Lipid Analysis in Oils and :Fats

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Lipid Analysis in Oils and Fats

Edited by

R. J. Hamilton Professor of Organic Chemistry

School of Pharmacy and Chemistry Liverpool John Moores University

Liverpool UK

m BLACKIE ACADEMIC & PROFESSIONAL

An Imprint of Chapman & Hall

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Published by Blackie Academic & Professional, an imprint of Thomson Science, 2-6 Boundary Row, London SEt 8HN

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Thomson Science, Suite 750, 400 Market Street, Philadelphia, PA 19106, USA

Thomson Science, Pappelallee 3, 69469 Weinheim, Germany

First edition 1998

© 1998 Chapman & Hall Softcover reprint of the hardcover 1 st edition

Thomson Science is a division of International Thomson Publishing I ® P ®

Typeset in 10/12 pt Times by Pure Tech India Ltd, Pondicherry

ISBN-13: 978-1-4612-8432-1

DOl: 10.1007/978-1-4613-1131-7 e-ISBN-13: 978-1-4613-1131-7

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A catalogue record for this book is available from the British Library

Library of Congress Catalog Card Number: 97-74428

§Printed on acid-free text paper, manufactured in accordance with ANSIINISO Z39.48-1992 (Permanence of Paper).

Contents

List of contributors x

Preface xii

List of abbreviations xiii

Introduction xix

1 Lipid analysis using thin-layer chromatography and the Iatroscan 1 N. C. SHANTHA and G. E. NAPOLITANO

1.1 Introduction 1 1.2 Principles of thin-layer chromatography 2

1.2.1 Stationary phases and their applications 2 1.2.2 Solvent systems and developments 7 1.2.3 Detection methods 11 1.2.4 Quantitation 16

1.3 Applications of thin-layer chromatography 17 1.4 Thin-layer chromatography-flame ionization detection for lipid analysis 19

1.4.1 Equipment 19 1.4.2 Optimization 20 1.4.3 Comparison of TLC-FID system with gas chromatography 22 1.4.4 Recent applications of TLC-FID Iatroscan system 23

1.5 Conclusions 28 Acknowledgements 28 References 28

2 Characterization of lipids by supercritical fluid chromatograpahy and supercritical fluid extraction 34 L. G. BLOMBERG, M. DEMIR BUKER and M. ANDERSSON

2.1 Introduction 2.2 Supercritical fluid chromatography

2.2.1 General aspects on the properties of supercritical media 2.2.2 Speed of analysis 2.2.3 Packed column diameter 2.2.4 Mobile phases for supercritical fluid chromatography 2.2.5 Stationary phases for supercritical fluid chromatography 2.2.6 Instrumental 2.2.7 Gradients 2.2.8 Widening the scope of supercritical fluid chromatography 2.2.9 Comparison of supercritical fluid chromatography with other

separation techniques for lipid characterization

34 35 35 37 40 40 42 45 47 49

49

vi

3

4

2.3 Supercritical fluid extraction 2.3.1 Analytical applications

CONTENTS

2.3.2 Semi-preparative applications 2.4 Reactions in supercritical media 2.5 Conclusions Acknowledgements References

Static heads pace gas chromatography in the analysis of oils and fats F. ULBERTH

3.1 Introduction 3.2 Theoretical background 3.3 Sampling systems 3.4 Applications of headspace gas chromatography in lipid chemistry

3.4.1 Volatile fatty acids 3.4.2 Oxidative deterioration of fats and oils 3.4.3 Flavour research 3.4.4 Residues of contaminants 3.4.5 Miscellaneous applications

3.5 Conclusions References

Multinuclear high-resolution nuclear magnetic resonance spectroscopy B. W. K. DIEHL

4.1 Introduction 4.2 The instrument 4.3 Principles 4.4 Spectra 4.5 Response 4.6 Reproducibility 4.7 Calibration 4.8 Applications

4.8.1 IH NMR spectroscopy of fatty oils 4.8.2 l3C NMR spectroscopy 4.8.3 31 P NMR spectroscopy of phospholipids 4.8.4 Ultra-high-resolution 31p NMR spectroscopy 4.8.5 Plasmalogens, O-alkyl-phospholipids and O-alkenyl-phospholipids

4.9 Quantitative determination of phospholipids: validation of the 31p NMR method 4.9.1 Test A: reproducibility 4.9.2 Test B: instrument precision 4.9.3 Test C: repeatability 4.9.4 Tests D-I: robustness 4.9.5 Validation summary

4.10 Instrument details Acknowledgements References

50 50 53 54 55 55 55

59

59 60 63 66 67 68 80 82 83 84 84

87

87 88 88 88 89 89 90 90 90 93

117 119 122

126 129 129 129 131 133 133 134 134

CONTENTS

5 Cyclic fatty acids: qualitative and quantitative analysis G. DOBSON

Vll

136

5.1 Introduction 136 5.2 Naturally occurring cyclic fatty acids 139

5.2.1 Cyclopentenyl fatty acids 139 5.2.2 Cyclopropane fatty acids 141 5.2.3 Cyclopropene fatty acids 147

5.3 Cyclic fatty acids formed in heated vegetable oils 152 5.3.1 Structural analysis of monomeric cyclic fatty acids 153 5.3.2 Quantification of monomeric cyclic fatty acids 171 5.3.3 Incorporation of monomeric cyclic fatty acids into biological material 175

5.4 Summary 176 References 176

6 Mass spectrometry of complex lipids A. KUKSIS

6.1 Introduction 6.2 Equipment and principles of soft ionization mass spectrometry

6.2.1 Direct probe mass spectrometry 6.2.2 Gas chromatography/mass spectrometry 6.2.3 Liquid chromatography/mass spectrometry

6.3 Applications 6.3.1 Fatty acids 6.3.2 Sterols and steryl esters 6.3.3 Neutral giycerolipids 6.3.4 Neutral giycerophospholipids 6.3.5 Acidic glycerophospholipids 6.3.6 Oxygenated glycerophospholipids 6.3.7 Glycosylated glycerophospholipids 6.3.8 Sphingomyelins, sphingolipids and gangliosides 6.3.9 Lipid A and other lipopolysaccharides

6.4 Summary and conclusions Acknowledgements References

7 Chromatography of food irradiation markers J.-T. MORSEL

7.1 Introduction 7.2 Chemical changes in irradiated foods

7.2.1 Lipids 7.2.2 Carbohydrates 7.2.3 Proteins

7.3 Determination of marker substances by gas chromatography 7.3.1 Determination of hydrocarbons 7.3.2 Determination of cyclobutanones 7.3.3 Determination of dose and method limits

7.4 Determination of marker substances by high-pressure liquid chromatography

7.5 Conclusions References

181

181 181 182 182 183 184 184 192 195 206 215 223 227 230 236 238 239 239

250

250 251 251 253 254 255 255 257 259

260 264 264

viii CONTENTS

8 Development of purity criteria for edible vegetable oils J. B. ROSSELL

8.1 Introduction 8.2 Materials and methods 8.3 Results and discussion

8.3.1 Fatty acid analyses 8.3.2 Other traditional analyses 8.3.3 Problems of maize oil analysis 8.3.4 Stable carbon isotope ratio analysis

Acknowledgements References

9 Analysis of intact polar lipids by high-pressure liquid chromatography-mass spectrometry/tandem mass spectrometry

265

265 266 270 270 277 282 283 288 288

with use of thermospray or atmospheric pressure ionization 290 A. A. KARLSSON

9.1 Introduction 9.2 Theory

9.2.1 Polar lipids 9.2.2 Liquid chromatography 9.2.3 Mass spectrometry 9.2.4 Liquid chromatography-mass spectrometry

9.3 Applications 9.3.1 Thermospray and plasma spray 9.3.2 Electrospray - atmospheric pressure chemical ionization

9.4 Practical experiences of analysis of polar lipids by means of liquid chromatography with (tandem) mass spectrometry 9.4.1 Polyetheretherketone compared with stainless-steel tubing 9.4.2 Column packing 9.4.3 Solvent quality 9.4.4 Adduct ions 9.4.5 Wasting

9.5 Summary References

10 The exploitation of chemometric methods in the analysis of spectroscopic data: application to olive oils A. JONES, A. D. SHAW, G. J. SALTER, G. BIANCHI and D. B. KELL

10.1 Introduction 10.2 Olive oil

10.2.1 Economics 10.2.2 Chemistry 10.2.3 Health aspects 10.2.4 Analysis

10.3 Data acquisition methods 10.3.1 Nuclear magnetic resonance 10.3.2 Pyrolysis mass spectrometry

10.4 Multivariate methods 10.4.1 Principal components analysis

290 291 291 292 294 295 296 296 301

311 312 312 312 313 313 313 314

317

317 318 318 318 320 322 326 326 328 335 335

CONTENTS U

10.4.2 Predictive models 339 10.4.3 Multiple linear regression 340 10.4.4 Ridge regression 342 10.4.5 Principal components regression 342 10.4.6 Latent variables 343 10.4.7 Validation 345 10.4.8 Partial least squares regression 350 10.4.9 Artificial neural networks 353 10.4.10 Chemometrics 357 10.4.11 Variable selection 358 10.4.12 Exploitation of multivariate spectroscopies in the identification

of the geographical origin of olive oils 363 10.5 Concluding remarks and future prospects 367 Acknowledgements 368 References 368

Index 377

Contributors

M. Andersson Department of Analytical Chemistry, Stockholm University, Arrhenius Laboratories for Natural Sciences, S-106G-91, Stockholm, Sweden

G. Bianchi Istituto Sperimentale per la Elaiotecnica, Contrada Fonte Umano, Cittit St. Angelo, Pescara, Italy

L. G. Blomberg Department of Chemistry, Karlstad University, S-651 88 Karlstad, Sweden

M. Demirbiiker Astra AB, S-151 85 S6dertalje, Sweden

B. W. K. Diehl Spectral Service GmbH, Laboratorium fUr Auftrags­analytik, Postfach 301186, D 50781, Koln, Germany

G. Dobson Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

R. J. Hamilton 10 Norris Way, Formby, Merseyside L37 8DB, UK

A. Jones Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DA, UK

A. A. Karlsson Nycomed Innovation AB, Ideon-Malmo, S-205 12 Malmo, Sweden

D. B. Ken

A. Kuksis

J.-T. Morsel

and Department of Chemical Ecology and Ecotoxicology, Lund University, S-223 62 Lund, Sweden

Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DA, UK

Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, 112 College Street, Toronto, Ontario, Canada M5G IL6

Technische Universitat Berlin, Institut fUr Lebensmittel­chemie, Gustav-Meyer Allee 25, 13355 Berlin, Germany

CONTRIBUTORS xi

G. E. Napolitano 1005 Brantley Drive, Knoxville, TN 37923-1709, USA

J. B. Rossell Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey KY22 7RY, UK

G. J. Salter Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DA, UK

N. C. Shantha Nestle Research and Development Center, PO Box 4002, 809 Collins Avenue, Marysville, OH 43040-4002, USA

A. D. Shaw Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DA, UK

F. Ulberth Department of Dairy Research and Bacteriology, Agri­cultural University, Gregor Mendel-Strasse 33, A-1180 Vienna, Austria

Preface

This book has been written to ensure that it will be of benefit to industrial analysts. Most chapters explain some of the relevant theory as well as give some historical references to place the technique in its proper context. In addition the book should appeal to academic scientists who require a good source of applications and a good set of references. Since lipids have many uses the appeal of the book will extend from the food industry to the pharmaceutical industry.

Acknowledgement

R. Hamilton Formby

June 1997

I would wish to acknowledge the considerable help and encouragement from my wife Shiela.

Abbreviations

The following are the abbreviations used within this book and do not necessarily represent convention or internationally accepted abbreviations.

AAPH Ac AchE ADC AI ALD AMPL amu AMVN ANN AOCS APCI APE API ARA ASG ASMS ATP BE CAD CBC CBO CCD CE CFAM CI CID CL Cn:m CR d.c. DCB DECB DGB DHA

2,2'-azobis (2-aminopropane )dihydrochloride acetyl acetylcholinesterase analog digital converter artificial intelligence aldehyde acetone mobile polar lipids atomic mass unit 2,2' -azo bis-2,4-dimethy lvaleronitrile artificial neural network American Oil Chemists' Society atmospheric pressure chemical ionization N-acyl-phosphatidylethanolamine atmospheric pressure ionization arachidonic acid acyl-sitosterylglyceride American Society for Mass Spectrometry adenosine triphosphate backward elimination collisionally activated dissociation cerebroside 13 sulphate Certified Brands of Origin (applied to Italian virgin olive oils) charge-coupled device capillary electrophoresis cyclic fatty acid monomer chemical ionization collision-induced dissociation cardiolipin Hydrocarbon with n carbon atoms and m double bonds continuum regression direct current 2-dodecylcyclobutanone 2-tetradecenylcyclobutanone digalactosyl-diacyl-glyceride 4,7, 10,13,16, 19-docosahexaenoic acid

xiv

DLCL DCI DG DGDG DGPP DGTS DHET DMOX DMSO DNPH DNPU DOC

DOP

DPG ECA ECL EDTA EET EI ELSD J.L-ELSD EPA ES FA FAB FAC FAME FAO FD FFA FFAP FID FOSFA FS FT FTID FTIR FT-NMR GalCer GC GL

ABBREVIA TIONS

dilysocardiolipin direct chemical ionization diacylglycerol digalactosyldi( acyl)glyceride diacylglycerol pyrophosphate diacylglyceryl-N,N,N,N-trimethylhomoserine dihydroxyeicosatrienoic acid dimethyloxazoline dimethylsulphoxide dinitrophenylhydrazone dinitrophenylurethane Denominazione di Origine Controllata (Controlled Denomina­tion of Origin, applied to Italian virgin olive oils; see also CBO) Denominazione di Origine Proteggetta (Protected Denomina­tion of Production; applied to Italian agricultural food pro­ducts) diphosphatidylglycerol equivalent carbon atom equivalent chain length eth ylenediaminetetraaceta te epoxyeicosatrienoic acid electron-impact ionization evaporative light-scattering detector miniaturized evaporative light-scattering detector 5,8,11,14, l7-eicosopentaenoic acid electrospray fatty acid fast atom bombardment fatty acid composition fatty acid methyl esters (UN) Food and Agriculture Organisation field desorption free fatty acid free fatty acid phase flame ionization detection Federation of Oils, Fats and Seed Associations Ltd. forward selection Fourier transform flame thermionic ionization detector Fourier transform infra-red Fourier transform nuclear magnetic resonance galactosyl ceramide gas chromatography glycolipid

GPC GPE GPI GPL GPS HAc HDL HETE HODE HPLC

HPTLC HS HSGC IAEA i.d. IFR ILPS IR ISF IV LC LCB LDL LPA LPC LPE LOD LOOH LOS LOQ LPS LSI LTB4 LTC4 LTD4 LTE4 MAFF Me ME MG MGDG MHE MI

ABBREVIA TIONS

glycerophosphatidylcholine glycerophosphoethanolamine glycerophosphatidylinositol or glycerophosphoinositol glycerophospholipid glycerophosphoserine acetic acid high-density lipoprotein hydroxyeicosatetraenoic acid hydroxy octadecadienoic acid

xv

high-pressure liquid chromatography (also referred to as high­performance liquid chromatography) high-performance thin-layer chromatography headspace headspace gas chromatography International Atomic Energy Association inner diameter Institute of Food Research International Lecithin and Phospholipid Society infra-red International Society for Fat Research iodine value liquid chromatography long-chain base low-density lipoprotein lyso-phosphatidic acid lyso-phosphatidylcholine lyso-phosphatidylethanolamine limit of detection linoleic acid hydroperoxide lipooligosaccharide limit of quantification lipopolysaccharide liquid secondary ion leukotriene B4 leukotriene C4 leukotriene D4 leukotriene E4 (UK) Ministry of Agriculture, Fisheries and Food methyl group methyl ester monoacylglycerol monogalactosyldiacylglyceride multiple headspace extraction matrix ionization

xvi

MIKE MLA MLCL MLR MRM MS MSD MSE MS-MS MUFA MW m/z NI NICI NIPALS OPLC OPTLC OTMS PA PAF PC PCA PCR PDB PE PFB PFBO PG PGD2 PGEn

PGF" PGF I "

PGF2 PGF2" PGH2 PI PIP PIP-2 PL PLS ppb ppm PS PSP

ABBREVIA TIONS

mass-analysed ion kinetic energy monophosphoryl lipid A monolysocardiolipin multiple linear regression multiple reaction mode mass spectrometry mass selective detector mean square error of prediction tandem mass spectrometry monounsaturated fatty acid molecular weight mass/charge negative ion negative ion chemical ionization non-linear iterative partial least squares over-pressure layer chromatography over-pressure thin-layer chromatography trimethylsilyl ether phosphatidic acid platelet activating factor phosphatidylcholine principal components analysis principal components regression Pee Dee Belemnite (ratio of 12C to l3C isotopes) phosphatidylethanolamine pentafluorobenzyl pentafluorobenzyloxime phosphatidylglycerol prostaglandin D2 prostaglandin En (n = 0, 1,2, 3) prostaglandin F" prostaglandin F I" prostaglandin F2 prostaglandin F2" prostaglandin H2 phosphatidylinositol phosphatidylinositol monophosphate phosphatidylinositol bisphosphate phospholipid partial least squares regression parts per billion parts per million phosphatidylserine plasmaspray

PTFE PUFA PYA PyMS Q QNP r.f. RMSEP RP RP-HPLC RR SCIR SFC SFS SG SIM SMLR SPH Suc CL t-BDMS TEA TG TH TLC TMS TNP TOF TMS TNT TPH TPP TS TTLC TXB2

UV VFA VHC VLDL WHO WMP

ABBREVIA TIONS

polytetrafluoroethylene polyunsaturated fatty acid polyvinyl alcohol pyrolysis mass spectrometry quadrupole quattro nuclei probe radio frequency root mean square error of prediction reversed phase reversed-phase high-pressure liquid chromatography ridge regression stable carbon isotope ratio [ 6(13C) ] supercritical fluid chromatography supercritical fluid extraction sitosterylglycoside single ion monitoring stepwise multiple linear regression sphingomyelin succinylated cardiolipin tert-butyldimethylsilyl triethylamine tri( acyl)glycerol a-tocopherol thin-layer chromatography trimethylsilyl trinitrobenzenesulphonic acid time of flight tetramethylsilane trinitrotoluene total petroleum hydrocarbon triphenylphosphate thermospray tubular thin-layer chromatography thromboxane B2 ultraviolet volatile fatty acid volatile hydrocarbon very low density lipoprotein W orid Health Organization whole milk powder

xvii

Introduction

The revolution in analysis begun with the two seminal papers by A. J. P. Martin in 1941 and 1952 had far-reaching consequences for our understand­ing of lipids in our everyday life. As chromatographic techniques matured, smaller and smaller quantities of lipids could be separated and detected, with the result that studies could be initiated into biological transformations and food constituents which had not been possible before 1950. Separation science and lipid analysis have been expanding in every decade since. New techniques have uncovered unexpected compounds and forced re-examina­tions of earlier theories. The present volume is the second in the series and concentrates on analysis of lipids and helps to explain the importance of some recent developments. The authors range from well-known experts to some newer workers in the field, all with a distinctive contribution to make in elucidating the range of techniques which are available to the lipid scientist.

In Chapter 1, Shantha of Nestle and Napolitano of Oak Ridge National Laboratory review the position of thin layer chromatography (TLC), describing commercial apparatus and highlighting how easy it is to modify TLC. Solvent systems, detectors and stationary phases are explained and a number of applications concerning adulteration, for example of cocoa but­ter, are mentioned. The authors then concentrate on the so-called weakness of the technique, namely quantitation. The authors comprehensively reject this claim and show how a TLC-flame ionization detector system gives acceptable quantitative results.

One of the newer derivatives of chromatography is supercritical fluid chromatography which Blomberg, Demirbuker and Andersson describe in Chapter 2. The theory is explained and the peculiar advantages which the supercritical fluid confers on the system are designated. Open tubular and packed columns can both be used and examples are shown.

In Chapter 3 Ulberth outlines the theory and practice of static headspace gas chromatography. He then considers the analysis of volatile lipids in the study of the digestive tract and in nutritional physiology. Equally important is the contribution headspace analysis can make in the study of oxidative rancidity. Ulberth contrasts static headspace gas chromatography with dynamic headspace analysis.

In industry, most lipid analysts are familiar with wide-line nuclear mag­netic resonance (NMR). In Chapter 4 Diehl describes the application of Fourier transform NMR spectroscopy and illustrates that the technique can be used in lipid chemistry both qualitatively and quantitatively. By means of

xx INTRODUCTION

correlation tables and typical spectra he shows how the composition of fat mixtures can be determined. He also outlines the value of 31 P NMR to detect phosphoglycerides. Although this NMR technique has not had a major impact on industrial analysis, the non-destructive nature of the technique may enable it to challenge gas chromatography (GC) in the future.

Dobson has described in Chapter 5 how the analysis of one group of lipids, namely the cyclic fatty acids, must be attacked. In this instance a combined approach using TLC, high-pressure liquid chromatography (HPLC), GC, gas chromatography-mass spectroscopy (GC-MS), NMR and Fourier transform infra-red (FTIR) spectroscopy as well as chemical modification is required. He illustrates the analysis of cyclic fatty acids from heat-treated linseed oil and from sunflower oil. The mechanism by which these acids are produced is outlined. Considerable interest has been gener­ated in these compounds because of the large amounts which can be formed during the frying of chips and snack foods.

In a wide-reaching chapter Kuksis explains the principles and apparatus used in soft ionization mass spectroscopy (Chapter 6). The ever increasing scope of these techniques has brought the analysis of complex high mole­cular weight molecules such as sphingomyelins within the reach of many scientists. He has shown that quadrupole instruments can be applied to a wide range of lipids, from hydroperoxy fatty acids through oxygenated sterols and serine glycerophospholipids to lipopolysaccharides. Sodiated adducts permit levels of 5 fmol to be detected.

Food irradiation may now be used in over 40 countries. The short chapter by Morsel describes the need to find markers which indicate that food has been irradiated (Chapter 7). The lipids which have been examined as poten­tial markers include hydroperoxides, alkenes, alkanes, aldehydes and cyclo­butanones. Derivative formation with cyclobutanones can increase the sensitivity of detection of butanones in irradiated food.

In Chapter 8 a leading expert in the detection of adulteration of oils and fats, Rossell, explains the economic advantages which tempt unscrupulous dealers to add cheap oils to high-value oils such as olive oil. He then describes a technique which he and his coworkers at Bristol have pioneered where stable carbon isotope ratio (SCIR) measurement has simplified the task of detecting adulteration. He compares sterol and fatty acid methyl ester analysis with SCIR and shows how clearly the latter detects maize oil.

In Chapter 9 Karlsson explains the importance of combined techniques, for example joint use of HPLC, flow or loop injection and MS or tandem MS in the analysis of lipids. The techniques of thermospray, electro spray and atmospheric pressure chemical ionization are described. Applications range from platelet activating factor to cardiolipin. Karlsson mentions some of the practical procedures which he adopts in his laboratory to ensure good reproducible analysis.

INTRODUCTION xxi

Finally in Chapter 10, Jones, Shaw, Salter, Bianchi and Kell, in a joint contribution from the University of Wales at Aberystwyth and the Istituto Sperimentale per la Elaiotecnica in Italy, give a thorough description of chemometric methods. They explain how multivariate analysis can be applied to NMR data and to pyrolysis mass spectrometry. They stress the relatively cheap methodology which the latter technique offers.