid) metabolites. Mean values of the neutral mycotoxins are presented in Table '7.30 and those of acidic mycotoxins in Table 7.31. The acidic mycotoxins were well separated on silica gel plates pre-treated with oxalic acid.

TABLE 7.30 Mean R x 100 values of neutral mycotoxins Adaptes from ref. 197. Mycotoxin

Aflatoxin B1 Sterigmatocystin Zearalenone Patulin T-2 toxin Roquefortine Penitrem A Fumitremorgin B Roridin A

Solvent system'

A

B

C

D

E

44

35 53

27 55

03

65 74 71 56 68 13 76 71 61

67

40 22 45

51 27 36

16 22

03

01

02

40 51 31

51

34 28 13

36 22

38

41 41 18 13 01 49 14 09

F

24 56

44 20

22 02

45 30

14

Solvent systems: A, chloroform-methanol (97:3); B, chloroform-acetone-nhexane (7:2:1): C, chloroform-acetone (9:l): D, ethyl acetate-nhexane (1:l): E l chloroform-acetone2-propanol (85:15:20); F, benzene-chloroform-acetone (45:40:15).

226

TABLE 7. 31 Mean RF x 100 values of acidic mycotoxins using pre-treated with oxalic acid Adapted from ref. 197. Mycotoxin

Citrinin Ochratoxin A a-Cyclopiazonic acid Penicillic acid

TLC plates

R F X 100

Chloroform-methanol

Chloroform-acetone

(98:2)

(9:1)

52 32 52 16

51 34 44 20

buraekova e t al. (130) presented a TLC systematic analysis for 37 mycotoxins and 6 other fungal metabolites in which Inchromatographic spectra" were generated for each toxin from their R values in eight solvent systems. The advantage of this system Ties in the comparisons of relative rather than absolute RF values, as the latter show greater variations than the former with changes in the conditions of the environment. This method was developed for the identification of known mycotoxins. The chromatographic spectrum of an unknown substance provides a preliminary identification by comparison with known chromatographic spectra or eliminates the known metabolites from the unknown. The method was extended to the detection of unknown mycotoxins by combining it with a bioassay to yield a bioautographic detection method (134). Lee e t al. (128) described a method for the simultaneous determination of thirteen mycotoxins by HPTLC. With seven continuous multiple developments with two solvent systems of different polarity, a baseline separation of sterigmatocystin, zearalenone, citrinin, ochratoxin A, patulin, penicillic acid, luteoskyrin and aflatoxins B1, B , G1, G2, M1 and M 2 was obtained. About 1 h was require3 for the separation and quantitation of all 13 mycotoxins from one spot. By using in s i t u scanning of the HPTLC plate, detection limits in the low nanogram range were obtained by UV-visible absorption and in the low picogram range by fluorescence, with a relative standard deviation of 0.7-2.2% in the nanogram range. Chromatography was performed on 10 x 10 cm HPTLC plates coated with silica gel 60 and impregnated with EDTA. The development stage and spectroscopic properties used for quantitative determination of the individual mycotoxins are given in Table 7.32. The mobile phase migration distance was 4 cm and was fixed by arranging for a portion of the plate to protrude through the top of the

221

saturated development chamber, at which point the solvent could freely evaporate. For the very complex sample of 13 mycotoxins, the use of continuous multiple development offerred certain advantages, such as the possibility of quantifying the components as they were separated, the use of more than one solvent system, and natural refocussing of the sample spot, which occurred when the plate was dried between developments. The resolution of sterigmatocystin, zearalenone and citrinin was obtained in the first continuous development. The plate was removed from the chamber and air-dried prior to making the quantitative measurement of the three separated toxins. The other toxins remained close to the origin. After a second and third development, ochratoxin A was separated sufficiently to be determined. A fourth development enabled penicillic acid, patulin and luteoskyrin to be determined. For the separation of aflatoxins, still remaining close to the origin, a second, more polar, solvent system was used. After three continuous developments with this new solvent system, the six aflatoxins were completely separated. TABLE 7.32 Development stages and spectroscopic detection of mycotoxins by HPTLC Adapted from ref. 128. Development stageX

Time Mycotoxin (min) separated

methods

for

the

Spectral characteristic used for detectionX

Tol-EtOAc-FA (30:6:0.5) 5.0 Sterigmatocystin 1st development Zearalenone Citrinin 2nd development 5 . 0 No measurement 3rd development 6.0 Ochratoxin A 4th development

used

6.0 Penicillic acid

Patulin Luteoskyrin Tol-EtOAC-FA (30:14:4.5) 5th development 8.0 No measurement 6th development 8.0 No measurement 8.0 Aflatoxins B1, B2, 7th development G1, G2, MI and M2

Ref. Flu.,, Flu.,,

at 324 nm at 313 nm at 460 nm

Flu.,, Flu.,, Ref. Ref. Ref.

at at at at at

Flu.,, Flu.,,

at 365 nm at 430 nm

313 460 240 280 440

nm nm nm nm nm

Abbreviations: Tol, toluene: EtOAc, ethyl acetate: FA, formic acid; Ref., reflectance; Flu., fluorescence: em, emission: ex, excitation.

228

At each scanning stage, the migration distance of the spot to be measured was maintained between 1 and 3 cm. Only patulin and luteoskyrin slightly overlapped each other, but as patulin does not show any absorption at the absorption maximum for luteoskyrin (440 nm), this was no problem. Hence the method described is capable of providing good resolution of complex mycotoxin mixtures. However, the authors used standard mycotoxin solutions and did not show whether comparable results could be obtained with samples extracted from natural commodities. HPTLC and reversed-phase TLC of 10 mycotoxins (ochratoxin A, aflatoxins Bl, B , G1 and G2, zearalenone, sterigmatocystin, T-2 toxin, diacegoxyscirpenol and vomitoxin) with the use of various normal- and reversed-phase solvents and UV detection were reported by Stahr and Domoto (516). Golinski and Grabarkiewicz-Szczesna (517) published chemical confirmatory tests for ochratoxin A, citrinin, penicillic acid, sterigmatocystin and zearalenone that are performed directly on TLC plates. Later Grabarkiewicz-Szczesna et a l . (518) reported a multi-detection procedure for the determination of 11 mycotoxins in cereals. A simultaneous TLC detection of aflatoxin B1 and zearalenone in mixed feed for pigs was described by Fulgeira and de Bracelenti (519). A quantitative TLC method for the analysis of aflatoxins, ochratoxin A, zearalenone, T-2 toxin and sterigmatocystin in foodstuffs was published by Tapia (520). Detections of Fusarium moniliforme toxins (493) and toxigenic Fusarium isolates (521) have been reported. A simple screening method for moulds producing the intracellular mycotoxins brevianamide A, citreoviridin, cyclopiazonic acid, luteoskyrin, penitrem A, roquefortine c, sterigmatocystin, verruculogen, viomellein and xanthomegnin was developed by Filtenborg et a l . (522). After removing an agar plug from the mould culture, the mycelium on the plug is wetted with a drop of methanol-chloroform (1:2). By this treatment the intracellular mycotoxins are extracted within a few seconds and transferred directly to a TLC plate by immediately placing the plug on the plate while the mycelium is still wet. After removal of the plug, known TLC procedures are carried out. The same procedure was applied to detect aflatoxins, ochratoxin A, citrinin, patulin and penicillic acid in solid substrates. In most screening procedures, extraction and clean-up techniques are applied prior to the TLC analysis. Krivobok et a l . (523) described rapid and sensitive methods for detecting toxigenic fungi producing aflatoxins, ochratoxin A, sterigmatocystin, patulin, citrinin, penicillic acid and zearalenone. The toxin-producing moulds tested produced detectable amounts of their respective mycotoxins within 2-4 days of incubation in a liquid medium. Sterigmatocystin had to be extracted from the mycelium and the rapid roduction of zearalenone needed to be temperature programmed (24I3C for growth

229

and 10°C for toxin production). Detection of the toxins by means of TLC was possible without extraction of the medium or after extraction without purification. The sensitivity of TLC detection and the recovery after extraction were good. An extraction, purification and separation diagram for mycotoxins from contaminated food was proposed by Hadidane et al. (524). Non-oleaginous and oleaginous solid samples and also oils were analysed. Four solvent systems and four detection techniques were used. Data for four aflatoxins, citrinin, and four Fusarium toxins were reported. A modification of these techniques and its use in monitoring and identification of fungal toxins in food products, animal fed and cereals in Tunisia was published more recently by Bacha et al. (525). The Fusariummycotoxins most frequently encountered in corn and often implicated in the natural causes of mycotoxicoses include: zearalenone, zearalenols, some trichothecenes and moniliformin. A multi-mycotoxin method for Fusarium isolates from corn kernels or tissues was published by Bottalico et al. (526). The isolates were grown on autoclaved corn kernels at 27OC for 4 weeks. Then the cultures were dried at 6OoC and finely ground. Samples (50 9 ) of dried corn (kernels or vegetative parts) or dried Fusarium cultures (20 9) were extracted with methanol-aqueous NaC1, defatted with hexane, and partitioned with dichloromethane. After the evaporation of the solvent, the residue was brought up to 2 mL with methanol-water (40:60), passed through a Sep-Pak C-18 cartridge, and eluted with a new portion (2 mL) of the methanol-water mixture representing the first pure fraction (fraction A ) . Further elution with methanol (2 x 2 mL) yielded fraction B. The two fractions were separately evaporated to near dryness and reconstituted with methanol (0.5 mL). Fraction A was examined for nivalenol, fusarenone, deoxynivalenol, 15-acetyldeoxynivalenol; and 3-acetyldeoxynivalenol, and fraction B was examined for diacetoxyscirpenol, T-2 toxin, zearalenone, and zearalenols (a and p ) . Analyses of zearalenone and trichothecenes were performed by TLC and GLC. Trichothecenes eluted in fraction A, as well as zearalenols, were confirmed and quantitated by HPLC. The separation of 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol was only possible by TLC or capillary GLC, and not by HPLC. Due to the low recovery of the extraction procedure for polar trichothecenes, particularlynivalenol, a HPLC method for nivalenol was used in few cases. Analysis of moniliformin was carried out in accordance with the method previously employed (527). Chakrabarti and Ghosal (528) used PLC in a study of the occurrence of free and conjugated 12,13-epoxytrichothecenes and zearalenone in banana fruits infected with Fusarium moniliforme. TLC was used by Mirocha et al. (529) to detect mycotoxin production by Fusarium oxysporum and F. sporotrichoides isolated

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from Baccharis spp. from Brazil (known to produce macrocyclic trichothecenes). Physical and chemical properties that may be used to determine the purity of several Fusarium mycotoxins have been investigated by Bennett and Shotwell (530). A combination of analytical procedures, which include HPTLC, liquid chromatography, gas chromatography, gas chromatography/mass spectrometry, ultraviolet spectrometry, and nuclear magnetic resonance spectrometry have been used to examine mycotoxin standards obtained from commercial sources and from laboratory fermentations. Data for the following mycotoxins were reported by these workers: a-zearalenol, f3-zearalenol, deoxynivalenol, T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, nivalenol and fusarenone X. 7.21 TLC IN CHEMOTAXONOMIC STUDIES OF TOXIGENIC FUNGI Multi-mycotoxin TLC has been introduced in chemotaxonomic studies of penicillia and other micromycetes. In addition to mycotoxins, other secondary metabolites have also been detected in such studies. The development of the methodology is given in this section. In addition to several physiological criteria, the pattern of extracellular metabolites (mostly mycotoxins) after TLC was used by Frisvad (531) as a chemotaxonomic criterion in identification of common asymmetric penicillia. Mycotoxin analyses were performed using an "agar plug methodmm.Agar plugs were cut out at the border of a colony grown on an agar medium with a flamed stainless-steel tube (inner diameter 0.4 cm). The plugs were placed directly on the pre-coated TLC plates and were removed after 10 s , and the application spots were allowed to dry. After spotting toxin standards (patulin, ochratoxin A, citrinin, penicillic acid, griseofulvin and penitrem A), the plates were developed in four solvent systems and each toxin was visualized in at least two ways. Later, Frisvad and Filtenborg (532) reported a classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites. In this study, extracellular and intracellular mycotoxins and other metabolites were analysed by means of TLC. Griseofulvin was used as an external standard in all analyses. Four solvent systems were used. The TLC plates were examined before and after chemical treatment under visible light and UV light at 366 nm. Frisvad (533) included TLC analyses of extracellular (ochratoxin A and citrinin) and intracellular mycotoxins (xanthomegnin and viomellein) in a screening of groups of toxigenic Penicillium viridicatum. Profiles of primary and/or secondary metabolites have been used by the same author in classification of various Penicillium and Emericella species (534-537). More recently, Frisvad and Thrane (538) published a general

23 1

standardized method for the analysis of 182 mycotoxins and other fungal metabolites, based on HPLC and combined with TLC in two different solvent systems using R values relative to griseofulvin. Data for the 182 metabolifes may be found in their paper. These metabolites include the best known mycotoxins, penicillin G , many alkaloids, polyketides and terpenes. A similar approach has been undertaken by Paterson (539). He presented standardized TLC data in two solvent systems for secondary metabolites of Penicillium and other fungi to assist in the identification of products of Penicillium species. Of 107 metabolites detected with TLC system /toluene-ethyl were named and 27 acetate-90% formic acid (5:4:1)/ 8 0 unidentified compounds were allotted reference numbers; in the case of the metabolites detected by system 2/chloroform-acetone2-propanol (85:15:20)/ the equivalent figures were 79 and 18, respectively. A chemotaxonomic study, confirming the production of a range of important mycotoxins by certain species of Penicillium, was reported by El-Banna et al. (540). One thousand four hundred Penicillium isolates were identified according to Pitt's classification. To confirm which species produce which mycotoxins, representative isolates were investigated for the synthesis of 18 mycotoxins. Isolates were grown on malt extract agar incubated for one to three weeks at 25OC. Thereafter the medium was extracted with chloroform, and the filtered, concentrated extracts used for mycotoxin analysis by TLC. The production of any particular mycotoxin was confirmed by using external standards in optimal developing systems with toxins visualized by the best visualizing methods. The following 18 mycotoxins investigated were produced by one or more Penicillium species: brevianamid A , citreoviridin, citrinin, cyclopiazonic acid, fumitremorgin B, griseofulvin, luteoskyrin, ochratoxin A , patulin, penicillic acid, penitrem A , PR toxin, roquefortine, rugulosin, verrucosidin, verruculogen, viridicatumtoxin and xanthomegnin. Procedures for the detection of the mycotoxins used in this work were described and included adsorbents, solvent systems, detection methods and colours of the mycotoxins after treatment. Mycotoxin production by the various species of Penicillium is quite distinctive and may be used as a valuable aid in their identification. 7.22 CONCLUSIONS

This chapter was written with the aim of demonstrating the scope of applications of TLC in the still developing field of mycotoxins. The "mycotoxin era" had its origins in the early sixties when the gradual decline of applications of paper chromatography was due to the rapid development of TLC. Hence, applications of PC in mycotoxicology are now interesting mostly

232

from a historical point of view and only some typical examples were mentioned in the Introduction. TLC is by far the most widely used chromatographic technique applied to mycotoxins owing to its relatively simple, fast and inexpensive character. As in most instances the mycotoxins to be analysed or purified by means of TLC are present in contaminated samples, they must be extracted and cleaned up prior to TLC if reliable results are to be obtained. Extraction procedures, reviewed in this chapter, include extractions of mycotoxins from feeds and foodstuffs, cultivation media and/or mycelia of toxigenic fungi. Extraction solvents include chloroform, methylene chloride, ethyl acetate, acetone, acetonitrile, methanol and their combinations. Clean-up procedures include CC (mostly using silica gel columns), gel-permeation chromatography, liquid-liquid partition and precipitation techniques. In these procedures, contaminating lipids, fatty acids, proteins and various pigments are mostly removed from the mycotoxin samples. Silica gel is the most commonly used adsorbent in the TLC of mycotoxins. With acidic toxins, better results are obtained when the silica gel plates are pre-treated with oxalic acid, tartaric acid or EDTA. Chemically bonded reversed-phase layers can be used in special applications. The variety of solvent systems is enormous. The most often used solvents combined in various ratios include benzene, chloroform, toluene, ethyl acetate, methylene chloride, acetone, methanol, formic acid and acetic acid. The detection techniques vary with the mycotoxins to be detected. Coloured toxins are examined under visible light, and fluorescent ones are revealed under short- and/or long-wave UV light. Colourless and non-fluorescent compounds can be detected by means of appropriate spray reagents producing colours or fluorescence. Bioautographic detections have also been described, using mostly Artemia salina larvae or microbial cultures. In addition to the classical one-dimensional TLC, two-dimensional TLC and HPTLC have been used by many researchers. With HPLC and in quantitations, TLC becomes more of densitometers and expensive owing to the need spectrophotometers. PLC has been used in the initial preparation of several mycot.oxins belonging to the aflatoxins, cytochalasans, hydroxyanthraquinones, indole-derived tremorgens, zearalenone and its derivatives, etc. The reviewed applications of the TLC of aflatoxins, sterigmatocystins and other aflatoxin intermediates, ochratoxins, rubratoxins, small lactones, trichothecenes, cytochalasans, tremorgenic mycotoxins, hydroxyanthraquinones, epipolythiopiperazine-3,6-diones, zearalenones, citrinin, a-cyclopiazonic cid, secalonic acids, PR toxin, roquefortine, xanthomegnin, viomellein, naphthopyrones and some peptidic mycotoxins emphasize the great importance of thin-layer

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chromatography in the relatively young field of mycotoxicology. However, other chromatographic techniques may be useful in such instances where TLC provides insufficient results. Examples may be found in chapters 8 and 9 of the present book. REFERENCES 1 K. Sargeant, A. Sheridan, J. OmKelly and R.B.A. Carnaghan, Nature (London), 192 (1961) 1095. 2 V. Betina (Editor), Mycotoxins - Production, Isolation, Separation and Purification, Elsevier, Amsterdam, 1984. 3 Y. Ueno, in V. Betina (Editor), Mycotoxins - Production, Isolation, Separation and Purification, Elsevier, Amsterdam, 1984, Ch. 15, p. 329. 4 Y. Ueno, in V. Betina (Editor), Mycotoxins - Production, Isolation, Separation and Purification, Elsevier, 1984, Ch. 24, p. 475. 5 V. Betina, J. Chromatogr., 477 (1989) 187. 6 J. Miyazaki, K. Omachi and T. Kamata, J. Antibiot., 6 (1953) 6. 7 J. Uri, Nature (London), 183 (1959) 1188. 8 P. Nemec, V. Betina and L. Kovaeieova, Folia Microbiol., 6 (1961) 277. 9 V. Betina, Nature (London), 182 (1958) 796. 10 V. Betina and P. Nemec, Nature (London), 187 (1960) 1111. 11 V. Betina, Chromatogr. Rev., 7 (1965) 119. 12 V. Betina, Methods Enzymol., 43 (1975) 100. 13 V. Betina, Chem. Zvesti (Bratislava), 15 (1961) 750. 14 V. Betina, Chem. Zvesti (Bratislava), 15 (1961) 859. 15 V. Betina, J. Chromatogr., 15 (1964) 379. 16 V. Betina, P. Nemec, M. Kutkova, J. Balan and 5 . Kovae, Chem. Zvesti (Bratislava), (1964) 128. 17 V. Betina, Antimicrobial Agents and Chemotherapy 1966, American Society for Microbiology, Washington, 1967, p. 637. 18 B.F. Nesbitt, J. O'Kelly, K. Sargeant and A. Sheridan, Nature (London), 195 (1962) 1062. 19 R.D. Hartley, B.F. Nesbitt and J. OIKelly, Nature (London), 198 (1963) 1056. 20 R.J. Cole (Editor), Modern Methods in the Analysis and

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250 494 J. Chelkowski, M. Zawadski, P. Zajkowski, A. Logrieco and A. Bottalico, Mycotoxin Res., 6 (1990) 41. 495 M.J. Shepherd and J. Gilbert, J. Chromatogr., 358 (1986) 415. 496 H.K. Abbas and C.J. Mirocha, Appl. Environ. Microbiol., 54 (1988) 1268. 497 R.F. Vesonder, R. Lambert, D.T. Wicklow and M.L. Biehl, Appl. Environ. Microbiol., 56 (1988) 830. 498 R.W. Jones and J.G. Hancock, Can. J. Microbiol., 33 (1987) 963. 499 H.R. Burmeister, M.D. Grove, R.E. Peterson, D. Weisleder and P.D. Plattner, Appl. Environ. Microbiol., 50 (1985) 311. Gelderblom, P.G. Thiel, W.F.O. Marasas and K.J. van 500 W.C.A. der Merwe, J. Agric. Food Chem., 32 (1984) 1064. 501 J.M. Farber and G.W. Sanders, Appl. Environ. Microbiol., 51 (1986) 381. 502 L.A. Wiebe and L.F. Bjeldanes, J. Food Sci., 46 (1981) 1424. 503 M.A. Jackson, P.J. Slininger and R.J. Bothast, Appl. Environ. Microbiol., 55 (1989) 649. 504 J.V. Edwards and E.B. Lillehoj, J. Assoc. Off. Anal. Chem., 70 (1987) 126. M ller, J. Assoc. Off. Anal. 505 B.G.E. Josefsson and T.E. Chem., 6 0 (1977) 1369. 506 Y. Takeda, E. Isohata, R. Amano and M. Uchiyama, J. Assoc. Off. Anal. Chem., 6 2 (1979) 573. 507 M.P. Whidden, N.N. Davis and B.A. Roberts, J. Agric. Food Chem., 28 (1980) 784. 508 D.S.P. Patterson, S.P. Derycks and U.L. Diener, J. Agric. Food Chem., 28 (190) 1265. 509 L. Fishbein and M.L. Falk, Chromatogr. Rev., 12 (1970) 42. 510 L. Stoloff, L. Nesheim, L. Yin, J.V. Rodricks, M. Stack and A.D. Campbell, J. Assoc. Off. Anal. Chem., 54 (1971) 91. Abdel-Kader and I.A. El-Kady, 511 A.H. Moubasher, M.I.A. Mycopathologia, 66 (1978) 187. 512 I. Coman, 0. Popescu, P. Halga, N. Grunberg and E. Ciudin, Rev. Cresterea Anim., 28 (1978) 58; C.A., 93 (1980) 68758d. 513 T. Ilus, M.L. Niku-Paavola and T.M. Enari, Eur. J. Appl. Microbiol., Biotechnol., 11 (1981) 244. 514 P. Nowotny, W. Baltes, W. Kroenert and R. Weber, Lebensmittelchem. Gerichtl. Chem., 37 (1983) 71. 515 A. Gimeno and M.L. Martins, J. Assoc. Off. Anal. Chem., 66 (1983) 85. Stahr and M. Domoto, Advances in Thin Layer 516 H.M. Chromatography (Proceedings of 2nd Bienn. Symp. 1980), 1982, p. 405. 517 P. Golinski and J. Grabarkiewicz-Szczesna, J. Assoc. Off. Anal. Chem., 6 7 (1984) 1106. 518 J. Grabarkiewicz-Szczesna, P. Golinski, J. Chelkowski and K. Szebiotko, Nahrung, 29 (1985) 229. Fulgeira and B.J. de Bracelenti, Rev. Latinam. 519 C. Microbiol., 29 (1987) 216. 520 M.O. Tapia, Rev. Argent. Microbiol., 17 (1986) 183. 521 U. Thrane, Lett. Appl. Microbiol., 3, No. 5 (1986) 93.

25 I 522 0. Filtenborg, J.C.

Frisvad and J.A. Svendsen, Appl. Environ. Microbiol., 45 (1983) 581. 523 S . Krivobok, F. Seigle-Murandi, R. Steiman and D. Marzin, J. Microbiol. Methods, 7 (1987) 29. 524 R. Hadidane, C. Roger-Regnault. H. Bouattour, F. Ellouze, H. Bacha, E.E. Creppy and G. Dirheimer, Human. Toxicol., 4 (1985) 491. 525 H. Bacha, R. Hadidane,

E.E. Creppy, C . Regnault, F. Ellouze and G. Dirheimer, J. Stored Prod. Res., 24 (1988) 199. 526 A. Bottalico, A. Logrieco and A . Visconti, Mycopathologia, 107 (1989) 85. 527 A. Bottalico, A. Visconti and M. Solfrizzo, Phytopath. Medit., 21 (1982) 105. 528 D.A. Chakrabarti and S . Ghosal, Appl. Environ. Microbiol., 51 (1986) 217. 529 C.J. Mirocha, H.K. Abbas, T. Kommendahl and B.B. Jarvis, Appl. Environ. Microbiol., 55 (1989) 254. 530 G.A. Bennett and O.L. Shotwell, J. Assoc. Off. Anal. Chem., 73 (1990) 270. 531 J.C. Frisvad, Appl. Environ. Microbiol., 41 (1981) 568. 532 J.C. Frisvad and 0. Filtenborg, Appl. Environ. Microbiol., 46 (1983) 1301. 533 J.C. Frisvad, J. Appl. Bacteriol., 54 (1983) 409. 534 R.A. Samson and J.I. Pitt (Editors), Advances in Penicillium

and Aspergillus Systematics, Plenum Press, Mew York, London, 1985. 535 J.C. Frisvad, 536 537 538 539 540

in R.A. Samson and J.I. Pitt (Editors), Advances in Penicillium and Aspergillus Systematics, Plenum Press, New York, London, 1985, p. 311. J.C. Frisvad, in R.A. Samson and J.I. Pitt (Editors), Advances in Penicillium and Aspergillus Systematics, Plenum Press, New York, London, 1985, p . 327. J.C. Frisvad, in R.A. Samson and J.I. Pitt (Editors), Advances in Penicillium and Aspergillus Systematics, Plenum Press, New York, London, 1985, p . 437. J.C. Frisvad and U. Thrane, J. Chromatogr., 404 (1987) 195. R.R.M. Paterson, J. Chromatogr., 368 (1986) 249. A.A. El-Banna, J.I. Pitt and L. Leistner, System. Appl. Microbiol., 10 (1987) 42.


253

Chapter 8 LIQUID COLUMN CHROMATOGRAPHY OF MYCOTOXINS J.C. FRISVAD and U. THRANE 8.1. INTRODUCTION

Liquid column chromatographic methods are by far the most important in preparative and analytical chemistry of non-volatile and non-antigenic natural products (1-10), even though developments in thin-layer techniques have been remarcable in recent years (1 1-13). Both types of methods are now used extensively in natural products chemistry and the combination of them is often rewarding. Being natural products from filamentous fungi that evoke a toxic response in vertebrates when introduced in low concentration by a natural route (14), mycotoxins are chemically very diverse. They may be polar (e.g. patulin), non-polar (e.g. aflatrem), acidic (e.g. citrinin), basic (e.9. roquefortine C), and they may contain chlorine (e.g. penitrem A), a nitro group (e.9. P-nitropropionic acid), a characteristic chromophore (e.g. luteoskyrin), be strongly fluorescing (e.g. territrems) etc. which are all attributes that are very important in the selection of separation and detection methods. Extraction and clean-up from a complex matrix (especially foods and feedstuffs) also depend heavily on the chemical nature of the mycotoxins. Thus because most of the known mycotoxins are present in the fungal membrane in conidia, ascomata, sclerotia, mycelium (15,16) they contain a non-polar moiety in the molecule and are often soluble in organic solvents, while other important very polar mycotoxins may have been partially neclected, because they were retained in the water or waterhnethanol fraction. Good examples of very polar compounds are the important carcinogens fumonisins (17) and islanditoxin and cyclochlorotine (18). However a substancial part of known mycotoxins have polar and non-polar portions in the molecule and will be

present both in the fungal thallus and excreted into the growth medium (i.e. food, feedstuff or fermentation broth). Most of those secondary metabolites are soluble in

254

chloroform or ethyl acetate. In the case of chloroform extractable mycotoxins fungal and food lipids will be a major analytical problem, while amino acids, carbohydrates, organic acids, nucleic acids etc. cause problems in water/methanol or acetonitrile extractions. Thus the structural diversity of the mycotoxins render the design of good general multi-mycotoxin methods difficult. In recent years a clear connection between fungal species and profiles of mycotoxins and other secondary metabolites have been apparent (16, 19-28). Furthermoreeach food commodity has its own associated spoilage mycoflora (29), i.e. fungi that actually grow on the product under natural conditions. This means that only few mycotoxins will be probable contaminants in different foods under specified environmental conditions. Such knowledge should be used more extensively in new multi-mycotoxin methods, but should of course be used with caution in compound feeds and blended foods. An updated list of the producers of important mycotoxins and other secondary metabolites is given in Table 8.1. A large number of producers of fungal metabolites have been misidentified (19, 26, 30) and the metabolites named after fungi that did not produce them. Together with data on the associated mycoflora of different types of foods and feedstuffs (29, 31), valuable information on the possible occurrence of mycotoxins may be drawn and this could help in simplifying clean-up procedures and HPLC methods for mycotoxins. A major part of known fungal secondary metabolites (32-34) are not classified as mycotoxins. They may have toxic effects on insects (insecticides), plants (herbicides) or microorganisms (antibiotics) or they may have pharmacological effects on vertebrates or act synergistically with known mycotoxins on vertebrates. Furthermore they may be good indicators of fungal contamination of foods and feeds or of mycotoxins produced in smaller amounts. Thus some of these fungal secondary metabolites will also be treated in this review. Several excellent reviews have been written on liquid chromatography of mycotoxins (3, 35-40), especially the aflatoxins, so recent advances in applications of liquid chromatography of mycotoxins will be emphasized in this chapter, especially analytical separation and detection methods.

255

TABLE 8.1. An updated list of mycotoxins and other secondary metabolites and their producers' Fungal metabolite

Known producers

4-acetamido-4-hydroxy-2-butenoic acid y-lactone (butenolide)

Fusarium acuminatum Fusarium cerealis Fusarium culmorum Fusarium graminearum Fusarium poae Fusarium sambucinum var. coeruleum Fusarium sporotrichioides Fusarium tricinctum

1'-acetoxypaxilline

Emericella striata

8a-acetoxyverrucarinJ

Myrothecium verrucaria

19-acetylchaetoglobosinA, B, D

Chaetomium globosum

3-acetyldeoxynivalenol

Fusarium cerealis (=F. crookwellense) Fusarium culmorum Fusarium graminearum Alternaria citri Fusarium culmorum Penicillium chrysogenum var. chrysogenum

Aculeasin A y Aflatoxiwl

B

Aspergillus aculeatus 2

Aflatoxin B,, B,

Aspergillus flaws Aspergillus nomius Aspergillus parasiticus

Aflatoxin G,, G2

Aspergillus nomius Aspergillus parasiticus

Aflatoxin

G,.


Aflatoxin M,, M2


Aflatrem

Aspergillus flaws

a-acetyl-y-methyl tetronic acid Altenuene

Alternaria alternata

Altenuisol


Altenusin


Alternariol

Alternaria alternata Alternaria brassicicola Alternaria cheiranti Alternaria citri Alternaria cucumerina

256

TABLE 8.1. (continued) Fungal metabolite

Known producers

Alternariol (continued)

Alternaria dauci Alternaria kikuchiana Alternaria longipes Alternaria porri Alternaria raphani Alternaria tenuissima Botrytis aclada (= 6. allii) Corynespora smithii Penicillium diversum Pleospora scrophulariae Talaromyces flavus

Alternariol-monomethylether

see alternariol

Altertoxin I, II

Alternaria alternata Alternaria cassiae Alternaria mali Alternaria tenuissima

Anhydrofusarubin

Fusarium solani Fusarium verticillioides

Antibiotic Y

Fusarium acuminatum Fusarium avenaceum Fusarium chlamydosporum Fusarium tricinctum

Aranotins

Amauroascus aureus Aspergillus terreus

Ascladiol

Aspergillus clavatus

Ascochalasin

Ascochyta heteromorpha

Ascochitine

Ascochyta fabae Ascochyta pisi

Asperfuran

Aspergillus oryzae Penicillium clavigerum Penicillium glandicola Neosartorya fennelliae

Asperlicin

Petromyces alliacea

Aspergillic acid

Aspergillus flavus Aspergillus nomius Aspergillus parasiticus Aspergillus sojae

Asperthecin

Emericella nidulans Emericella quadrilineata Emericella rugulosa

Aspochalasin A-D

Aspergillus microcysticus

257


Known producers

Asteltoxin

Emericella variecolor

Asterric acid

Aspergillus terreus Penicillium glabrum Penicillium vulpinum

Auranthine

Penicillium aurantiogriseum var. aurantiogriseum chernotype I

Aurantiarnine

Penicilliurnaurantiogriseurn var. aurantiogriseum chernotype I var. aurantiogriseum chernotype I I var . neoechinulaturn

Aurovertin B

Calcariosporium arbuscula

Austalides

Aspergillus ustus

Austamide

Aspergillus ustus

Austin

Aspergillus ustus Emericella variecolor Penicillium diversum

Austdiol

Aspergillus ustus

Austocystins

Aspergillus puniceus Aspergillus ustus

A verufin

see sterigmatocystin Cercospora arachidicola Cercospora smilacis

Barnol

Eupenicillium baarnense

Benzoic acid

Aspergillus raperi Fusarium oxysporum Rhizoctonia leguminiwla Rhizoctonia solani

Bostrycin

Alternaria eichhorniae Bostriconema alpestre

Bostrycoidin

Fusarium oxysporum Fusarium solani

Botryodiploidin

Apiosordaria sp. Botryodiplodia theobromae Lacunospora tetraspora Penicillium brevicompactum Penicillium roqueforti var. carneum Talaromyces stipitatus Triangularia bambusae Zopfiella maisushimae

258


Known producers

Brefeldin A

Alternaria carihami Curvularia lunata var. lunata Cylindrocarpondestructansvar .destructans Eupenicillium brefeldianum Eupenicillium ehrlichii Eupenicillium ludwigii Penicillium cremeogriseum Penicillium onobense Penicillium piscarium Phoma medicaginis var. medicaginis

Brevianarnide A, B

Penicillium aurantiogriseum var. viridicatum chemotype I Penicillium brevicompactum

Byssochlamic acid

Byssochlamys fulva Byssochlamys nivea Paecilomyces variotii

Byssotoxin

Byssochlarnys fulva

Canadensolide

Aspergillus tamarii Neosariorya stramenia Penicillium arenicola

Calonectrin

Monographella nivalis

Canescin

Aspergillus fumigatus Penicillium canescens Penicillium smithii

Carlosic acid

Penicillium charlesii

Carolic acid

Penicillium charlesii

Catenarin

Drechslera catenaria Drechslera grarninea Drechslera tritici-repentis Eurotium acutum Eurotium amstelodami Eurotium chevalieri Eurotium cristatum Eurotium echinulatum Eurotium glabrum Eurotium leucocarpum Eurotium niveoglaucum Eurotium repens Eurotium rubrum Helminthosporium velutinum Penicillium islandicum Pyrenophora graminea Pyrenophora tritici-repentis Talaromyces stipitatus

259


Known producers

Chaetochromin

Chaetomium caprinum Chaetomium gracile Chaetomium tetraspermum Chaetomium thielavioideum

Chaetocin

Chaetomium minutum Chaetomium tenuissimum Chaetomium thielavioideum Farrowia sp.

Chaetoglobosin A-E

Chaetomium cochliodes Chaetomium globosum var. globosum Chaetomium globosum var. rectum Chaetomium mollipilium Chaetomium subaffine Penicillium echinulatum var. discolor Pencillium expansum

Chaetoglobosin F, G, J

Chaetomium globosum var. globosum

Chaetoglobosin K, L, M

Diplodia macrospora

Chetomin

Chaetomium cochliodes Chaetomium funicola Chaetomium globosum var. globosum Chaetomium subglobosum Chaetomium tenuissimum Chaetomium umbonatum

6a-chlamydosporol

Fusarium acuminatum Fusarium avenaceum Fusarium chlamydosporum

6p-chlamydosporol

see 6a-chlamydosporol

Chromanol 1, 2, 3

Aspergillus duricaulis

Chrysarin Chrysogine

Alternaria citri Aspergillus parasiticus Penicillium chrysogenum var. chrysogenum

Chrysophanol

Ascochyta pisi Chaetomium elatum Drechslera catenaria Hypocrea austro-grandis Penicillium islandicum Phoma foveata Pseudospiropes simplex Sepedonium ampullosporum Trichoderma hamatum

260

TABLE 8.1 . (continued) Fungal metabolite

Known producers

Citreomontanin

Penicillium manginii

Citreoviridin A

Eupenicillium ochrosalmoneum Penicillium citreonigrum Penicillium rnanginii Penicillium miczynskii Penciillium smithii

Citreoviridin

see citreoviridin A

Citrinin

Aspergillus carneus Aspergillus terreus Ceuthospora sp. Clavariopsis aquatica Penicillium citrinum Penicillium expansum Penicillium hirsutum var. albocoremium Penicillium lividum Penicillium verrucosum chemotype II Penicillium westlingii Pythium ultimum

Citromycetin

lllosporium olivatrum Penicilliurn glabrum Penicillium roseopurpureum Penicillium steckii

Clad0fulvin

Fulvia fulva

Cladosporin

Aspergillus proliferans Cladosporium cladosporioides Eurotium glabrum Eurotium pseudoglaucum Eurotium repens Penicillium daleae Penicillium selandiae

Clerocidin

Oidiodendron truncatum

Cochliodinol

Chaetomium cochliodes Chaetomium elatum Chaetomium globosum

Compactin

Aspergillus terreus Penicillium solitum

Crotocin

Acremonium crotocinigenum Trichothecium roseum

Culmorin

Fusarium cerealis Fusarium culmorum Fusarium graminearum

26 1

TABLE 8.1. (continued) ~~

Fungal metabolite

Known producers

Curvularin

Alternaria cinerariae Alternaria zinniae Bipolaris nodulosa Bipolaris papendorfii Bipolaris spicifera Drechslera australiensis Eupenicillium senticosum Penicillium roseopurpureum Penicillium steckii Pseudodiplodia obiones

Curvulinic acid

Bipolaris ellisii Bipolaris papendorfii Microdiplodia microsporella Penicilliumjanczewskii Penicillium novae-zeelandiae

Cyclochlorotine

Penicillium islandicum

Cyclopaldic acid

Aspergillus duricaulis Aspergillus puniceus Penicillium commune chernotype I & II Penicillium mononematosum Pestalotia palmarum Neosadorya quadricincta

Cyclopenin

Penicillium aurantiogriseum var. aurantiogriseum chernotype II var. neoechinulatum var. polonicum var. viridicatum chernotype II Penicillium commune chernotype II Penicillium crustosum Penicillium echinulatum var. echinulatum var. discolor Penicillium hirsutum var. albocoremium var. allii var. venetum Penicillium solitum Penicillium vulpinum

Cyclopenol

see cyclopenin

Cyclopeptin

see cyclopenin

Cyclopiamide

Penicillium griseofulvum var. griseofulvum

Cyclopiamine

Aspergillus caespitosus Penicillium griseofulvum var. griseofulvum

262


Known producers

Cyclopiazonic acid

Aspergillus flavus Aspergillus otyzae Aspergillus tamarii Penicillium camemberti Penicillium commune chemotype I 8 II Penicillium griseofulvum var. griseofulvum

Cyclosporin A

Beauveria nivea Cylindrocarpon lucidum Tolypocladium inflatum

Cynodontin

Bipolaris cynodontis Sipolaris euchlaenae Bipolaris oryzae Bipolaris sorokiniana Bipolaris speciiera Bipolaris victoriae Curvularia lunata var. lunata Curvularia pallescens Cycloconium olieagineum Drechslera avenae Exserohilum rostratum Mycocentrospora acerina Phoma terrestris

Cytochalasin A

Ascochyta heteromorpha Curvularia lunata var. lunata Drechslera biseptata Drechslera dematioidea Gnomonia erythrostoma Hypomyces odoratus Phoma exigua var. exigua

Cytochalasin B

Ascochyta heteromorpha Curvularia lunata var. lunata Drechslera dematioidea Hormiscium sp. Phorna exigua var. exigua

Cytochalasin C

Metarrhizium anisopliae Hypoxylon terricola

Cytochalasin D

Coriolus vernicipes Engleromyces goetzii Metarrhizium anisopliae Zygosporium masonii Hypoxylon terricola Microporus afinis

263


Known producers

Cytochalasin E

Aspergillus clavatus Aspergillus terreus Drechslera dernatioidia Rosellinia necatrix

Cytochalasin F

Drechslera dernatioidia

Cytochalasin G

Nigrosabulum sp.

Cytochalasin H

Phomopsis paspali

Cytochalasin K, L, M

Chalara microspora

Cytochalasin K

Aspergillus clavatus

Cytochalasin N, 0, P, Q, R, S

Phomopsis sp.

Cytochalasin N’, 0:P’, Q’, R’

Hypoxylon terricola

bis-dechlorogeodin

Penicillium glabrum

Dechlorogriseofulvin

see griseofulvin

Dechloronidulin

Emericella unguis

Dehydrocarolic acid

Penicillium adametzii Penicillium charlesii

Dehydrocurvularin

Alternaria cinerariae Alternaria citri Alternaria cucumerina Alternaria dauchi Alternaria macrospora Alternaria scirpimla Alternaria tomato Alternaria zinniae Aspergillus aureofulgens Drechslera australiensis Penicillium restricturn Penicillium steckii Pseudodiplodia obiones

Dehydrocyclopeptin

see cyclopenin Emericella striafa

Dehydropaxilline Dehydroustic acid


Z’-dehydroverrucarin A


Demethoxyviridiol

Nodulisporium hinnuleum Trichoderma viride

Deoxybostrycin

Alternaria eichhorniae

Deoxaphomin

Phoma exigua var. exigua Ascochyta heteromorpha

264


Known producers

Deoxynivalenol

Fusarium cerealis Fusarium culmorum Fusariurn graminearum

1-deoxypebrolide

Penicillium brevicompactum

12,13-deoxyverrucarinA


Dermoglaucin

Cortinarius sanguineus

Desacetylpebrolide


Desertorin A, B, C

Emericella desertorurn

Destrutoxins

Metarrhizium anisopliae

Dethiosecoemestrin

Emericella striata

Diacetoxyscirpenol

Fusarium acuminatum Fusarium equiseti Fusarium sambucinum var. sambucinum Fusarium sporotrichioides

2',3', ip,8p-diepoxyroridin H

Cylindrocarpon sp.

Diethylphthalate

Penicillium funiculosum

Dihydrocytochalasin B

Drechslera dematioidea

22,23-dihydr0-24,25-dehydr02 1-oxo-aflavinine

Aspergillus niger

2,3-dihydro-3,6-dihydroxy2-methyl-4-pyrone

Penicilliurn restricturn

Dihydroergotamin

Claviceps paspali Claviceps purpurea

cis-dihydrofusarubin


trans-dihydrofusarubin

see cis-dihydrofusarubin

5,6-dihydro-4-methoxy-2Hpyran-2-0118

Penicillium italicum

2 ',3'-dihydrosorbicillin

Verticillium intertexturn

Dihydrosterigmatocystin

Aspergillus versicolor

Dihydroxyaflavinine

Aspergillus flavus

2,4-dihydroxy-6-(1,2-dioxopropyl) benzoic acid


2,4-dihydroxy-6-(1-hydroxy2-oxopropyl) benzoic acid


2,4-dihydroxy-6-(2-oxopropyl) benzoic acid


265


Known producers

2,7-dimethoxy-6-( 1-acetoxyethyl)-juglone 2,7-dimethy1-6-ethyljuglone

Nattrassia mangiferae

3,5-dimethyl-6-hydroxyphthalic acid

Penicillium gladioli

Dimethylphthalate

Penicillium funiculosum

Dipicolinic acid

Fusarium reticulaturn Beauveria bassiana Paecilomyces furnosoroseus Penicillium citreonigrum Verticilium lecanii

Diploidiatoxin

Diplodia maydis

Dithiosilvatin

Aspergillus silvaticus

Dothistromin

Cercospora arachidicola Cercospora microsora Cercospora rosicola Cercospora smilacis Cercosporidium personaturn Mycovellosiella ferruginea Scirrhia pini Sirosporium di ffusum

Duclauxin

Penicillium duclauxii Penicillium herquei Talaromyces macrosporus Talaromyces stipitatus

Echinulin

Eurotium amstelodami Eurotium chevalieri Eurotium echinulatum Eurotium heterocaryoticum Eurotium repens Eurotium rubrum

Emestrin

Emericella acristata Emericella foveolata Emericella parvathecia Emericella quadrilienata Emericella striata

Emestrin B

Emericella quadrilineata Emericella striata

Emindol DA

Emericella desertorurn Emericella quadrilineata

Emindol SA

Emericella striata

Nattrassia mangiferae

266


Known producers

Emodin

Aspergillus aculeatus Aspergillus ochraceus (= A. alutaceus) Aspergillus wentii Acroschyphus sphaerophoroides Caloplaca sp. Cetraria cullulata Cortinarius sanguineus Drechslera catenaria Eurotium chevalieri Eurotium cristatum Eurotium echinulatum Fulvia fulva Hamigera avellanea Nephroma laevigata Peniciliopsis clavariaeformis Penicillium brunneum Penicillium islandicum Penicillium tardum Hypocrea austro-grandis Phoma foveata Pyrenochaeta terrestris Talaromyces stipitatus Valsonia rubricosa Xanthoria fallax

Engleromycin

Engleromyces goetzii

Enniatins

Fusarium acuminatum Fusarium avenaceum Fusarium oxysporum Fusarium sambucinum var. sarnbucinum

€pi- & fagi-cladosporic acid

Cladosporium herbarum

Epicorazine A, 6

Epicoccum nigrum

1Cepi- 14-hydroxy-10,23-dihydro24,25-dehydro-aflavinine

Aspergillus f l a w Aspergillus niger Aspergillus parasiticus

€pi- 10-verruculotoxin

Penicillim brasilianum

Epoxycytochalasin H, J

Phomposis sojae

$,@-epoxyisororidin E

Cylindrocarpon sp.

Tp,Bp-epoxyroridinH

Cylindrocarpon sp.

Equisetin

Fusarium equiseti Fusarium pallidoroseurn

Eremofortins

Penicillium roqueforti

261


Known producers

Ergocristine

Claviceps paspali Claviceps purpurea

Ergocryptin

see ergocristine

Ergometrin

see ergocristine

Ergotamin

see ergocristine

Ergosterol

nearly all fungi

Erythroglaucin

Alternaria porri Dermocybe cinnabarina Drechslera catenaria Eurotium acutum Eurotium appendiculatum Eurotium chevalieri Eurotium cristatum Eurotium echinulatum Eurotium glabrum Eurotium herbariorum Eurotium intermedium Eurotium leucocarpum Eurotium niveoglaucum Eurotium pseudoglaucum Eurotium repens Eurotium rubrum Eurotium spiculosum Talaromyces stipitatus Xanthoria fallax Xanthoria mandschurica

Eryirhoskyrine


Ethisolide

Micropera caespitosa Penicillium decumbens

Expansolide

Penicillium expansum

Ferulic acid

Rhizoctonia leguminicola Rhizoctonia solani

Flavipin

Acrospheira sp. Aspergillus flavipes Epicoccum nigrum

Flavoglaucin

Eurotium amstelodami Eurotium chevalieri Eurotium echinulatum Eurotium herbariorum Eurotium heterocaryoticum Eurotium niveoglaucum

268


Known producers

Flavoglaucin (continued)

Eurotium pseudoglaucum Eurotium repens Eurotium rubrum

Frequentin

Penicillium commune chemotype I & II Penicillium ierlikowskii

Fructigenine A

Penicillium vulpinum

Fulvic acid

Eupenicillium brefeldianum Eupenicillium ehrlichii Penicillium cremeogriseum Penicillium glabrum Penicillium griseofulvum var. griseofulvum Penicillium hirsutum var. allii Penicillium piscarium

Fumagillin

Aspergillus fumigatus Penicillium scabrosum

Fumigaclavine A, B, C

Aspergillus fumigatus

Fumigatin

Aspergillus fumigatus

Fumitremorgin A, B, C

Aspergillus caespitosus Aspergillus fumigatus Neosartorya fischeri var. fischeri Penicillium brasilianum Penicillium graminicola Penicillium mononematosum

Fumonisin B,, B,

Fusarium proliferatum Fusarium verticillioides

Fusarenone X

s0e 3-acetyldeoxynivalenol

Fusaric acid

Fusarium lateritium Fusarium oxysporum Fusarium solani Fusarium verticillioides Peziza atrovinosa

Fusarin C

Fusarium avenaceum Fusarium cerealis Fusarium culmorum Fusarium graminearum Fusarium oxysporum Fusarium poae Fusarium sambucinum var. sambucinum Fusarium sporotrichioides Fusarium tricinctum Fusarium verticillioides

269


Known producers

Fusarochromanone

Fusarium equiseti

Fusarubin


Fusidic acid

Acremonium fusioides Acremonium strictum Calcarisporium arbuscula Gabarnaudia tholispora lsaria kogane Mortierella ramanniana Verticillium lamellicola

Gallic acid

Phycomyces blakesleanus

Gentisylalcohol

see patulin

Gibberellic acid

Gibberella fujikuroi

Gladiolic acid

Penicillium gladioli

Glauconic acid

Penicillium purpurogenum Talaromyces assiutensis Talaromyces ohiensis Talaromyces panasenkoi Talaromyces trachyspermus

Gliotoxin

Aspergillus fumigatus Aspergillus terreus Aspergillus ustus Eurotium chevalieri Eurotium rubrum Gliocladium virens Penicillium adametzii Penicillium turbatum Rosellinia necatrix Trichoderma lignorum Trichoderma hamatum

Gregatins

Aspergillus panamensis Phialophora gregata

Griseofulvin

Khuskia oryzae Khuskia sacchari Penicillium aethiopicum Penicillium canescens Penicillium coprophilum Penicillium griseofulvum var. griseofulvum var . dipodomyimla Penicillium janczewskii Penicillium jensenii Penicillium lanosum

270


Known producers

Griseofulvin (continued)

Penicillium nodusitatum Penicillium raistrickii Penicillium sclerotigenum

Griseophenone C

see griseofulvin

Hadacidin

Byssochlamys nivea P enicillium camemberti Penicillium crustosum Penicillium glabrum Penicillium hispanicum Penicillium lividum P enicillium purpurescens Penicillium simplicissirnum Penicillium spinulosum Penicillium turbatum

Helminthosporin

Bipolaris cynodontis Drechslera catenaria Drechslera graminea Drechslera tritici-repentis

Helvolic acid

Aspergillus fumigatus Emericellopsis pusilla Emericellopsis terricola Gliocladium sp. Mammaria echinobotryoides Metarrhizium anisopliae Neosartorya aurata Sarocladium oryzae Stilbella eryihrocephala Verticillium epiphytum Verticillium lecanii

Hyalodendrin

Hyalodendron sp.

5'-hydroxyasperentin

see cladosporin

para-hydroxybenzoic acid

Eurotium echinulatum Lambertella corni-maris Penicillium griseofulvum var. griseofulvurn Polyporus tumulosus Rhizoctonia leguminicola Rhizoctonia solani

Hydroxyisocanadensicacid

see canadensolide

5-hydroxymaltol

Penicillium sp.

4-hydroxymellein

Apiospora camptosporas Aspergillus melleus Cercospora taiwanensis Lasiodiplodia theobromae

27 I


Known producers

o-hydroxVpachybasin

see pachybasin

Bp-hydroxyroridin E

Myrothecium roridum

8a-hydroxyverrucarinJ


2’-hydroxy-2’-(E)-verrucarinJ

Myrothecium roridum

lndoleacetic acid

Aureobasidium pullulans Cladosporium herbarum Epicoccum nigrum Fusarium spp.

lpomeamarones

Ceratocystis fimbriata Fusarium oxysporum (both on sweet poatatoes)

lslandicin


Isochromantoxin

Penicillium mononematosum Penicillium steckii

Isocochliodinol

Chaetomium murorum

lsoemodin lsomarticin

Neocosmospora sp.

Isororidin E

Cylindrocarpon sp. Myrothecium roridum Myrothecium verrucaria

lsosatratoxin H

Stachybotfys atra

ltalicic acid


ltalinic acid


ltalinic acid methylester


Janthitrem B

Eupenicillium zonatum Penicillium piscarium

Javanicin


Kojic acid

Aspergillus flavus Aspergillus oryzae Aspergillus nomius Aspergillus parasiticus Aspergillus sojae Penicillium lanosum

Kotanin

Aspergillus clavatus Eurotium sp.

Lambertellin

Lambertella corni-maris Lambertella hicoricae Pseudospiropes simplex

212

TABLE 8.1. (continued) fungal metabolite

Known producers

Lapidosin

Eupenicillium lapidosum

Leucinostatins

Paecilomyces silacinus

Lichexanthone


Luteoskyrin


Macrosporin

Alternaria porri Alternaria solani

Malformin C

Byssochlarnys nivea Aspergillus niger Thielava sepedonium

Maltoryzin

Aspergillus flavus

Marticin

see javanicin

Meleagrin

Penicillium chrysogenum Penicillium conferturn Penicillium coprophilum Penicillium glandicola var. glandicola var. glaucovenetum Penicillium hirsuturn var. albocoremiurn

Melinacidins

Chaetomium retardaturn Verticillium cinnabarinum Verticillium tenerum

6-methoxymellein

Aspergillus caespitosus Penicillium thomii Sporormia affinis Sporormia bipartis

Methoxysterigmatocystin

see sterigmatocystin see cyclopenin

3-methoxyviridica tin

Methylhydroquinone

Nectria erubescens

6-methylsalicylic acid

see patulin

Mevinolin

Aspergillus terreus Monascus purpureus Monascus ruber

Mitorubrin

Hypoxylon fragiforme Penicillium crateriforme Talaromyces flavus Talaromyces rnacrosporus Talaromyces rnimosinus Talaromyces udagawae Talaromyces wortmannii

Mitorubrinic acid

s0e mitorubrin

273


Known producers

Mitorubrinol

see mitorubrin

Mitorubrinol acetate

see mitorubrin

Mollicellins

Chaetomium amygdalisporum Chaetomium mollicellum

Mollisin

Mollisia caesia Mollisia gallens

Moniliformin

Fusarium anthophilum Fusarium avenaceum Fusarium chlamydosporum Fusarium oxysporum Fusarium proliferaturn Fusarium sacchari Fusarium verticillioides

Mono-methoxycurvulinicacid

see curvulinic acid

Monorden

Cylindrocarpon destructans Monocillium nordinii Penicillium resedanum Verticillium chlamydosporum

Mycelianamide


Mycochromenic acid

Penicillium brevicompacturn

Mycophenolic acid

Leptographium abientinum Penciillium brevicompactum Penciilliurn raciborskii Penicillium roqueforti var. roqueforti var. carneum Phaerosphaeria nodorum

Myrotoxin A, B, C, D

Myrothecium roridum

Myioxin A, B, C

Myrothecium roridum

Naphthalic anhydride

Aspergillus silvaficus Godronia cassandrae Penicillium herquei Roesleria pallida

"Naphthoy-quinones, toxic"

Aspergillus carbonarius Aspergillus niger

Nalgiolaxin

Penicillium nalgiovense

Nalgiovensin

Penicillium nalgiovense

Nectriafurone

Fusarium solani

Neocochliodinol

Chaetomium amydalisporum

214


Known producers

Neoxaline

Aspergillus aculeatus

Neosolaniol

Fusarium acuminatum Fusarium sporotrichioides

Nidulin

Emericella unguis

Nidulotoxin

Aspergillus sydowii Aspergillus versicolor Emericella nidulans

Nigragillin

Aspergillus niger

P-nitropropionic acid

Arthrinium phaeospermum Arthrinium sacchari Arthrinium saccharicola Aspergillus avenaceus Aspergillus flavus Aspergillus oryzae Aspergillus wentii Penicillium atrovenetum

Nivalenol

see 3-acetyldeoxynivalenol

Nominine

Aspergillus nomius

Norjavanicin

see javanicin

Norlichexanthone

see griseofulvin

Norsolorinic acid

see sterigmatocystin

Nortryptoquivaline

Aspergillus clavatus Aspergillus furnigatus

Ochratoxin A,

B, C

Aspergillus ochraceus Aspergillus melleus Aspergillus petrakii Aspergillus sclerotiorum Aspergillus fresenii Penicillium verrucosum chemotype I & II Petromyces alliacea

Oosporein

Acremonium sp. Beauveria bassiana Chaetomium aureum Chaetomium trilaterale Phlebia albida Phlebia mellea Verticillium psalliotae

Orsellinic acid

widespread precursor

275


Known producers

Oxalic acid

Aspergillus niger Penicillium oxalicum Penicillium verrucosum Whetzelinia sclerotiorum and many other fungi

Oxaline

Penicillium atramentosum Penicillium aurantiogriseum var. melanoconidium Penicillium coprophilum Penicillium glandicola var. glandicola Penicillium oxalicum Penicillium vulpinum

Pachybasic acid

see pachybasin Aspergillus crystallinus Trichoderma viride

Pachybasin Palitantin

Eupenicillium brefeldianum Eupenicillium ehrlichii Penicillium commune Penicillium echinulatum var. echinulatum var. discolor Penicillium solitum

Paraherquamide

Penicillium brasilianum Penicillium charlesii

Parasitic01


Paspaline

Aspergillus clavatoflavus Aspergillus leporis Claviceps paspali

Paspalinine

Aspergillus flavus Claviceps paspali

Paspalitrems

Claviceps paspali

Patulin

Aspergillus clavatus Aspergillus giganteus Aspergillus terreus Byssochlamys fulva Byssochlamys nivea Paecilomyces variotii Penicillium clavigerum Penicillium coprobium Penicillium expansum

216


Known producers

Patulin (continued)

Penicillium glandicola var. glandicola var. glaucovenetum Penicillium griseofulvum var. griseofulvum var. dipodomyicola Penicillium melinii Penicillium novae-zeelandiae Penicillium selandiae Penicillium vulpinum

Paxillin

Acremonium loliae Aspergillus clavatoflavus Emericella desertorum Emericella striafa Eupenicillium tularense Penicilliurn paxilli

PD 113,325

Myrothecium roridum

Pebrolide


Penicillic acid

Aspergillus ochraceus Aspergillus auricomus Aspergillus fresenii Aspergillus melleus Aspergillus ostianus Aspergillus sderotiorum Eupenicillium baarnense Eupenicillium ehrlichii Paecilomyces lilacinus Penicilliurn aurantiogriseurn var. aurantiogriseurn var. melanoconidium var. neoechinulatum var. polonicum var. viridicatum Penicillium brasilianum Penicillium fennelliae Penicillium hirsuturn var. albocoremium Penicillium janczewskii Penicillium matriti Penicillium megasporurn Penicillium pulvillorum Penicillium raistrickii Penicillium rolfsii Penicillium roqueforti var. carneum Petromyces alliacea

277


Known producers

Penicillin G

Acremonium chrysogenum Aspergillus caespitosus Emericella nidulans Penicillium chrysogenum var. chrysogenum var. dipodomyis Penicillium matriti Penicillium turbatum

Penitrem A, 8, C, 0,E, F

Penicillium aurantiogriseum var. melanoconidium Penicillium clavigerum Penicillium crustosum Penicillium glandicola var. glandicola var. glaucovenetum Penicillium hirsutum var. albocoremium Penicilliumjanczewskii

Phoenicin

Eupenicillium cinnamopurpureum Penicillium chermesinum Penicillium crateriforme Penicillium atrosanguineum

Phomarin

Phoma foveata

Phomopsins

Phomopsis lepstromiformis

Physcion

Achaetomium cristalliferum Alternaria porri Aspergillus wentii Caloplaca murorum Cetraria cullulata Dermocybe cinnabarine Eurotium acutum Eurotium amstelodami Eurotium appendiculatum Eurotium carnoyi Eurotium chavalieri Eurotium cristatum Eurotium echinulatum Eurotium glabrum Eurotium herbariorum Eurotium intermedium Eurotium leucocarpum Eurotium niveoglaucum Eurotium pseudoglaucum Eurotium repens Eurotium rubrum Eurotium spiculosum

278


Known producers

Physcion (continued)

Eurotium tonophilum Penicillium herquei Physcia sp. Xanthoria fallax Xanthoria mandschurica

PI-3


PR-toxin

Penicillium roqueforti var. roqueforti

PR- 1636

Aspergillus candidus Aspergillus ustus

Preechinulin

Eurotium amstelodami Eurotium chevalieri Eurotium repens

Protophomin

Phoma exigua var. exigua

Proxiphomin

Phoma exigua var. exigua

Puberulonic acid

Penicillium aurantiogriseum var. aurantiogriseum

Purpurogenone

Penicillium purpurogenum

Pyrichalasin H

Pyricularia grisea

Pyrogallol

Penicillium griseofulvum

2-pyrovo ylaminobenzamide

Alternaria citri Fusarium culmorum Neosartorya fischeri var. spinosa Penicillium chrysogenum

Questin

Aspergillus terreus Chrysosporiurn merdarium Dermocybe cinnarnomeolutea Eurotium cristatum Eurotium glabrum Eurotium repens Eurotium rubrum Monascus ruber Penicillium glabrum

Questinol

see questin

Ravenelin

Bipolaris ravenelii

Regulin

Aspergillus restrictus

Restrictocin

Aspergillus restrictus

Roquefortine A, B

Penicillium roqueforti

219


Known producers

Roquefortine C

Penicillium atramentosum (trace? Penicillium aurantiogriseum var. melanoconidium (trace) Penicillium chrysogenum var. chrysogenum Penicillium confertum (trace) Penicillium coprobium (trace) Penicillium coprophilum Penicillium crustosum Penicillium expansum Penicillium glandicola var . glandicola var glaucovenetum Penicillium griseofulvum var. griseo fulvum Penicillium hirsutum var. hirsutum var. albocoremium var. allii var. hordei var. venetum Penicillium oxalicum (trace) Penicillium roqueforti var. roquefodi var. carneum Penicillium sclerotigenum Penicillium vulpinum

.

Roquefortine D Roridin A

Roridin D

see roquefortine C Cryptomela acutispora Cylindrocarpon sp. Dendrodochium toxicum Myrothecium roridum Myrothecium verrucaria Phomposis lepstromiformis Cryptomela acutispora Cylindrocarpon sp. Myrothecium roridum Myrothecium verrucaria

Roridin E

Myrothecium roridum Myrothecium verrucaria Stachybotrys atra Stachybotrys karnpalensis

Roridin t i

Cylindrocarpon sp. Myrothecium roridum Myrothecium verrucaria

Roridin J


280

TABLE 8.1 . (continued) Fungal metabolite

Known producers

Roridin K acetat


Roritoxin A, B, C, D

Myrothecium roridum

Roseopurpurin

Penicillium roseopurpureum

Roseotoxin B

Trichotheciurnroseum

Rubratoxin A, B

Penicillium crateriforme

Rugulosin

Acroschyphus sphaerosporoides Cryphonectria parasitica Endothia coccolobii Endothia fluens Endothia gyrosa Endothia japonica Endothia macrospora Endothia viridistroma Penicillium brunneum Penicillium concavorugulosum Penicillium islandicum Penicillium piceum Penicillium rugulosum Penicillium tardum Penicillium variabile Sepedonium ampullosporum Talaromyces rotundus Talaromyces wortmannii

Rugulovasine A

Gloeophyllum trabeum Pellicularia filamentosa Penicillium atramentosum Penicillium corylophiloides Penicillium commune Penicillium concavorugulosum Penicillium crateriforme Pulcherricium caeruleum

Satratoxin F, G, H

Stachybotrys albipes Stachybotrys atra Stachybofrys kampalensis Stachybotrys microspora

Scytalidine

Scytalidium album

Scytalone

Penicillium aurantiogriseum Phialophora lagerbergii Scytalidium album Thielaviopsis basicola Verticillium dahlia8

28 I


Known producers

Secalonic acid A

Aspergillus ochraceus Claviceps purpurea Parmelia entotheiochroa Phoma terrestris

Secalonic acid B

Aspergilus aculeatus Claviceps purpurea

Secalonic acid D

Aspergillus aculeatus Claviceps purpurea Penicillium isariiforme Penicillium oxalicum

Secalonic acid F

Aspergillus aculeatus Claviceps purpurea

Shikimic acid

widespread precursor

Simatoxin


Sirodesmin

Sirodesmium diversum

Skyrin

Acroscyphus sphaerophoroides Cryphonectria parasitica Endothia fluens Endothia gyrosa Endothia havananensis Endothia japonica Endothia longirostris Endothia rnacrospora Endothia radicalis Endothia singularis Endothia tropicalis Hypomyces lactifluorurn Hypomyces trichothecioides Penicilliopsis clavariaeforrnis Penicillium brunneum Penicillium concavorugulosum Penicillium islandicum Penicillium piceum Penciillium rugulosum Penicillium variabile Physcia obscura var. endococcina Pyxine endochrysina Sepedonium ampullosporum Trypetheliopsis boninensis

Slaframin

Rhizoctonia leguminicola

Solaniol

see fusarubin

Soranjidiol

282


Known producers

Sorbicillin

Penicillium chrysogenum Verticillium intertexturn

Spiculisporic acid

Penicillium crateriforme Penicillium minioluteum Talaromyces panasenkoi Talaromyces trachyspermus

Spinulosin

Aspergillus fumigatus Penicillium spinulosum

Sporidesmin

Leptosphaerulina chartarum Pithomyces chartarum

Steckiin

Penicillium steckii

Sterigmatocystin

Aspergillus flavus Aspergillus multicolor Aspergillus nomius Aspergillus parasiticus Aspergillus versicolor Bipolaris nodulosa Chaetomium thielavioideum Chaetomium udagawae Emericella acristata Emericella aurantiobrunnea Emericella bicolor Emericella cleistominuta Emericella corrugata Emericella dentata Emericella echinulata Emericella falconensis Emericella foveolata Emericella heterothallica Emericella lata Emericella navahoensis Emericella nidulans Emericella parvathecia Emericella purpurea Emericella quadrilineata Emericella rugulosa Emericella spectabilis Emericella striata Emericella unguis Emericella variecolor Farrowia malaysiensis Monocillium nordinii

Stipitatic acid

Talaromyces stipitatus

283


Known producers

Sulochrin

Aspergillus fumigatus Aspergillus terreus Aspergillus wentii Oospora sulphurea-ochracea Penicillium glabrum

Sydowic acid

Aspergillus sydowii

T-2 toxin

Fusarium acuminatum Fusarium poae Fusarium sporotrichioides

Talaromycins

Talaromyces stipitatus

Tenuazonic acid

Alternaria alternata Alternaria brassicae Alternaria brassicicola Alternaria cheiranti Alternaria citri Alternaria japonica Alternaria kikuchiana Alternaria longipes Alternaria mali Alternaria oryzae Alternaria porri Alternaria raphani Alternaria solani Alternaria tenuissima Aspergillus nomius Phoma sorghina Pyricularia oryzae

Terphenyllin

Aspergillus candidus

Terrecyclic acid

Aspergillus terreus

Terreic acid

Aspergillus terreus

Terrein

Aspergillus terreus Neosartorya fischeri var. spinosa Penicillium soppii

Terrestric acid

Penicillium aurantiogriseum var. aurantiogriseum chernotype I Penicillium crustosum Penicillium hirsutum var. hirsutum var. albocoremium var. hordei var. venetum Pyricularia oryzae

Terretonin

Aspergillus terreus

284


Known producers

Territrerns

Aspergillus terreus Penicilliurn echinulaturn var. echinulaturn

para- toluquinone

Nectria erubescens

Torreyol

Clitocybe illudens

TR-2

see verrucologen

Trichoderrnin

Trichoderma viride

TrichorzianinesA

Trichoderma haRianUm

Trichorzianines B

Trichoderma harzianum

Trichothecolone

Trichotheciurnroseum

3,4,5-trihydroxy-7-rnethoxy 2-methylanthraquinone

Alternaria porri

Trypacicin

Aspergillus furnigatus Aspergillus ochraceus Neosartorya fenneliae

Tryptoquivalines

Aspergillus furnigatus Neosartorya aureola Neosartorya fischeri var. fischeri var. glabra chemotype 111 var. spinosa Penicillium aethiopicurn Penicilliurn digitaturn

Tryptoquivalones

see tryptiquivalins

Tubingensin A, 6

Aspergillus niger

Ustic acid


Verrnicellin

Penicillium aculeaturn Penicillium panamense Talarornyces flaws

Verrniculin

Penicilliurn crateriforrne Penicilliurn pinophilurn Talarornyces flaws Talarornyces ohiensis

Verrucarin A

Dendrodochiurn toxicurn Myrotheciurn leucotrichurn Myrotheciurn roridum Myrotheciurn verrucaria

Verrucarin 6

Myrotheciurn roridurn Myrotheciurn verrucaria Stachybotrys atra

285


Known producers

Verrucarin J

Myrothecium roridum Myrothecium verrucaria Stachybotrys albipes Stachybotrys atra Stachybotrys kampalensis Stachybotrys microspora

Verrucofortine

Penicillium aurantiogriseum var. aurantiogriseum var. polonicum var. viridicatum

Verrucolon

Penicillium verrucosum

Verrucosidin

Penicillium aurantiogriseum var. aurantiogriseum var. melanoconidium var. polonicum

Verrucologen

Aspergillus caespitosus Aspergillus fumigatus Eupenicillium crustaceum Penicillium brasilianum Penicillium graminicola Penicillium mononematosum

Verruculotoxin

Penicillium brasilianum

Versicolorin A

Aspergillus flavus Aspergillus multicolor Aspergillus parasiticus Aspergillus puniceus Aspergillus ustus Aspergillus versicolor Drechslera sorokiniana Emericella nidulans

Verticillin A

Verticillium sp.

Vertinolide

Verticillium intefiextum

Vertisporin A

Verticimonosporium diffractum

Violaceic acid

Emericella striata

Viomellein

Aspergillus alutaceus Aspergillus auricomus Aspergillus melleus Aspergillus ostianus Aspergillus sulphureus Eupenicillium javanicum

286


Known producers

Viomellein (continued)

Penicillium aurantiogriseum var. aurantiogriseum chernotype II var. melanoconidium var. viridicatum Penicillium clavigerum Penicillium mariaecrucis Penicillium simplicissimum Nannizzia cajetanii Trichophyion megninii Trichophyton rubrum Trichophyton violaceurn

Viridamine

Penicillium aurantiogriseum var. viridicatum

Viridic acid

Penicillium aurantiogriseum var. viridicatum

Viridicatic acid

see terrestric acid

Viridicatin

see cyclopenin

Viridicatol

see cyclopenin

Viridicatumtoxin

Penicillium aethiopicurn Penicillium brasilianum Neosartorya fennelliae

Viriditoxin

Aspergillus viridi-nutans Neosartorya aureola Paecilomyces variotii Penicillium mononematosum

Wentilacton

Aspergillus wentii

Wortmannin

Aspergillus janus Fusarium sarnbucinum var. coeruleum Myrothecium roridum Penicillium proteolyticum Talaromyces flavus

Xanthoascin

Aspergillus candidus

Xanthocillin X

Eupenicillium egyptiacum Eurotium chevalieri Neosartorya spathulata Penicillium chrysogenum Penicillium italicurn

Xanthomegnin

see viomellein

Zearalenol

see zearalenone

287


Known producers

Zearalenone

Fusarium cerealis Fusarium culmorum Fusarium equiseti Fusarium graminearum Fusarium pallidoroseum

Zygosporin 0,E, F, G

Zygosporium masonii

' List of references on data presented in this tabel can be obtained from the authors upon request

* Presumably a fungal metabolite and/or unknown producer Production of roquefortine C in trace amounts

8.2. COLUMN CHROMATOGRAPHY In most cases column chromatography has been applied for large scale separation of standards of mycotoxins, for analytical standards, or for toxicological testing (41-44). The principles of low, medium or high pressure liquid chromatography are quite analogeous and one of the most important principles in these techniques for producing pure analytical standards is the combination of different types of columns. Of the four most important principles in column chromatography, adsorption, partition, ion-exchange and gel filtration, the first is most widely used. lon-exchange chromatography is suited for acidic mycotoxins like P-nitropropionicacid, ochratoxin A, citrinin, penicillic acid, terrestric acid, secalonic acid, cyclopiazonic acid, fumonisins, mycophenolic acid, viridicatumtoxin and tenuazonic acid, but may also be used for ionic mycotoxins like moniliformin or basic mycotoxins like roquefortine C and ergot alkaloids. These ionic molecules have been quite difficult to analyze using common TLC or HPLC systems, e.g. some acids (secalonic acid, viridicatumtoxin) or bases (roquefortine C. meleagrin, oxaline) will not elute on silica gel using the common TLC eluent toluene/ethyl acetate/formic acid (45) or they will show tailing spots (cyclopiazonic acid, citrinin, rugulosin, luteoskyrin). Other acidic (terrestric acid) or basic (roquefortine C, meleagrin, oxaline) fungal secondary metabolites will give broad

288

peaks, even in acidic HPLC eluents (42-48). As many filamentous fungi are able to produce either acidic or basic metabolites or both (16, 19-27, 31, 49) this fact should be considered carefully when extracting and purifying secondary metabolites from fungal cultures or foods. It is preferable to use two different types of columns (especially normal phase

followed by reversed phase column material) instead of two consequetive elutions on the same column material with different eluents. Column material of many different kinds are now available among them silica, silicic acid, alumina, magnesium oxide, magnesium silicate, calcium hydrogenphosphate, calcium sulphate, charcoal, diatomaceous earth, cellulose, and silica bonded with non-polar phases e.g. phenyl, cyclohexyl, octyl (CJ, octadecyl (C,J, ethyl (C,); polar phases, i.e. diol, cyanopropyl (CN), aminopropyl, N-propylethylenediamine or

ion-exchange phases, e.g.

benzenesulphenylpropyl, trimethylaminopropyl and carboxymethyl. Multi-mycotoxin methods may require a combination of two extraction methods, one for polar mycotoxins, usually using methanol/water or ethanol/water and one for non-polar mycotoxins usually chloroform, dichloromethaneor ethylacetate. The polar fraction will also contain carbohydrates, amino acids, organic acids, purines and pyrimidines etc., while the non-polar fraction will contain lipids such as mono-, di- and tri-glycerides, sterols, waxes and phospholipids, carotenes etc. The polar compounds may be fractionated by different ion-exchangers(50) and many lipids in non-polar solvents may be removed for example by partition between methanol (after evaporation of first extraction solvent and redissolving in the alcohol) and hexane. Most known fungal secondary metabolites can be extracted by chloroform or ethyl acetate in an acidified system, but important compounds such as the fumonisins and P-nitropropionic acid will remain in the water/alcohol phase (17). Much more specific extraction and purification procedures may be selected for a single rnycotoxin or a group of chemically related mycotoxins. Flash column chromatography has been used for the purification and separation of trichothecene mycotoxins (51, 52) Dry column chromatography has also been used instead of preparative TLC. Much less organic solvent is used and the individual toxins can be cut as slices of the columns. In a separation of aflatoxin B,,B, G, and G, Megalla (53) used a layer of neutral alumina (5 cm) and silica gel (25 cm) in a cellophane bag. A similar method was used by McKinney (54) for the purification of aflatoxins. The use of polythene bags

289

also allows a chemical confirmation by dipping the columns in reagent solutions such as mineral acids (55). 8.3. MINI-COLUMN CHROMATOGRAPHY Mini-columns have been used for clean-up of many mycotoxins and they are part of some of the analytical methods published by the AOAC (Association of Official Analytical Chemists)(56). The Romer and Holaday-Velasco mini-columns are, for example, packed with calcium sulphate, florisil, silica gel, neutral alumina and calcium sulphate (5657). Often these procedures are necessary because of the high content of lipid in the foods and feeds they are used for, e.g. groundnuts, corn and milk, or because of coloured interfering compounds. The many types of pre-packed cartridges with the different types of adsorbents mentioned above have raised the efficiency, repeatability and quality of clean-up procedures and the final analytical result. A combination of these mini-columns may be used for a very efficient clean-up of different types of mycotoxins, especially from complex matrices such as foods, feedstuffs, blood and urine. Mini-columns are used in many methods for aflatoxins including aflatoxin M,. Both silica gel (58-73) and reversed phase (66,68,74-80), florisil (70,81) and gel-permeation (82) mini-columns have been used. Often silica gel rinse up involve application of a chloroform or dichloromethane fraction to a column (often hexane-solvated), washing with hexane or diethyl ether and eluting with strong eluents such as chloroform or dichloromethane

- ethanol or acetone mixtures. Differenttypes of (mini)-columnshave also been used for other mycotoxins such as sterigmatocystin (cupric carbonat

- diatomaceous earth, florisil and polyamide,

69,83), trichothecenes (silica gel (84), reversed phase (85-89), cyano (71,go), charcoal (91), florisil (92) or other phases (93)), zearalenone (amino (94) or florisil (95-96)), ochratoxin A (cupric carbonate - diatonaceous earth (70), cyano (81), reversed phase (97) or XAD-2 (98)) and moniliformin (amberlite IRC50 (99)). Combinations or sequences of charcoal, alumina, florisil, CN and C, or C,, mini-columns have been used for an efficient clean-up of primarily aflatoxins and trichothecenes from biological material (40, 100-103).

290

A very simple and efficient method for the determination of aflatoxin M, in milk was based on immunoaffinity column clean-up (104) and this kind of method is especially suited for a single mycotoxin often giving very "clean chromatograms" (105-106). lmmunoaffinitycleanup has also been applied to aflatoxin B, (107-108) and ochratoxin A (109) analysis. There is little doubt that a large number of future mycotoxin analyses in foods and feeds will involve clean-up with disposable mini-columns. It is not clear yet which clean-up methods are most efficient in multi-mycotoxin methods. The sample may be subdivided into for example acidic, neutral/polar, neutraVapolar and basic fractions at all stages and extracted, cleaned-up, separated and detected accordingly. This approach may be an advantage for detectors such as diode array detectors (DAD) or mass selective detectors (MSD) (see later). However in many cases only a single analytical procedure is feasible and an analytical compromise between chemically quite different mycotoxins is necessary. More research is needed to evaluate which types of minicolumns and eluents are best in separating co-occurring mycotoxins from the background matrix in one general procedure. 8.4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 8.4.1. Aflatoxins Determination of aflatoxins by HPLC has been reviewed extensively by Scott (110) Shepherd (37), Betina (39), Coker and Jones (40),Beaver (111) and Shepherd

ef a/. (1 12) and determination of aflatoxin M, by Scott (1 13), so this topic will not be reviewed extensively here. However some important applications of HPLC in aflatoxin analysis will be summarized below. 8.4.1 . I . HPLC solvents and sample introduction. It is generally recognized that the injection solvent should be close or equal to the eluent. In the case of aflatoxin M, (convertedto the more fluorescing aflatoxin M2,), Beaver

(1 14)

has

shown

that

in

reversed-phase

(RP)-HPLC

using

water/isopropanoI/acetonitrile as the eluent, the aflatoxin M, peak exhibited 25.000

theoretical plates when injected dissolved in 30% aqueous acetonitrile and 10.000 plates when injected dissolved in water alone, compared to for example only 2000

29 1

plates when injected into a water/ acetonitrile/methanoIeluent in a 30% acetonitrile in water. solution. This clearly shows that careful choice of sample solvent can improve the quality of an analysis considerably. In broader analysis, involving several chemically different mycotoxins the choice of solvent are more restricted because of poor solubility in for example water. Also degradation of aflatoxins depend heavily on the solvent used (1 15). Exposure to light in solvents without acetic acid resulted in significant aflatoxin degradation and even at -1 8 "C, aflatoxin degraded when acetic acid was absent (1 15). An optimally stable solvent was acetonitrile/water/aceticacid

(50:50:0.5),but aflatoxins were also stable in crude peanut extracts (1 15). Our experience with crude fungal extracts dissolved in methanol and stored at -1 8 "C has been very good, probably because of "co-stabilization" caused by the many different secondary metabolites in one extract (Frisvad and Thrane, unpublished). A new method for aflatoxin M, in milk, which can be automated, using on-line dialysis and trace enrichment on a RP column, and back-flush to the analytical column, has been developed by Tuinstra et a/. (1 16-1 17). The recovery was over 50% at the 50 ng/kg level in automated analysis and is proposed for automated screening of large

numbers of milk samples at 100 ppt level or higher. The combination of flow injection analytical (FIA) methods and HPLC in the analysis of mycotoxins may be the basis of several future methods. The FIA technique may be used for detection of the total of several related mycotoxins (e.g. total aflatoxins) and as a post-column reactor/detector (1 18-119). 8.4.1.2. Analytical separation of aflatoxins.

A large number of column types and materials have been used for an efficient separation of the aflatoxins. In most applications normal phase (NP) or reversed phase (RP) silica gel based materials in 10-30 cm long columns have been used, but rapid methods using radial compression columns have also been applied with success for separation of the aflatoxins (1 20-1 21). Effective separation of the aflatoxin have been achieved both by using NP- and RP-HPLC. A major problem of the latter methods are that eluents always contain water, which will quench the fluorescence of aflatoxin B, and G,. Therefore pre- or post-column reactions or silica gel-filled detector cells are often a part of such systems (see below). NP-HPLC systems have been used in several methods (e.g. 59, 122-142). One advantage of the NP systems is that

292

transformation to water adducts (from B,, G, and M, to B,

G, and M ,, respectively)

is avoided, but the chloroform/cyclohexane/acetonitrile/isopropanol(73:22:3:2.5) eluent most often used (134) may cause separation problems because of chloroform humidity fluctuations and isopropanol content (142). Tutelyan et a/. (141-142) suggested a low viscosity eluent, ether/methanol/water(95:4:1) causing better separations and shorter retention times than the chloroform containing eluent mentioned above. The method of Tutelyan eta/. (141-142) allows determination of both B,, B, G,, G, and the more polar M,, M ,,

and B,= Shorter retention times of the latter polar aflatoxins could be

obtained with a more polar eluent ether/methanol/water (90:8:2). The stability of dynamically modified silica gel (much less dependent on brand

of stationary phase) can be used to reduce inter-laboratory performance differences (143). Unfortunately the equilibration time can be 12 to 18 hours, but after further

method development these methods may be of great value in the development of highly reproducible mycotoxin analysis. The number of applications of RP-HPLC methods in aflatoxin analysis is now much greater than the number of NP applications (121, 123-124, 126, 129, 132, 138, 144-191). These methods often involve use of acetonitrile and/or methanol and water,

in isocratic or gradient elutions. Because of water quenching of aflatoxin B, and G, fluorescence these are often treated with acid to give the B, and G,

derivatives.

However, B, and G, derivatives are unstable in methanol (191), which is a very much used as part of many eluents. Among eluents used, the most common are

water/acetonitrile/methanoImixtures (75, 126, 138, 144, 146-148, 151-152, 156, 162163, 167-168, 170, 172, 175, 180, 182, 186-187, 190), water/acetonitrile (121, 146, 148, 155, 159-160, 165, 169, 171, 174) and water/methanol (148, 153-154, 157-158, 173, 179, 185, 188). Some of these eluents are added acetic acid (121, 171, 186),

phosphate buffers (182, 188) or sodium chloride (152, 167). Few of these analysis are gradient elutions (4, 47, 156-157, 165). The gradient elutions are required in multimycotoxin analysis, but excellent separation of the aflatoxins have been achieved with the isocratic eluents refered to above. Injection with a water/acetonitrile soluent and elution with either water/acetonitrile or water/acetonitrile/methanol seem to be the best choices for general aflatoxin analysis. The reversed phase column brand seems to be less important, however a new column should always be tested with a mixture of the important aflatoxins and separation optimized by minor adjustments in the eluent

293

composition. Flow rate is mostly dictated by optimization due to a common wish for short analysis time (high flow rate), use of as little eluent as possible (e.g. microbore columns) and possible interface to a mass selective detector, which usually requires low flow rates. In the applications above flow rates from 0.5 to 3 ml/min have been used. 8.4.1.3. Detection of aflatoxins. Most detectors for aflatoxins take advantage of the strong fluorescence, but if aflatoxins are present in more than trace levels UV detection is an alternative or both methods can be used for confirmation of identity. Diode array detection will reveal the very characteristic UV spectra of the aflatoxins (4,47), but at a cost of sensitivity. Fluorescence detection of aflatoxins in NP-HPLC applications was intially used by e.g. Hsieh el a/. (193) and Johnson eta/. (194). Manabe eta/. (195) examined the influence of different eluents on fluorescence quenching and recommended an eluent containing toluene, ethyl acetate, formic acid and methanol. Panalaks and Scott (124) and Zimmerli (149-150) developed silica gel-packed flow cells for the sensitive detection of aflatoxins for NP-HPLC applications, because aflatoxin B, and B, fluoresce poorly compared to GIand G,in NP systems (196-197). The methods of Panalaks and Scott (124) has since been used by Pons (130) and Thean etal. (59) for determination of aflatoxins in corn and Francis etal. (134) for determination of aflatoxins in peanut butter. The major disadvantage of the silica gel-packed flow cells is the instability of the packed cell with time (191). The fluorescence of aflatoxin B, and G, is poor in aqueous systems (used in RP-HPLC systems), so treatment with hydrochloric acid (167) or trifluoroacetic acid (TFA) to convert aflatoxin B, and G, to their B, recommended. Aflatoxin B, and

and G ,,

derivatives has been

G, are left unaltered. Diebold and co-workers (144-

145) proposed hydrochloricacid as the derivatisation reagent, because TFA according to them caused additional unidentified peaks and they used a very sensitive laser

fluorometric detection method for the aflatoxins. However in most cases TFA has been used for the formation of highly fluorescent hemiacetal derivatives (62, 75, 121, 127, 138, 147-148, 151, 156, 159-160, 162, 164, 166, 168, 171, 178, 186, 187, 198-199).

Davis and Diener proposed that iodine could be used for post-column derivatisation of aflatoxins for enhanced flourescence (158). This principle is now used

294

in several methods for aflatoxin using either iodine (174, 176, 180, 182, 165,200-204), bromine (1 19, 181) or chloramine (1 19) as the oxidizing agent. The methods based on TFA or iodine require an extra chemical conversion step and an extra pump respectively, but at this stage these two methods appear to be the best available RPHPLC methods concerning accurate and sensitive detection of aflatoxins (189). Cyclodextrin has also been used to enhance the fluorescence of the aflatoxins (205) and synchronous fluorescence spectrophometry has been introduced (206). Another method such as electrochemical detection (207) may also be basis for new methods, even though the latter method is not particularily sentitive. HPLC methods have been compared to ELSA (enzyme linked immuno sorbent assay) techniques and for screening purposes (89, 189, 203, 208-210). The ELISA techniques appear to be very good and simple, but they compared most favorably to HPLC methods with lipid containing products (corn, nuts, peanuts) and less favorably for cereals and grain samples (210). Immunological methods seem to be of use both in the clean-up phase (211) and in the final confirmation phase, but use of ELISA cards is also as very simple screening technique, which can be used in "the field". 8.4.1.4. Aflatoxin determination in different products

Most methods for aflatoxins have been developed for their determination in lipid containing products such as corn (59, 121, 130, 136, 159, 175, 182, 185, 187, 192, 204,212), cottonseeds (131,186,214-215) and peanuts, peanut butterand nuts (121, 125, 134, 151, 152, 154, 175, 180, 182, 187, 203-204, 209-210, 212, 215). However

methods have also been proposed for

COCO

beans (167), feedstuffs (126, 128, 159,

174, 189, 210, 217), eggs (216), wine (147-148),soy products (132) and spices (131).

Finally methods have been proposed for human and animal tissue both for metabolic and medical studies, but also for the analysis of meat (76, 135, 157, 164, 166, 218), serum (138, 168, 173, 188) and urin (137, 165, 168, 190). Studies on milk and milk products have been reviewed extensively by Scott (89) and include ref. 75, 139-140, 155-156, 160, 163, 169-172 and 219. Of particular interest is the new hydroxy derivative of aflatoxin M,, aflatoxin M, (220-221). This metabolic product is more carcinogenic in rainbow trout than aflatoxin B, or M, and more emphasis should be given to analytical methods forthis important new derivative.

295

8.4.2. Sterigmatocystin and related compounds Sterigmatocystin is a carcinogenic mycotoxin produced by a series of both related and quite unrelated fungi (Table 8.1). Fortunatelyonly three species (Aspergilus flavus, A. nomius and A. parasiticus) have the enzymes needed for the production of the next biosynthetic steps towards aflatoxin G, (via methoxy-sterigmatocystin and aflatoxin B,).There is an interest in sterigmatocystin both because it is a precursor of aflatoxin nut also because it is produced by common fungi in foods, notably Aspergillus versicolorand Emericella nidulans. For the first objective analysis of other biosynthetic intermediates are also interesting and the HPLC methods should be developed accordingly. For the second objective HPLC methods may be more directed towards very sensitive and specific analysis, even though sterigmatocystin is among the mycotoxins which quite often have been included in multi-mycotoxin HPLC methods. Few NP- and several RP-HPLC applications have been developed for the analysis of sterigmatocystinin foods and feedstuffs and fungal cultures (205,222-235). Furthermore several multi-mycotoxin methods including sterigmatocystin have been proposed, either comprising both related and unrelated mycotoxins (4, 47, 236-238) or secondary metabolites biosyntheticallyrelated to sterigmatocystin (239-243). Some of the early methods employed NP separations using eluents containg two or more of the following eluents hexane, chloroform, dichloromethane, ethyl acetate, n-propanol combined with acetic acid (e.g. 239-240) after silica gel (240) rinse up. The RP methods employ simple silica gel (232) or gel permeation rinse up (224) and the same kind of eluents as those used for aflatoxin analysis, i.e. methanol and or acetonitrile combined with water, the latter often acidified with acetic acid (234,237,242-243) or buffered with phosphate or phosphoric acid (224,230). Other methods employ

methanoI/tetrahydrofuran/aceticacid (242). Hurst et a/. (238) obtained good results for several mycotoxins using a cyano column and hexaneh-propanoVglacial acetic acid. Water/acetonitrile gradients have been shown to be of general use (4,47,226) for a large number of mycotoxins, especially in acidic gradients (4,47). Most detection methods for sterigmatocystin have been based on the UV maximum for sterigmatocystin at 325 nm (e.g. 230), but interfering compounds from foods or feeds reduce the reproducibility of the methods. Two derivatisation methods clearly improve the specific detection of sterigmatocystin:Abramson and Thorsteinson (232) acetylated sterigmatocystinin pyridin and acetic anhydride for 3 hours at 100 "C

296

and they achieved to diminish the observed background interference from barley considerably. Neely and Emerson (235) considered the "relatively long reaction time, the sensitivity of the reation to water and the gradual decomposition of the acetyl derivative" a problem and suggested the use of an aluminium chloride post column derivatisation of sterigmatocystin and fluorescence detection (exitation 254 nm and emission 455 nm). The latter method was developed for fermentation broth analysis, but it may be combined with parts of the method of Abramson and Thorsteinson (232) to a good general method for the detection of sterigmatocystin in cereals.

Two papers on the NP-HPLC separation of secondary metabolites related to sterigmatocystin were published in 1976 (239-240), but interestingly only the most predominant metabolites (sterigmatocystin, demethylsterigmatocystin

and

5-

methoxysterigmatocystin) were analysed by both groups of researchers, while sterigmatin, 6-deoxyversicolorinA, 6,8-O-dimethylaverufin,6,8-O-dimethylversicolonn A and aversin were analysed by Ito et a/. (240), the metabolites also found in Aspergillus parasiticus, versicolorin A & C, averufin and avermutin were analysed by Kingston et a/. (239). 8.4.3. Trichothecenes The trichothecenes are among the most important mycotoxins, but the poor UV absorption of tnchothecenes without an enone chromophore (type A trichothecenes, e.g. T-2 toxin, HT-2 toxin diacetoxyscirpenol (DAS)) makes HPLC analysis a less applicable methodthan gas chromatography or mass spectrometry (39-40,89,244-248).

8.4.3.1. Non-macrocyclictrichothecenes At least 80 different non-macrocyclic trichothecenes have been structureelucidated (249). It has been possible to analyze underivatized T-2 toxin and othertype A trichothecenes by HPLC (4,47), but such methods are only applicable to extracts containing very high concentrations of type A trichothecenes, and are thus only of practical use in few cases. Sensitive HPLC methods for type A trichothecenes require effective clean-up and derivatisation. Type B trichothecenes (e.g. nivalenol (NIV), deoxynivalenol (DON) = vomitoxin, fusarenone X (FUS-X), 3-acetyl deoxynivalenol(3AC DON)) have an absorption maximum at 219-221 nm (34,47) and may be more

297

easily detected by HPLC-UV. Lanin et a/. (250-252) have examined the influence of different eluents on the separation of five type B trichothecenes (NIV, DON, 3-AC DON, 15-AC DON and 7desoxy DON) on a RP-column and found that the best separation under isochratic conditions was achieved with waterltetrahydrofurane (76:24). Eluents with acetonitrile gave better separations than eluents with ethanol, but both gave insufficient separations of DON and 7-desoxy DON and 3-AC DON and 15-AC DON. Electrochemical detection has been used for the detection of DON (253-255). The electrochemical detection method used by Sylvia et a/. (254) improved sensitivity 12 times compared to UV detection. A C,, reversed phase system was used with

methanoVwater (35:65) as the eluent. The method could not be used for T-2 toxin. The method of Childress eta/. (255) employed photolysis before electrochemical detection. Because of the poor UV absorption of type A trichothecenes and the difference between type A and B trichothecenes, several chemical post-column derivatization methods have been developed. The post-column derivatization method developed by Sano et a/. (256) can only be used for type B trichothecenes however, because it is based on production of formaldehyde from the ketogroup after warm alkali treatment, followed by reactions with methyl acetoacetate and ammonium acetate. Other postcolumn derivatization methods have been based on p-nitrobenzylchloride (257), diphenylindenonesulphonyl esters (258)or anthracene-9-carbonylchloride (259). The method of Yagan et a/. (258) is very sensitive and is applicable to type A trichothecenes. The method of Bayliss eta/. (259) is also sensitive, but anthracene-9carbonyl chloride also react with the hydroxy groups in for example (fungal) sterols and may be a problem in extracts which will usually contain several such compounds. An elegant method based on ELlSA as an post column monitoring system for type A trichothecenes has been developed by Chu and Lee (260). Coupled with non-aqueous size-exclusion chromatography rinse up systems (261) or other column clean-up systems (262), immunological detection methods may result in very sensitive and specific analyses. Thus post-column derivatization or sensitive immunological monitoring has meant a significant improvement of analysis of type A trichothecenes compared to earlier methods (263). It is typical for most applications of HPLC in trichothecene analysis that reversed phase systems (&,

or occasionally C),

and

gradients of methanoVwater or acetonitrile /water are used, even though

298

tetrahydrofuran may have the best separation ability at isocratic conditions (252). The most common type B trichothecenes were baseline separated using acetonitrile /water gradients (4,47). Cereals are often invaded by Fusarium species both before and after harvest. The risk of trichothecene contamination of cereals is therefore of great concern. The methods available are often based on column clean-up (93,100,253,256,264-275) and have been developed both for carbohydrate rich (wheat, rice) and fat rich cereals, especially corn (271-275). Lauren and Agnew (275) suggested to hydrolyze the major trichothecenes to four basic families of trichothecens with the basic trichothecene skeletons NIV, DON, scirpentriol and T-2 tetraol respectively and they improved and extended the methods developed by Rood et a/. (276-277) and Kroll (278) and included moniliformin and zearalenone in their analysis. Methods have been developed for the analysis of trichothecenes in animal tissues (279-281) and plasma and urine (258,276). Like the methods for cereal products the trichothecene analysis are based on column chromatography clean-up and chemical derivatizations. 8.4.3.2. Macrocyclic trichothecenes A large number of the macrocyclic trichothecenes have absorption maxima in the range of 217-259 nm (34) and can thus be detected by UV detectors. Roridin A and verrucarin A had retention indices of 1013 and 1022, respectively, in the acidic water/acetonitrilegradient used by Frisvad and Thrane (47) and could be determined at their absorption maxima of 245 and 259 nm. HPLC and flash chromatography have been used extensively in the purification and monitoring of the synthesis of baccharins and related toxic secondary metabolites from Brazilian plants (52, 282-283). Normal phase systems using ethyl acetate and hexane were used for the analysis of verrucarins and roridins (284-285), but the same toxins are also easily separated by

RP systems using water/acetonitrile gradients (47). Satratoxin G and H and trichoverrols were determined in conidia and cereals grains respectively by Sorenson et a/. (286) and Stack and Eppley (287) respectively. Being very important in the

airspora of houses and factories Stachybotrys atra conidia may be of more concern as airborne contaminants. The same fungus has only been sporadically reported from cereals. It is expected that more multi-toxin methods will be developed for the

299

macrocyclic trichothecenes, probably based on water/acetonitrile or water/methanol gradients and diode array detection. 8.4.4. Small lactones A quite large number of filamentous fungi produce small lactones or related

compounds (Table 8.1) which are either generally toxic, e.g. patulin and penicillic acid (34,42-43), neurotoxins, such as citreoviridin and verrucosidin (34), good chelators of metal ions, such as the Raistrick phenols (288), mycophenolic acid, terrein, terrestric acid, kojic acid, cyclopaldic acid etc. (48), or they have other occasionally unknown biological effects. Most HPLC methods have been developed for patulin and penicillic acid, but all the fungal secondary metabolites listed above can be analyzed by general methods such as that of Frisvad and Thrane (47). 8.4.4.1. Patulin Patulin is produced by Penicillium expansum in fruits and fruit products, but may also be produced by P. griseofulvum in cereals and P. glandicola, P. roqueforti var.

carneum,Paecilomyces variotii, Byssochlamys nivea, B. fulva,Aspergillus clavatus and

A. ferreus in silage, malt or airtight storage (31). Several TLC and GC methods are available for patulin analysis, but most methods are now based on HPLC, occasionally with TLC or GC-MS confirmation. In general the strong UV absorbtion at 275-276 nm is used for detection. Patulin and griseofulvin produced in culture by Penicillium griseofulvum (= P.

urticae = P. pafulum) can be analyzed directly from the fermentation broth after SepPak NP clean-up (289-290), after chloroform/ethyl acetate and/or ethyl acetate extraction (47) or diethylether extraction (290). The method of Priest and Light (291) includes an effective separation of a series of the biosynthetic intermediates in the patulin biosynthesis by using RP-HPLC and a gradient of buffered methanoVwater. Several of the same intermediatesare also separated in the acidified acetonitrilelwater gradient used by Frisvad and Thrane (47). Most HPLC methods for patulin have been developed for its determination in apple juice and other fruit products. The first methods were based on NP columns (292-305), but since 1980 RP columns have been used almost exclusively (305-319). The separation from 5-hydroxymethylfurfuralis important in these analyses (308,318-

300

321) and Sep-Pak clean-up is very often employed after extraction with ethyl acetate. In the reversed phase applications,water (306), water/acetonitrile (4,47,307,313,316317), water/tetrahydrofuran (312,315) or watedmethanol (314) are all used with success. Patulin is unstable in cheese, other milk products, and meat (322), but has been included in several multi-mycotoxinscreening methods in fungal cultures or foods such as cocoa beans (4,47, 236-238,323-324). 8.4.4.2. Penicillic acid The most important penicillic acid producers in food are Penicillium

aurantiogriseum (var. aurantiogriseum,var. polonicum, var. melanoconidiumand var. viridicatum) and members of Aspergillus subgenus Circumdati section Circumdati (formerly the Aspergillus ochraceus group). Other reported producers are quite uncommon and apparently only as superficial contaminants in foods and feedstuffs (Frisvad and Filtenborg, unpublished data). Penicillic acid has been isolated from corn (325-326), poultry feed (327), dried beans (328) and tobacco products (329), but interestingly not from cereals with a low lipid content such as wheat and barley. The poor stability of penicillic acid in the presence of -SH groups may explain the absense from both wheat, meat and cheese (330-333). Penicillic acid is best determined at its absorption maximum at 226 nm (34,47), but in several applications its UV absorbance is determined at 254 nm (323-324,334336) or at 245 nm (238). The reason for this may be that a large number of compounds have a maximum absorbance at 225 nm, while fewer compounds have a maximum at 254 nm. It has been analyzed together with patulin, zearalenone, and sterigmatocystinon a cyano column with hexane/l -propanol/acetic acid as the eluent (238) or on RP columns using acidified acetonitrile/water as eluent (47,237, 334) or neutral acetonitrile/water(238,335-336).Extraction methods and purificationof penicillic acid from biological tissues were developed by Chan et a/. (334) and Hanna et al. (336), but were as simple as those used for fungal cultures (47). However, for cocoa beans a silica Bond-elut columns clean-up step was included (238). As Penicillium

aurantiogriseum varieties (Table 8.1) are extremely common in cereals (31), more cereal samples should be screened for the presence of penicillic acid.

30 1

8.4.4.3. Mycophenolic acid

Mycophenolic acid is produced by three species of Penicillium and Lepfographium abienfinum (Table 8.1). It is however only P. brevicompacfum and P. roqueforti var. roqueforti and var. carneum that are relevant producers of this

apparently only weakly toxic secondary metabolite concerning foods and feedstuffs. It elutes as a sharp peak in the general HPLC screening system of Frisvad and Thrane (47, illustrated in ref. 45) and it is easily separated from the many other secondary

metabolites produced by Penicillium species. Neely and Parks (336a) developed a simple HPLC method for analysis mycophenolic acid in fermentation broth. 8.4.4.4. Butenolide

Butenolide is a short and too general abbreviation for 4-acetamido-4-hydroxy-2butenoic acid y lactone. This mycotoxin is also included in the general HPLC screening system of Frisvad and Thrane (47), but poor absorption at 225 nm and high polarity require a better HPLC method for an optimal detection, especially in foods and feedstuffs. 8.4.4.5. Verrucosidin

Verrucosidin, a neurotoxin, is produced by Penicillium auranfiogriseum var. auranfiogriseum chemotype I, var. polonicum and var. melanoconidium. It was first

described as a tremorgen from a strain of P. verrucosum var. cyclopium (337-338). This strain was later examined taxonomically and found to be P. auranfiogriseumvar. polonicum (16). Verrucosidin is also included among the mycotoxins in the multi-

mycotoxin method of Frisvad and Thrane (47), but again it seems likely that other more specific analytical LC methods can be developed for this mycotoxin. It should be among the mycotoxins screened for in cereals, as the three varieties of P. aurantiogriseum,very common in cereals, are consistent producers of this toxin (16). 8.4.4.6. Citreoviridin

Citreoviridin is produced by Penicillium citreonigrum, P. manginii, P. miczynskii, P. smifhii and Eupenicillium ochrosalmoneum (Table 8.1). The first and the latter

species and their neurotoxins may be of importance as they occur frequenly in rice in Taiwan and Japan (43) and pecans in U.S.A. (P. citreonigrum) (15,339) and corn in

302

U.S.A (Eupenicillium ochrosalmoneum)(l5,19,340,341). Cole et a/. (339) used a reversed phase column and methanol/water (65:35)at 1.5 ml/min as eluent, but Stubblefield et a/. (342)developed a normal phase method for determination of citreoviridin in corn and rice. This method was based on dichloromethane extraction, silica and amino-column clean-up and ethyl acetatdn-hexane (7:3)elution at 1.5 mllmin. The neurotoxin was detected by UV absorbtion at 388 nm. The chemically related secondary metabolites citreoviridin, citreomontanin, asteltoxin and aurovertin B were also included in the multi-mycotoxinHPLC method of Frisvad and Thrane (47).

The very characteristic UV spectra of these metabolites make their analysis specific.

8.4.5.Macrocyclic lactones The most important mycotoxin in this group of secondary metabolites are zearalenone and zearalenol, but several other compounds have been described in this The producers of these estrogenic compounds are biosynthetic family (43,p. 275,47). listed in Table 1 , and they are all quite frequently occuring and actively growing in cereals. Other compounds in this group include monorden, brefeldin A and curvularin. These compounds are produced by certain Penicillium species that are quite uncommon in foods and feedstuffs, but other species, often "field fungi" may produce them (see Table 8.1).It is not clear whether any of these compounds are mycotoxins sensu stricto, but they can all be separated by HPLC in an acidic water/acetonitrile gradient (47). Several methods have been developed for the detection of zearalenone in different foods, feeds and animal tissues, blood and urine. In most cases fluorescence detection has been used (exitation wavelenth 236 or 280 nm, emmission 41 8 or 470 but electrochemicaldetection (343),voltametric detection (344)and nm)(356,372,363), UV detection have also been used (4,47,237,275). Most methods have been developed for corn and other cereals (266,275,323,345-363). Trenholm et a/. (354) used zearalenone oxime as an internal standard for their analysis of zearalenone in wheat. However because of its hormone like activity and carry-over of the toxin, analytical methods have also been developed for the detection of zearalenone in milk (368),blood (369-372)and urine (372-374). animal tissue (364-367),

303

Rinsing up of zearalenone has included addition of diatomaceous earth, extraction with chloroform or dichloromethane, occasionally silica cartridge rinse up (372), extraction into base, acidification of the water phase and reextraction with chloroform or dichloromethane (356,363,372). NP applications have included eluents such as water-saturated dichloromethane containing 2 Yo 1-propano1 (372) and RP applications have included eluents such as acetonitrile/water (94:6) (363), acetonitrile/water/acetic acid (55:45:2) (237), methanol/acetonitrile/water(5:8:10) (356), methanol/ 1 Yo acetic acid (62:38) (275) and methanol/water (7:3) or acetonitrile /water/acid gradients (4,47,236). An amino column was used by Rannft et a/. (363).

8.4.6. Ochratoxins and related compounds 8.4.6.1. Ochratoxin A Being both a nephrotoxin and a carcinogen, ochratoxin A is considered one of the most important mycotoxins (42-43). Ochratoxin A has been found naturally occuring in barley, wheat, rye, oats, corn, sorghum, peanuts, coffee beans in Denmark, Canada, U.S.A., France, Sweden, Poland, Yugoslavia, Great Britain and India (43). In addition to Aspergillus species (Table 8.1) several species of Penicillium have been reported to be producers of ochratoxin A, but only one species have been found to produce this nephrotoxin: Penicillium verrucosum (16). The culture ex type of this species and cultures of its synonyms, such as P. caseiand P. nordicum are very good producers of this toxin. P. verfucosum has been isolated from all barley samples of 77 tested leading to porcine nephropathy in Denmark (Frisvad, unpublished results) and no other species were able to produce ochratoxin A. Other Penicillium species reported to produce ochratoxin A were misidentified (16,19,375-377) or the metabolite detected was another bluish green flourescing secondary metabolite (19). It should be further investigatedwhether simultaneous productionof ochratoxin A and citrinin by Penicillium

verrucosum chemotype I in cereals or ochratoxin A production by P. vefrucosum chemotype I in meat products such as salami are bofh causes of ochratoxin A contamination and human and animal health problems. Like in the case of zearalenone, rubratoxin and citrinin, the analysis of ochratoxin A in biological matrices is greatly improved by using acids in the eluent.

304

Severe peak broadening and/or binding to the column, often dependent on the batch

of reversed phase column (236) havelhas been observed in neutral eluents because of the carboxylic and/or phenolic groups in these mycotoxins. Therefore most multimycotoxin methods have included acids like phosphoric acid, acetic acid, formic acid or triflouracetic acid (4,47). The acidic groups in these molecules also suggest a partition into bicarbonate solution after organic solvent extraction, and a re-extraction into organic solvent after acidification of the water phase as a very efficient clean-up method. Several general multi-mycotoxin methods that include ochratoxin A have been developed (e.g. 4,47,236-237). These methods often depend on a general detection method, i.8. UV detection. For most dedicated applications of ochratoxin A analysis, however, fluorescence detection is much more sensitive and has been applied almost exclusively. Applications have been developed for cereals and feedsstuffs (90,347, 362,378-388), coffee and cocoa beans (389-392), foods and feedsstuffs (393-399), cheese (400), eggs (401), tissues, liver and kidneys (402-410), milk (411-412), serum and blood (413-415), urine (416), and rumen fluid (417). Reversed phase columns (C,, C, (362,413) or C, (403)) have also been used nearly universally for ochratoxin A (all above except 395) with an eluent of either acidified methanowwater (350,362,392, 394,396), buffered methanol/water (415), acidified acetonitrilelwater (394,387388,400,403-405,413,416-417, isopropanol added also in ref. 387-388 and 400),

bufferedacetonitrile/water (391,397-398,402) or acidified acetone/water(310). Gradient elution has been used when several mycotoxins have been analyzed together with ochratoxin A (4,47,386). Chamkasem e l al. (386) used phosphate buffer and methanoVacetonitrile in their gradient elution method for aflatoxins, ochratoxin A and zearalenone in grains, oilseeds and animal feeds. 8.4.6.2. Citrinin Citrinin is also a nephrotoxic mycotoxin and it is produced simultaneously in several cases with ochratoxin A by P. verrucosum. However several other Penicilium species have been shown to be producers of citrinin. Of these (see table 1) only Penicillium verrucosum, P. expansum, P. hirsutum var. albocoremium, P. citrinum and Aspergillus terreus are known to be active colonizers of foods and feeds (16,19,29,49).

305

Citrinin has been found as a natural contaminant of barley, wheat, rye, oats in Canada (418), barley and oats in Denmark (419) and rice in Japan (420).

Only quite few analytical HPLC methods exist for citrinin compared to the large number of HPLC methods for ochratoxin A. Citrinin is a stonger acid than ochratoxin A and is more difficult to analyze without ion-pairing agents, buffers or acids. In most

cases citrinin is extracted by organic solvents and rinsed up by partition into bicarbonate. Most application have been based on reversed phase columns but Dick et a/. (421) developed a sensitive NP-HPLC method for citrinin in cereals using

hexane/chloroform (6:4). RP-HPLC applications include eluents such as 0.25 N phosphoric acid and methanol or acetonitrile (422), 0.25N phosphoric acid/acetonitrile/ isopropanol (387,423), water/acetic acid/acetonitrile (40:59:1) containing 0.025 M tetrabutylammonium phosphate (424) or ion-pair partition chromatography (425) or other acidic eluents (4,47,426). However Zimmerli et a/. (427) claimed that these methods worked poorly for them, except the ion-pairing method (425). The latter method gave problems with lost fluorescence, which could be overcome by post column addition of acid (427). Zimmerli etal. (427) thus developed a sensitive method based on an acid-buffered silica gel column (428) using the same eluent as in their earlier method (421). UV detection at 340 nm has been used in some cases (423), but fluorescense detection (387,421-422,427) (exitation 340-360 nm, emission 500 nm) is much more sensitive (424). It seems that either a acid-buffered NP column (427) or a tetramethylammonium phosphate buffered RP system (424) are the only analytical systems giving consistently sharp peaks of citrinin independent of the brand of column. HPLC methods for citrinin has been developed for cereals (387,421 -422,425427), fermentation broth (424) or biological fluids (423). For broth and fluids, rinsing

up may not be necessary at all. Improved methods for citrinin and other acidic mycotoxins such as ochratoxin A, terrestric acid, penicillic acid, rubratoxin B and zearalenone in cereals may be based on the method developed by Vail and Homann (424) or the NP method of Zimmerli etal. (427). 8.4.6.3. Xanthomegnin, viomellein and related compounds.

In foods the most important producers of xanthomegnin and viomellein and the related viriditoxin are several varieties of Penicillium aurantiogriseum, Aspergillus ochraceus and Paecilomyces variotii (Table 8.1). Xanthomegnin and viomellein has

306

been found to occur naturally in barley in Denmark (429) and wheat in Great Britain

(430). Xanthomegnin and viomellein have been considereddifficult to analyze because of binding to HPLC columns (431). Earlier methods were based on normal phase separations. Because of the acidity of the phenolic groups in xanthomegnin and viomellein acid should be added to the eluent (432-435).The first methods developed used NP columns and either chloroform/methanol/ acetic acid (98:l:l)(432-434) or toluene with 1% acetic acid/ methanol (493:7)(435).Reversed phase applications were developed by Carmen el a/. (436-438)using acidified water/acetonitrile as eluent and modified by Wall and Lillehoj (431) by adding sodium dodecyl sulphate to avoid irreversible binding to the RP column. Preparative HPLC methods for ochratoxin A, viomellein and xanthomegnin have also been developed (439-442). Xanthomegnin and viomellein have been detected by UV absorption at 405 nm giving a detection limit of 12 ng xanthomegnin (431-436), electrochemical detection, with 0.5 ng xanthomegnin as the detection limit (438) or fluorometric detection after reaction with ammonia and hydrogen peroxide (exitation 340 nm and emission 445

nm)(437). Applying the last method as little as 0.1 ng of xanthomegnin could be detected (437). It is known that Penicillium verrucosum producing ochratoxin A and citrinin and varieties of P. auranfiogriseum producing penicillic acid, xanthomegnin and viomellein are co-occurring in cereals (1 6,49),but until now no multi-mycotoxinmethod has been developed for all these five nephro- and hepato-toxins in cereals. All these toxins were detected by Frisvad (48) in cultures of Aspergillus ochraceus by an acidic gradient elution method and diode-array detection, but this method has not been further developed for cereals.

8.4.7. Rubratoxins The only confirmed producer of rubratoxin is Penicillium crateriforme (formerly called P. rubrum)(49).This species also produce another acidic mycotoxin spiculisporic acid (49).The closely related species P. purpurogenum produce other chemical related acids (glauconic and glaucanic acid) but not rubratoxins (1 9,20). P. crateriforme has been found in corn and is probably able to produce rubratoxin B on that substrate

(444-445). Rubratoxin elutes as a sharp peak in RP systems with acidic acetonitrile/water

307

gradients (47,236-237) using UV detection at 254 nm or diode-array detection. Unger and Hayes (446) developed a RP-HPLC method for rubratoxin B in plasma and urine using an eluent of water/acetonitrile/ethyl acetate (9.9:11:3). Engstrom and Richard (447) developed a NP-HPLC method for rubratoxin B in mixed feed based on acidic

ethyl acetate extraction, cool and dark handling and storage using ethyl acetatekh loroform/acetic acid (80:20:1) as the eluent.

8.4.8. Hydroxyanthraquinonesand xanthones

Several monomeric anthraquinones have been characterized from filamentous fungi (Table 8.1), but only emodin (448) and physcion (449-451) have been suggested as mycotoxins. Penicillium islandicum and several other penicillia produce bianthraquinone mycotoxins such as rugulosin and luteoskyrin ("Yellow rice toxins"), while other species produce a family of bixanthones including the mycotoxin secalonic acid D. Other related anthraquinonesand xanthones are treated under Alfemaria toxins (see below). 8.4.8.1. Emodin and physcion

These anthraquinonesare producedby several species of filamentous fungi, but also by lichens and plants (Table 1). Most HPLC applications have been developed for anthraquinones as extracted from plants such as rhubarb (452-457), sometimes as glycosides (458). However they are also easily determined by the general HPLC method of Frisvad and Thrane (47), even though the anthraquinones elute quite late in that system. Matthees (459) developed a NP and RP analytical HPLC system for emodin in feeds based on extraction into aqueous acetonitrile, partitioning into chloroform and NP-HPLC using isooctan/isopropanol/acetic acid (95:5:1) or water/methanol/acetic acid (20:80:1) for RP-HPLC and UV detection at 280 nm. 8.4.8.2. Rugulosin and luteoskyrin

Rugulosin is produced by food and feed-borne fungal species such as Penicillium islandicum, P. rugulosum and P. piceum (19) and luteoskyrin is produced

by P. islandicum common species in rice (29). Rugulosin and luteoskyrin eluted quite

308

late in the HPLC method of Frisvad and Thrane (47), and dedicated methods based on HPLC should be developed for these important carcinogenic mycotoxins. 8.4.8.3. Secalonic acids Secalonic acid D is produced in corn by Penici//iumoxalicum, but it may also be produced naturally by other species, e.g. by Claviceps purpufea in rye. A HPLC method for this mycotoxin was developed by Reddy et a/. (460) and secalonic acid D was detected for the first time a natural contaminant of corn dust in 1982 by Ehrlich

eta/. (461). Two eluents were used in a RP-HPLC system using UV detection at 340 nm: water/acetonitrile/ acetic acidnetrahydrofuran (6:lO:l:l) or (6:8:1:1) and applied on biological fluids (460). Secalonic acid D could also be determined in fungal cultures by the HPLC method of Frisvad and Thrane (47), but like other quinones and xanthones it also eluted quite late. 8.4.9. Epipolythiopiperazine-3,6-diones. The most important toxins in this class are gliotoxin, sporidesmin and emestrin. The most important producers of these toxins are Aspergillus fumigatus (gliotoxin),

Pithomyces chartarum(sporidesmin)and Emericella striata (emestrin). HPLC methods have been developed for gliotoxin and sporidesmin. 8.4.9.1. Gliotoxin Gliotoxin extracted from fungal cultures eluted as a sharp peak (47), but a dedicated method has also been developed for gliotoxin in rice (462). After chloroform extraction and partial clean-up by petroleum benzine precipitation and gel permeation chromatography, gliotoxin was analyzed by RP-HPLC using water/methanol(57:43) as eluent and UV detection at 254 nm. 8.4.9.2. Sporidesmin Sporidesmin has been detected by UV at 254 nm by a cyano column and hexane/isopropanolor hexane/chloroform (for preparative purposes) (463) or on RP columns (464-466)using water/methanolas eluent for analytical separations. Different very complex clean-up procedures have been suggested to avoid sporidesrnolides, polyphenoles and other interfering substances (463-466), including ethyl acetate

309

extraction and clean-up by chloroform/hexaneelution on a Lipidex 5000 column (463), acetonitrile/benzene extraction, evaporation to

dryness and dissolving in

methanoVwater, removing lipids with hexane and reextraction of the water phase with benzene (464), extraction with diethylether, partitioning into hydrochloric acid, addition of water and back-extraction into diethylether, followed by partitioning into sodium bicarbonate/hydroxide solution, neutralized, evaporated to dryness and dissolved in chloroform followed by preparative HPLC with chloroform with 0.8% ethanol as eluent (465). Separation from the interfering substances may also be obtained by gradient

elution and less complex rinsing up. 8.4.10. Tremorgenic mycotoxins

A large number of tremorgenicfungalsecondary metabolites have been isolates, including those with a tryptophan nucleus, penitrems, janthitrems, lolitrems, aflatrems, paxilline, paspaline, paspalicine, paspalitrems, verrucologens, and the tryptoquivalins, but also the territrems and verrucosidin, lacking any nitrogen in the molecule. 8.4.10.1. Penitrems

The most important producer of penitrem A in foods and feeds is Penicillium crustosum, but other producers such as f .glandicola (formerly f .granulatum) and f . aurantiogfiseumvar. melanoconidiummay also play a role (16). Penitrem A has been

found naturally occuring in refrigerated cream cheese, where it caused intoxication of two dogs (467) or mouldy walnuts, where it caused toxicosis in a dog (468) and it may also have been present in a sample of beer which caused tremors in a man (469). Even though the toxin was not found originally in the isolate of f . cfustosum, we have later examined the strain and found that it produced large amounts of penitrem A. Even though the penitrems were nor actually found in the samples all evidence indicates that these tremorgens were involved in a toxic syndrome of sheep and horses (470) and corn infected with f . cfustosum caused a natural intoxication of cattle (471). Maes et a/. (472) developed a RP method for penitrem A to F using water/methanol (22:78) at a flow rate of 1.5 ml/min (and a column temperature of 40

"C).As an internal standard they prepared penitrem A monoacetate. Even though UV absorbance is higher at 233 nm than at 296 nm, the latter wavelength was selected

310

for monitoring because of the greater selectivity. The method of Maes eta/. (472) was also used by Dorner et a/. (471) and in a modified form by Mantle et a/. (473). The latter authors used water/methanol (1:5) as an eluent at a flow rate of 2.5 ml/min and UV detection at 335 nm. di Menna et a/.(474) used a combination of a RP system (C,)

with a gradient from water/methanol (28:72) to (8:92) at a flow rate of 1.2 ml/min and UV detection at 230 nm with a NP system monitored at 290 nm and an eluent

consisting of dichloromethane/acetonitrile (92.5:7.5). The latter NP system was used mainly for confirmation of identity of the penitrems. The penitrems appear to be sensitive to light and acids (472). 8.4.1 0.2. Janthitrems The janthitrems are produced by Eupenicillium zonatum and Penicillium janfhinellum, which are not particularily common in feeds or foods. They were

considered to be involved in ryegrass staggers, but this neurological disease in cattle and sheep is now believed to be caused by endophytes producing lolitrems. One method has been developed for the HPLC determination of the janthitrems (473). RPHPLC (C,) (preferred for a NP and a CN column) (473) was used to separate the janthitrems produced in laboratory media using an eluent of water/methanol (20:80) and UV detection at 265 nm. In this system janthitrem A, B, C, verrucologen and fumitremorgen

D, penitrem A,

B could be separated. For fungal extracts

water/methanol (36:64) for 10 min followed by a linear gradient over 5 min to water/ methanol (20:80) was used with detection at 330 nm. Fluorescencedetection was used to confirm identity of the janthitrems and increased (50 fold) sensitivity (exitation 254

nm and cutoff emission at 370 nm). The fluoresecence was only high in the RP system, and poor in the NP system (eluent hexane/ethylacetate/methanol,85:14.7:0.3) or the CN system (eluent hexane/isopropanol,9:l).The janthitrems appear quite late in the HPLC system of Frisvad and Thrane (47). 8.4.1 0.3. Lolitrems and paxilline The lolitrems are important tremorgens involved in ryegrass staggers (474) and paxilline, produced by several filamentous fungi (Table 8.1), appears to be a precursor of lolitrems.Weedon and Mantle (475) used the HPLC system of Gallagher eta/. (476) to quantify lolitrem 8, i.e. a NP silica column and a mobile phase of

31 1

dichloromethane/acetonitrile (1 5 : l ) at 2 ml/min and fluorescence detection (exitation

268 nm, emission 450 nm). Paxilline was anlyzed on a NH, column using dichloromethane/isopropanol (10O:l)at a flow rate of 4 ml/min and UV detection at 281 nm. Paxilline and l-acetoxy paxilline had retention indices of 1291 and 1386 in an acidified water/ acetonitrile gradient HPLC system (47).

8.4.10.4. Aflatrem, paspaline, paspalicine and paspalitrem A series of indol and carbazole alkaloids have been isolated from Aspergillus

flaws (477-481), A. nomius (482), A. leporis (483) and A. tubingensis (484-486). All these metabolites have been separated using HPLC. Cole et a/. (477) separated aflatrem, paspalinine and dihydroxyaflavinine by RP-HPLC gradient elution (water/acetonitrile 80:20 to 20:80). Gloer and TePaske and co-workers (478,481-486) used RP-HPLC to separate several aflavinines, nominine, leporin, aflavazol and tubingensins. The conditions were alike: Watedmethanol (30:70 or 10:90) was used as eluent at 2 ml/min and the metaboliteswere monitored at 215 nm, occasionally also employing diode array detection to find new metabolites with similar chromophores. Nozawa and coworkes used either NP separation (using hexane/ ethylacetate, 4:l)

(479) of the aflatrem, paspalinine and aflavinines, but later employed RP- HPLC for separation (480). Paspalinin, paspalin and aflatrem had retention indices of 1332,161 7 and 1514, respectively in a acidic water/acetonitrile gradient (47).

8.4.10.5. Fumitremorgins and verrucologen Verrucologen and other fumitremorgins are produced by Aspergillus fumigatus,

Neosartorya fischeri var. fischeri, Penicillium brasilianum,P. graminicolaand other less common species (Table 8.1). These tremorgens and fumigaclavine A, B, and C may have been implicated in mouldy corn silage intoxication of cattle, in which A. fumigatus is particularily common (487). Di Menna et a/. (474) used HPLC to separate verrucologen and fumitremorgen B. The fungal cultures were analyzed by a RP HPLC column using water/methanol (28:72) as eluent and monitored at 230 nm. The results were

validated

by

employing

NP-HPLC

of

the

same

extracts

in

dichloromethane/acetonitrilecontaining 0.5 % acetic acid and UV detection at 230 nm

and by comparison to standards. Nielsen eta/. (488-490) used RP-HPLC to separate verrucologen and fumitremorgen A, B and C. Their method was modified from the

312

method described by Frisvad and Thrane (47) by avoiding trifluoroacetic acid, which is not necessary for good separation and peak shape of these tremorgens. 8.4.10.6. Tryptoquivalins Tryptoquivaline and tryptoquivalone may be implicated in Aspergillus clavatus malt intoxication of different animals. HPLC has been used in the separation of these tremorgens (47,491-492), but no dedicated method has been developed for them. They are separated easily by using gradient elution using water/acetonitrile (47). 8.4.10.7. Territrems The territrems are some of the few known fungal tremorgens without nitrogen in the molecule. They were originally isolated from a strain of Aspergillus ferreus (493496), and apparently only produced by the original isolate (497), but it appears to be consistently produced by Penicillium echinulatum var. echinulatum (498), a species common on lipid-containing foods. The territrem are strongly fluorescing like the aflatoxins and therefore Ling et al (499) used both TLC and HPLC to differentiate between these toxins. Using a NP column and water saturated chloroform/cyclohexane/acetonitrile(25:7.5:1) with 0.25% ethanol at a flow rate of 2 ml/min, aflatoxin B, and B, could not be fully separated from territrem A and B, and the authors adviced to verify the presence of the aflatoxins by measuring the ratio of peak heights at 365 nm compared to 335 nm (UV detection). Later Ling (personal communication) used RP-HPLC using 60% acetonitrile in water with 0.1 N acetic acid at a flow rate of 1 ml/min to separate territrem A,

B and C

(retention time 9.36, 8.37, and 5.3, respectively). 8.4.1 1. Alternaria toxins Species of Alternaria produce a series of chemically different secondary metabolites (Table 8.1) and several of these are considered as mycotoxins (500-501).

Alternaria alternata, a very common fungus in plants, is often capable of producing large amounts of tenuazonic acid, an important mycotoxin (500).

A series of host-selectivesecondary metabolites have been analyzed by HPLC, e.g. macrosporin, altersolanol A and alterporriols (502-504) but several eluents were used to separate these metabolites on RP columns (502): 0.05 M ammonium

313

dihydrogen phosphate and phosphoric acid (pH 2.5) in water/acetonitrile (7:3, 4:l or

1:l) at a flow rate of 1 ml/min. Maleyl amide derivatives of some host selective phytotoxins from Altefnaria alternata fsp. lycopefsici were prepared and analyzed at

250 nrn in a RP gradient system (503). However most interest have been invested in the mycotoxic secondary metabolites of Alfernaria.

8.4.11 . 1 Tenuazonic acid Tenuazonic acid, cyclopiazonic acid and terrestric acid will give quite broad peaks in most chromatographicsystems (47,505)and may cause trouble because they are strong acids and extremely efficient metal chelators (505). Several methods for tenuazonic acid have been suggested, often in analytical procedures involving other

Ahernaria toxins such as alternariol (AOH), alternariol monornethylether (AME), altenuene (ALT) and altertoxin I and II (ATX-I & ATX-11). Scott and Kanhere (505) tested several HPLC systems for the analysis of tenuazonic acid. A RP system using methanoVwater with 0.1 % phosphoric acid gave very broad peaks. Two other RP systems were also tested but interfered with constituents from tomato paste. The HPLC system advocated was based on RP column coated with C12 dien (4-

dodecyldiethylene-triamin) and a eluent consisting of methanoVwater with 0.001 M ZnSO,. However Heisler and co-workers reported on good results for tenuazonic acid using a RP separation based on a water/ methanol (1 :9) at a flow rate of 2 ml/rnin in fruit and vegetable products (506-508).Later Stack eta/. (509) developed a RP HPLC method for tenuazonic acid (and alternaflol) using rnethanoVwater (85:15) containing

300 rng ZnSO,/I as eluent. Tenuazonic acid eluted as a broad but symmetric peak in the system of Frisvad and Thrane (47, see ref. 510, fig. 3 for illustration). Some of the most efficient analytical RP-HPLC systems for tenuazonic acid were developed by Lebrun eta/. (51 1-512). Ion-pair (5 mM cetrimid in water/methanol (45:55))and ligandexchange chromatography (5 mM C, dien and 5 mM ZnSO, added to waterimethanol

(25:75)bufferedwith 30 mM ammonium acetate, pH 6) could be applied for an efficient quantification of tenuazonic acid in Pyricularia oryzae infected leaves and were preferrred for anion-exchange chromatography. However the latter methods may not be suitable for LC-MS because several constituents are not volatile.

3 14

8.4.1 1.2. Alternariols, altenuenes and altertoxins.

Alternariols and related mycotoxins are more easily analyzed by HPLC than tenuazonic acid (513). Chu and Bennett (514) developed a method for preparing large amounts of alternariol by preparative NP-HPLC using different ratios of hexane and ethyl acetate as eluents. However for analytical HPLC methods RP columns have been used in most cases. However,Ozcelik eta/. (515) preferred an NP-HPLC system after comparing with several RP techniques for tenuazonic acid and several alternariols. This system involved chloroform/methanol(95:5)at a flow rate of 0.7 ml/min and UV detection at 280 nm as in many other applications. However RP systems has been advocated by

other

authors

(506-507, 509316-520).

MethanoVwater or

acetonitrilelwater eluents have often been used in RP applications of analysis of AMernaria toxins (47,506-507,509,516-518). Frisvad and Thrane (47) used a water/acetonitrile gradient with trifluoroacetic acid, while Palmisano el a/. (518) used a water/methanol gradient with phosphoric acid for diode array applications. Both systems are generally applicable for all kinds of mycotoxins, but the former has the advantage of low UV absorbtion and volatility and low corrosiveness of trifluoroacetic acid in contrast to phosphoric acid. A HPLC system of high selectivity was developed by Palmisano and Visconti and co-workers (519-520) using electrochemical detection after post-column addition of bromine (519) for altenuene and isoaltenuene. The methods involved addition of either sodium nitrate, sodium bromine and nitric acid (519) (altenuenes) or just nitrate and nitric acid (520) (altertoxins) to an eluent of water and methanol. 8.4.12. Toxic peptides

Only few analytical methods have been proposed for other peptide mycotoxins than cyclosporin and toxins from fleshy fungi. The latter toxins were reviewed in detail by Betina (39). The method of Edwards and Lillehoj (521) for cyclosporin in rice was based on gel permeation chromatography, followed by RP HPLC using water/acetonitrile (1:l) at a flow rate of 1 ml/min, and monitored at 212 nm. Cyclosporin was also analyzed by TLC and the identity was further confirmed by infrared spectroscopy. Samuels et a/. (522) used a related method for cyclodepsipeptides from Metarhizium anisopliae but they used cation exchange chromatography followed by a similar RP HPLC analysis and confirmed their results

315

by TLC and fast-atom bombardment mass spectrometry. A method developed for phomopsinA in lupin stubble also employedcation exchange chromatography followed by RP HPLC after methanoVwater extraction and purification by partitioning between n-butanol and water (523). These three methods have a lot in common and could be used for important toxins such as cyclochlorotine, for which only TLC methods exist

(524) to the authors knowledge. The nephrotoxic glycopeptides from Penicillium auranfiogriseum var. auranfiogriseum possibly involved in Balkan endemic nephropathy were purified by a

procedure used for proteins, i.e. employing water extraction, cation exchange, anion exchange, size exclusion chromatography, reverse phase Sep-Pak mini column chromatography, followed by RP gradient HPLC and finally isocratic RP HPLC (525). For RP-HPLC water/acetonitrile with trifluoroacetic acid were used as eluents and UV monitoring was at 226 or 210 nm. Analysis for penicillin often follow the same kind of analytical RP-HPLC procedures as those outlined above, often using eluents containing water with phosphate buffer and acetonitrile (526-527) ocasionally using post-column reactions

(527-528). 8.4.13. Fusarium toxins other than trichothecenes and zearalenones A series of toxins have been proposed to be implicated in equine leukoencephalomalaciaand other diseases (moniliformin,fusarins and fumonisins) and several HPLC methods have been developed for these mycotoxins. It is now believed that the cancerogenic fumonisins are the principal causes of several diseases and the fumonisins have been found to occur naturally (529). However other mycotoxins have also been analyzed by HPLC such as the fusarochromanones and fusaric acid.

8.4.13.1. Fumonisins The fumonisins may be purified by ion exchange followed by preparative RP HPLC using watedmethanol containing trifluoroacetic acid and/or acetic acid (530) and confirmed analytically by a series of chromatographic and spectroscopic techniques

(17,531),such as TLC, GC-MS and HPLC. Shepherd eta/.(532) developed a HPLC method for the fumonisins based on methanoVwater (3:l) extraction, ion-exchange mini-column chromatography and pre-column derivatizationwith ortho-phthaldialdehyde

316

followed by separation by RP-HPLC using water with 0.1 M sodium dihydrogen phosphate adjusted to pH 3.3 with oftho-phosphoric acid/methanol (20:80)at a flow rate of 1 ml/min. The derivatives of the fumonisins were detected by fluorescence (exitation 335

nm

and

emission

440 nm).

This

method

and

other

chromatographidspectroscopicmethods were used by Gelderblom and co-workersand Plattner and co-workers to analyze for fumonisins in foods and feeds (17,533-536).

8.4.13.2.Fusarochromanone Fusarochromanone has been found naturally occuring in cereal feed associated with tibia1 dyschondroplasia(537)and it was produced by isolates of fusafium equiseti

(26,538).The fusarochromanones could be separated in the RP-HPLC system of Frisvad and Thrane (47),and similar methods were used by Wu eta/. (539)and Yu and Chu (540).Yu and Chu (540)used water with trifluoroacetic acid/acetonitrile (4:6) at 1 ml/rnin and immunodetection for determination of fusarochromanone in cereals

and Wu el a/. (539)used water/methanol/acetic acid (20:120:1)at 1 ml/min and UV detection at 254 nm.

8.4.13.3. Fusarins The fusarins, especially fusarin C, are mutagenic mycotoxins once believed to be involved in leukoencephalomalaciain horses, esophageal cancer in humans and hepatocarcinomasin ducks and mice, but these clinical effects are now believed to be caused by the fumonisins. The fusarins have been found to be naturally occuring in corn (541)and have been analyzed by TLC and HPLC (541-549). Gelderblom e l a/.

(548)and Jackson et a/. (547)used a NP-HPLC method both for preparative and analytical HPLC. They employed chloroform/methanol (19:l)as eluent at 1.5 ml/min. This eluent or methylenechloride/methanoI(l9:1) have been used for the determination of fusarin C in cereals. Fusarin C was detected at 365 nm or 350 nm. However fusarin C is quite unstable at some conditions and should be analyzed accordingly (545,550). In the system of Frisvad and Thrane (47)the UV break-down products reported by Scott eta/. (545)were not observed (546).The break-down products have another UV spectrum which could be detected easily by diode array detection, if they were present. The break-down products, never observed by us, may have been caused by chloroform.

317

8.4.1 3.4. Moniliformin Moniliformin has a characteristic UV spectrum (maxima at 227 and 261 nm) and can be detected by RP-HPLC-diode array detection using water/acetonitrile with trifluroacetic acid (47). However in that HPLC system the retention is weak and more dedicated methods have been developed for this mycotoxin, especially based on ionpairing extraction and chromatography (275,549,551-552). Eluents based on water/methanol or water/acetonitrile have been used together with different ion-pairing reagents (tetra-n-butyl-ammoniumhydroxide), and phosphate buffers (551-552). 8.4.13.5. Fusaric acid Like moniliformin, cyclopiazonic acid, terrestric acid, tenuazonic acid and dipicolinic acid, fusaric acid has also been analysed by ion-pairing HPLC (553-554). It is not known if fusaric acid is an important in any mycotoxicosis. 8.4.13.6. Gibberellins The gibberellins are phytotoxic secondary metabolites from Gibberella and

Fusarium species, but it is not known, whether they have any role in mycotoxicosis. HPLC methods for gibberellins have been summarized by Lin eta/. (555), which used a gradient RP-HPLC method from 35

methanol in water, containing 0.05 % acetic

acid to 100 Yo methanol at a flow rate of 1 ml/min. They included 66 different gibberellins in their assay. 8.4.14. Miscellaneous toxins 8.4.14.1. Fumagillin Fumagillin, a secondary metabolite of strains of Aspergi//usfumigaius, has been analyzed by RP HPLC using water/acetonitrile/ acetic acid (500:500:1.5), and UV detection at 351 nm (556-557). The HPLC method of Assil and Sporns (557) also included an ELSA screening technique for this antiprotozoan metabolite in honey. 8.4.1 4.2. P-nitropropionic acid This mycotoxin has recently been reported to be produced by Arthrinium

sacchari, A. saccharicola and A. phaerospermum in sugarcane causing severe

318

poisoning in humans (558), and it may also be produced by an artificially inoculated strain of Aspergillus oryzaeon cooked sweet potato, white potato, banana and cheddar cheese (559). RP-HPLC was employed for the analysis of P-nitropropionic acid in plasma after perchloric acid treatment, using an isocratic 0.15% phosphoric acid eluent at 0.9 ml/min followed by rinsing of the column by a methanol (0-35 %) gradient and detected at 210 nm (560-561), but the method was later modified to use a rinsing gradient of acetonitrile (040%) (562). 8.4.14.3. Cyclopiazonic acid The principal producers of cyclopiazonic acid in foods and feedstuffs are Penicillium commune, P. griseofulvum, Aspergillus flavusand A. tamarii.These species

are very common and cyclopiazonic acid has been found as a natural contaminant of corn, cheese, peanuts and millet (563). Peterson et al. (563) developed a HPLC method for purification of cyclopiazonic acid and they used NP-HPLC for preparative purification based on an eluent of chloroform/methanol(99:1or 99.5:0.5) on a silica gel column preparated with oxalic acid. For analytical HPLC they used an amino column and an eluent consisting of 25 mM potassium dihydrogen phosphate/methanol (223). UV detection at 282 nm was necessary using the chloroform containing eluents,

whereas the stronger absorption at 225 nm could be used with the buffer-methanol eluent. Lansden (564) developed a RP HPLC method for cyclopiazonic acid, based on the method for tenuazonic acid (505) and this method was later modified by Norred et al. (565). However the peak of cyclopiazonic acid is still quite broad in the system of Lasnsden and co-workers (564-565) and Frisvad and Thrane (47). Goto et al. (566) developed a dedicated sensitive NP- HPLC method for cyclopiazonic acid, using an eluent more like those used in NP-TLC: ethyl acetate /isopropanol/25% aqueous ammonia . This is one of the few systems employing bases in the eluent, but it may be efficient for alkaloids. However for LC-diode array detection or LC-MS RP-HPLC systems with volatile main eluents and acids, bases or buffers are preferred. 8.4.14.4. Roquefortine C Roquefortine C is produced by a large number of Penicillium species, some of which occur very regularily in foods and feedstuffs (Table 8.1). It has been found in feed grain causing mycotoxicosis (567). Roquefortine C and the related secondary

319

metabolites meleagrin and oxaline are difficult to analyze by TLC and HPLC (47). For TLC the eluents chloroform/acetone/isopropanol or chloroform/ammonia/methanolare very efficient for separation of roquefortine C,meleagrin and oxaline (45) but for diode array detection RP system are preferred. Ware et a/. (568) developed a RP HPLC system for roquefortine C in blue cheese using ethyl acetate extraction of melted cheese added diatomaceous earth and partition into a hydrochloric acid solution. After neutralization roquefortine C was extracted back with ethyl acetate and the toxin was analyzed by HPLC in an eluent of a 0.05 M monobasic ammonium acetate in water/methanol (1 :I). Danieli eta/. (569) used RP-HPLC to analyze for roquefortine C in cheese employing a gradient of buffered water (pH 4)/acetonitrile. Experience in our laboratory has shown that trifluoroacetic acid in both the water and acetonitrile part of the gradient will give a good peak shapes of roquefortine C, meleagrin and oxaline. 8.4.14.5. PR-toxin PR-toxin has only been found in Penicillium roqueforti var. roqueforti (16). Moreau el a/. (570) developed a NP-HPLC method for PR-toxin and eremofortins A,

B and C using chloroform as the eluent and thsi method was also used later (571-572). Danieli el a/. (573) later developed a RP-HPLC method for PR-toxin using water/acetonitrile (65:45) as eluent and UV detection at 250 nm. Frisvad and Thrane (47) (see illustration in ref. 45) used a water/acetonitrile gradient to separate several toxins from P. roquefortivar. roqueforti. 8.4.15. Multi-mycotoxin analyses by HPLC

A series of papers have been published on HPLC multi-mycotoxin analysis and several

of

those

have

been

mentioned

above

(4,46-47,161,236-238,

386,392,396,545,574-577). They often cover chemically related mycotoxins or mycotoxins that are present in the same commodities. The associated mycoflora (29) of different foods and feeds may help in determining the mycotoxins that should be included in a particular HPLC multi-mycotoxinmethod (31). It is characteristic for most of these methods that they employ acidic extraction with either acetonitrile, chloroform or ethyl acetate, followed by partition with petroleum benzine or hexane to remove lipids and often mini-column clean-up steps. Most applications use RP determinations with either methanol or acetonitrile with acidified water (4,46,47,161,236,237). The

320

method of Hurst et a/. (238,392)for patulin, penicillic acid, zearalenone, sterigmatocystin and ochratoxin A in cocoa beans employs a cyano column and an eluent consisting of hexaneln-propanollaceticacid with UV detection at 245 and 280 nm. Most multi-toxin methods should cover several chemically different mycotoxins and therefore a diode-array detector or a mass selective detector may be particularily well suited for these analysis. This require, however, volatile buffers or acids and eluents with low absorbtion in the spectral range from 200-600 nm, hence in general one should chose among the following eluents and buffers: methanol, ethanol, tetrahydrofuran, acetonitrile, water, trifluoroacetic acid, acetic acid, triethylamin and ammonium acetate (see below). The HPLC method of Frisvad and Thrane (47)now include approximately400 standards of mycotoxinsand secondary metabolites (Table

8.2)and is thus of very general applicability. Retention times of the different mycotoxins will vary significantly between different batches and brands of columns (46).Hill et a/. (236)therefore suggested to use an alkylphenone retention index system for mycotoxins. This was taken up by Frisvad and Thrane (47)and later Paterson and Kemmelmeier (578)and Kuronen (4). Kuronen (4)pointed out that alkylphenone retention indices may be less accurate with compounds outside the range of the index compounds. The compounds he suggested, 1-[4-(2,3-dihydroxy-propoxy)phenyl]-I-alkanones, may be slightly better for the

purpose, but are not commercially available. Experience has shown that the alkylphenones work excellently, but a little less precise for very fast and slow eluting compounds. However such compounds are always those for which better analytical methods could be developed anyway. In practice the compounds with less precise retention indices may then be recognized by their UV-VIS spectra (diode array detection). The method of Frisvad and Thrane (47)also included a confirmation of identity by using NP-TLC in two different eluents. Even though retention indices may be more stable than retentiontimes, an extra correction may be necessary (579).Sole reliance on retention indices may cause misidentification of unknown secondary metabolites and mycotoxins. For example Paterson and Kemmelmeier (578)reported, based on retention index data, that the unrelated Penicillium brevicompactum and P. citrinum produced mycotoxins such as ochratoxin A, viridicatum-toxin, griseofulvin,

xanthomegnin, viomellein, and several other metabolites which are not produced by these species (16,19,21). This emphasizes the importance of using standards and

32 1

confirmatory tests. Inter-laboratorystudies may be necessary to standardize retention index data to a level where preliminary identification can be suggested. However confirmations of identity are still of major importance. 8.5. INFORMATIVE ON-LINE DETECTION METHODS 8.5.1. Applications of HPLC diode array detection Diode array detection (DAD) giving full UV-VIS spectra in the range of (190-) 200-600 nm, have put a new dimension into HPLC analysis. A large number of mycotoxins and other fungal secondary metabolites, but also food constituents, have very characteristic spectra (47,538) which can be used for confirmation of identity, peak purity determination, peak "unmixing" and optimal selection of detection wavelength. Fortunatelymany of the mycotoxins with weak uncharacteristic UV spectra can often be analyzed more efficiently by e.g. gas chromatography (e.g. the type A trichothecenes). HPLC-DAD has been used to analyze very complex mixtures of secondary metabolites from fungi (1 6,20-27,47-46,510,518,546) but can also be used for foods and feedstuffs (518,577, Frisvad, unpublished). Frisvad and Thrane (47) reported on TLC data, HPLC retention indices and UV maxima for 182 mycotoxins and fungal secondary metabolite data. This data base has now been expanded considerably,including more precise UV-VISdata. These data are presented in Table 8.2, which includes retention indices and UV spectral data reported as all maxima, minima and shoulders and their relative absorptions related to the largest absorbtion (100%). Many of the spectra have been plotted in ref. 538. The original data-base (47) was quite meagre concerning UV data as only maxima and an indication of the largest and next largest absorption were given. It is well known that UV spectra, in contrast to IR and MS spectra, are often dependent of pH and type of eluent (580-582). This make comparisons between literature data taken in methanol, ethanol and data recorded by the diode array detector difficult in several cases. Again it is ernphazised that standards should always be used to verify identity of a mycotoxin and not just by comparison to literature data on UV spectra. The gradient used by Frisvad and Thrane (47) will start with a pH value of ca. 3.5 and end with a pH of ca. 2.7, but this little change does not affect the chromophore absorbtions for compounds eluting early and late in the 50 min analytical run-time. However a change from acetonitrile to methanol or especially neutral or

322

TABLE 8.2. Retention indices (RI) and UV data of fungal metabolites as measured on-line with a diode array detector in the HPLC system of Frisvad and Thrane (47). Absorbing wavelengths are given in nm. Wavelengths marked are maxima, those marked 's' are shoulders and the other figures are minima. After each wavelength (i.e. after ' I ') the relative absorption in % of the maximal absorption is given. In case the only absorption maximum is below 200 nm, UV data is presented as 'end'. I*'

Fungal metabolite 4-ace famido-4-hydroxyFbutenoic acid y-lactone (butenolide)

7 '-acetoxypaxilline

RI

673

7386

UV Data

........

........

230'1 700

2651 77 280'1 27

201') 700

2061 45

15-acetoxyscirpenol

783

end

3-acetyldeoxynivalenol

747

279'1 700

33-acetyldiacetoxyscipenol

986

end

a-acetyl-y-methyl-tetronic acid

669

2071 74

237'195

244155 263'1 700

756

207'1 700

2394 35

2671 79 306'1 29

Acetyl T-2 toxin

7 757

end

Aculeasin A y

7216

220sI 28

250)3

274') 4

AflatoxicolB

929

207'1 700

2391 75

257~127 261 '1 26 2751 4 333'1 37

Aflatoxin 8,

895

2721 57

225'1 68

257 I26

267'1 44 2881 6 362'1 74 Aflatoxin 8,

867

2121 59

278'1 67

2334 54 253 I 28 267'1 44 2881 6 364'1 82

323


RI

UV Data

........

....,...

G,

865

201'1 100 2101 82

218'1 87 242s I 36 251 I28 265'1 37 2831 3 368'1 64

Aflatoxin G2

834

2081 85

216'1 93

242'1 44 2531 34 265'1 40 2841 3 368'1 79

Aflatoxin GZa

769

2081 83

216'1 88

242'1 41 2551 32 265'1 34 2841 3 366'1 71

Aflatoxin M,

820

2161 55

229'1 71

251 I28 265' I 42 2851 7 358*161

Aflatoxin Mz

785

216'1 53

2481 21

251 *I 23 2531 21 263'1 29 2851 4 356'1 53

1514

2141 65

231'1 100

2651 27 283'1 30

Agroclavine

754

2081 79

223'1 100

2461 6 275~121 281 '1 22 290s I 18

Altenuene

839

214 I 24

240'1 100

261 I20 281 *I 34 3041 16 319'1 18

Aflatoxin

Aflatrem

324

TABLE 8.2. (continued) Fungal metabolite Aiternariol

RI

........

........

276~149 237132

255'1 700 2751 77 288'1 27 2941 79 298'1 27 3081 70 337'1 23

7074

2331 22

257'1 55

2791 72 287'1 74 2941 72 298'1 73 3701 7 339'1 74

902

203165

273'187

237 I 30 259'1 700 2791 45 285'1 48 3731 6 355'1 76

Anhydrofusarubin

7766

278155

237'169

2551 44 290'1 68 245s I 7 7 3991 0 540'1 33

para-anisaide hyde

798

208137

223'157

257 I 2 288'1 79

Antibiotic Y

957

276.~173 227167

243'1 83 2631 67 273s I 67 287'1 77 306s I 43 3791 38 347'1 57 3541 48 364' I 53

Ascochitine

7080

2031 76

2331 59 263 *I 92 279'1 700 341'1 75 3561 70 476'1 39

A Iternariol-monomethylether

Alterfoxin I

935

UV Data

275'190

325

TABLE 8.2. (continued) ~

~~~

RI

UV Data

........

........

1881

2161 51

221 *( 51

2701 6 316'1 16

Asperthecin

885

2181 49

237'1 88

2461 68 263'1 100 2791 52 287'1 56 3021 31 317'1 35 351 16 482'1 55 509s 1 46

As teltoxin

983

272~131 2351 18

Fungal metabolite Aspergillic acid

273 I 93

2961 15 366'1 700

Asterric acid

969

212'1 100

239 I 23

251'1 27 2791 4 315'1 15

Aurantiamine

868

2121 42

231 *I 54

269 1 24 321'1 100

758

2121 75

225'1 100

251 15 285'1 24 292s I 23

1129

272s 1 33

2351 20

273'1 84 2941 7 364'1 100

Austamide

907

2031 64

214sl 72

233'1 100 2551 47 265'1 48 285s I 35 341 14 395'1 11

Austdiol

700

203'1 34

225)8

257'1 51 3101 3 381'1 100

Aurantioclavine

Aurovertin B

326

TABLE 8.2. (continued) RI

UV Data

........

Averufin

7 342

2091 60

224'1 700 247 I36 257s I 48 269'1 55 2731 54 293' I 98 3751 26 322'1 26 3531 70 453'1 37

Barnol

837

207'1 700 225~127 267 I 2 275'1 2

Benzoic acid

747

2701 32

237'1 97

2591 6 275'1 8

Bostrycin

750

208'1 60

2721 59

227'1 700 2651 74 302'1 30 257 12 480 I 24 503 I 26 540'1 76

Fungal metabolite

........

Bostrycoidin

1046

207'1 700 2251 37

257'1 95 2921 72 323 I 22 3871 5 488'1 28 522.~178

Brassicasterol

2060

2721 65

2221 65 227'1 66

Brefeldin A

97 7

214sl 65

Brevianamide A

869

2071 66

233'1 700 265s I 22 2881 7 407'1 73

7097

205s I 95

233s I 4 7

897

207'1 700

Byssochlamic acid Canadensolide

276'1 66

253s I 28

327


UV Data

........

........

Canescin

912

2071 15

244'1 100

2771 74 277'1 15 287s I 11 2981 4 337'1 13

Carlosic acid

690

2031 72

233'187

246154 265'1 100

Carolic acid

677

2001 15

231'1 90

2441 51 265'1 700

Catenarin

7 798

2081 39

231'1 100

2441 41 257*150 2631 46 277'1 54 2921 31 304'1 33 3431 4 477s I 36 488' I 42 520s I 27

Chaetoglobosin C

7176

2081 79

221'1 700

240126 251 7 27 279~117 288~114

706

208 I 78

223'1 100

244 I 6 273~127 281 *I 23 290s I 20

201'1 100

223s 64

2631 72

Fungal metabolite

Chanoclavine

Chetomin

7 137

287'1 13 6a-chlamydosporol

729

205'1 100

233s 8

24214 285* I 30

6Q-chlamydosporol

720

205'1 100

233s 8

2421 4 285'1 30

Chromanol 7

917

227'1 100

2441 16

267'145

Chromano12

909

223'1 100

2441 76

269'1 45

Chromanol3

835

227'1 100

2441 16

269'147

328


UVData

,

.......

........

1124

2101 58

223' I 79

235 I 26 255'1 44 283'1 21 3081 3 428'1 19

719

201 *I 83

2121 52

229'1 100 251 I 19 265' 1 25 273s 1 23 2871 10 304'1 16 313sl 13

Chrysophanol

1244

2081 51

225') 100

237133 259'1 65 2751 30 279'1 31 2831 30 287'1 31 3081 2 428'1 30

Citreomontanin

1679

229s 1 28

2481 18

267'121 2851 15 317*122 3411 18 413'1 84

Citreoviridin

1051

205'1 58

2231 21

239'1 27 2591 21 288s I 89 294'1 97 321 I24 388'1 100 403s I 94

Citreoviridin A

1070

201'1 53

221 I 16

237'1 21 261 I 12 285s I 48 294 I 53 3191 13 366.~173 387'1 100

Fungal metabolite

Chrysazin

Chrysogine

329


UV Data

........

........

7074

2081 22

276'125

2351 77 273'1 87 3001 13 368'1 700

Citrinin

867

2031 78

214'1 100

2784 97 246s I 56 2791 9 327'1 44

Citromycetin

697

214'1 700 2421 52

251 *I 53 283)27 302'1 37 323 I 26 358'1 48

2791 53

2471 47 265sl 72 269'1 75 294s I 38 3331 5 445. I 33

Fungal metabolite Citreoviridin X

Clad0fulvin

7260

Cladosporin

986

274'1 700 2401 72

Clerocidin

960

2031 76

235'1 700

Compactin

7208

203 1 24

237'1 92

23-3197 237') 700 244s I 68

Curvularin

976

2731 62

227'1 65

233~157 257 I20 277'1 34 2881 25 300'1 26

Cyclochlorotine

828

207'1 100

Cyclopaldic acid

833

2151 9

245'1 100

273.~129 323s'l 7

233'1 74

267'1 63 288 1 24 298 I 26

330


UV Data

........

......

Cyclopenin

863

203) 93

210'1 100

231~159 251s 1 25 2751 5 288'1 7

Cyclopenol

771

201'1 100

214.~167

233~135 2691 5 285'1 8

Fungal metabolite

Cyclopiazonic acid

1169

2081 65

225'1 100

246123 281 '1 51

Cynodonfin

1369

2231 22

239'1 35

2514 16 2691 5 290'1 6 3681 1 516'1 12 5281 10 542'1 1 1 5481 1 1 550'1 72

Cytochalasin A

1129

216s I 48

229s I 32

Cytochalasin B

1015

212.~166 231~124

Cytochalasin C

1074

end

Cytochalasin D

1004

end

Cytochalasin E

1058

end

Cytochalasin H

1004

end

Cytochalasin J

900

end

bis-dechlorogeodin

919

207'1 100

216~182 2511 15 285'1 61 339s I 13

1076

205'1 100

255~130

201 122

248'1 100

Dechloronidulin Dehydrocarolic acid

68 1

271 I34 296 I 49

33 I


RI

Dehydrocurvularin

854

UV Data

........

........

216~163 233~146 2531 21 2831 54 304'1 70

Dehydropaxilline

1398

2081 49

232'1 100

248~148 279s I 23

Demethoxyviridiol

809

2191 21

253'

100

2831 15 321'1 49

Deoxybostrycin

84 1

2121 51

227'

100

2671 12 304'1 29 3521 2 471~123 501'1 27 536~117

Deoxynivalenol

685

218'1 100

Dermoglaucin

1078

2031 85

211'1 100

233~148 2461 38 265s I 56 283' I 92 3371 5 430'1 37

Desacetylpebrolide

9 6

2101 5

232'148

26313 273'1 4

Desertorin A

958

210*) 100

237~142 2651 12 294s I 36 308'1 48 319~144

Desertorin 6

1044

210'1 100

237s 41

2621 13 298s I 39 308') 48 319~144

Desertorin C

1111

210'1 100

223s 78

237~147 2651 16 296~141 308'1 50 319~143

Desmosterol

1920

end

332


UV Data

........

........

Dethiosecoemestrin

1568

2121 57

225'1 78

2481 5 283'1 13 290.~112

Diacetoxyscirpenol

866

end

4,15-diacetylverrucarol

95 1

end

Diethylphthalate

996

205s I 94

Fungal metabolite

Dihydrocytochalasin 6

1127

208'

I 100

2161 83 221 *I 87 223 I 86 231 * ( 93 2631 12 277'1 17

end

Dihydroergotamin

96 1

2141 72

219'1 74

2441 5 279'1 14

cis-dihydrofusarubin

803

208s I 76

227 I 44

244'1 86 261 I25 277'1 34 302'1 24 3331 9 391'1 41

trans-dihydrofusarubin

846

see above

2',3'-dihydrosorbicillin

1194

2031 74

216'1 100

23 1s55 2481 7 285'1 76 3171 28 329 I 29

Dihydroxyaflavinine

1056

21 1 I 71

225'1 100

2571 10 277.~116 283'1 18 288s I 76

2,4-dihydroxy-6-(1,Pdioxopropyl) benzoic acid

680

214'1 100 2391 18

261 *I 46 2791 15 296'1 23

333


UV Data

........

........

2,4-dihydroxy-6-(1,2-dioxopropyl) benzoic acid, lactol

855

221 150

239'1 100

253~167 2751 18 298') 25 3231 18 347'1 22

2,4-dihydroxy-6-(1-hydroxy-2. oxopropyl)benzoic acid

717

212'1 100

240122

261 *I 33 2871 18 298' I 20

2,4-dihydroxy-6-(1-hydroxy-2oxopropyl) benzoic acid, lactol

698

212'1 100

227~157 2421 15 269'1 48 2881 24 302'1 28

2,4-dihydroxy-6-(2-oxopropyl) benzoic acid

719

214'1 100

2391 18

2,4-dihydroxy-6-(2-oxopropyl) benzoic acid, lactol

807

214'1 100

228.~163 2401 13 271 *I 63 2921 26 300'1 28

Dihydroxysterigmatocystin

1069

207.~176

221 I59

233' I 77 2351 77 249'1 100 2791 11 327' I 50

2,7-dimethoxy-6-(1-acetoxyethyl)-juglone

1015

218'1 100

239126

263'1 51 2791 9 308'1 33 3431 4 427') 13

1150

218'1 100

237125

263 I 59 2791 9 310'1 33 3451 3 426'1 15

Fungal metabolite

902

205~194 209'1 100

263'1 37 2871 18 298'1 21

229 1 23 241 *I 33 261 I 1 298'1 14

334


RI

UV Data

........

........

Dimethylphthalate

851

2031 93

208'1 100

2161 85 223s 1 88 231 *I 95 2631 12 277'1 18

Dipicolinic acid

675

223s I 26

2461 6

267.~112 272'1 14 281sJ10

Dithiosilvatin

1152

221 I33

227'1 36

233s I 3 1 251 1 10 273'1 21

Dothistromin

1061

2161 41

223'1 42

2421 16 257sl 18 267s I 20 292') 33 317sl 10 3491 5 455'1 12

Duclauxin

1137

203' I 76

2111 68

229'1 100 265s I 33 3061 11 321'1 13 345s I 11

Echinulin

1370

2141 80

231'1 95

2571 1 1 283 I 22 294s) 18

693

2071 86

223'1 100

2461 8 275s 1 24 281'1 25 288s I 2 1

Emestrin

1036

227s I 51

259s I 27

267s I 25 283~116

Emestrin B

1050

225s)53

248)28

265') 34 285s I 25

Emindole DA

1560

211162

225'1 95

2481 5 279~116 283') 16 288s)15

Elymoclavine

335


UV Data

........

........

Emindole DB

1557

201'1 85

2081 69

223'1 100 2481 6 275s I 20 283'1 22 290s I 20

Emodin

1132

2071 58

223'1 100

2371 37 255s I 51 267'1 56 275)52 288'1 60 331 14 441 *I 33

Epicorazine A

847

2081 94

218'1 99

271~15

Epoxyagroclavine

704

2071 55

223'1 100

2461 12 275s I 22 281'1 23 288s I 20

Epoxyagroclavine-AgroclavineN,N-dimer

850

221sl 78

2481 15

265s I 23 273'1 26 283s I 24 292s I 21

Epoxyagroclavine-N,N-dimer

806

216~170

2481 12

265~119 273'1 21 283s I 19 292s I 16

Epoxysuccinic acid

677

end

Fungal metabolite

1381

221 I 28

235'1 37

261 I25 294 1 59

Ergocristine

991

239s I 4 1

271 I 4

319'1 17

a-ergokryptin

970

221.~163 239s I 53

271 15 319'1 22

Ergometrin

715

2141 88

227'1 96

237s I 88 2691 8 313'1 36

Equisetin

336


RI

Ergosterol

1585

Ergotamin

947

Eryf hroglaucin

7428

UV Data

........

.......

2461 9

265sl 7 7

273'1 f 4 2771 13 283'1 14 294~19

237~147

271 I 4

319'1 77

2741 44

237'1 68

2441 28 257'1 34 265)30 275'1 34 2921 79 303 I 20 347 13 488' I 28

2271 60 237'1 66 263 I 24 296s I 87 323'1 100

Ethisolide

73 1

203'1 700

Expansolide A

978

end

Expansolide 6

1008

end

I

Ferulic acid

749

2071 57

278'1 71

Festuclavine

762

2081 60

223'1 100 2441 8 273s I 20 279'1 21 288~178

Flavoglaucin

1557

2271 27

239'1 37

9 76

207 I 34

237'1 700

Frequentin Fructigenine A

1728

205~199 2271 72

2561 14 277'1 30 3081 7 389'1 74

246'125 279s 1 4 285s) 4

337


UV Data

........

Fulvic acid

939

2031 96

208'1 700 2271 62 233 1 64 271s I 22 288)15 337'1 42 3451 41 389'1 78

Fumigaclavine A

725

2071 53

223'1 100

Fumigaclavine B

690

2071 52

223'1 700 2421 6 279'1 79 290sl 15

Fumigaclavine C

881

2701 55

229'1 100 24818 283'1 25

Fumitremorgen A

7387

2191 68

225'169

Fungal metabolite

Furnitremorgen B

7 797

2751 77

Fumitremorgen C

956

278'1 73

Fusarenone X

706

227'1 100

Fusaric acid

77 7

Fusarin C Fusarochromanone

229'1 86

........

2421 5 273~179 279'1 20 288~117

2551 273'1 2831 292'1

70 14 72

2571 279'1 2851 296'1

77 78

72

17 20

269'1 77

290'1 75

274 I 28

227'1 34

2461 70 273'1 46

7 060

2571 73

368'1 700

727

272'1 82

2331 37

257'1 700 3691 35 281'1 42 370sJ 75 3331 5 387'1 55

338

TABLE 8.2. (continued) Fungal metabolite Fusarubin

RI

UV Data

........

........

90 1

212157

227'1 100

2671 13 302') 32 3521 2 469s I 23 496'1 26 530s I 16

1313

218.~165

Gallic acid

68 1

214'1 100

2401 10

271'141

Genfisylalcohol

669

221sI 26

2531 2

292'1 17

Gibberellic acid

736

203'1 100

Gladiolic acid

771

2051 32

231 *I 100

2571 31 267'1 35 302.~113

Glauconic acid

93 1

212'1 100

Gliotoxin

833

2441 27

269'1 34

Griseofulvin

999

212'1 98

2271 81

Griseophenone C

936

205'1 100

227~144 2481 5 297'1 50 337sl 15

HT-2 toxin

928

end

Hadacidin, Na'

669

end

Fusidic acid

Helminthosporin

1317

211145

231'1 100

Helvolic acid

1260

214 I 68

233'1 81

237'1 88 251~169 271 I47 292'1 100 325s I 23

242134 255'1 49 281 I20 288 * I 23 3191 3 484'1 30 518sl 18

339

TABLE 8.2.(continued) RI

UV Data

........

........

5 '-hydroxyasperentin

825

274'1 700

2401 72

267'1 63 288 1 24 300'1 26

para-hydroxybenzoic acid

676

208s I 89

2271 78

255'1 97

Hydroxyisocanadensicacid

728

225'1 700

5-hydroxymalfol

692

276s I 59

2351 72

263~142 287'1 66

4-hydroxymellein

755

208'1 700

2291 72

246'1 22 2751 2 373'1 75

w -hydroxypachybasin

978

274 I 58

227'1 60

237 I48 257s I 88 259'1 700 279s I 42 3041 6 335s I 7 0 403'1 20

lndolacetic acid

766

2041 67

278'1 700

2451 8 280'1 27 289~177

7349

2741 48

237'1 83

2421 46 253'1 56 2751 75 290'1 20 3251 3 465s I 24 488 I 29 524s I 78

Isochromantoxin

882

227s I 20

2531 7

283'16

lsoemodin

985

2081 46

225'1 700

2371 34 257'1 65 279s 1 29 287s I 29 2081 2 429 I 30

Fungal metabolite

lslandicin

340

TABLE 8.2. (continued)

.,......

RI

UV Data

........

lsomarticin

972

210158

227'1 100 2671 74 304'1 29 357 I2 480s I 24 498'1 25 534~175

ltalicic acid

907

270120

237~142 240'150 2571 42 269'1 45 292 I 29 335'1 700 352sl 65

ltalicic acid-methylester

7052

272123

239'150

Janfhifrem 6

7172

237~133 240126

Fungal metabolite

257139 271 *I 47 285s I 38 294 I 29 335' I 700 352s I 68 263'157 287 I 9 331 *I 34

Ja vanicin

968

212157

227'1 100 267)73 305'1 37 3561 2 475s 1 23 502'1 27 536s I 7 7

Kojic acid

673

203165

276'1 700 235140 244s145 269'1 73

1097

2031 92

208'1 700 2374 35 2591 75 296s)36 306'1 41 377~135

Kotanin

341

TABLE 8.2. (continued) Fungal metabolite Larnbertellin

RI

UV Data

........

........

961

2051 92

211'195

233~171 261 I48 283~167 290 I 69 298s I 27 3351 4 432'1 27

Lanosterol

1962

Lapidosin

898

216'1 100

231 I 78

239'1 81 2651 46 273'1 48 321 I 13 335'1 14

Lichexanthone

1377

207sl 83

221 151

242'1 100 267~133 281 I22 309 I 63 339s I 24

Luieoskyrin

1269

2331 12

253.91 17

261'120 2671 18 273'1 19 2851 14 296*1 17 3251 9 445'1 100

Macrosporin

1133

2181 53

225'1 56

243 I 27 285'1 100 306s I 38 341 I 14 381'1 21

Malformin A

1031

end

Malforrnin B

1047

end

Malformin C

1036

end 227'1 100

2671 13 304'1 31 351 12 4 75s I 24 498'1 26 532s I 16

Marticin

942

end

2121 58

342

TABLE 8.2.(continued) Fungal metabolite Meleagrin

Methoxysterigmatocystin

RI

UV Data

........

........

849

2181 60

229'1 67

259 I 20 283s I 25 329'1 66

1072

207'1 82

2781 50

239'1 700 2751 70 315'1 42

3-methoxyviridica tin

995

203'1 100 2121 88

221 '1 98 2631 16 281 *I 21 3001 14 315.~179 323 I 23 335s I 17

6-methylsalicylic acid

760

207'1 100

238~177

2651 2 300'1 9

Mevinolin

7274

2031 27

233'1 89

2351 88 239'1 700 246s I 67

Mitorubrin

7 098

2031 74

273'1 85

2351 42 267'1 100 292s I 63 3701 47 351'1 88

Mitorubrinic acid

946

212'1 78

239) 47

273'1 700 300s I 59 3171 49 349'1 66 366s I 52 395s I 27 422s I 27 455sl9

Mitorubrinol

936

2031 74

213'1 85

2351 45 265'1 100 292s 1 64 3701 46 351 *I 86 364~179

343


UV Data

........

........

Mitorubrinol acetate

7 059

2041 72

274'1 83

2371 52 265'1 700 292s)62 3061 46 349'1 84

Mollisin

7 756

207' 700 233130

Fungal metabolite

259'1 67 277s I 42 3061 3 478'1 72

Moniliformin

670

2031 75

Monorden

923

205'1 100 274sl 77

2421 27 275'1 52

2294 31

2651 75

379'1 55

274'1 700 2351 74

249'1 22 2731 2 303'1 7 7

7442

2701 72

274'1 75

2371 37 255'1 65 288.~176 3731 6 356' I 34 374s I 27

Nectriafurone

949

2781 57

240'1 74

257 I 77 259'1 73 2871 72 323' 1 23 3771 7 1 443'1 49

Neosolaniol

723

end

205'1 700 2764 89

2571 78 267'1 79

Mycelianamide Mycophenolic acid

Naphthalic anhydride

Nidulin

7202 977

7487

Nigragillin

773

269'1 700

P-nitropropionicacid

678

208'1 700

Nivalenol

676

227'1 700

227'1 700 2451 26 267'1 30

344

TABLE 8.2. (continued) Fungal metabolite Norninine

Norjavanicin

RI

UV Data

........

........

7 620

2721 57

227'1 82

2481 4 277sl 73 284'1 74 290s 1 13

89 1

2741 57

223'1 62

2671 72 298'1 27 34.31 3 497'1 77

Norlichexanthone

7 000

205.~168 2781 47

Norsolorinic acid

7524

2731 69

Nortryptoquivaline

1763

208'1 700 2271 75

229'1 87 253s 1 38 267s I 24 275s 1 20 2981 7 304'1 8 377sl 7

Ochratoxin A

7091

2051 94

275'1 98

248s I 2 7 2831 2 332'1 77

Oosporein

667

205'1 99

237 I37

257~139 289'1 700

Orsellinic acid

777

272'1 700 2351 79

Oxalic acid

676

203'1 99

247'1 700 267s I 26 2871 79 373'1 60 346s I 20

235'1 700 2551 56 275~179 304'1 87 352sl 78 366sl 77 3971 74 463'1 36

246sl 7

253'1 33 2771 9 292'1 74

345 TABLE 8.2. (continued) RI

UV Data

........

........

883

2181 59

229'1 65

2571 21 283s I 26 327'1 66 339s I 57

Pachybasic acid

1004

205'1 87

221 I69

225'1 69 2371 56 259'1 100 279s I 36 3001 6 337'1 10 3491 10 403'1 21

Pachybasin

1232

2141 56

223'1 60

231 I52 248~184 259 I 98 277s I 47 3021 6 333'1 10 351 19 405'1 20

Fungal metabolite Oxaline

Palitantin

886

231'1 100

Parasiiicol

880

205'1 100 216~157 2421 15 255s I 21 263'1 23 2751 4 329'1 27

Paspaline

1617

2081 53

231'1 100 251 18 281 *I 24

Paspalinin

1332

2101 59

231'1 100 273~126

680

2051 10

230~124 277'1 100

1291

2081 49

231'1 100 2691 20 281 *I 22

Penicillic acid

715

2051 45

229'1 100

Penicillin G

669

208'1 100 2441 4

259'1 4

Penitrem A

1342

225 I 46

259 I 7 296'1 17

Patulin Paxilline

235'1 50

346


RI

UV Data

........

L,L-phenylalanine anhydride

867

2441 1

259'1 2

Phoenicin

721

2051 74

214'1 79

........

2351 43 267'1 100

3391 1 488'1 7

Phomarin

1097

2051 59

218'1 94

239 I 30 269'1 100 294s I 48 3251 7 413'1 23

Physcion

1340

2101 63

223'1 82

2391 37 253s I 40 267'1 43 2751 40 287'1 43 300s 1 29 3331 5 439'1 27

218'1 100

257~130 2671 18 292s I 30 323'1 40 354s I 7 1

PI-3

PR-1635

802

PR-toxin

86 1

2781 23

249'1 100

Preechinulin

890

2121 70

225'1 85

2531 10 283'1 20 288~118

1319

212.5 73

2231 53

239'1 73 2481 66 267'1 77 3131 11 351'1 17 395)4 501'1 17 536sl 11

Purpurugenone

2831 79 363'1 100

347

TABLE 8.2. [continued) Fungal metabolite

RI

UV Data

........

........

Pyrogallol

679

203~197 207'1 100

2-pyruvoylaminobenzamide

679

218'1 100

244.~128 27314 296'1 8

1027

2081 67

223'1 100

239136 251s I 39 285. I 62 3231 6 436'1 26

Questinol

867

2071 67

223'1 700

239141 246'1 44 2551 42 269s I 52 285') 63 3231 6 434'1 28

Ravenelin

1089

221 1 45

233'1 62

2371 61 261'1 700 2881 10 339'1 37 3771 70 397'1 11

Riboflavin

786

201 I41

218'1 100

231 I45 248s I 84 261'1 100 2851 2 352'1 30 387s I 22

Roquefortine A

743

2071 55

223'1 100

2441 5 281 * ) 22

Roquefortine B

686

2071 55

223'1 100

2421 6 281 *I 21 288.~118

Roquefortine C

922

205~192 2231 33

Quesfin

225.~154 2481 4 269'1 6

233') 34 2631 19 304'1 54

348

TABLE 8.2. (continued) Fungal metabolite Roquefortine D

........

RI

UV Data

........

686

2031 67

218'1 100 251 ( 2 1 283s143 288'1 45 294 I 44 302'1 45

Roridin A Roseopurpurin

1013 866

245'1 100 2071 71

221'1 100

2371 47 249'1 56 2591 53 269s I 56 285'1 65 3251 4 434 I 29

Rubratoxin B

1076

205~195 2351 23

Rugulosin

1132

2291 49

251'1 83

273~164 3151 38 391 *I 81

Rugulosuvine

859

208184

216'190

24817 273.~114 281'1 14 288~112

Rugulovasine A

711

216' 100

24616

285'1 17

Scytalidine

1301

Scytalone

711

Secalonic acid D

Shikimic acid

1190

678

249'1 29

end 218' 93

231~166 24718 283'1 100 317.~139

216~155 233~140 2551 30 263'1 31 2871 19 337'1 71 377s I 17 210'1 700

349


UV Data

........

........

1349

2161 39

223'1 40

2421 23 255'1 27 2791 15 298'1 17 331 13 457'1 12

958

2121 50

229'1 100

2691 11 308'1 29 3561 2 480s I 22 505'1 27 5301 17 540'1 18

Soranjidiol

1119

2051 59

219'1 90

2391 31 269'1 100 292s I 50 3191 7 413'1 25

Sorbicillin

1172

203'1 100

233s I 30

2551 13 325*1 76

Spinulosin

700

208s I 80

2531 11

296'1 100

Steckiin

792

2051 97

21091 100

2251 56 233 I 64 2501 10 283'1 76 304s I 47

Sferigmafocystin

1104

205s I 80

2181 54

233s I 84 248'1 roo 2791 10 327'1 46

Stigmasterol

1916

end

Stipitatic acid

686

2081 19

259'1 100

2941 10 327'1 13 3391 12 355'1 14

Sulochrin

919

207'1 100

2431 16

283'1 39 319sl 17

Fungal metabolite Skyrin

Solaniol

350 TABLE 8.2.(continued) Fungal metabolite T-2 toxin

RI 1025

UV Data

........

........

2331 44 248.~150 279'1 100

end

Tenuazonic acid

810

2081 41

223'1 47

Terrein

682

216) 9

281.1 100

Terrestric acid

711

212s I 35

231'1 64

Terretonin

1043

2331 19

277'1 100

Territrern A

1135

2071 85

216'1 96

2751 7 339'1 39

Territrern B

1114

212.~161

2751 5

331'1 16

Territrern C

1033

210.~165

2831 4

341'1 14

2081 43

221.1 51

242s I 18 2651 8 288 I 22

Toluhydroquinone

Torreyol Trichoderrnin

689

1299

end

993

end

2441 39 269'1 100

Trichorzianines A'

1379

2161 43

221'144

24813 281'1 7 290sl6

Trichorzianines B Ila

1377

221.5 41

24813

271.~15 279'1 6 290.~15

Trichorzianines B lllc

1393

2161 42

221 '1 42

251 I 3 281 *I 6 290s I 5

Trichorzianines B IVb

1375

223~135 25113

Trichorzianines B Vb

1383

end

Trichorzianines B Vla

1468

251 13

283'1 3

271~15 281 '1 5 290~14

288.~12

35 I TABLE 8.2. (continued) Fungal metabolite

RI

........

........

2141 60

229'1 80

2481 48 261~152 281'1 100 313~136 3451 8 430'1 41 287'1 57 321sl 18

UV Data

Trichonianines B Vlb

1415

end

Trichonianines 6 Vll

1446

end

Trichothecin

1004

214'1 100

730

231'1 100

Trichothecolone 3,4,5-trihydroxy-7-methoxy2-methyl-anthraquinone

1155

Trypacidin

988

207'1 100

2491 11

Vermiculin

839

2051 86

221'1 100

1032

221 I25

261 *I 100

Verrucarin A Verrucarol

715

end

1072

205s I 91

2271 14

68 1

2031 13

237'1 100

Verrucosidin

1214

2251 53

239'1 61

269 I 25 294 * I 39

Verruculogen

1137

2161 75

225'1 76

2551 11 275'1 15 2871 13 294'1 13

Verruculotoxin

766

205s I 86

2401 1

257'1 1

epi- 10-verruculotoxin

715

end

Vertinolide

85 1

2071 20

235'1 50

2461 45 279'1 100

Violaceic acid

838

2181 72

229'1 79

2451 52 263') 66 283s I 52

Verrucofortine Verrucolon

246'1 26 275~16 283s)5

352


UV Data

........

........

Viomellein

1235

2031 42

223'1 60

2371 52 265'1 100 294s I 21 3231 8 371 *I 22 409s I 10

Vioxanthin

1369

2121 31

221 '1 32

233 I 27 269'1 100 306sl 13 3331 8 374'1 24

Viridamine

897

216)48

225'1 49

2631 23 373'1 100

Viridicatic acid

687

2051 17

233'1 87

244 I 53 265'1 100

Viridicatin

988

205'1 83

2101 81

223'1 100 239s 1 55 2651 14 288'1 21 2971 20 308s I 24 318'1 29 329s I 22

Viridicatumtoxin

1206

221 I47

239'1 75

251 I62 267sl 76 285'1 94 331sl 1 I 3581 5 434'1 28

Viriditoxin

1286

2161 51

221 *I 51

2371 36 263'1 91 3001 12 377'1 26

Woltmannin

946

2351 31

259 * 55

283 I 29 295'1 35

Xanthocillin X

1110

221 I 18

239' 21

2651 5 294s I 12 362'1 100

Fungal metabolite

353

TABLE 8.2. (continued) Fungal metabolite Xanthomegnin

Zearalenol

Zearalenone

RI

UV Data

........

..,.....

1110

203152

237'1 100

281129 292'1 31 337 16 403'1 19

973

1075

208~183 221 I77

237'1 94 2591 44 267'1 45 308.~115

2071 30

255129 273'1 44 2991 17 315'1 19

237'1 100

' Major of several peaks

slightly basic conditions may change some UV spectra drastically. These problems were misinterpreted by Paterson and Kemmelmeier (583), when they claimed that the small change in pH during the elution should affect the UV spectra of similar chromophores. Their comparisons of spectra taken in neutral or basic solvents taken by a stand-alone spectrophometer to spectra taken on-line by the diode array detector in acidic water/acetonitrile mixtures as occuring in gradients were simply not relevant as such spectra are known to be different in many cases (580-582). Furthermore neutral solvents or even worse very basic eluents could not be used generally because of problems with peak broadening of basic or acidic mycotoxins and corrosiveness of sodium hydroxide (pH approx. 12.4). We have made a library of the many compounds (authentic standards) listed in Table 2 and this work excellently for peak identification in the system we use. A large change system would mean that the standards should be run again and new UV spectra taken in that system. The differences between spectrataken in acidic methanoVwatergradients and acidic acetonitrile/ water gradients are very small however (Frisvad and Thrane, unpublished observations). Diode array

354

detection, especially in connection with retention index data and confirmed by TLC data, using authentic standards is a very efficient method for reliable mycotoxin analysis. After screening using such a system, more dedicated sensitive methods may developed for those mycotoxins that are considered a problem.

8.5.2.Applications of HPLC mass spectrometry HPLC mass spectrometry (HPLC-MS) have a great potential for very specific analysis but may be more difficult to apply to more broad screening-like analysis (584-

586). Five interfaces between the liquid chromatograph and the mass selective detector are of interest for mycotoxins at present: Direct inlet, thermospray/ plasmaspray, fast atom bombardment, particle beam and electrospray. Particle beam are giving the most informative spectra but are best for quite apolar molecules whereas electrospray is most suited for very polar molecules (584-586). Thermospray is in an intermediate position between those extremes, but can be used for "normal" flow rates at 1 mWmin. However the spectra from thermospray applications only contain little structural information and they are dependent of the eluent (586a). Tiebach et a/. (587-588)used a direct inlet technique to analyze aflatoxin, nivalenol and deoxynivalenol by micro HPLC-MS, using acetonitrile/water (1 :I ), flow rate 5 pl/min, as eluent and using chemical ionisation and both positive and negative ion detection. The separation of compounds was poor but the mass spectra quite informative and the authors claimed that the method could be used for foods. Thermospray have been the most widely used interface between the liquid chromatograph and the mass selective detector (e.g. 577,589-593).Usually acidified (phosphoric acid) water/ acetonitrile (577)or water/methanol isocratic runs (589), added ammoniumacetate to generate ions, on RP columns have been used. Carlson

et a/. (592)used a plasmaspray interface and a isocratic waterlacetonitrile (30:70)at 1 ml/min for prehelminthosporol. RajakylP et a/. (577) used an acidified water

acetonitrile gradient and thermospray and combinedthe HPLC-MS analysis with HPLCDAD.

One of the most interesting interfaces for LC-MS is fast atom bombardment which have been used directly on a mixture of fungal metabolites (594)or after HPLC separation. Kostiainen et a/. (595)used a water/methanol gradient and post-column addition of glycerol and obtained very informative mass spectra with glycerol adducts

355

for trichothecenes. A large number of new developments will probably be seen in the years to come in the LC-MS area of mycotoxin analysis, especially the electrospray interface (596). 8.6. CONCLUSIONS

HPLC is probably the most valuable method for mycotoxin analysis, however both selectivily, sensitivity and confirmation of identity should be considered. Based on the many applications listed above it may be concluded that the most general applications are those that involve gradient elutions using acidified water/acetonitrile or acidified waterlmethanol, especially if diode array detection and/or mass spectrometric detectors are available. In the latter case acids like acetic acid, trifluroacetic acid or other volatile acids should be used. It is also recommended to used a retention index series and to use authentic standards, and a great number of standards are now commercially available. Identifications should be confirmed by normal phase TLC if RP-HPLC is used or vice versa, rather than using a series of eluents. It is more difficult to propose a general method for individual mycotoxins in differentkinds of foods. Here one should consider all the available chemical information and design an optimally sensitive method accordingly. Knowledge of the associated mycoflora of the foods or feedstuffs may help in deciding which method should be used. For these more dedicated HPLC analysis both reversed phase, normal phase, cyano, amino etc. columns could be considered and several ion-pairing reagents, eluents etc. Also the actual extraction method and final detection method may be based on chemical and biological knowledge of the fungi, their toxins and the commodity they grow in.

356

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371 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584

585 586 586a 587 588 589 590

J.-T. Lin, A.E. Stafford, G.L. Steffens and N. Murofushi, J. Chromatogr. 543 (1991) 471. J.M. Brackett, M.D. Arguello and J.C. Schaar, J. Agric. Food Chem. 36 (1988) 762. H.I. Assil and P. Sporns, J. Agric. Food Chem. 39 (1991) 2206. X.J. Liu, X.Y. Luo and W.J. Hu, in: S. Natori, K. hashimoto and Y. Ueno (Eds.), Mycotoxins and phycotoxins '88. Elsevier, Amsterdam, 1989, p. 109. A.J. Penel and F.V. Kosikowski, J. Food Prot. 53 (1990) 321. A.D. Muir and W. Majak, Toxicol. Lett. 20 (1984) 133. A.D. Muir, W. Majak, M.A. Pass and G.S. Yost, Toxicol. Lett. 20 (1984) 137. W. Majak and R.E. McDiarrnid, Toxicol. Lett. 50 (1990) 213. R.E. Peterson, G.M. Shannon and O.L. Shotwell, J. Assoc. Off. Anal. Chem. 72 (1989) 332. J.A. Lansden, J. Assoc. Off. Anal. Chem. 67 (1984) 728. W.P. Norred, R.J. Cole, J.W. Dorner and J.A. Lansden, J. Assoc. Off.Anal. Chem. 70 (1987) 121. T. Goto, E. Shinshi, K. Tanaka and M. Manabe, Agric. Biol. Chem. 51 (1987) 2581. P. Haggblom, Appl. Environ. Microbiol. 56 (1990) 2924. G.M. Ware, C.W. Thorpe and A.E. Pohland, J. Assoc. Off. Anal. Chem. 63 (1980) 637. B. Danieli, M. Magri and D. Lavezzari, in: XXI International Dairy Congress. Vol. 1. Book 2. Moscow, USSR, 1982, p. 170. S. Moreau, A. Masset and J. Biguet, Appl. Environ. Microbiol. 37 (1979) 1059. S. Moreau, A. Combier-Lablanche and J. Biguet, Appl. Environ. Micobiol. 39 (1980) 770. S.-C. Chang, Y.-H. Wei, D.-L. Wei, Y.-Y. Chen and S.-C. Jong, Appl. Environ. Microbiol. 57 (1991) 2581. B. Danieli, B. Bianchi-Salvadori and A.V. Zambrini, Milchwissenschaft 35 (1980) 423. M. Li and F. Jia, Shipin Kexue 23 (1981) 33. G.F. Griffin, S.C. Bennett and F.S. Chu, J. Chromatogr. 280 (1983) 363 F.G. Thiel, C.J. Meyer and W.F.O. Marasas, J. Agric. Food Chem. 30 (1982) 308. E. Rajakyla, K. Leasasenako and P.J.D. Sakkers, J. Chromatogr. 384 (1987) 391. R.R.M. Paterson and C. Kemmelmeier, J. Chromatogr. 483 (1989) 153. M. Bogusz, J. Chromatogr. 387 (1987) 401. C. Reichardt, Solvent and solvent effects in organic Chemistry, VCH, Weinheim, 1988. A.I. Scott, Interpretation of the ultraviolet spectra of natural products, Pergamon Press, Oxford, 1964. M. Jaquet and P. Laszlo, in: A. Weissberger (Ed.), Techniques of chemistry, Vol. VIII, Solutions and solubilities, John Wiley and Sons, New York, 1975. R.R.M. Paterson and C. Kemmelmeier, J. Chromatogr. 511 (1990) 195. A.L. Yergey, C.G. Edmonds, I.A.S. Lewis and M.L. Vestal, Liquid chromatographylmass spectrometry - Techniques and applications, Plenum Press, New York, 1990. W.M.A. Niessen and J. van der Greef, Liquid chromatography -mass spectrometry Principles and applications, Marcel Dekker, New York, 1990. M.A. Brown (Ed.), Liquid chromatographylmass spectrometry - Applications in agricultural, pharmaceutical and environmental chemistry, ACS Symposium Series 420, American Chemical Society, Washington D.C., 1990. E.R.J. Wils and A.G. Hulst, Fres. 2. Anal. Chem. 342 (1992) 749. R. Tiebach, W. Blaas and M. Kellert, J. Chromatogr. 323 (1985) 121. R. Tiebach, W. Blaas, M. Kellert, S. Steinmeyer and R. Weber, J. Chromatogr. 318 (1985) 103. T. Krishnamurthy, D.J. Beck and R.K. Isensee, Biomed. Environ. Mass Spetrom. 18 (1989) 287. R.D. Voyksner, W.M. Hagler and S.P. Swanson, J. Chromatogr. 394 (1987) 183.

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R.D. Voyksner, W.M. Hagler, K. Tyczkowska and C.A. Haney, J. High Resolution Chromatogr. Chromatogr. Commun. 8 (1985) 119. H. Carlson, P. Nillson, H.-B. Jansson and G. Odham, J. Microbiol. Methods 13 (1991) 259. T. Krishnamurthy, D.J. Beck, R.K. lsensee and B.B. Jarvis, J. Chromatogr. 469 (1989) 209. J.R.J. Pare, R. Greenhalgh, P. Lafontaine and J.W. Apsimon, Anal Chem. 57 (1985) 1472. R. Kostiainen, K. Matsuura and K. Nojima, J. Chromatogr. 538 (1991) 323. W.P. Korfmacher, M.P. Chiarelli, J.O. Lay, J. Bloom and M. Holcomb, Rapid Comm. Mass Spetrometry 5 (1991) 463.

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Chapter 9 GAS CHROMATOGRAPHY OF MYCOTOXINS PETER M. SCOTT 9.1. INTRODUCTION

Gas chromatography (GC) may be used as an analytical technique for mycotoxins that can be volatilized in a heated GC column or possess at least one functional group allowing conversion of the mycotoxin into a volatile derivative. In practice, the reactive group is in almost all cases a hydroxyl group and the derivatives formed are usually trimethylsilyl (TMS) ethers or heptafluorobutyryl (HFB), pentafluoropropionyl (PFP) or trifluoroacetyl (TFA) esters. The first mycotoxin (apart from oxalic acid) to be analysed by GC was zearalenone (F-2) as reported in 1967 by Mirocha et al. (1), followed by patulin, mycophenolic acid, griseofulvin, koj ic acid, terreic acid and terrein in 1970 (2-5) and in 1971 by certain ergoline alkaloids, sterigmatocystin, alternariol and related Alternaria toxins, penicillic acid and various trichothecenes (610). Since then the application of GC to mycotoxin analysis has grown tremendously, mainly because of the interest in trichothecenes, butthere appears to be only one review specifically devoted to this topic (11). Reviews on chromatographic analysis of mycotoxins in general have, of course, included GC (12-15). GC has a major advantage over other forms of chromatography, liquid chromatography (LC)-mass spectrometry (MS) notwithstanding, in that it can be readily coupled to a mass spectrometer to enable more specific detection and determination of mycotoxins, as well as their identification. Vesonder and Rohwedder (16) have reviewed this specialized technique of GC-MS and its application to mycotoxin analysis. Flame ionization detection (FID) and electron capture detection (ECD) are the main other techniques used for GC of mycotoxins. The trichothecenes are the only mycotoxins for which GC is widely used. There is one Official Method of the Association of Official Analytical Chemists (AOAC) that employs GC for mycotoxin determination, viz. for deoxynivalenol (DON) in wheat. 9.2. TRICHOTHECENES 9.2.1 Introduction

The total number of trichothecenes isolated from natural sources (mainly fungal) was 148 at last count (17), comprising 83 non-macrocyclic and 65 macrocyclic compounds. Only a few of these, in particular DON, nivalenol (NIV), T-2 toxin (T-2), HT-2 toxin (HT2) and diacetoxyscirpenol (DAS) (Fig. 9.1) have been detected so far as naturally occurring contaminants in foodstuffs (18).

374

CH20H

I1

I

Fig. 9.1. Examples of trichothecenes. Type A: diacetoxyscirpenol ( I , R'=H; R2=OCOCH,) , T-2 toxin (I,R'= (CH,) ,CHCH,COO : R2=OCOCH,) , HT-2 toxin (I,R1=(CHS)2CHCH2COO;R2=OH). Type B: nivalenol (11, R=OH) , deoxynivalenol (11, R=H)

.

GC is the most commonly used means of separating and identifying trichothecenes, not only in extracts of foodstuffs but also in biological fluids and tissues. These applications will be discussed later (see Sections 9.2.3-9.2.6). Several types of internal standards (e.9. deuterated DAS and HT-2, methoxychlor, 7hydroxy DAS, 16-hydroxyverrucarol, isoT-2) have been used in GC determinations of trichothecenes (19-25, inter alia) ; they have been added to the sample before extraction, before derivatization or after derivatization. Derivatization and detection Drocedures for trichothecenes 9.2.2.1 No derivatization. It is normal to derivatize the hydroxyl group(s) of trichothecenes for GC in order to attain the volatility and sensitivity needed for trace analysis. However, several workers have omitted the derivatization step. Stahr et al. (26,27) demonstrated that GC of underivatized T-2, DAS and other trichothecenes was possible, with a sensitivity of 10-100 ng on a packed column using FID. Bijl et al. (28) also detected ng amounts of T-2 and DAS, as well as trichothecin (which has no free hydroxyl group), by capillary GC with FID. D'Agostino et al. (29) performed capillary GC on underivatized verrucarol, DON, DAS, T-2, HT-2 and T-2 triol using both FID and ammonia chemical ionization (CI) MS detection. Co-injection of standards with an acetone plug improved peak shape. T-2, HT-2 and T-2 triol did not separate on DB-1 but there were slight differences in retention time on a DB-5 column. MS detection, particularly negative ion (NI) CI (30-33), has in fact been the preferred means of detection for GC of underivatized trichothecenes. Detection limits for 9 trichothecenes detected by oxygen NICI capillary GC-MS ranged from 50 pg for DON, monitored at masses of 284 and 295, to 9.2.2

3 75

375 pg for neosolaniol (NS) and T-2 trio1 at a signal-to-noise ratio of 1O:l (32). Lau et al. (34) determined eight underivatized trichothecenes by capillary column GC-CI tandem mass spectrometry (MS/MS) with detection limits in the range 10-67 pg except for HT-2 (305 pg): HT-2 and T-2 did not separate on the 15 m DB-5 capillary column used. GC of underivatized trichothecenes has been particularly useful for their characterization in extracts of fungal cultures. Capillary columns have been employed, with detection by FID or MS (operated in the electron impact (EI) mode) (35-38). The GC-MS technique was most useful for identification of new trichothecenes. However, Plattner et al. (39) noted losses and reproducibility problems with underivatized T-2 and NS, but not with DAS, when these trichothecenes were introduced into the mass spectrometer by GC for MS/MS analysis. On-column injection of underivatized DON caused up to 8% degradation, principally to isoDON, when assay was made by capillary GC-MS (35). As previously mentioned, not all trichothecenes possess derivatizable hydroxyl groups. Thus when GC of acetyl T-2 in the presence of trichothecenes that had been trimethylsilylated (40,41), heptafluorobutyrylated (41) or trifluoroacetylated (42) was reported, it was of course not being chromatographed as a derivative. Similary, triacetoxyscirpenol, trichodermin, crotocin and trichothecin were necessarily chromatographed underivatized, with FID detection, in the presence of TMS derivatives of trichothecenes possessing free hydroxyl groups (10,43). Trichothecin, together with its de-esterified analog trichothecolone, has been chromatographed in the absence of derivatizing agent and detected by FID (28,44,45). Ishii et al. (46) measured trichothecin in wheat by GC-MS (EI mode) using ions at m/z 246, 203 and 175. 9.2.2.2 Trimethylsilylation. The very first report on GC of trichothecenes was by Ikediobi et al. (10) who formed TMS derivatives of a number of trichothecenes with derivatizable hydroxyl groups, plus four that would not have derivatized (see section 9.2.2.1) and were not affected by the silylation reagents used. These reagents were (i) hexamethyldisilazane (HMDS) trimethylchlorosilane(TMCS)-pyrid~ne(2+1+7) and (ii) N,Obis(trimethylsily1)acetamide (BSA) - pyridine (4+1), used at room or refrigerator temperature. The second reagent was preferred as the derivatives were stable for at least two weeks at room temperature and much longer at -2OOC. Low pg quantities of trichothecenes were detected by FID. Subsequently, TMS derivatives have been the ones most frequently used for GC of trichothecenes (particularly for type B trichothecenes possessing 7-hydroxyl and conjugated 8-carboxyl groups) (Fig. 9.1). Various reagent mixtures have been employed. Type A trichothecenes such as DAS and T-2 are readily derivatized, even with BSA alone (47.48). However, Tanaka et al. (49) showed

376

that HMDS-TMCS-pyridine gave two peaks with diacetyl NIV and fusarenone-X (FX), both type B trichothecenes, after 0.25 or 6 hours at room temperature. Nakahara and Tatsuno (50) trimethylsilylated NIV, another type B trichothecene, with a BSA-TMCS (1+1) reagent and also obtained two peaks due to incomplete derivatization after 20 minutes at 75'C. None of these type B trichothecenes had been tested by Ikediobi et al. (10). Bis(trimethylsily1)trifluoroacetamide (BSTFA), a reagent used by some researchers (51,52), did not give any fully derivatized DON, nor did a mixture with TMCS (3+2), even after heating at 100°C for 30 minutes (53). Variable results with BSA-TMCS (5+1) (Tri-Silo BT) and BSTFA-TMCS (5+1) for DON and NIV were also reported by Kientz and Verweij (54). Gilbert et al. (53) theorized that in type B trichothecenes it is the 7-hydroxyl group that is difficult to derivatize because of hydrogen bonding to the adjacent 8-carbonyl group. In support of this, tris-TMS NIV was shown to contain an unreacted 7-hydroxyl group based on nuclear magnetic spectroscopic evidence (50). However, reaction of DON with BSTFA and BSTFA-TMCS (4+1) formed two bis-TMS derivatives, identified by GC-MS, at least one of which must logically have had a derivatized 7-hydroxyl group (54). Tanaka et al. (49) were the first to show that trimethylsilylimidazole (TMSI) was a necessary ingredient for complete trimethylsilylation of type B trichothecenes; they chose TMSI-TMCS-pyridine (5+1+45) as reagent. A mixture of TMSI, TMCS and a suitable solvent has been the preferred reagent of a number of workers subsequently (55-65). Gilbert et al. (53) studied the optimum conditions for forming TMS derivatives of trichothecenes. Whereas TMSI alone brought about complete derivatization of DON, it was not a convenient reagent to use, causing damage to the capillary column unless removed by washing the reaction mixture with water (56). Regisil 323 (BSTFA-TMCS-TMSI, 3+2+3) gave 100% tris-TMS DON at room temperature (53). Tri-Sil. TBT is another commercial formulation, consisting of TMSI-BSA-TMCS (3+3+2), that readily derivatizes type B and other trichothecenes (66-75): Ohta et al. (76) used a reagent ratio of (5+5+1). Again it is preferable to wash the reaction mixture with an aqueous solution before GC (72). Trichothecolone, possessing only a 4-hydroxyl group, and T-2 have been trimethylsilylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) (77,78). TMS derivatization of macrocyclic trichothecenes - verrucarin A, roridins A and E, satratoxins G and H and baccharin B5 - was accomplished with BSTFA (at 90°C) by Rosen et al. (79). Except for roridin E and satratoxin G, they could be detected in the 1-10 ng range by GC-MS with selected ion monitoring (SIM) using a short (1 m) capillary column. A more general approach to detection of macrocyclic trichothecenes is alkaline hydrolysis to the parent alcohol verrucarol which is treated with BSTFA or Tri-Silo BT to form the TMS derivative and determined by GC-FID or GC-MS (80-82) (see also Section 9.2.2.3).

377

TMS ethers of trichothecenes are generally stable once formed (10,58,60). They can even be purified by thin layer chromatography (TLC) (83). TMS ethers of trichothecenes are usually detected by FID, ECD or MS (SIM) Kuroda et al. (60) were the first to show that ECD is more sensitive than FID for determining TMS ethers of both type A and B trichothecenes, but particularly so for the latter because of their conjugated 8-carbonyl group. A s little as 2-4 pg of DON, NIV or FX separated on a packed column could be detected by ECD, compared to 5-10 ng with FID: corresponding detectable quantities for the type A trichothecenes DAS, NS and T-2 were 200-400 pg by ECD and 30-60 ng by FID. In other laboratories, the superiority of ECD over FID for detecting trichothecene TMS derivatives (59) and the increased ECD sensitivity of TMS derivatives of type B compared to type A trichothecenes have been confirmed (59,84,85). Detection and determination of TMS derivatives of trichothecenes by MS, particularly after capillary GC, is the favoured technique if the instrumentation is available (86). The specificity of MS gives reliable identifications in grains, biological fluids and other matrices and it has been often used to confirm results obtained with other detection techniques. The EI mode is commonly employed with single or multiple ion monitoring (19,53,55,57,75,78,84,87-97). Examples of ions that may be monitored are m/z 512, 497, and 422 for DON TMS ether: 600, 510, 482, and 379 for NIV TMS ether: 480 for FX TMS ether: 436 for T-2 TMS ether: and 496, 481 and 406 for the TMS ether of the DON metabolite DOM-1. GC-MS of macrocyclic trichothecene TMS ethers has been previously mentioned (79). Full EI mass spectra of trichothecene TMS ethers have been published in several of these papers. The positive or negative ion CI MS modes have also been used for GC analysis of trichothecene TMS ethers (69,87,98-101). Characteristic ions are 513, 497 and 423 for DON TMS ether and 601, 585, 511 and 289 for NIV TMS ether in the positive ion (PI) CI mode: and 512, 305 and 297 for DON TMS ether and 600, 303, 298 and 297 for NIV TMS ether in the NICI mode (87,98,99,101). GC-tandem MS of T-2 TMS ether was carried out by Desjardins et al. (102). TMS derivatives of DON and NIV have been also identified by matrix isolation/Fourier transform infrared spectrometric analysis following capillary GC (103). Separation of TMS derivatives of several trichothecenes of both A and B types is readily achieved on a packed or capillary GC column (preferablytemperatureprogrammed) (19,49,51,56,59,60,84,85). Some variation of elution order for five type A and five type B trichothecene TMS derivatives with column polarity was noted by Scott and Kanhere (85) in a study using six different fused silica capillary columns. Generally, complete resolution was obtained, with a few exceptions: e.g. on DB-1701, 3- and 15-acetyl DON were not separated and FX, NIV and 15-monoacetoxyscirpenol (MAS) formed an almost unresolved peak. Separation on SE-30 is shown in Fig. 9.2.

.

378

SE- 30 TMS 3-ADON

0'

I

I

10

I

I

I

20

I

I

30

RETENTION TIME(MIN)

Fig. 9.2. Ca illary GC-ECD of trichothecene TMS ethers on SE-30; ca. 30 p g eaci of DON, 3-acetyl DON (3-ADON), 15-acetyl DON (15ADON) X and NIV and 1000 pg each of 15-MASI DAS, NS, T-2 and HT-2 injecked (85). 9.2.2.3 Hevtafluorobutvrvlation. In order to utilize the sensitivity of ECD, heptafluorobutyrate (HFB) derivatives are commonly used for determination of trichothecenes. The first application was with T-2 and DAS, which react very readily at room temperature with heptafluorobutyrylimidazole (HFBI) (21,104). Later, DON HFB was formed with this reagent by heating at 60'C for one hour (105) Other trichothecenes that have been derivatized with HFBI include HT-2, verrucarol, 4- and 15-acetoxyscirpendiol (monoacetoxyscirpenol, MAS), NS, NIV and FX (22,23,72,105-111). Reaction with type A trichothecenes proceeds at room temperature as indicated above for T-2 and DAS, while temperatures used for DON and other type B trichothecenes have ranged from 45' (112) to 110' (23). Luo et al. (108) found that l0O'C caused loss of NIV HFB. Reaction mixtures are washed with aqueous sodium bicarbonate (which may be followed by a water wash) , water (113) or phosphate buffer (pH 7.0)

.

379 (72) in order to remove excess reagent; Muszkat et al. (114) noted less interference after two washes with sodium bicarbonate solution. Heptafluorobutyrylation of trichothecenes is also carried out using heptafluorobutyric anhydride (HFBA) with 4-dimethylaminopyridine (4-DMAP) or trimethylamine as catalysts dissolved in an organic solvent (24,25,115). Again the reaction mixture is usually washed with aqueous sodium bicarbonate solution. Faster derivatization of DON at 6OoC is achieved with HFBA/4-DMAP than with HFBI (115). HT-2 and a demethylated analogue have been derivatized with HFBA at 6OoC without a catalyst (116). No catalyst was used by Muiioz et al. (117) to derivatize DON either although the extent of heptafluorobutyrylation was not indicated. Partial derivatization of DON and NIV could be observed in a recent report on the use of a polymer-bound 4-(N-benzyl-N-methylamino)pyridine solid catalyst with HFBA: DON bis-HFB and NIV tris-HFB were identified by GC-MS (118). The unreacted hydroxyl group was presumed to be the 7-hydroxyl group by analogy with acetylation studies on DON and 3-acetyl DON (119,120) and the similar slow heptafluorobutyrylation of 3,15-diacetyl DON (118). HFB derivatives of trichothecenes are determined by ECD or MS (SIM) Low picogram quantities can be detected by both techniques, even on a packed column. Using ECD, sensitivities are generally worse for derivatized DAS and T-2, which are later eluting and only contain one HFB grouping per molecule, than for derivatized DON and NIV, which elute early in the chromatograms and possess three and four HFB groups, respectively (85). Heptafluorobutyrylation is also advantageous for MS detection of trichothecenes because the high molecular weight of the HFB derivative offers greater specificity for GC-MS(S1M) than the TMS ether. For example, DON tris-HFB has a molecular ion at m/z 884 in the EI mass spectrum and the limit of detection is 1-3 pg on a packed column (105). Other packed column detection limits for GC-MS have been reported as 13-80 pg for DAS and HT-2 but about 1 ng for T-2 (monitored at m/z 602) (105). EI mass spectra of HFB derivatives of 12 trichothecenes have been published by Krishnamurthy et al. (23) , in addition to the PI- and NICI mass spectra. The latter mode of ionization gives very high sensitivity with the electronegative HFB groups and is about 5000 times more sensitive than PIC1 for DON HFB derivative (0.1 pg was measurable) (121). Minimum amounts of HFB derivatives detectable by capillary GC-NICI MS as reported by Krishnamurthy et al. (23) ranged from 0.1 pg (DON) to 2.0 pg (T-2) and confirmable limits were 1-5 pg using five or six ions. Macrocyclic trichothecenes (see also Section 9.2.2.2) were analyzed, after alkaline hydrolysis, by capillary GCNICI MS of the resulting verrucarols as their HFB derivatives (201000 pg) (107). Detailed studies on capillary GC-NICI tandem MS of HFB derivatives of 7 trichothecenes were reported by Kostiainen et al. (122) and Kostiainen and Rizzo (24); high selectivity and

.

380

sensitivity down to 0.1-2 pg were achieved by this MS detection technique also. HFB derivatives of trichothecenes are generally stable for several days, with the exception of FX (23), NS (123) and NIV (110); deterioration of HT-2 HFB was mentioned in an earlier publication (105) but was not a problem according to others (23, 110). Increased stability of DON HFB derivative was observed if silylated glassware was used (124). Double peaks with NIV and DON HFB have been encountered under certain conditions (22,72,110,111) as well as a shoulder on the DON HFB peak (110). The formation of two isomeric tetrakis-HFB derivatives of NIV has been shovn by GC-MS (72,125). Separation of all of several trichothecene HFB derivatives may not be complete on a packed column (126) but generally is on a capillary column (85,108,127), although 3- and 15-acetyl DON derivatives do not resolve on some phases (85). Elution order can vary according to column polarity; separation on DB-1701 is shown in Fig. 9.3 (85).

25 DON

DB-1701 HFB

MAS 15-ADON 3.ADON

HT.2 I

20

x

15

cl

z

u)

g 2a a

0 UJ

10 w

'I

I

0

I

10

I

I

20

I

I

1

30

TIME (MIN)

Fig 9.3. Capillar GC-ECD of trichothecene HFB derivatives on DB1701; 20 p of eacg injected, except for DAS and T-2 (40 pg) (85). Peak markel MAS is for 15-MAS.

38 1

9.2.2.4 PentafluoroDroDionvl derivatives. Analogous to HFB derivatives, pentafluoropropionyl (PFP) derivatives of trichothecenes are formed with pentafluoropropionic anhydride and triethylamine or 4-DMAP (123,128) or with pentafluoropropionylimidazole (129). They have the advantage over HFB derivatives for MS detection using instruments with an upper mass limit of about m/z 1000 that the molecular ions are lower (

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