Compound-Specific and Enantioselective Determination of 2- and 3-Hydroxy Fatty Acids in Food

Compound-Specific and Enantioselective Determination of 2- and 3-Hydroxy Fatty Acids in Food Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Fakultät Naturwissenschaften Universität Hohenheim Institut für Lebensmittelchemie vorgelegt von Ramona Jenske aus Waren/Müritz 2009

Dekan: Prof. Dr. Heinz Breer 1. berichtende Person, 1. Prüfer: Prof. Dr. Walter Vetter 2. berichtende Person, 2. Prüfer: Prof. Dr. Lutz Graeve 3. Prüfer: Prof. Dr. Heinz-Dieter Isengard Eingereicht am: 08. April 2009 Mündliche Prüfung am: 28. Juli 2009 Die vorliegende Arbeit wurde am 17. Juni 2009 von der Fakultät Naturwissenschaften der Universität Hohenheim als Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften angenommen.

Berichte aus der Chemie Ramona Jenske Compound-Specific and Enantioselective Determination of 2- and 3-Hydroxy Fatty Acids in Food D 100 (Diss. Universität Hohenheim) Shaker Verlag Aachen 2009

Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de. Zugl.: Hohenheim, Univ., Diss., 2009 Copyright Shaker Verlag 2009 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 permission of the publishers. Printed in Germany. ISBN 978-3-8322-8468-8 ISSN 0945-070X Shaker Verlag GmbH P.O. BOX 101818 D-52018 Aachen Phone: 0049/2407/9596-0 Telefax: 0049/2407/9596-9 Internet: www.shaker.de e-mail: info@shaker.de

DANKSAGUNG Herrn Prof. Dr. Walter Vetter danke ich sehr herzlich für die interessante Themenstellung, die exzellente Betreuung und seine ständige Diskussionsbereitschaft. Weiterhin bedanke ich mich bei ihm für das harmonische Arbeitsverhältnis und die hervorragenden Arbeitsbedingungen am Institut für Lebensmittelchemie der Universität Hohenheim. Herrn Prof. Dr. Lutz Graeve für die Übernahme des Mitberichts. Bei allen Kolleginnen und Kollegen am Institut für Lebensmittelchemie der Universität Hohenheim, insbesondere bei Simon Gaul, Simone Hauff, Thomas Kapp, Katja Lehnert, Joachim Melcher und Natalie Rosenfelder, möchte ich mich für das freundschaftliche und angenehme Arbeitsklima bedanken. Ich bedanke mich vor allem für viele fachliche Diskussionen und die stete Hilfsbereitschaft. Mein ganz besonderer Dank gilt meinen Eltern, die mir diesen Weg ermöglicht haben und mich immer unterstützt haben.

PRELIMINARY REMARKS The work presented in this doctoral thesis was carried out under the supervision of Prof. Dr. W. Vetter at the Institute of Food Chemistry, University of Hohenheim, from January 2005 to October 2007. Parts of this work have already been published in international peer-reviewed journals: 1) Jenske, R. and Vetter, W. Gas chromatography/electron-capture negative-ion mass spectrometry for the quantitative determination of 2- and 3-hydroxy fatty acids in bovine milk fat. J. Agric. Food Chem. 2008, 56, 5500-5505. 2) Jenske, R. and Vetter, W. Concentrations of medium-chain 2- and 3-hydroxy fatty acids in foodstuffs. Food Chem. 2009, 114, 1122-1129. 3) Jenske, R. and Vetter, W. Highly selective and sensitive gas chromatography electron-capture negative-ion mass spectrometry method for the indirect enantioselective analysis of 2- and 3-hydroxy fatty acids in food and biological samples. J. Chromatogr. A 2007, 1146, 225-231. 4) Jenske, R. and Vetter, W. Enantioselective analysis of 2- and 3-hydroxy fatty acids in food samples. J. Agric. Food Chem. 2008, 56, 11578-11583. 5) Jenske, R.; Lindström, F.; Gröbner, G. and Vetter, W. Impact of free hydroxylated and methyl-branched fatty acids on the organization of lipid membranes. Chem. Phys. Lipids 2008, 154, 26-32. or were presented at international scientific conferences: Thurnhofer, S.; Jenske, R. and Vetter, W. Quantitative Bestimmung von methyl- und hydroxy-substituierten Fettsäuren in verschiedenen Lebensmitteln, Regionaltagung der Lebensmittelchemischen Gesellschaft Süd-West 2006, Karlsruhe, Lebensmittelchemie 2006, 60, 159. I

Jenske, R.; Thurnhofer, S. and Vetter, W. Gaschromatographische Enantiomerentrennung von chiralen substituierten Fettsäuren, Regionaltagung der Lebensmittelchemischen Gesellschaft Süd-West 2006, Karlsruhe, Lebensmittelchemie 2006, 60, 159-160. Jenske, R. and Vetter, W. Enantioselektive Bestimmung von 2- und 3- Hydroxyfettsäuren mittels GC/ECD, 35. Deutscher Lebensmittelchemikertag 2006, Dresden, Lebensmittelchemie 2007, 61, 123-124. Jenske, R. and Vetter, W. Enantioselective GC/ECD determination of 2- and 3- hydroxy fatty acids after derivatization with Mosher s reagent, 4 th EuroFedLipid Congress 2006, Madrid/Spain. Lindström, F.; Jenske, R.; Thurnhofer, S.; Vetter, W. and Gröbner, G. Monitoring the effect of branched-chain fatty acids on phospholipid membranes by DSC and solidstate NMR, 51 st Annual Meeting of the Biopysical Society 2007, Baltimore/USA. Jenske, R. and Vetter, W. GC/NCI-MS-Nachweis von 2-Hydroxyfettsäuren in Lebensmitteln nach Derivatisierung mit Mosher Reagenz, Regionaltagung der Lebensmittelchemischen Gesellschaft Süd-West 2007, Gießen, Lebensmittelchemie 2008, 62, 6-7. Jenske, R. and Vetter, W. Enantioselektive und sensitive Bestimmung von 2- und 3- Hydroxyfettsäuren nach Derivatisierung mit Mosher-Reagenz mittels GC/NCI-MS, ANAKON 2007, Jena. Jenske, R. and Vetter, W. Bestimmung von 2- und 3-Hydroxyfettsäuren in Milch und Milchprodukten mittels GC/NCI-MS, 36. Deutscher Lebensmittelchemikertag 2007, Nürnberg-Erlangen, Lebensmittelchemie 2008, 62, 40-41. Jenske, R.; Matz, D.; Lindström, F.; Gröbner, G. and Vetter, W. Einfluss freier Hydroxyfettsäuren auf Lipidmembranen, 36. Deutscher Lebensmittelchemikertag 2007, Nürnberg-Erlangen, Lebensmittelchemie 2008, 62, 88. II

Chapters 2-6 of this doctoral thesis are in form identical with the full publications 1-5. Recurring figures have been omitted, tables and chemical structures have been numbered consecutively, and a combined directory of references was created. To clarify the participation and contributions of each author, named in the publications, it is explained in the following: All essential practical and analytical work was performed by Ms. Ramona Jenske. This included the developing of two highly sensitive GC/ECNI-MS-SIM methods, one for quantification of 2- and 3-OH-FAs in food, and one for the enantioselective determination of 2- and 3-OH-FAs in food. Furthermore, Ms. Jenske performed the synthesis of the internal standard 2-OH-18:0-ME-9,10-d 2 and the generation of enantiopure 2-OH-18:0-ME, accomplished all DSC-measurements, and carried out the sample preparations, derivatizations and analyses with GC/ECD and GC/ECNI-MS. The developed methods were used to generate data regarding the amounts as well as the enantiomeric distributions of 2- and 3-OH-FAs in different food samples. The analysis and interpretation of the obtained data was carried out by her, as well as the preparation of the complete manuscripts including the tables and figures. Prof. Dr. Walter Vetter was the supervisor of this work. Proof-reading of the manuscripts and corrections in terms of formal and textural aspects were done by him. Prof. Dr. W. Vetter also functioned as an advisor throughout the publications processes and was responsible for all formal aspects of the publications. He also co-wrote the DAAD/STINT proposal on DSC analysis of fatty acids. Dr. Fredrick Lindström introduced Ms. Jenske in the theory of DSC. Prof. Dr. Gerhard Gröbner co-wrote the DAAD/STINT proposal and functioned as an advisor throughout the DSC studies carried out at the University of Umeå (Sweden). III

ABBREVIATIONS ACP AFA AOAC AOCS ASE BSE CD CNS CoA CSP DHA DMAP DMOX DMPC DSC ECD ECNI EE EF EI EU FAD FAEE FAME FAS FID gal GC glc H cal HPLC I rel i.d. acyl carrier protein anteiso-fatty acid Association of Official Analytical Chemists American Oil Chemists` Society accelerated solvent extraction bovine spongiform encephalopathy cyclodextrin central nervous system coenzyme A chiral stationary phase (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid 4-dimethylaminopyridine 4,4-dimethyloxazoline dimyristoyl-sn-glycero-3-phosphatidylcholine differential scanning calorimetry electron capture detection electron capture negative ion ethyl ester enantiomer fraction electron ionization European Union flavin adenine dinucleotide fatty acid ethyl ester fatty acid methyl ester fatty acid synthase flame ionization detection galactosyl gas chromatography glucosyl phase transition enthalpy high-performance liquid chromatography relative intensity internal diameter IV

ISTD IUPAC LPS l.w. MAE MBFA ME MOX MS MTPA NAD + NADP + nd NMR OH OH-FA PDO PFBO PPCD PSE R S RRF RRI SIM SPE T m TBDM TBME TEA TFA TLC TMS t R tr internal standard International Union of Pure and Applied Chemistry lipopolysaccharide lipid weight microwave-assisted extraction monomethyl-branched fatty acid methyl ester 4-methyloxazoline mass spectrometry -methoxy- -trifluoromethylphenylacetyl nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate not detectable nuclear magnetic resonance hydroxyl group hydroxy fatty acid protected designation of origin pentafluorobenzoyl perpentylated cyclodextrin pressurized solvent extraction chiral resolution relative response factor relative retention index selected ion monitoring solid phase extraction phase transition temperature 6-O-tert.-butyldimethyl-2,3-di-O-methylsilyl tert.-butyl methyl ether triethylamine trifluoroacetyl thin layer chromatography trimethylsilyl retention time trans V

TABLE OF CONTENTS 1 GENERAL INTRODUCTION... 1 1.1 Lipids and lipid classes... 1 1.1.1 Acylglycerides... 1 1.1.2 Glycerophospholipids and sphingolipids... 2 1.1.3 Lipopolysaccharides... 4 1.1.4 Further lipid classes... 5 1.2 Hydroxy fatty acids... 7 1.2.1 Structure and nomenclature of fatty acids... 9 1.2.2 2- and 3-hydroxy fatty acids... 9 1.2.2.1 Occurrence and relevance... 10 1.2.2.2 Formation... 12 1.2.2.2.1 de novo biosynthesis... 12 1.2.2.2.2 -oxidation... 14 1.2.2.2.3 -oxidation... 15 1.2.2.3 Properties... 16 1.2.2.4 Chirality... 18 1.2.3 Further hydroxy fatty acids... 19 1.2.3.1 4- and 5-hydroxy fatty acids ( - and -lactones)... 19 1.2.3.2 Additional hydroxy fatty acids... 19 1.3 Analysis of 2- and 3-hydroxy fatty acids... 22 1.3.1 Derivatization procedures for qualitative and quantitative gas chromatographicmass spectrometric analyses of hydroxy fatty acids... 22 1.3.1.1 Derivatization of the carboxylic group formation of fatty acid methyl esters... 22 1.3.1.1.1 Electron ionization mass spectra (EI-MS) of 2- and 3-hydroxy fatty acid methyl esters... 23 1.3.1.2 Additional derivatization of the hydroxyl group in 2- and 3-hydroxy fatty acid methyl esters... 24 1.3.1.2.1 Formation and GC/EI-MS of trimethylsilylethers of 2- and 3-hydroxy fatty acid methyl esters... 24 VI

1.3.1.2.2 Formation and mass spectra of trifluoroacetyl and pentafluorobenzoyl derivatives of 2- and 3-hydroxy fatty acid methyl esters... 25 1.3.2 Electron capture negative-ion mass spectrometry (ECNI-MS)... 28 1.3.2.1 Formation of negative ions... 28 1.3.2.2 Selectivity of ECNI-MS... 29 1.3.3 Sample preparation for the determination of hydroxy fatty acids in food... 30 1.3.3.1 Extraction of lipids... 30 1.3.3.2 Enrichment of hydroxy fatty acids from extracted lipids... 33 1.3.4 Internal standards... 34 1.3.5 Enantioselective analyses of hydroxy fatty acids... 36 1.3.5.1 Direct gas chromatographic enantioseparation of hydroxy fatty acids... 37 1.3.5.1.1 Preface... 37 1.3.5.1.2 Application to 2- and 3-hydroxy fatty acids... 40 1.3.5.2 Indirect gas chromatographic enantioseparation of hydroxy fatty acids... 42 1.3.5.2.1 Preface... 42 1.3.5.2.2 Application to 2- and 3-hydroxy fatty acids... 42 1.3.5.3 Enantiopure standards of 2- and 3-hydroxy fatty acids... 47 1.3.6 Differential scanning calorimetry (DSC)... 49 1.4 Aims of the study... 52 2 GAS CHROMATOGRAPHY/ELECTRON-CAPTURE NEGATIVE ION MASS SPECTROMETRY FOR THE QUANTITATIVE DETERMINATION OF 2- AND 3-HYDROXY FATTY ACIDS IN BOVINE MILK FAT... 53 2.1 Abstract... 53 2.2 Introduction... 54 2.3 Materials and methods... 55 2.3.1 Materials and chemicals... 55 2.3.2 Synthesis of 9,10-dideutero-2-hydroxyoctadecanoic acid methyl ester... 55 2.3.3 Standard solutions... 56 2.3.4 Sample preparation... 57 2.3.5 PFBO derivatization... 57 2.3.6 Gas chromatography with electron-capture negative ion mass spectrometry. 57 2.4 Results and discussion... 58 2.4.1 GC/ECNI-MS capability of PFBO derivatives... 58 VII

2.4.2 Sample preparation for bovine milk fat samples... 61 2.4.3 Selection of internal standards... 62 2.4.4 Application of the method to bovine milk fat... 63 3 CONCENTRATIONS OF MEDIUM-CHAIN 2- AND 3-HYDROXY FATTY ACIDS IN FOODSTUFFS... 67 3.1 Abstract... 67 3.2 Introduction... 67 3.3 Materials and methods... 69 3.3.1 Samples and chemicals... 69 3.3.2 Sample preparation... 70 3.3.3 Gas chromatography with electron-capture negative ion mass spectrometry (GC/ECNI-MS)... 71 3.4 Results and discussion... 72 3.4.1 Accuracy of the determinations... 72 3.4.2 Concentrations of 2- and 3-hydroxy fatty acids in milk and cheese samples.. 74 3.4.3 Concentrations of 2- and 3-hydroxy fatty acids in animal products and human vernix caseosa... 76 3.4.4 Concentrations of 2- and 3-hydroxy fatty acids in vegetable oils... 79 3.5 Conclusions... 80 4 HIGHLY SELECTIVE AND SENSITIVE GAS CHROMATOGRAPHY- ELECTRON-CAPTURE NEGATIVE-ION MASS SPECTROMETRY METHOD FOR THE INDIRECT ENANTIOSELECTIVE IDENTIFICATION OF 2- AND 3- HYDROXY FATTY ACIDS IN FOOD AND BIOLOGICAL SAMPLES... 82 4.1 Abstract... 82 4.2 Introduction... 82 4.3 Experimental... 84 4.3.1 Materials... 84 4.3.2 Derivatization reactions... 84 4.3.3 Sample preparation... 85 4.3.4 Gas chromatography with electron-capture detection (GC/ECD)... 85 4.3.5 Gas chromatography-mass spectrometry (GC/MS) analysis... 86 VIII

4.4 Results and discussion... 87 4.4.1 Enantioseparation of 2- and 3-hydroxy fatty acid methyl esters and ethyl esters as diastereomeric MTPA derivatives on non-chiral stationary phases... 87 4.4.2 Enantioselective identification of 2-hydroxy fatty acids in samples... 91 4.5 Conclusion... 94 5 ENANTIOSELECTIVE ANALYSIS OF 2- AND 3-HYDROXY FATTY ACIDS IN FOOD SAMPLES... 96 5.1 Abstract... 96 5.2 Introduction... 96 5.3 Materials and methods... 98 5.3.1 Samples and chemicals... 98 5.3.2 Sample preparation and derivatization... 98 5.3.3 Gas chromatography with electron capture negative ion mass spectrometry (GC/ECNI-MS)...100 5.4 Results and discussion...100 5.4.1 Quality of the method...100 5.4.2 Enantiomeric composition of 2-OH-FAs in food samples...102 5.4.3 Enantiomeric composition of 3-OH-FAs in food samples...105 5.4.4 Potential for using enantiomer fractions (EF) of 2- and 3-OH-FAs in authenticity control...107 6 IMPACT OF FREE HYDROXYLATED AND METHYL-BRANCHED FATTY ACIDS ON THE ORGANIZATION OF LIPID MEMBRANES...109 6.1 Abstract...109 6.2 Introduction...109 6.3 Experimental...111 6.3.1 Chemicals and standards...111 6.3.2 Esterification of free fatty acids...112 6.3.3 Generation of enantiopure 2-hydroxyoctadecanoic acid methyl ester...112 6.3.4 Sample preparation...112 6.3.5 Differential scanning calorimetry (DSC) measurements...113 6.4 Results...113 IX

6.4.1 Membrane phase behavior...113 6.4.2 Free fatty acids with a single double bond and/or a hydroxyl group...116 6.4.3 Free hydroxy fatty acids with different chain length...118 6.4.4 Influence of the position of the hydroxyl group...118 6.4.5 Racemic vs. enantiopure fatty acids...119 6.5 Discussion...121 6.6 Conclusion...123 7 CONCLUDING REMARKS...124 8 SUMMARY...126 9 ZUSAMMENFASSUNG...130 10 REFERENCES...134 X