MASS SPECTROMETRY IN GRAPE AND WINE CHEMISTRY. PART I: POLYPHENOLS

Transcription

1 MASS SPECTROMETRY IN GRAPE AND WINE CHEMISTRY. PART I: POLYPHENOLS Riccardo Flamini* Istituto Sperimentale per la Viticoltura, Viale XXVIII Aprile 26, I Conegliano (TV), Italy Received 11 February 2003; revised 28 April 2003; accepted 28 April 2003 I. Introduction II. Quality Improvement III. Grape and Wine Polyphenols IV. Mass Spectrometry in the Study of Polyphenols and Procyanidins V. Mass Spectrometry in the Study of Anthocyanins and their Derivatives VI. Mass Spectrometry in the Study of Structures Formed by Polymerization of Anthocyanins and Flavan-3-ols VII. Mass Spectrometry and Grape and Wine Resveratrol VIII. Application of MALDI in the Study of Polyphenols IX. Mass Spectrometry Applied to the Study of Wine Polyphenols from Cork Bottle Stoppers and Oak Barrels X. Conclusions References Mass spectrometry, had and still has, a very important role for research and quality control in the viticulture and enology field, and its analytical power is relevant for structural studies on aroma and polyphenolic compounds. Polyphenols are responsible for the taste and color of wine, and confer astringency and structure to the beverage. The knowledge of the anthocyanic structure is very important to predict the aging attitude of wine, and to attempt to resolve problems about color stability. Moreover, polyphenols are the main compounds related to the benefits of wine consumption in the diet, because of their properties in the treatment of circulatory disorders such as capillary fragility, peripheral chronic venous insufficiency, and microangiopathy of the retina. Liquid Chromatography-Mass Spectrometry (LC-MS) techniques are nowadays the best analytical approach to study polyphenols in grape extracts and wine, and are the most effective tool in the study of the structure of anthocyanins. The MS/MS approach is a very powerful tool that permits anthocyanin aglycone and sugar moiety characterization. LC-MS allows the characterization of complex structures of grape polyphenols, such as procyanidins, proanthocyanidins, prodelphinidins, and tannins, and provides experimental evidence for structures that were previously only hypothesized. The matrix-assisted-laser-desorption-ionizationtime-of-flight (MALDI-TOF) technique is suitable to determine the presence of molecules of higher molecular weight with high accuracy, and it has been applied with success to study procyanidin oligomers up to heptamers in the reflectron mode, and up to nonamers in the linear mode. The levels of resveratrol in wine, an important polyphenol well-known for its beneficial effects, have been determined by SPME and LC-MS, and the former approach led to the best results in terms of sensitivity. # 2003 Wiley Periodicals, Inc., Mass Spec Rev 22: , 2003; Published online in Wiley InterScience ( DOI /mas Keywords: grape and wine polyphenols; anthocyanins; procyanidins and tannins; resveratrol; mass spectrometry; liquid chromatography; MALDI *Correspondence to: Riccardo Flamini, Istituto Sperimentale per la Viticoltura, Viale XXVIII Aprile 26, I Conegliano (TV), Italy. flamini@mail.emmenet.it I. INTRODUCTION On the basis of data reported from the Office International De la Vigne et Du Vin (O.I.V.) The State of Vitiviniculture in the World and Statistical Information in 1999 Mass Spectrometry Reviews, 2003, 22, # 2003 by Wiley Periodicals, Inc.

2 MS IN GRAPE AND WINE CHEMISTRY & in the years a large increase of production and consumption of table grape was registered; that increase was also encouraged by the amply demonstrated beneficial effects of this food on the human health. Also, the worldwide wine production registered a sensible increase, favored by the suppression of measures that encouraged the permanent uprooting implemented in recent years by European Union, and by recent plantations in certain number of non-european countries with potentially high yields (Dutruc-Rosset, 1999). Trends of grape and wine worldwide production of these years are reported the histograms of Figure 1a,b. The particular increase in 1999 can be observed. The worldwide market has been influenced by the repercussions on the media of seminars and scientific symposia in the field of research, medicine, toxicology of foods, and human health. In particular, the beneficial effects of moderate wine consumption on certain categories of diseases, such as cardiovascular diseases, brain degeneration from aging, and certain carcinogenic diseases, have been discussed. The report of O.I.V. also indicates raisins as an interesting means by which to fight against hunger in the world. As a consequence of these trends, efforts of the largest grape- and wine-producer countries (Argentina, France, Italy, South Africa, Spain, United States) are addressed to improve the product quality, rather than to increase production, so as to remain competitive on emergent countries by the growth of systematic positioning in the market niches of premium and super premium wines. The main efforts of researchers and Organisms of Control are addressed to develop new methods to detect the product origin (Ogrinc et al., 2001), the detection of adulteration involving sugar-beet, cane sugar, or ethanol addition and watering (Guillou et al., 2001), the protection of consumer health through determination of food contaminants such as heavy metals, toxins, and pesticides (Szpunar et al., 1998; MacDonald et al., 1999; Wong & Halverson, 1999), and the study of plant metabolism and diseases (Pérez, Viani, & Retamales, 2000; Tabacchi et al., 2000). For the improvement of product quality, aroma and polyphenolic compounds have been widely studied. The numerous classes of grape polyphenols transferred to the wine are responsible for the taste and color of beverage. In this review, the important role of mass spectrometry in the study of grape and wine polyphenols is discussed a field where a rapid increase of knowledge has been observed, due also to the development of the new technologies introduced in the recent years. II. QUALITY IMPROVEMENT To improve the final product the wine the research in viticulture is addressed to improve the quality of the grape through the study of grape-ripening, which involves cultural techniques, the selection of clones and varieties of best potentiality (clonal selection), and the study of environmental influence on the vineyard (zoning). In the enology field, main efforts are devoted to optimize industrial processes finalized to obtain products with peculiar characteristics. In this frame, (i) the inoculum of selected yeast permits regular fermentation with minimum secondary processes by other microorganisms, (ii) the use of selected enzymes leads to a better extraction of grape components, (iii) the maceration of grape skins is performed in controlled conditions of temperature and atmosphere, (iv) malolactic fermentation is employed to improve organoleptic characteristics and to confer microbiological stability to the wine, and, finally, (v) the barreland bottle-aging refine the final product. To reach the proposed aims and to be able to estimate the potentiality of starting material and how it can be transferred to the final product, a good knowledge of grape chemistry is essential. FIGURE 1. Trends of total world-wide wine (a) and grape productions (b) for the period

3 & FLAMINI To define characteristics and identity of product, the research in viticulture attempts to determine origin parameters. For variety characterization (chemotaxonomic classification), DNA, amphelography, isoenzymes, and secondary metabolites of plant are studied (Costacurta et al., 2001). Secondary metabolites in the grape are compounds (terpenes and terpenols, methoxypyrazines, volatile sulfur compounds, benzenoids, norisoprenoids, and polyphenols) mainly linked to the variety but not indispensable for the plant survivor, also whether environmental and climatic variables can influence their contents in the fruit (Di Stefano, 1996; Flamini, Dalla Vedova, & Calò, 2001). III. GRAPE AND WINE POLYPHENOLS In the winemaking, grape compounds are transferred to the must and to the wine, which contain several polyphenols at different degree of polymerization. The simplest compounds are mono-, di-, and tri-phenols [phenol, pyrocatechol (1), resorcinol (2), hydroquinone (3), phloroglucinol (4)], phenolic aldehydes such as vanillin (5), p-hydroxybenzaldehyde (6), syringic aldehyde (7), coniferyl aldehyde (8), benzoic acids such as gentisic acid (9), gallic acid (10), vanillic acid (11), salicilic acid (12), and syringic acid (13). Also the hydroxycinnamic acids (HCA) caffeic (14), ferulic (15), and p-coumaric (cis- and trans-isomers) (16) and their esters formed by condensation with tartaric acid (hydroxycinnamoyltartaric acids HCTA) (17) are present in grape and wine in considerable amounts. In order to give to the reader a general view of the chemistry involved in this context, the structure of these molecules are reported in Figure 2. More-complex grape polyphenols contain two or more aromatic rings (cumarines, benzopyrones, and flavilium ions) to form flavanols (18), flavonols (19), and anthocyanins (20) (Macheix, Fleuriet, & Billot, 1990) (see Fig. 3). These molecules are present in the grape mainly in the monoglycoside form, with the sugar residue linked to the hydroxyl group in position C-3 of the O-containing ring. The glycoside flavonols kaempferol (19a), quercetin (19b), and myricitin (19c) (Fig. 3) form co-pigments with anthocyanins (in red wines); they, together with oxidation products of tannins, are in the main responsible for the color of white grapes and wines (Cheynier & Rigaud, 1986; Usseglio-Tomasset, 1995). Anthocyanins contain in their skeleton the benzopyrilium ion as base molecule, which is responsible for the color of red berry varieties and red wines. They are present in the grape as mono- or diglucosides, depending on variety, with the second glucose molecule linked to the C-5 hydroxyl group. The flavan-3- ols (þ)-catechin, (þ)-gallocatechin, ( )-epicatechin, and ( )-epigallocatechin are present in the grape as monomers, FIGURE 2. Structures of mono-, di-, and tri-phenols present in grape: (1) pyrocatechol, (2) resorcinol, (3) hydroquinone, (4) phloroglucinol, (5) vanillin, (6) p-hydroxybenzaldehyde, (7) syringic aldehyde, (8) coniferyl aldehyde, (9) gentisic acid, (10) gallic acid, (11) vanillic acid, (12) salicilic acid, (13) syringic acid, (14) caffeic acid, (15) ferulic acid, (16) p-coumaric acid, and (17) hydroxycinnamoyltartaric acids. FIGURE 3. Chemical structures of polyphenol aglycones present in grape: (18) flavanols, (19) flavonols, and (20) anthocyanins. 220

4 MS IN GRAPE AND WINE CHEMISTRY & or linked between them to form procyanidins, proanthocyanidins and tannins of type reported in Figure 4. With grape pressing, polyphenols are released in the must from the different parts of berry: HCTA, phenolic acids, and aldehydes from juice and pulp; HCTA, phenolic acids, anthocyanins, procyanidins and proanthocyanidins, and flavonols from skin; and tannins, procyanidins and proanthocyanidins, gallic acid, catechin, and epicatechin from seeds (Figs. 2 and 4). Moreover, these molecules can undergo condensation and polymerization processes during the winemaking and wine aging, to produce new structures. Polyphenols play an important role in the organoleptic characteristics of wine; in particular, tannins confer FIGURE 4. Structures of catechins, and procyanidin dimers and trimers in grape seeds. (Reprinted from Phytochemistry 49, de Freitas et al., Characterization of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry, p. 1436, Copyright 1998, with permission from Elsevier.) 221

5 & FLAMINI astringency and structure to the beverage by formation of complexes with the proteins of saliva. Their knowledge is very important to predict the aging attitude of wine, and to attempt to resolve problems about color stability in particular in the case of premium red wines that are destined to long aging periods (e.g., Brunello di Montalcino, Chianti, Barolo). Polyphenols are the principal compounds related to the benefits of wine consuming in the diet because of the properties attributed to them. Procyanidins and proanthocyanidins from Vitis vinifera seeds are used as active ingredients in medicinal products for the treatment of circulatory disorders such as capillary fragility, peripheral chronic venous insufficiency, and microangiopathy of the retina. Pharmacological properties of selected proanthocyanidins from grape seeds (Leucoselect TM ) are related to an increase of tonicity and resistance of capillary walls, as well as to radical scavenging and inhibition of superoxide ion formation. Proanthocyanidins supplementation in the rat, made the heart less susceptible to the ischemia/ reperfusion damage, and increased the total antioxidant plasma capacity and the ascorbic acid plasma level (Maffei Facino et al., 1996, 1998). Additionally, proanthocyanidins from grape seeds decreased the susceptibility of healthy cells to toxic and carcinogenic agents. Phenolic compounds of the grape have been associated with cardiovascular benefits, a reduction of platelet aggregation, and a modulation of eicosanoid synthesis. Recently, the antioxidant activity of phenolic compounds from 12 different varieties of grape toward human low-density lipoprotein (LDL) in vitro has been evaluated (Maffei Facino et al., 1994; Frankel, Waterhouse, & Teissedre, 1995; Bagchi et al., 1997; Meyer et al., 1997; Waterhouse & Walzem, 1997; Joshi et al., 1998; Schramm et al., 1998; Maffei Facino et al., 1999). The flavonol quercetin blocked the aggregation of human platelets by ADP and thrombin, and this compound has gained considerable prominence as an inhibitor of carcinogens and of cancer cell growth in many experimental and human tumors (Goldberg et al., 1998). Furthermore, the anthocyanin profile is a useful tool to characterize and to determine the origin of products, and in the identification of possible adulterations. For example, in some countries the production and commercialization of wine from not Vitis vinifera grape are prohibited. Hybrid grapes are characterized by peculiar anthocyanin 3,5-O-diglucoside contents, which are practically absent in grapes from Vitis vinifera, and these compounds can be consequently employed for the identification of possible adulterations. Finally, anthocyanins from grape are also important in the synthetic colorants market, in particular in the food industry (Hong & Wrolstad, 1990a). Traditional methods to determine polyphenols and anthocyanins in natural extracts are usually performed by liquid chromatography (LC) analysis and spectrophotometric measurements. The LC methods are mainly applied for chemotaxonomic studies by determination of polyphenol and anthocyanin profiles of grape extracts (Flamini & Tomasi, 2000). Several methods by spectrophotometric measurements have been developed to determine indexes related to the different class of polyphenols in the grape and wine and to their polymerization state; some of them are easy and fast to perform and are usually applied to monitor processes in the winemaking (Paronetto, 1977; Di Stefano, Cravero, & Gentilini, 1989; Di Stefano et al., 2000). IV. MASS SPECTROMETRY IN THE STUDY OF POLYPHENOLS AND PROCYANIDINS Gas Chromatography-Mass Spectrometry (GC-MS) has been applied in the field of grape and wine aroma since the seventies, but because of the low volatility of polyphenols, significant papers on their characterization in grape and wine by the use of this technique have not been found in the literature. To increase the volatility of these polar compounds, the sample derivatization must be performed, but often structure of derivatives can not be determined by GC/ MS. Their high molecular weight (MW) exceeds the mass range available for the most common GC/MS systems, thus making this approach ineffective. Moreover, derivatization leads to a more difficult interpretation of fragmentation patterns, also for simple procyanidin monomers with a C 15 (C 6 -C 3 -C 6 ) skeleton. Consequently, in the early investigations structural characterization of grape and wine polyphenols was usually performed by hydrolysis or thiolysis, steps and the subsequent identification of hydrolysis products by LC, spectrophotometric analysis, or thin layer chromatography (TLC) methods (Wulf & Nagel, 1978; Hebrero, Santos-Buelga, & Rivas-Gonzalo, 1988; Hebrero et al., 1989; Hong & Wrolstad, 1990b; Lee & Jaworski, 1990). One of the earlier studies by mass spectrometry on grape underivatized polyphenols was published in In that research Fast Atom Bombardment (FAB) (De Pauw, 1986; De Pauw, Agnello, & Derwa, 1991) in the positiveand negative-ion modes was used to perform analysis of grape extract samples with glycerol as matrix. The catechin-gallate (Fig. 4) (identification of ion [M H] at m/z 441, ions at m/z 151 and 137 as qualifiers), catechincatechin-gallate (Fig. 4) (ion [M H] at m/z 729, ion m/z 577 corresponding to the loss of gallic acid fragment) and gallocatechin-gallate (Fig. 4) (ion at m/z 460) were identified in extracts from Niagara Grapes (Lee & Jaworski, 1990). In the nineties, the development and the availability of effective Liquid Chromatography-Mass Spectrometry (LC-MS) and the Multiple Mass Spectrometry (MS/MS and MS n ) systems (Niessen & Tinke, 1995; de Hoffmann, 222

6 MS IN GRAPE AND WINE CHEMISTRY & 1996; Abian, 1999) supplied very useful tools to study the polyphenol structures as well as the mechanisms in which they are involved in winemaking and wine aging. In this frame, Cheynier et al. studied tannins (oligomers and polymers of flavan-3-ols, Fig. 4) in grape seed extracts by a simple LC-MS system equipped with an Electrospray Ionization (ESI) source (Fenn et al., 1990; Gaskell, 1997; Cole, 2000; Cooks & Caprioli, 2000) operated in the negative-ion mode and a quadrupole mass analyzer. They determined a series of peaks attributed to non-substituted procyanidins from trimers [detected as [M H] ] to hexadecamers [detected as [M 3H] 3 ] and their acylated derivatives that contained one, two, or three gallic acid residues (Cheynier et al., 1997). The mass spectrum of a grape-seed extract is reported in Figure 5. They also proposed the fragmentation of B-type and A-type dimers and trimers of catechin (structures reported in Fig. 6), previously studied by Karchesy et al. with FAB (Karchesy et al., 1986). The analysis of the collision data generated by increasing the orifice voltage showed two different fragmentation patterns for the two trimeric species that were detected. Collision spectra are reported in Figure 7 together with the related fragmentation patterns. The ion at m/z 863, corresponding to the trimer A-type, leads to the formation of two different dimeric ions at m/z 575 and 573, and undergoes the Retro-Diels-Alder (RDA) fragmentation process to produce the ions at m/z 711. Furthermore, the loss of a neutral fragment of 152 Da, corresponding to 3,4- dihydroxy-a-hydroxystyrene, and the formation of two fragments at m/z 285 and 289, generated by cleavage of the A-type interflavanic linkage, are also observed. A study of oligomeric and polymeric procyanidins (structures formed by linkage of (þ)-catechins and ( )- epicatechins units) present in grape seed extracts was reported a year later. Extracts were previously fractionated by gel chromatography, and the m/z values of deprotonated molecules [M H] were determined by FAB. The related spectra are reported in Figure 8 (de Freitas et al., 1998). Oligomeric (þ)-catechin, ( )-epicatechin, (þ)-catechin gallate, ( )-epicatechin gallate, up to decamers, were identified (m/z values between 290 and 3100 Da). The authors emphasized the advantages of FAB as a rapid technique that required only little amounts of sample for analysis and without derivatization. In that investigation, the MW of oligomeric procyanidins that contained up to seven catechin units were determined for the first time. Atmospheric-Pressure-Chemical-Ionization (APCI) (Wachs et al., 1991) and Electrospray-Ionization (ESI) techniques were used to study a series of low-molecular mass phenols and polyphenols present in wine, such as vanillin (5), syringic aldehyde (7), gallic acid (10), vanillic acid (11), caffeic acid (14), ferulic acid (15), p-coumaric acid (16), (þ)-catechin, ( )-epicatechin, ( )-epigallocatechin, ( )-epicatechin-3-o-gallate, and epigallocatechin- 3-O-gallate (see Figs. 2 and 4) (Pérez-Magariño et al., 1999). The investigation was performed at different cone voltages (60, 120, 180, and 210 V) in the positive- and negative-ion modes, and ESI was a particularly effective technique for the analysis of flavan-3-ols in both modes. In the negative-ion mode with a cone voltage of 60 V, the lowmolecular mass phenols were identified by the production of very abundant deprotonated molecules. The increase of cone voltage up to 120 V caused a reduction of the molecular species intensity, and the most abundant peaks were due to fragments that originated from the losses of carboxyl [M H-45], hydroxyl [M H-17], or/and aldehyde [M H-30] groups. A higher cone voltage leads to quite complex mass spectra, with many peaks due to either fragment ions or polymeric adducts. In particular, the latter phenomenon was observed for flavan-3-ols, due to their high self-polymerization capability. On the contrary of what was observed in ESI conditions, the APCI method exhibited a lower sensitivity and did not lead to relevant results in the positive- and negative-ion mode. Only operating in positive-ion mode (APIþ) with a cone voltage of 60 Vand injecting solutions that contained 4.5% formic acid, the protonated molecule ion, [M þ H] þ, of flavan-3-ols was obtained with a better FIGURE 5. ESI mass spectra (negative-ion mode) of grape seed extract. (Reprinted from Analusis Magazine 25, Cheynier et al., ESI-MS analysis of polyphenolic oligomers and polymers, p. 35, Copyright 1997, with permission from EDP Sciences.) 223

7 & FLAMINI FIGURE 6. Structures of B-type (a) and A-type (b) dimer of flavan-3- ols. (Reprinted from Journal of Agricultural and Food Chemistry 47, Lazarus et al., High-performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in foods and beverages, p. 3693, Copyright 1999, with permission from American Chemical Society.) sensitivity than that achieved in the negative-ion mode. However, because usually acid and non-acid compounds are both present in natural samples, and acid compounds are detected in low yield in the positive-ion mode, the negative-ion mode was proposed as more suitable for the analysis of the natural extracts of interest. In the same year, using a LC-ESI-quadrupole analyzer system that operated in the negative-ion mode, Fulcrand et al. (1999) characterized tannins of a Cabernet Sauvignon wine (vintage 1994). The dealcoholized wine sample was fractionated on a Fractogel column by elution with an ethanol/water/trifluoroacetic acid mixture. After thiolysis, fractions were analyzed, and deprotonated molecules of oligomers up to pentamers (based on flavanol units with trihydroxylated ß-ring, prodelphinidins), were identified; pentamers and larger oligomers were detected as doubly charged anions. Heptamer species corresponded to the highest mass detected. The results showed that condensed tannins present in wine consist of procyanidins, prodelphinidins, and polymers that contain di- and tri-hydroxylated flavanol units. Lazarus et al. (1999) studied proanthocyanidins from grape seeds extracts, in grape juice, and in the Pinot Noir wine. Differently from other authors, they performed LC/ MS analyses in normal phase chromatography with a silica column. The normal phase chromatography had previously showed to be the better method to obtain a satisfactory separation of proanthocyanidin oligomers on the basis of their MW. Analyses were performed in ESI conditions in the negative-ion mode with NH 4 þ OH as buffer. [M H], FIGURE 7. Mass spectra of A-type procyanidin trimers obtained by LC-ESI-MS in the negative-ion mode and related fragmentation patterns. (Reprinted from Analusis Magazine 25, Cheynier et al., ESI-MS analysis of polyphenolic oligomers and polymers, p. 34, Copyright 1997, with permission from EDP Sciences.) 224

8 MS IN GRAPE AND WINE CHEMISTRY & FIGURE 8. FAB spectrum of oligomeric and polymeric procyanidins in grape seed extracts. (Reprinted from Phytochemistry 49, de Freitas et al., Characterization of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry, p. 1438, Copyright 1998, with permission from Elsevier.) 225

9 & FLAMINI [M 2H] 2 and [M 3H] 3 ions of the different compounds were detected. An ion at m/z 881 was also present in the spectrum, and it was suggested to correspond to two different isobaric compounds: the dimer of epicatechingallate or the trimer epicatechin-epicatechin-epigallocatechin. Because a MS/MS system (by which to perform collisional experiments) was not available, the authors assigned this peak to the dimer on the basis of retention times. The ion at m/z 325 was identified as [M H] of feruloyltartaric acid (structure 17 in Fig. 2). Differently from the grape seeds extracts, galloylated oligomers were not observed in the red wine, so authors hypothesized that they are poorly released from the grape in the winemaking. The reaction between (þ)-catechin and glyoxylic acid in a model solution system was investigated by LC/ESI- MS-quadrupole analyzer system that operated in the positive- and negative-ion modes (Es-Safi et al., 2000a). Glyoxylic acid is formed in the wine by an oxidation of tartaric acid, and it has been demonstrated that it reacts with (þ)-catechin to give colorless compounds that consist of oligomeric molecules, where flavanol units are linked between them by carboxymethine groups (Fulcrand et al., 1997; Es-Safi et al., 2000b). Structures with one or two formyl groups in positions C-6, C-8 or C-6, and C-8 of (þ)- catechin ([M H] ions at m/z 317 and 345) and dimers formed by a two (þ)-catechin linkage through a methine group ([M H] ions m/z at 587) were identified. Methyl and ethyl esters of xanthylium compounds (m/z at 629 and 643) with adsorption maximum at 450 nm were also identified. The authors suggested that these compounds could play an important role in white wines and in grapederived food browning, and could be involved in red-wine aging. Some structures identified by Es-Safi et al. (2000b) are reported in Figure 9. A study on the characterization of proanthocynidins contained in the Leuconoselect TM commercial batch (from Vitis vinifera seeds) by the use of LC coupled with thermospray has been published in It was based on the fractionament over Sephadex 1 LH-20 resin column of Leuconoselect TM, and the analysis of fractions by the employment of a triple-quadrupole mass spectrometer that recorded positive ions from m/z 160 to 1200 (Gabetta et al., 2000). Signals that corresponded to the protonated molecule ions, [M þ H] þ, of catechin (m/z 291), epicatechin gallate (m/z 443), and flavan-3-ol dimers (m/z 579), and to cationized molecules [M þ Na] þ of flavan-3-ol dimers (m/z 601) and flavan-3-ol dimer galloylated (m/z 731) were identified (see Fig. 10). In that study an ESI method was also developed, and by this approach, [M þ H] þ ions of dimers, trimers, and tetramers of catechin (m/z 579, 867, 1155), their mono- and di-galloyl derivatives (m/z 731, 1019, 1307, 883, 1171, 1459), and the trigalloyl derivatives of trimers and tetramers (m/z 1323 and 1611) were easily found. Identification of [M þ H] þ ions of flavan-3-ols pentamers, hexamers, and heptamers (m/z 1443, 1731, 2019), of their monogalloyl FIGURE 9. Structures of the xanthylium salts (b) and with (þ)-catechin substituted at positions C-6, C-8 or C-6, and C-8 by formyl groups (a), identified in a model solution by LC/ESI-MS. These compounds could play an important role in white wine and grape-derived food browning, and in red wine aging. (Reprinted from Journal of Agricultural and Food Chemistry 48, Es-Safi et al., New phenolic compounds formed by evolution of (þ)-catechin and glyoxylic acid in hydroalcoholic solution and their implication in color changes of grape-derived foods, pp and 4236, Copyright 2000, with permission from American Chemical Society.) 226

10 MS IN GRAPE AND WINE CHEMISTRY & FIGURE 10. Identification of flavan-3-ols and flavan-3-ol dimers in the HPLC thermospray-ms profile of grape seeds extract: D, dimer; C, (þ)-catechin; E, ( )-epicatechin; DG, dimer gallate; EG, ( )-epicatechin 3-O-gallate. (Reprinted from Fitoterapia 71, Gabetta et al., Characterization of proanthocyanidins from grape seeds, p. 171, Copyright 2000, with permission from Elsevier.) derivatives (m/z 1595, 1883, 2171), of pentamers and hexamers digalloyl derivatives (m/z 1747 and 2035), of pentamers and hexamers trigalloyl derivatives (m/z 1899 and 2187) was also reported (see Fig. 11). In a recent study, ESI coupled with Fourier Transform Mass Spectrometry (Amster, 1996) was applied to determine polyphenolic fingerprints of five different wines on the basis of accurate mass values; the analysis was performed without any prior separation or purification step (Cooper & Marshall, 2001). This study was possible because of the high mass-resolving power (typically m/ Dm 50% 80000) and mass accuracy (1 ppm) typical of this technique. The method was ideal for the study of complex mixtures such as wine, because the accurate mass 227

11 & FLAMINI FIGURE 11. Positive ESI mass spectrum of a fraction of Leuconoselect TM after fractionation over a Sephadex LH-20 resin column. (Reprinted from Fitoterapia 71, Gabetta et al., Characterization of proanthocyanidins from grape seeds, p. 172, Copyright 2000, with permission from Elsevier.) of the different components are simultaneously determined to allow an immediate elemental composition assignment. The study was performed on California Red, Corbiere, Zinfandel, Beaujolais, and Sauvignon Blanc wines; the instrument was operated in the positive- and negative-ion modes. The positive-ion mass spectra were dominated by sucrose and anthocyanin signals, but more than 30 compounds such as anthocyanins, tannins, procyanidins, flavonols and flavanonols, HCTA, and carbohydrates were identified. Peaks that corresponded to protonated and cationized (K þ ) homodimers of catechin or epicatechin (theoretical mass of and Da, structure 21 in Fig. 12) and an ion at m/z (proposed to be a protonated heterodimer that contained one monohydroxydimethoxylated flavan-3-ol group and one trimethoxylated flavan-3-ol group) were determined in the positiveion mass spectra of red wines. Peaks that corresponded to protonated and cationized (K þ ) heterodimers that consisted of one dihydroxylated and one trihydroxylated flavan-3-ol units [M 1 þ M 2 þ H] þ and [M 1 þ M 2 þ K] þ (theoretical mass of and Da, respectively), and a protonated heterodimer that contained two hydroxyl and three methoxyl constituents (theoretical mass of Da), were identified in the Corbiere spectrum. One must emphasize that these species have been found in wine samples for the first time. Peaks of esculin sodiate (theoretical mass of Da, structure 22 in Fig. 12) and a series of glucosyl-p-coumaric acids that contained hydroxyl and methoxyl substituents at aromatic ring were also identified. As flavanonols and flavanols, peaks that corresponded to cationized (K þ ) 1-hydroxy flavanonol (taxifolin) glucoside ([M þ K] þ, theoretical mass of FIGURE 12. Structures of cationized (K þ ) homodimers of catechin or epicatechin (21), esculin sodiate (22), and cationized (K þ ) 1-hydroxy flavanonol (taxifolin) glucoside (23) identified in wine by ESI-FT-MS. (Reprinted from Journal of Agricultural and Food Chemistry 49, Cooper & Marshall, Electrospray ionization Fourier transform mass spectrometric analysis of wine, pp , Copyright 2001, with permission from American Chemical Society.) 228

12 MS IN GRAPE AND WINE CHEMISTRY & Da, structure 23 in Fig. 12), cationized (K þ )1- methoxy-2-hydroxy flavanonol glucoside (theoretical mass of Da), and cationized (K þ ) 2-methoxy-1- hydroxy flavonol glucoside (theoretical mass of Da) were evidenced. The negative-ion spectra showed peaks that corresponded to a large number of compounds, and showed far greater differences among different wines, with respect to either different components or the relative abundance of common ones. Consequently, the authors proposed that, for classification of wines by ESI FT-ICR, the negative-ion mode is preferable than the positive-ion one, as was wellevidenced by the results shown in Figure 13. V. MASS SPECTROMETRY IN THE STUDY OF ANTHOCYANINS AND THEIR DERIVATIVES The five common anthocyanins in the grape from Vitis vinifera are delphinidin (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn), and malvidin (Mv) present as 3-Omonoglucosides, 3-O-acetylmonoglucosides, and 3-O-(6- O-p-coumaroyl)monoglucosides. In the case of Mv, the 3-O-(6-O-caffeoyl)monoglucoside is also present (see Fig. 14). In the not Vitis vinifera grape, anthocyanins with a second glucose linked to the C-5 hydroxyl group may be present. Reversed-phase LC and detection at wavelength 520 nm is usually employed in the study of grape anthocyanin profile, and leads to results analogous to those reported in Figure 15 for a not Vitis vinifera extract. Of course, the peak assignment is based only on the retention time and the chromatographic sequence of the different components. To achieve more confident data, mass spectrometry seemed to be highly attractive in this theme. One of the earlier studies on grape anthocyanins by mass spectrometry was performed by Bakker & Timberlake (1985). In that research, skin extracts of 16 grape cultivars grown in the Douro Valley in Northern Portugal used for Port Wine production were analyzed by FAB-MS, and confirmed the compound identification obtained with LC. Molecular ions of the Dp, Pt, Pn, and Mv 3-O- FIGURE 13. Negative-ion (a) and positive-ion (b) ESI FT-ICR mass spectrum of five different wines. (Reprinted from Journal of Agricultural and Food Chemistry 49, Cooper & Marshall, Electrospray ionization Fourier transform mass spectrometric analysis of wine, pp , Copyright 2001, with permission from American Chemical Society.) 229

13 & FLAMINI FIGURE 14. Structure of grape anthocyanins. According to the chemical nature of the compounds, the glucose residue can be also linked to an acetyl, coumaroyl or caffeoyl group. monoglucosides, the Mv acetylglucoside, and the Mv p- coumaroylglucoside were easily identified (Bakker & Timberlake, 1985). Tamura et al. (1994) reported the use of LC-MS and continuous-flow fast atom bombardment (CF-FAB) to separate and identify anthocyanins in the Japanese grape Muscat Bailey A. For the analysis of grape extracts, reversed-phase chromatography was used to perform gradient elution by a water/acetonitrile/trifluoroacetic acid solvent mixed with a methanolic solution to which glycerol and dimethyl sulfoxide were added. The positive molecular ions of five Mv derivatives were detected, together with FIGURE 15. HPLC anthocyanin profile of the crude extract of hybrid grape Clinton (Vitis labrusca Vitis riparia) recorded at wavelength 520 nm. Compound identification is reported in Table 1. (Reprinted from American Journal of Enology and Viticulture 51:1, 2000, Favretto & Flamini, Application of electrospray ionization mass spectrometry to the study of grape anthocyanins, p. 57, with permission from the American Society for Enology and Viticolture, Copyright 2000.) 230

14 MS IN GRAPE AND WINE CHEMISTRY & signals of several fragments and the M þ of aglycone (m/z 331). Until a few years ago, FAB was the most common mass spectrometry technique used to achieve structural information on aglycone and sugar residue of anthocyanins. A disadvantage of this technique is that analyses must necessarily be preceded by purification and dissolution of sample in a polar matrix. In recent years, ESI has been shown to be suitable for the analysis of polar compounds in aqueous solution without any previous sample derivatization, and the relatively soft API method (atmospheric pressure, room temperature) provides information on the compounds without any interference of signals due to the formation of thermal degradation compounds. As consequence, several studies on grape and wine anthocyanins have been performed by these approaches. Baldi et al. (1995) used the LC/MS-API method to study grape anthocyanins from Vitis vinifera Sangiovese and Colorino varieties. In that study Dp, Cy, Pt, Pn, and Mv 3-O-monoglucosides, 3-O-acetylmonoglucosides, and p-coumaroylmonoglucosides, and Mv and Pn caffeoylmonoglucosides, were identified. Operating in the positive-ion mode and in acid medium (with 7% v/v formic acid), anthocyanins were detected as flavylium cations, M þ,to provide signals of high intensity. Spectra of 3-O-monoglucosides were characterized by M þ and [M 162] þ [loss of sugar residue by a rearrangement reaction, the ion labeled Y 0 þ by nomenclature of glycoconjugates introduced from Domon and Costello (Domon & Costello, 1988)] ions, those of 3-O-acetylmonoglucosides by M þ and [M-204] þ (loss of acetylglucose residue) ions, and those of p-coumaroylmonoglucosides by M þ and [M- 308] þ (loss of p-coumaroylglucose residue) ions. Peaks at m/z 655 and 625 were also present and were identified as the M þ ion of Mv and Pn caffeoylmonoglucosides, respectively (the latter co-eluting from the HPLC column with Mv 3-O-acetylmonoglucoside). After backgroundnoise suppression, the mass spectrum appeared to be welldefined, and the authors confirmed the Mv and Pn caffeoylmonoglucosides identification on the basis of isotopic peaks ( 13 C and 12 C) ratio. Ions [M þ H] þ of anthocyanins Dp and Pt 3,5-O-diglucosides (m/z at 627 and 641) were determined, and confirmed that, differently from the belief until that time, low levels of diglucoside anthocyanins may be present also in Vitis vinifera grapes. In 1999, a study on LC-MS methods for the detection and identification of anthocyanins in methanolic extracts of Tinto Fino and Cabernet Sauvignon (Vitis vinifera) grape skins and in Cabernet Sauvignon, Graciano, Garnacha, and Tinto Fino (young and 2-year bottle-aged) red wines, was published (Revilla et al., 1999). By an analysis of standard compounds in the positive- and negative-ion modes, the power of APCI and ESI was investigated. The former technique showed poor sensitivity, and the best conditions for analysis were achieved in the ESI positive mode, in acidified medium (10% v/v formic acid) with a cone voltage of 60 V. The authors emphasized that the behavior under negative- and positive-ion modes was similar, but in the former conditions a lower sensitivity was observed. In the LC-MS chromatogram of wines, other than the five anthocyanin monoglucosides and their acetyl and p-coumaroyl derivatives, a number of pigments formed by the aging reactions undergone by monomer anthocyanin were identified. They were vitisin A and vitisin B (the structures are reported in Fig. 19), a series of compounds that the authors proposed formed by a condensation between Dp, Pt, and Pn and pyruvic acid (pyruvic derivatives), and five other unrecognized pigments. For two components, the authors proposed structures that consisted of two dimers of Mv monoglucoside and catechin linked by an ethyl bridge. In the same year, anthocyanins in Concord (Vitis labrusca) grape juice extracts were characterized by ESI in the positive-ion mode and tandem mass spectrometry (MS- MS) (Giusti et al., 1999). Collision-induced dissociation experiments were performed on a triple quadrupole, using Ar as the target gas. Analyses were performed by the direct injection of samples into the mass spectrometer in either an aqueous or methanolic solution. With this approach, the five anthocyanins Dp, Cy, Pt, Pn, and Mv as 3-O-monoglucosides, p-coumaroylmonoglucosides, 3,5-O-diglucosides, and 3-(6-O-p-coumaroyl),5-O-diglucosides were easily identified. The authors emphasized that the collision energy strongly affected the relative abundance of diagnostic fragments, and that, for the acylated derivatives, any cleavage mechanism that involved the cleavage of an ester group was not observed. Favretto and Flamini developed an ESI-MS/MS method to gain structural and semi-quantitative information on grape anthocyanin contents. The anthocyanin composition of the two hybrid grape Clinton (Vitis labrusca Vitis riparia) and Isabella (Vitis vinifera Vitis Vitis labrusca) varieties (both characterized by the presence of a large number of different anthocyanins) and of the Vitis vinifera Cabernet Franc variety were studied (Favretto & Flamini, 2000). To develop a fast and lowsolvent consuming method, analyses were performed by direct injection in the ESI source of crude extract, previously purified by solid-phase-extraction (SPE), without performing LC separation. First, to obtain a valid MS n database to allow the unequivocal identification of the different compounds, the anthocyanin extract was fractionated by off-line LC semipreparative chromatography, and the structural characterization of each compound was obtained with multiple-step mass spectrometric (MS n ) analysis by ion trap. The crude extract was directly injected into the ESI source, to produce the spectrum shown in Figure 16. With this approach, the identification of the components reported in Table 1 was easily achieved. 231

15 & FLAMINI FIGURE 16. Positive-ion ESI mass spectrum of the crude extract of hybrid grape Clinton (Vitis labrusca Vitis riparia). Identification of compounds is reported in Table 1. (Reprinted from American Journal of Enology and Viticulture 51:1, 2000, Favretto & Flamini, Application of electrospray ionization mass spectrometry to the study of grape anthocyanins, p. 60, with permission from the American Society for Enology and Viticolture, Copyright 2000.) Isobaric compounds are present, but MS n was in general highly effective for their differentiation. The detection of fragment ions [M-162] þ (Y 0 þ ), [M-324] þ (due to consecutive losses of two sugar residues), [M-204] þ, [M-308] þ, and [M-470] þ (due to consecutive losses of p-coumaroylglucose and sugar residues) allowed the characterization of the five monoglucoside and diglucoside anthocyanins. However, an MS n experiment was not effective in the differentiation between two isobaric compounds Mv diglucoside and Mv caffeoylmonoglucoside, because of their identical molecular mass and the identical aglycone moiety. To achieve their characterization, deuterium-exchange experiments were successfully employed: samples were dissolved in deuterated water, and different mass shifts were observed in agreement with the different number of exchangeable, acidic protons present in the molecules (see Fig. 17). Finally, a semi-quantitative procedure to estimate the amounts and percentage of anthocyanin monoglucosides and diglucosides in the extracts was developed. The Mv- 3,5-O-diglucoside (m/z 655) and Mv-3-O-monoglucoside (m/z 493) were isolated from an extract, and calibration curves were calculated by the loop injection of standard solutions at different concentrations. The areas obtained by recording the time versus ion current for the molecular ion at m/z 655 and 493 in the SIM mode were measured, and the relative abundance of the M þ species in the ESI spectrum were used in order to obtain plots that represented the qualitative and semi-quantitative anthocyanin profile of cultivar. Asenstorfer et al. studied oligomeric wine pigments in grape marc extracts and 4-year-old wine from Vitis vinifera cv. Shiraz by ESI in the positive- and negative-ion modes. Analyses were performed by direct injection of sample in to the mass spectrometer and by HPLC separation (Asenstorfer, Hayasaka, & Jones, 2001). Before analysis, the isolation of compounds by cation-exchange chromatography, in the absence and presence of a bisulfite buffer, was performed. Bisulfite excess was used to take advantage of the resistance of the 4-substituted anthocyanins to form an anionic bisulfite adduct, and to allow their separation from other compounds. Structures of anthocyanidin C-4 substituted with vinyl group linked to the hydroxyl group at C-5 such as vitisin B (24), Mv-3-O-glucoside 4-vinylguaiacol (35), Mv 4-vinylphenol (31), also with acetyl or p- coumaroyl group linked to glucose, and a series of structures with vinyl malvidin linked to monomer, dimer and trimer of catechin (28), were proposed (see Fig. 18). The mass spectral data of the proposed pigments isolated from grape marc and wine are reported in Table 2. Recently, Hayasaka and Asenstorfer studied the evolution of wine pigments in a 3-year-old Shiraz wine by ESI- MS/MS and nanoelectrospray tandem mass spectrometry (Nano-ESI-MS/MS), on a triple-quadrupole mass spectro- 232

16 MS IN GRAPE AND WINE CHEMISTRY & TABLE 1. Fragmentation of the M þ of anthocyanin compounds Compounds present in the direct infusion ESI mass spectrum of Clinton extract obtained by multiple step mass spectrometric (MS n ) analysis. Reprinted from American Journal of Enology and Viticulture 51:1, 2000, Favretto & Flamini, Application of electrospray ionization mass spectrometry to the study of grape anthocyanins, p. 59, with permission from the American Society for Enology and Viticolture, Copyright meter (Hayasaka & Asenstorfer, 2002). The structures of anthocyanidin C-4 substituted with vinyl linkage previously proposed were confirmed, and new structures were also identified. The authors emphasized the advantages of the nano-esi-ms/ms technique over conventional ESI mass spectrometry, due to a higher sensitivity and smaller sample-size requirement (typically 1 ml). Sample solution is directly infused into the ESI source (without LC separation) and loaded into a glass capillary coated with gold used as nano-esi needle at a voltage of 700 V. Fragmentation patterns of aglycone cations were studied by in-source collisions. Aglycone cations of Mv always follow the same fragmentation pattern, regardless of the modification of the Mv moiety; a fingerprint of the five anthocyanins and their derivatives could be determined on the basis of neutral fragments. Structures of 10 vinyl aglycones of Mv, two of Pn and two of Pt were proposed, including Mv vinylmethyl, Mv vinylhydroxyl and Mv, Pt, and Pn vinylformic adducts. The former, together with the vinylmethyl (29) and vinylformic (25) derivatives of Mv-3-O-acetylglucoside, Pt-3-O-monoglucoside (26), and Pn-3-O-monoglucoside (27), were previously identified by LC-MS in grape skin extracts from Cabernet Franc and Carignane Vitis vinifera varieties (Benabdeljalil et al., 2000). Also, structures with Mv linked to 4-vinylcathecol (34) and to 4-vinylsyringol (36), and with Mv, Pt, and Pn 3-O-monoglucosides linked to 4-vinylphenol (31,32,33) were proposed. Mv-3-Omonoglucoside 4-vinylphenol, formed by condensation of Mv with the 4-vinylphenol produced by enzymatic decarboxylation of p-coumaric acid, was previously identified by Fulcrand et al., 1996a in wines from Carignane grape. Structures of anthocyanin derivatives identified are reported in Figure 18. The characterization and fragmentation patterns of vitisin A, vitisin B, and their 3-O-acetylglucoside derivatives were studied by Bakker and Timberlake by FAB-MS, and the authors proposed the structures reported in Figure 19 (Bakker & Timberlake, 1997; Bakker et al., 1997). Analyses were performed in the positive-ion mode with a glycerol matrix. The molecular ion (M þ ) of two vitisins at m/z 517 and 561 and of their acetyl derivatives (m/z 559 and 603, respectively) were identified, and were accompanied by ions due to the loss of a neutral fragment 233

17 & FLAMINI FIGURE 17. Positive-ion ESI mass spectrum of a pure sample of Mv-3-O-(6-O-caffeoyl) monoglucoside (above) and a pure sample of Mv-3,5-O-diglucoside (below) dissolved in D 2 O. (Reprinted from American Journal of Enology and Viticulture 51:1, 2000, Favretto & Flamini, Application of electrospray ionization mass spectrometry to the study of grape anthocyanins, p. 62, with permission from the American Society for Enology and Viticolture, Copyright 2000.) of mass 162 and 204, respectively. The two vitisins are distinguished by a higher resistance to color losses when adding SO 2, a greater color expression at higher ph values, and a stability higher than those exhibited by anthocyanin glucosides. The year after Fulcrand et al. (1998) isolated and characterized, in a model solution prepared with pigments extracted from V. vinifera Carignane grape, a pigment similar to vitisin A; data suggested that the condensation of Mv with pyruvic acid probably proceeding with the mechanism postulated from the authors reported in Figure 20. The Mv vinylformic adduct was studied in an LC/ESI- MS system operated in the negative-ion mode; the pseudomolecular ion [M þ 2H þ ] at m/z was identified. VI. MASS SPECTROMETRY IN THE STUDY OF STRUCTURES FORMED BY POLYMERIZATION OF ANTHOCYANINS AND FLAVAN-3-OLS The changes in the wine color that occur during aging were hypothesized to be related to the formation of products of 234

18 MS IN GRAPE AND WINE CHEMISTRY & FIGURE 18. Structures proposed of anthocyanins C-4 substituted in 4-years-old Shiraz wine. (Reprinted from Journal of Agricultural and Food Chemistry 50, Hayasaka & Asenstorfer, Screening for potential pigments derived from anthocyanins in red wine using nanoelectrospray tandem mass spectrometry, p. 757, Copyright 2002, with permission from American Chemical Society.) condensation between anthocyanins and flavan-3-ols. In the seventies, two different structural models were hypothesized: in the first, proposed by Somers, anthocyanin is linked by the C-4 position directly to the C-8 of flavan-3-ol (Somers, 1971); in the second, anthocyanin is linked to a flavan-3-ol molecule through an ethyl bridge (see Fig. 21). The latter model was proposed by Timberlake & Bridle (1976) after the observation that the addition of acetaldehyde to mixtures of anthocyanins and flavan-3-ols caused a rapid color augmentation with a shift toward the violet, and consequently the authors suggested the mechanism of polymerization between anthocyanins and flavan-3-ols promoted by acetaldehyde. Bakker, Picinelli, & Bridle (1993) studied, the anthocyanins present in the Touriga Nacional grape-skin extract with FAB mass spectrometry. They observed a peak at m/z 809, which could correspond to the molecular ion of a dimeric species formed by a linkage of Mv 3-O-monoglucoside with catechin through an acetaldehyde bridge of the type proposed by Timberlake & Bridle. In the study of the loss of astringency that was observed during the wine aging, and suspected as a result of TABLE 2. ESI-MS spectral data of pigments isolated from Shiraz marc and wine, and corresponding proposed compounds nd, not detected. Reprinted from Journal of Agricultural and Food Chemistry 49, Asenstorfer et al., Isolation and structures of oligomeric wine pigments by bisulfite-mediated ion-exchange chromatography, p. 5959, Copyright 2001, with permission from American Chemical Society. 235

19 & FLAMINI FIGURE 19. Structures of vitisin A (1) and vitisin B (2), and the isomeric structures of the flavylium forms. (Reprinted from Journal of Agricultural and Food Chemistry 45, Bakker & Timberlake, Isolation, identification, and characterization of new color-stable anthocyanins that occur in some red wines, p. 38, Copyright 1997, with permission from American Chemical Society.) the insolubilization of tannins by a reaction with acetaldehyde, the mechanism of polymerization between flavan-3- ols was evaluated (Fulcrand et al., 1996b). Ion-spray mass spectrometry in the negative-ion mode (LC-ISP-MS) was applied to study reactions in model wine solutions that contained (þ)-catechin or ( )-epicatechin and acetaldehyde, and the mechanism of polymerization reported in Figure 22 was proposed. In that study, ion spray (other than to take advantage of the soft sample ionization) permitted one to determine oligomers up than hexamers as singlecharged ions, and to characterize oligomers with similar chromophores that co-eluted from the LC column and that were undistinguishable by UV-Vis detection. The LC-ISP- MS approach was more suitable than FAB-MS: for FAB- MS, sample purification before analysis was required, and made it difficult to apply to these studies for the rate of polymerization and the instability of products. The year after the results obtained in the study of the evolution of polyphenols during aging of the Cabernet Sauvignon wine were published, evidence was reported of products formed by polymerization of two (þ)-catechins by an ethyl bridge (Saucier, Little, & Glories, 1997). The structure was characterized by ESI in the positive-ion mode by an application of rapid cone-voltage switching from 70 to 25 V to obtain alternate mass scans of fragmented (70 V) and non-fragmented (25 V) ions. In the low cone-voltage mode, only the molecular ion (m/z 607) was detected, whereas, at high cone-voltage, characteristic fragmentations were activated, with the formation of ions at m/z 317 and 291, which corresponded FIGURE 20. Mechanism postulated for the formation of Mv vinylformic derivate. (Reprinted from Phytochemistry 47, Fulcrand et al., A new class of wine pigments generated by reaction between pyruvic acid and grape anthocyanins, p.1406, Copyright 1998, with permission from Elsevier.) 236

20 MS IN GRAPE AND WINE CHEMISTRY & FIGURE 21. Structure formed by polymerization of anthocyanin and flavan-3-ol by an ethyl bridge; proposed by Timberlake & Bridle. (Reprinted from Journal of Agricultural and Food Chemistry 45, Francia- Aricha et al., New anthocyanin pigments formed after condensation with flavanols, p. 2263, Copyright 1997, with permission from American Chemical Society.) FIGURE 22. Mechanism of polymerization of flavan-3-ols induced by acetaldehyde in model wine solution; proposed by Fulcrand et al. (Reprinted from Journal of Chromatography A 752, Fulcrand et al., Study of the acetaldehyde induced polymerization of flavan-3-ols by liquid chromatography-ion spray mass spectrometry, p. 89, Copyright 1996, with permission from Elsevier.) to vinyl-catechin and catechin fragments, respectively (see Fig. 23). The same year Cheynier et al. (1997), by ESI in the positive- and negative-ion modes, confirmed the identification of the signal at m/z 809 as a product of polymerization between epicatechin and Mv 3-O-monoglucoside by an ethyl bridge. Signals at m/z 605 and 921 of the [M H] ions extracted from the total ion chromatogram (obtained in the negative-ion mode) of a red wine sample were attributed to ethyl-linked catechin dimers and trimers even on the basis of their retention times. In another paper Francia-Aricha et al. (1997) studied with LC/MS in the positive-ion mode the pigments formed during aging in model wine solutions that contained Mv monoglucoside, acetaldehyde, and procyanidin B2 (dimer of ( )-epicatechin) isolated from grape seeds. A peak at m/z 1097, corresponding to the two different enantiomers formed by an epicatechin dimer linked to Mv monoglucoside by an ethyl bridge, and a peak at m/z 1093, assigned to pigment B2-III (Mv-3-O-glucoside-vinylcatechin-epicatechin), were determined. Similar pigments were found in solutions that contained (þ)-catechin, ( )-epicatechin, or procyanidin B3 (dimer of (þ)-catechin). With LC-MS, a study on copigmentation (hydrophobic association of an anthocyanin chromophore with the planar electronically unsatured part of a copigment) in the wine was also performed. The transformations that occurred in hydroalcoholic synthetic solutions that contained Mv-3-Oglucoside as pigment and ( )-epicatechin as copigment were investigated, and the influence of acetaldehyde on the formation of covalent compounds was evaluated (Mirabel et al., 1999). Operating in the positive-ion mode, peaks at m/z 809, m/z 783 (probably corresponding to the molecular ion M þ of compound formed by direct linkage of Mv-3-Oglucoside with flavan-3-ol), and m/z 621 and 469 (corresponding to fragments formed by glucose residue loss and the consecutive RDA) were detected. The fragmentation pattern of molecular ion m/z 783 and the structure proposed for the molecular ion at m/z 809 are reported in Figure 24. Also, a peak at m/z 453 was present, and the authors hypothesized that it corresponded to either a possible yellow-orange product formed by Mv oxidation or a copigment flavonol-anthocyanin complex. A peak at m/z 809 was found also in non-acetaldehyde solutions confirming that acetaldehyde is formed by ethanol oxidation. Remy et al. (2000) studied the molecular species formed by anthocyanin-tannin condensation in solutions from 2-year-old Cabernet Sauvignon wine obtained after fractionament on a Toypearl TSK gel HW-50 (F) column. Thiolysis of the polymeric fractions was performed, and products were analyzed by LC-API in the negative-ion mode. Compounds were detected as their [M H] deprotonated molecules, or [M þ 2H] in the case of 237

21 & FLAMINI FIGURE 23. ESI positive-ion mode mass spectrum of the compound reported in Figure 22. In low cone voltage mode (25 V) molecular ion at m/z 607 is detected; in high cone voltage mode (70 V) the formation of characteristic fragments at m/z 291 and m/z 317 is activated. (Reprinted from American Journal of Enology and Viticulture 48:3, 1997, Saucier et al., First evidence of acetaldehyde-flavanol condensation products in red wine, p. 371, with permission from the American Society for Enology and Viticolture, Copyright 1997.) flavylium. Thiolysis experiments confirmed the presence of a structure with position C-6 or C-8 of Mv-3-Omonoglucoside linked to C-4 of flavan-3-ol to form a dimer of sequence T-A type (A, anthocyanin; T, tannin) (see Fig. 25a). The formation of this compound follows a mechanism that involves the nucleophilic attack of an aromatic ring of Mv to the carbonium ion of flavan-3-ol liberated by acidic cleavage of a proanthocyanidin interflavanic bond. Also, the structure previously hypothesized from Somers was confirmed by this study, and the results indicated that acetaldehyde-mediated condensation is not a prevailing mechanism involved in the transformation of tannin in wine (Somers, 1971). LC/MS Analyses also revealed the presence of an [M H] species at m/z 781 that corresponded to two different chromatographic peaks that were tentatively attributed to a flavene or a bicyclic structure that originated from the direct reaction of flavonol position C-6 or C-8 with the C-4 of Mv-3-O-monoglucoside with the formation of a A-T type dimer (see Fig. 25b). The presence of those structures implies a reaction mechanism, where the flavilium ion is subjected to a nucleophilic attack from the aromatic ring of flavan-3-ol; however, these structures were not confirmed by thiolysis experiments. Finally, the authors reported that structures T- A and A-T appeared to be predominantly associated with lower-mw flavanols. In 4-year-old Shiraz wines, Asenstorfer et al. identified a series of structures, where Mv-3-O-monoglucoside, Mv- 3-O-acetylglucoside and Mv-3-O-p-coumaroylglucoside are linked to monomer, dimer and trimer of catechin by a vinyl group, to produce the compounds that are reported in Table 2. The presence of these types of structures in Shiraz wines was also confirmed 1 year later by Hayasaka and 238

22 MS IN GRAPE AND WINE CHEMISTRY & FIGURE 24. ESI-MS positive-ion mode fragmentation pattern proposed for molecular ion at m/z 783. Below, the structure proposed for compound corresponding to molecular ion at m/z 809. (Reprinted from American Journal of Enology and Viticulture 50:2, 1999, Mirabel et al., Copigmentation in model wine solutions: occurrence and relation to wine aging, p. 217, with permission from the American Society for Enology and Viticolture, Copyright 1999.) FIGURE 25. a: structure determined in 2-year-old Cabernet Sauvignon wine by LC-API negative-ion mode, and confirmed by thiolysis experiments with an equilibrium between the hydrated and flavylium forms (dimer T-A type: A, anthocyanin; T, tannin). b: structures proposed for the signal at m/z 781 (dimer A- T type). (Reprinted from Journal of the Science of Food and Agriculture 80, Remy et al., First confirmation in red wine of products resulting from direct anthocyanin-tannin reactions, p. 748, Copyright 2000, with permission of John Wiley & Sons Limited.) 239

23 & FLAMINI Asenstorfer, with the identification of Mv-3-O-glucoside vinylcatechin (Asenstorfer, Hayasaka, & Jones, 2001; Hayasaka & Asenstorfer, 2002). VII. MASS SPECTROMETRY AND GRAPE AND WINE RESVERATROL Resveratrol (3,5,4 0 -trihydroxystilbene) is a polyphenol that is mostly present in red grapes and wines as cis-and transisomers in the free or glycosidically bonded form (Fig. 26). In the last 10 years, it has become an important qualitative parameter because of the several beneficial effects on human health that were revealed by recent studies: anticancer activity, cardioprotection, antioxidant activity, inhibition of platelet aggregation, and anti-inflammatory activity (Frankel, Waterhouse, & Kinsella, 1993; Bertelli et al., 1995; Pace-Asciak et al., 1995; Jang et al., 1997; Fremont, Belguendou, & Delpal, 1999; Hung et al., 2000). The total resveratrol content in a wine may be higher than 30 mg/l (Paronetto & Mattivi, 1999). As a consequence, the interest for analytical applications to determine resveratrol in grape and wine was increased, and several studies have been done to develop suitable analytical methods, some of which are based on mass spectrometry. One of the first applications of mass spectrometry to determine resveratrol in grape juice and wine was based on GC-MS, and the method permitted one to determine simultaneously cis- and trans-isomers (Soleas et al., 1995). Preparation of samples by SPE with C 18 cartridges, and derivatization by bis-[trimethylsilyl]-trifluoroacetamide (BSTFA), was performed. For analyses, a DB-5HT (5% phenyl, 95% methyl) column was used. The electron ionization (EI) 70 ev fragmentation spectra of tristrimethylsilyl trans-resveratrol derivative is reported in Figure 27. For quantitative evaluation, the signal at m/z 444 was employed for SIM analysis, and under those conditions, a limit of detection of ca. 10 mg/l was obtained. For comparative evaluation, it is to be emphasized that LC methods, which require sample preparation by multiple solvent extractions, exhibit a limit of detection between 1 and 50 mg/l. Wines from different zones of Canada were analyzed, and samples from Ontario showed higher contents of cis- and trans-isomers. Because the cis-resveratrol FIGURE 26. Trans-(a) and cis-(b) isomers of resveratrol. was not detected in grape skins or juices, the formation of this isomer by isomerization of the trans, or by a breakdown of resveratrol polymers during fermentation, has been hypothesized. The hydroxylated stilbene contents of a number of commercial American red wines were determined by GC- MS by performing sample preparation and analyses under conditions that were similar to those reported in the previous paper (Lamikanra et al., 1996). Six stilbenols (cisand trans-resveratrol and others mono-, di-, tri-, and tetrahydroxystilbenes) in wines from different grape varieties were determined as trimethylsilyl derivatives. Tris-[trimethylsilyl] stilbene was still quantified on the basis of the ion at m/z 444. Standards of mono-, di-, and tetrahydroxystilbene were not available, and their structural characterization was based on their molecular ions and fragmentation patterns. Ions at m/z 268 (tentatively identified as monohydroxystilbene), m/z 356 (tentatively identified as dihydroxystilbene), and m/z 532 (tentatively identified as tetrahydroxystilbene) were employed for SIM. Retention times were compared to those of cis- and trans-resveratrol, and compounds proposed as dihydroxy and tetrahydroxy stilbenes were eluted from the column before and after cis- and trans-resveratrol, respectively. Higher levels of cis isomer (between 0.1 and 31.9 ppm) with respect to the trans were registered; the latter isomer was absent in some samples. The concentrations of resveratrol in wine from Vitis rotundifolia (muscardina) grape were higher than those present in wine from Vitis vinifera and Vitis labruscana (Concord) grape. A year later, four different methods, two based on LC and two on GC-MS, to assay cis- and trans-resveratrol in wine samples were compared in an analysis of 169 different wines (Soleas et al., 1997). The two LC methods used, respectively, a normal phase (isocratic elution) and a reversed-phase (gradient elution) chromatography with spectrophotometric detection at 306 nm. The two GC-MS methods (flow charts of Fig. 28) both required a previous sample preparation with SPE on a C 18 cartridge: the first analysis was direct injection of sample on a DB-17 HT (diphenyl dimethyl polysiloxane) column, and recording signals in the SIM mode at m/z 228 (M þ ), m/z 227 ([M H] þ ), and m/z 229 ( 13 C-isotope containing species) as qualifiers; the second was the derivatization of sample with BSTFA, and recording signals at m/z 444 of the molecular ion, and m/z 445 and m/z 446 ( 30 Si-isotope containing species) as qualifiers. By direct injection (i.e., without derivatization), higher levels of trans-resveratrol were found, this result was, at least in part, attributed to the thermal decomposition of resveratrol glucosides. On the contrary, the method that implyed the derivatization procedure showed a tendency to overestimate the cis-resveratrol and underestimate the trans, possibly as a consequence of the trans-to-cis 240

24 MS IN GRAPE AND WINE CHEMISTRY & FIGURE 27. GC-MS electron ionization (70 ev) mass spectrum of tris-trimethylsilyl trans-resveratrol. isomerization that occurred during the derivatization. Results of analysis of 90 different wines with and without previous sample derivatization with BSTFA are compared in Figure 29. The greater advantage of GC methods was the very shorter analysis time (ca. 10 min), whereas the LC methods required 48 min in the normal phase and 33 min in the reversed phase. A method based on solid-phase-micro-extraction (SPME) for the trace analysis of trans-resveratrol in wine has been recently developed in order to eliminate the laborious and time-consuming SPE procedure for the sample pretreatment (Luan, Li, & Zhang, 2000). To perform GC- MS analysis, the sample was derivatized with BSTFA. After sampling by immersion of the SPME fiber (polar FIGURE 28. Flow-chart of the two GC-MS methods that were compared by Soleas et al. (1997) to determine cis- and trans-resveratrol in the wine: (a) direct injection; (b) derivatization with BSTFA. 241

25 & FLAMINI FIGURE 29. Comparison of wine analysis between direct injection GC-MS and BSTFA-derivatization GC-MS. A: cis-resveratrol; (B) trans-resveratrol; (C) total resveratrol. Ninety red wines were analyzed. (Reprinted from American Journal of Enology and Viticulture 48:2, 1997, Soleas et al., Comparative evaluation of four methods for assay of cis- and trans-resveratrol, p. 170, with permission from the American Society for Enology and Viticolture, Copyright 1997.) 242

26 MS IN GRAPE AND WINE CHEMISTRY & FIGURE 30. Negative-ion ESI mass spectra of trans-resveratrol recorded at a low cone-voltage (25 35 V). (Reprinted from Rapid Communications in Mass Spectrometry, Gamoh & Nakashima, Liquid chromatography/mass spectrometric determination of trans-resveratrol in wine using a tandem solid-phase extraction method, p. 1114, Copyright 1999, with permission of John Wiley & Sons Limited.) polyacrilate, 85 mm thickness) directly into the wine, the derivatization step was performed in gas phase by exposing the fiber in the head space of silylating agent, so as to avoid its hydrolysis. Analyses were performed in the SIM mode, by recording the signal at m/z 444; a linear response over a concentration range of 10 ng/l to 1 mg/l with a detection limit of 5 ng/l (about 2000-times lower than that reported by Soleas et al. for the SPE method), were obtained. Results confirmed the wider presence of the trans-isomer in grape with respect the cis-isomer, where their concentrations were similar in wines. In 1999, a method to determine trans-resveratrol in wine with ESI LC-MS in the negative-ion mode (capillary voltage 4 kv) was developed (Gamoh & Nakashima, 1999). The chromatography was performed on a C 18 column with a CH 3 OH/ammonium acetate 20 mm (ph 5.5) (55:45 v/v) eluent. For quantitative analysis, the SIM method on the ion at m/z 227, corresponding to the [M H] ion of resveratrol, was used. At low cone voltage (25 35 V), the molecular species (m/z 227) predominates, and at higher cone-voltage values (45 55 V), diagnostic fragment ions are observed (see Fig. 30). Sample preparation was performed by reversed-phase SPE, and trihydroxyflavone was used as the internal standard due to its high ionization efficiency in ESI conditions and its hydrophobic properties comparable with those of resveratrol. The best condition of cone voltage for analysis was 35 V, and the SIM detection limit was 200 pg injected on-column (volume injected 10 ml of a 20 mg/l solution, signal/noise ratio 3). Recently, Wang et al. (2002) studied by LC-MS the cisand trans-resveratrol contents of grape juice and wines from Vitis vinifera Negroamaro. The glucoside resveratrol fraction was determined in the grape by an extraction with methanol, and in the wine by an extraction with ethyl acetate. Enzymatic hydrolysis of extracts was performed to liberate aglycones, and APCI in the positive-ion mode was used. The reversed-phase chromatography was performed with CH 3 OH/HCOOH as eluent; the formic acid was added to the mobile phase to suppress an on-column ionic dissociation of resveratrol due to the acidic character of the hydroxyl groups. SIM of the [M þ H] þ ion (m/z 229) was used for quantitative determination. Cis- and trans-isomers were determined with a limit of detection of 70 pg injected on-column with a signal/noise ratio of 10. VIII. APPLICATION OF MALDI IN THE STUDY OF POLYPHENOLS The first application of MALDI in wine chemistry was by Szilágyi et al. (1996) and was devoted to the study of wine protein. Only more recently, some studies on grape polyphenols with matrix-assisted-laser-desorption-ionizationtime-of-flight (MALDI-TOF) (Karas & Bahr, 1991; Sundqvist, 1992; Busch, 1995; Guilhaus, 1995; Gluckmann & Karas, 1999; Karas, Gluckmann, & Shafer, 2000) mass spectrometry have been published. The MALDI-TOF technique has advantages over other mass spectrometric systems, such as FAB, in sensitivity and mass range, and produces only a single ionization event (Krueger et al., 2000). Furthermore, it is highly tolerant toward contaminants, and is thus particularly suitable for the direct analysis of complex mixtures (Yang & Chien, 2000). Sugui et al. (1999) studied by MALDI-TOF the anthocyanic profile of methanol extracts from five different 243

27 & FLAMINI French hybrid grape varieties: Chancellor, Marechal Foch, MN 1095, Cynthiana and Concord. A system with a nitrogen laser that operated at 337 nm, and an accelerating voltage 28 kv, was used, and a-cyano-4-hydroxycinnamic acid (CCA) was employed as the matrix. The five anthocyanins Dp, Cy, Pt, Pn, and Mv as 3-O-monoglucosides, acetylmonoglucosides, p-coumaroylmonoglucosides, 3,5- O-diglucosides, and p-coumaroyldiglucosides were easily identified. The MALDI spectra of five grape extracts are reported in Figure 31. FIGURE 31. MALDI anthocyanic profile of methanolic extract of five red grape varieties: (A) Chancellor; (B) Marechal Foch; (C) MN 1095; (D) Cynthiana; (E) Concord. (Reprinted from American Journal of Enology and Viticulture 50:2, 1999, Sugui et al., Matrix-assisted laser desorption ionization mass spectrometry analysis of grape anthocyanins, p. 201, with permission from the American Society for Enology and Viticolture, Copyright 1999.) 244

28 MS IN GRAPE AND WINE CHEMISTRY & For the considerable laser shot-to-laser-shot variability, the average value of relative ion abundance was calculated for three different MALDI analyses. Simple MALDI-MS cannot differentiate isobaric compounds; in particular, Mv-3-O-caffeoylmonoglucoside and Mv diglucoside could not be distinguished, and only after HPLC analysis could the absence of the former be verified. A qualitative and quantitative MALDI-TOF study of anthocyanins in red wine and fruit juice was reported in the same year (Wang & Sporns, 1999). Analyses of Merlot, Pinot Noir, Zinfandel, and Cabernet Sauvignon wines and of Concord grape juice anthocyanins were performed by the co-crystallization of extracts with 2,4,6-trihydroxyacetophenone (THAP) in acetone. The THAP matrix produced the best spot-to-spot reproducibility. The system was operated in the linear-mode and positive-ion modes. With this approach, the five anthocyanin 3-O-monoglucosides and their derivatives were identified, and quantification was performed with cyanidin 3-rutinoside as internal standard. In the quantitative analysis of monoglucoside anthocyanins, the responses were only slightly different; however, for diglucosides and anthocyanin with a second carbohydrate moiety to a 3-glucoside (e.g., cyanidin 3- rutinoside), the relative molar response was only ca. onefourth of that noted for monoglucosides. MALDI-MS responses were linear for groups of chemically similar anthocyanins, and experiments showed that the internal standard addition had no effect on the relative responses of the other anthocyanins. The MALDI-TOF technique has also been applied to characterize polygalloyl polyflavan-3-ols (PGPF) in grape seed extracts dissolved in a trans-3-indolacrylic acid (IAA) matrix (Krueger et al., 2000). Ions that corresponded to a series of PGPF units up to nonamers were observed in the positive-ion reflectron mode, and ions up to undecamers were observed in the positive-ion linear mode, also with lower resolution. The highest galloylation degree found was six, and sodium adducts [M þ Na] þ were revealed in the reflectron and linear modes. The positive-ion reflectron mode permitted one to identify a series of compounds with MWs two mass units lower than those of the abovedescribed compounds, and that corresponded to polycatechins A-type (see Fig. 6). On the basis of the galloylated structures, an equation was developed to predict the mass distribution of PGPF in grape seed extracts: 290 þ 288c þ 152g þ 23, where 290 is the MW of the terminal catechin/epicatechin unit, c is the degree of polymerization, g is the number of galloyl esters, and 23 is the atomic mass of sodium. With this equation, an easy description of the MS data can be achieved. In the same year, Yang and Chien published a study, where LC/MS and MALDI-TOF (operating in the reflectron and linear modes) were applied to characterize procyanidins in grape seeds extracts, with a critical comparison of the results achieved by two techniques (Yang & Chien, 2000). LC/MS analyses were performed with normal-phase and reversed-phase columns, with APCI in the negative-ion mode. MALDI-TOF analyses were performed with 2,5-dihydroxybenzoic acid (DHB) or trans-3- indoleacrylic acid (IAA) as the matrix. LC-MS methods did not permit the separation and identification of oligomers higher than pentamers because, as the MW increases, the number of diastereomers becomes so large that separation of individual isomers becomes impossible. On the other hand, MALDI-TOF in the positive-ion reflectron mode was a rapid method of analysis that allowed one to determine oligomers that contained (þ)-catechin, ( )-epicatechin, and their galloylated derivatives up to heptamers with resolution higher than This resolution allowed the separation of individual ions of different isotopic composition: for example, the ion at m/z was further resolved into a group of four peaks, as shown in the expanded view of the spectrum in Figure 32. The application of MALDI-TOF MS in the linear mode permitted one to detect oligomers up to nonamers as sodium adducts. The lower sensitivity of the reflectron mode for the large ions is reasonably due to their collisionally induced decompositions that occur in the flight path. The authors reported that DHB as a matrix leads to the best analytical conditions for the detection of procyanidins in reflectron mode to provide the broadest mass range with the least background noise. IX. MASS SPECTROMETRY APPLIED TO THE STUDY OF WINE POLYPHENOLS FROM CORK BOTTLE STOPPERS AND OAK BARRELS The several compounds released in to the wine from the wood materials used in the wine storage and aging provide an important contribution to the organoleptic characteristics of the final product. Between them, a number of simple phenols and polyphenols were identified, in particular by GC-MS, and some of these studies are discussed here. The volatile compounds released in to the wine from the different types of oak woods that are used in the construction of barrels that are used for the aging of wine and brandy have been studied by a number of researchers. GC- MS analysis of alcoholic wood extracts identified different classes of compounds such as ketones, aldehydes, esters and lactones, alcohols, furanic compounds, fat acids, and phenols. Phenols such as vanillin, syringol and syringaldehyde, coniferyl alcohol and aldehyde (compounds 5, 7, and 8 in Fig. 2), guaiacol (37), 4-methylguaiacol (38), 4- ethylguaiacol (39), 4-vinylguaiacol (40), eugenol (41), isoeugenol (42), acetovanillone (43), and cresols were identified. In particular, vanillin provides an important 245

29 & FLAMINI FIGURE 32. Positive-ion MALDI-MS mass spectrum of grape seed extract (DHB matrix, reflectron mode). (Reprinted from Journal of Agricultural and Food Chemistry 48, Yang & Chien, Characterization of grape procyanidins using high-performance liquid chromatography/mass spectrometry and matrix-assisted laser desorption time-of-flight mass spectrometry, p. 3993, Copyright 2000, with permission from American Chemical Society.) contribution to the aroma of barrel-aged wine. Structures of those compounds are reported in Figure 33. Those compounds are released in different amounts, depending on the heat treatment of the wood (Cutzach et al., 1997; Moio et al., 1999; Pérez-Coello, Sanz, & Cabezudo, 1999; Cerdán, Goñi, & Azpilicueta, 2002). Galletti, Bocchini, & Antonelli (1996) characterized the structural polymers of cork bottle stoppers by pyrolysis/ GC/MS. Pyrolysis at 6008C for 5 s in an inert atmosphere induced the thermal fission of cork, and the phenolic macromolecules released from lignin and suberin were analyzed by GC-MS. X. CONCLUSIONS Mass spectrometry has a very important role for research and quality control in the viticulture and enology field. After the first applications by GC-MS on grape and wine aroma and FAB for polyphenol characterization, LC-MS FIGURE 33. Some phenol compounds that were present in the wine and released from barrel-oak wood: (37) guaiacol; (38) 4-methylguaiacol; (39) 4-ethylguaiacol; (40) 4-vinylguaiacol; (41) eugenol; (42) isoeugenol; (43) acetovanillone. The Vineyards of Cartizze Zone in Northern Italy are held in high esteem for the production of Prosecco sparkling wines (by permission of the Author Dr. S. Cancellier.) 246