ir å il f. lÌ{Sl Íl'U ft liorinnlt APPLICATION OF FAST ATOM BOMBARDMBNT MASS SPECTROMETRY TO THE EXAMINATION OF GLYCOCONJUGATES IN GRAPE JUICE AND WINE by Vassilios A. Marinos, BSc Thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide (Faculty of Agricultural and Natural Resources) August 1992 TABLE OF CONTENTS page Table of contents 1 List of figures iii List of tables v Abstract vi Declaration ix Acknowledgements x Publications xii Abbreviations xiii 1 Introduction and general literature review 1 1.1 Wine composition and flavour 1 1.1.1 Classification of wine constituents in terms of their metabolic origin 2 1.1.2 Sensory evaluation of wine metabolites 2 1.1.3 Classification of wine flavour constituents 3 1.1.4 The accumulation of flavour components originating from $apes 4 1.1.5 The flavour precursor analysis approach J 1.2 Volatile secondary metabolites in grape juice and wine 5 1.2.1 Terpenoids 5 1.2.2 Monoterpenes 6 1.2.3 Norisoprenoids 11 1.2.4 Shikimic acid derived volatiles 18 1.3 Glycosidically bound secondary metabolites 20 1.3.1 Biological formation of glycosides 20 1.3.2 The composition of glycoconjugated precursors in fruits 2t t.4 The analysis of flavour precursors 23 1.4.1 New dimensions in flavour precursor analysis 25 1.5 Fast atom bombardment-mass spectrometry (FAB-MS) 26 1.5.1 Instrumentation and principles 26 1.5.2 The sputtering phenomenon 28 1.5.3 Matrix support 30 1.5.4 Characteristics of FAB-MS 32 1.5.5 Mechanism of ionization 33 i page 1.5.6 Sensitivity 35 t.5.7 Application of FAB-MS to structure elucidation of glycoconjugates 36 1.6 Employment of FAB-MS in a multidimensional analysis 4L 1,7 Aims of this thesis 42 2 Late-eluting, relatively non-polar wine glucosides 43 2.1 Introduction 43 2.2 Results and discussion 44 3 Early-eluting polar monoglucosides 56 3.1 Introduction 56 3.2 Results and discussion 56 4 Early-eluting disaccharide glycosides 67 4.t Introduction 67 4.2 Results and discussion 67 5 Very early-eluting glycoconjugates 79 5.1 Introduction 79 5.2 Results and discussion 79 6 p-Carbolines: polar, non-volatile materiat in Riesting wine 89 6.1 Introduction 89 6.2 Results and discussion 89 7 Conclusions 96 I Experimental 100 8.1 Chapter 1. General 100 8.2 Chapter 2. Late-eluting, relatively non-pola¡ wine glucosides 105 8.3 Chapter 3. Early-eluting polar monoglucosides 110 8.4 Chapter 4. Early-elutin g disaccharide glycoside s 113 8.5 Chapter 5. Very early-eluting glycoconjugates tt9 8.6 Chapter 6. B-Carbolines: polar, non-volatile material in Riesling wine I23 References 126 ll LIST OF FIGURES Figure page 1 Key volatile monoterpene compounds 6 2 Structures of some monoterpenes identified in grapes and wines 8 3 Monoterpenes induced by acid hydrolysis 10 4 p-Menthenediols and ø-terpineol derivatives 10 5 Carotenoid degradation 11 6 Norisoprenoids identified as free volatiles in grapes and wines L2 7 TDN and vitispirane precursors in wine L4 I p-Damascenone precursors 15 9 The actinidols and actinidol precursors 15 10 Norisoprenoids identified in the enzymic hydrolysate of glycosidic isolates of premium varietes 17 11 Shikimate-derived volatile phenols identified in juices and wines 19 L2 Transglycosylation reaction for the formation of glycosidicatly bound secondary metabolites 20 L3 Overall diagram of a FAB-MS ion source 27 1,4 Regimes of sputtering by elastic collisions 29 15 Positive and negative ion mode FAB-MS spocta of glycosidic mixture fl,. frs.j-I2 45 16 Positive and negative ion mode FAB-MS/IVIS spectra of glycosides with Mr 332 47 t7 cls-þran linalool oxide-B-D-glucopyranoside and its acetylated derivative 53 18 Some Riesling wine norisoprenoid constituents and their glycosidic derivatives 55 t9 2-[2-Methoxy-4-(chydroxypropyl)-phenoxy]-propan-1,3-diol 60 20 1,2,3,4-Tetahydro-7-hydroxy- 1 - (4'-hydroxy-3'-methoxyphenyl)-6- methoxy-2,3-napthalenedimethanol (i.e. isolariciresinol) 63 2l 2,3-Bis(4'-hydroxy-3'-methoxybenzyl)butane- 1,4-diol (i.e. seco- isolariciresinol), its glucosidic derivative and some of the positive ion mode mass spectral fragmentations of the glucosidic derivative 65 22 Benzyl alcohol-B-D-rutinoside and its per-O-acetylated derivative 70 23 @)-2,6-Dimetþlocta-2,7-diene-1,6-diol 72 24 (E,E)-2,6-Dimetþlocta-2,6-diene-l,8-diol andits glycosidic derivatives 74 25 @)-3,7-Dimethylocta-2-ene-l,7-diolanditsglycosidicderivatives 75 111 Figure page 26 p-Menth- 1 -ene-7-(B-D-apiofuranosyl-( 1 -+6)-B-D-glucopyranosyl)-8-ol, vomofoliol-9-O-arabinofuranosyl-O-glucopyranoside and their per-O- acetylated derivatives 77 27 2, 3 -Dihydro-7-hydroxy-2- (4' -O- P- glucopyranosyl- 3'-methoxyphenyl) - 3- hydroxymethyl-5-benzofuranpropanol, a novel Riesling wine constituent and its per-O-acetylated derivative 84 28 2,3-Dihydro-7-hydroxy -2- (4' -hy droxyl-3'-methoxybenzyl)-3- hydroxymethyl-5-benzofuranpropanol 84 29 @)-2,6-Dimethylocta-2,7-diene- 1,6-diol- 1 -O-B-D-glucopyranosyl-O- arabinofuranosyl-O-B-glucopyranoside and its per-O-acetylated derivetive 86 30 1-Methyl- 1,2,3,4-tetrahydro-B-carboline-3-carboxylic acid: a novel constituent of Riesling wine 90 3L p-Carboline derivatives in Riesling wine and some of their mass spectral 94 fragmentations 1V LIST OF TABLES Table page 1 Glycosidically bound volatile secondary metabolites identified in grapes 23 and wines 2 lH NIrrlR data of compounds 85a, 85b 49 3 1g Nn¡n data of compounds 86, 30a,30b 51 4 lff NUn data of compounds 90, 90a 59 5 lu NvtR data of compounds 91, 91a and some of the positive ion mode mass spectral fragmentations of 91 62 6 lff NUR data of compound s 94,94a 7L 7 lH Nn¡n data of compounds 95a, 96a,97a 78 8 lfr NUn data of compounds 99a, 100a 88 9 1ff NUR data of naturally occurring and synthetic diastereoisomers of 1 -methyl- 1,2,3,4-teûahydro-p-carboline-3-carboxylic acid 9l 10 13C NUn data of naturally occurring and synthetic diastereoisomers of 1 -methyl- 1,2,3,4-tetahydro- p-carboline-3-carboxylic acid 93 v Abstract Flavour is probably the single most important factor influencing wine quality and is related to the chemical composition of both the grape juice and the wine. Flavour- important compounds are accumulated in glapes in two different fotms, i.e. as free volatile compounds and as non-volatile polyols or glycosidically bound metabolites. For the study of these compounds a technique named the flavour precursor analysis approach has been developed. The progress made in grape juice and wine compositional analysis through application of this approach has been reviewed and the limitations of the techniques involved are discussed. The introduction of fast atom bombardment mass spectrometry (FAB-MS) together with the improved mass ranges of magnetic sector and quadrupole instruments has opened new horizons for the analysis of glycoconjugates and other polar and non-volatile compounds of biological importance. A protocol which combines partition liquid chromatography (e.g. droplet counter curent chromatography (DCCC) and high performance liquid chromatogtaphy (HPLC)) and soft ionization mass spectrometry (i.e. FAB-MS) has been developed for the direct analysis of wine and juice glycoconjugates without prior derivatization of these compounds. This protocol offers an alternative to overcome the limitations of the flavour precursor analysis approach in the investigation of polar, non-volatile and thermally labile material. Application of the protocol to pre-fractionated wine glycosidic mixtures of different degtees of polarity demonsüated that the protocol can be successfully employed as a technique to screen for flavour precursors and other glycosidically bound secondary metabolites present in wine. In the relatively non-polar glycosidic fraction (i.e. late-eluting DCCC fractions L90-220), the p-D-glucopyranosides of ethyl indole lactate, uroterpenol and cis-pyran linalool oxide have been identified. The presence in the same glycosidic fraction of hexopyranosides of 3-hydroxy-5,6-epoxymegastigm-Zine-9-one and of a mixture of diastereoisomeric 3,4- dihydroxy-7,8-dihydro-B-ionone, was observed. As a consequence of these investigations, a new class of wine glucopyranosides with phenylpropanoids and lignans as aglycones has been recognized in the medium polarity v1 fractions (i.e. early-eluting DCCC fractions 90-100). 2-Methoxy-4-(ohydroxypropyl)- phenoxypropan-3-ol, isolariciresinol and søco-isolariciresinol B-D-glucopyranosides have been isolated from this fraction and characterized by spectroscopic and spectrometric techniques. This class of compound, although without an apparent flavour precursor role, it does contain compounds which have been suggested as precursors in the formation of biologic ally active compounds, e. g. seco-isolariciresinol- p-D - glucopyranos ide. In this same polarity group of fractions, a new class of flavour precursor material has been recognized. This class comprises a mixture of monoterpene B-D-apiosyl-P-D- glucopyranosides. These compounds include (E)-2,6-dimethylocta-2,7-diene- 1,6-diol, (E,E)-2,6-dimethylocta-2,6-diene-1,8-diol and p-menth-1-ene-7,8-diol apiosylglucosides which have been isolated and subsequently identified by specúometric and specroscopic techniques. A new arabinoglucoside of (E)-3,7-dimethylocta-2-ene-L,7-diol together with two other known flavour precursors (i.e benzyl alcohol-p-D-rutinoside and vomifoliol arabinoglucoside), were also identified in this fraction. Investigation of the most polar glycosidic constituents of wine (i.e. very early-eluting DCCC fractions 60-75), has shown that the bulk of the material was made up of monoglucosidic derivatives of very polar aglycones such as the 4'-O-B-D-glucopyranoside of the neolignan 2,3-dihydro-7-hydroxy-2-(4'-hydroxy-3'-methoxybenzyl)-3- hydroxymethyl-S-benzofuranpropanol (i.e.