Maktub II - bivir

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

Riccardo Flamini*Istituto Sperimentale per la Viticoltura, Viale XXVIII Aprile 26, I-31015Conegliano (TV), Italy

Received 11 February 2003; revised 28 April 2003; accepted 28 April 2003

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

II. Quality Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

III. Grape and Wine Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

IV. Mass Spectrometry in the Study of Polyphenols and Procyanidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

V. Mass Spectrometry in the Study of Anthocyanins and their Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

VI. Mass Spectrometry in the Study of Structures Formed by Polymerization of Anthocyanins and Flavan-3-ols . . . . . 234

VII. Mass Spectrometry and Grape and Wine Resveratrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

VIII. Application of MALDI in the Study of Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

IX. Mass Spectrometry Applied to the Study of Wine Polyphenols from Cork Bottle Stoppers and Oak Barrels . . . . . 245

X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Mass spectrometry, had and still has, a very important role forresearch and quality control in the viticulture and enology field,and its analytical power is relevant for structural studies onaroma and polyphenolic compounds. Polyphenols are respon-sible for the taste and color of wine, and confer astringency andstructure to the beverage. The knowledge of the anthocyanicstructure is very important to predict the aging attitude ofwine, and to attempt to resolve problems about color stability.Moreover, polyphenols are the main compounds related to thebenefits of wine consumption in the diet, because of theirproperties in the treatment of circulatory disorders such ascapillary fragility, peripheral chronic venous insufficiency, andmicroangiopathy of the retina. Liquid Chromatography-MassSpectrometry (LC-MS) techniques are nowadays the bestanalytical approach to study polyphenols in grape extractsand wine, and are the most effective tool in the study of thestructure of anthocyanins. The MS/MS approach is a verypowerful tool that permits anthocyanin aglycone and sugarmoiety characterization. LC-MS allows the characterization of

complex structures of grape polyphenols, such as procyanidins,proanthocyanidins, prodelphinidins, and tannins, and providesexperimental evidence for structures that were previously onlyhypothesized. The matrix-assisted-laser-desorption-ionization-time-of-flight (MALDI-TOF) technique is suitable to determinethe presence of molecules of higher molecular weight with highaccuracy, and it has been applied with success to studyprocyanidin oligomers up to heptamers in the reflectron mode,and up to nonamers in the linear mode. The levels ofresveratrol in wine, an important polyphenol well-known for itsbeneficial effects, have been determined by SPME and LC-MS,and the former approach led to the best results in terms ofsensitivity. # 2003 Wiley Periodicals, Inc., Mass Spec Rev22:218–250, 2003; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10.1002/mas.10052Keywords: grape and wine polyphenols; anthocyanins;procyanidins and tannins; resveratrol; mass spectrometry;liquid chromatography; MALDI

I. INTRODUCTION

On the basis of data reported from the Office InternationalDe la Vigne et Du Vin (O.I.V.)—The State of Vitivini-culture in the World and Statistical Information in 1999—

Mass Spectrometry Reviews, 2003, 22, 218– 250# 2003 by Wiley Periodicals, Inc.

————*Correspondence to: Riccardo Flamini, Istituto Sperimentale per la

Viticoltura, Viale XXVIII Aprile 26, I-31015 Conegliano (TV), Italy.

E-mail: [email protected]

in the years 1990–1999 a large increase of productionand consumption of table grape was registered; that in-crease was also encouraged by the amply demonstratedbeneficial effects of this food on the human health. Also, theworldwide wine production registered a sensible increase,favored by the suppression of measures that encouraged thepermanent uprooting implemented in recent years byEuropean Union, and by recent plantations in certainnumber of non-European countries with potentially highyields (Dutruc-Rosset, 1999).

Trends of grape and wine worldwide production ofthese years are reported the histograms of Figure 1a,b. Theparticular increase in 1999 can be observed.

The worldwide market has been influenced by therepercussions on the media of seminars and scientificsymposia in the field of research, medicine, toxicology offoods, and human health. In particular, the beneficialeffects of moderate wine consumption on certain cate-gories of diseases, such as cardiovascular diseases, braindegeneration from aging, and certain carcinogenic dis-eases, have been discussed. The report of O.I.V. alsoindicates raisins as an interesting means by which to fightagainst hunger in the world.

As a consequence of these trends, efforts of the largestgrape- and wine-producer countries (Argentina, France,Italy, South Africa, Spain, United States) are addressed toimprove the product quality, rather than to increase pro-duction, so as to remain competitive on emergent countriesby the growth of systematic positioning in the marketniches of ‘‘premium’’ and ‘‘super premium’’ wines. Themain efforts of researchers and Organisms of Control areaddressed to develop new methods to detect the productorigin (Ogrinc et al., 2001), the detection of adulterationinvolving sugar-beet, cane sugar, or ethanol addition andwatering (Guillou et al., 2001), the protection of consumerhealth through determination of food contaminants such asheavy metals, toxins, and pesticides (Szpunar et al., 1998;

MacDonald et al., 1999; Wong & Halverson, 1999), and thestudy of plant metabolism and diseases (Perez, Viani, &Retamales, 2000; Tabacchi et al., 2000).

For the improvement of product quality, aroma andpolyphenolic compounds have been widely studied. Thenumerous classes of grape polyphenols transferred to thewine are responsible for the taste and color of beverage. Inthis review, the important role of mass spectrometry in thestudy of grape and wine polyphenols is discussed—a fieldwhere a rapid increase of knowledge has been observed,due also to the development of the new technologiesintroduced in the recent years.

II. QUALITY IMPROVEMENT

To improve the final product—the wine—the research inviticulture is addressed to improve the quality of the grapethrough the study of grape-ripening, which involvescultural techniques, the selection of clones and varietiesof best potentiality (clonal selection), and the study ofenvironmental influence on the vineyard (zoning). In theenology field, main efforts are devoted to optimizeindustrial processes finalized to obtain products withpeculiar characteristics. In this frame, (i) the inoculum ofselected yeast permits regular fermentation with minimumsecondary processes by other microorganisms, (ii) the useof selected enzymes leads to a better extraction of grapecomponents, (iii) the maceration of grape skins isperformed in controlled conditions of temperature andatmosphere, (iv) malolactic fermentation is employed toimprove organoleptic characteristics and to confer micro-biological stability to the wine, and, finally, (v) the barrel-and bottle-aging refine the final product. To reach theproposed aims and to be able to estimate the potentiality ofstarting material and how it can be transferred to the finalproduct, a good knowledge of grape chemistry is essential.

FIGURE 1. Trends of total world-wide wine (a) and grape productions (b) for the period 1991–1999.

MS IN GRAPE AND WINE CHEMISTRY &

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To define characteristics and identity of product,the research in viticulture attempts to determine originparameters. For variety characterization (chemotaxonomicclassification), DNA, amphelography, isoenzymes, andsecondary metabolites of plant are studied (Costacurtaet al., 2001).

Secondary metabolites in the grape are compounds(terpenes and terpenols, methoxypyrazines, volatile sulfurcompounds, benzenoids, norisoprenoids, and polyphenols)mainly linked to the variety but not indispensable for theplant survivor, also whether environmental and climaticvariables can influence their contents in the fruit (DiStefano, 1996; Flamini, Dalla Vedova, & Calo, 2001).

III. GRAPE AND WINE POLYPHENOLS

In the winemaking, grape compounds are transferred to themust and to the wine, which contain several polyphenols atdifferent degree of polymerization. The simplest com-pounds are mono-, di-, and tri-phenols [phenol, pyroca-techol (1), resorcinol (2), hydroquinone (3), phloroglucinol(4)], phenolic aldehydes such as vanillin (5), p-hydro-xybenzaldehyde (6), syringic aldehyde (7), coniferylaldehyde (8), benzoic acids such as gentisic acid (9), gallicacid (10), vanillic acid (11), salicilic acid (12), and syringicacid (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 tartaricacid (hydroxycinnamoyltartaric acids HCTA) (17) arepresent in grape and wine in considerable amounts. In orderto give to the reader a general view of the chemistry in-volved in this context, the structure of these molecules arereported in Figure 2.

More-complex grape polyphenols contain two or morearomatic rings (cumarines, benzopyrones, and flaviliumions) to form flavanols (18), flavonols (19), and anthocya-nins (20) (Macheix, Fleuriet, & Billot, 1990) (see Fig. 3).

These molecules are present in the grape mainly in themonoglycoside form, with the sugar residue linked to thehydroxyl 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 withanthocyanins (in red wines); they, together with oxidationproducts of tannins, are in the main responsible for thecolor of white grapes and wines (Cheynier & Rigaud, 1986;Usseglio-Tomasset, 1995). Anthocyanins contain in theirskeleton the benzopyrilium ion as base molecule, which isresponsible for the color of red berry varieties and redwines. They are present in the grape as mono- or di-glucosides, depending on variety, with the second glucosemolecule 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.

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or linked between them to form procyanidins, proantho-cyanidins and tannins of type reported in Figure 4.

With grape pressing, polyphenols are released in themust from the different parts of berry: HCTA, phenolicacids, and aldehydes from juice and pulp; HCTA, phenolicacids, anthocyanins, procyanidins and proanthocyanidins,and flavonols from skin; and tannins, procyanidins and

proanthocyanidins, gallic acid, catechin, and epicatechinfrom seeds (Figs. 2 and 4). Moreover, these moleculescan undergo condensation and polymerization processesduring the winemaking and wine aging, to produce newstructures.

Polyphenols play an important role in the organo-leptic 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.)


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astringency and structure to the beverage by formation ofcomplexes with the proteins of saliva. Their knowledge isvery important to predict the aging attitude of wine, and toattempt to resolve problems about color stability—in parti-cular in the case of premium red wines that are destined tolong aging periods (e.g., Brunello di Montalcino, Chianti,Barolo).

Polyphenols are the principal compounds related tothe benefits of wine consuming in the diet because of theproperties attributed to them. Procyanidins and proantho-cyanidins from Vitis vinifera seeds are used as activeingredients in medicinal products for the treatment ofcirculatory disorders such as capillary fragility, peripheralchronic venous insufficiency, and microangiopathy of theretina. Pharmacological properties of selected proantho-cyanidins from grape seeds (LeucoselectTM) are related toan increase of tonicity and resistance of capillary walls,as well as to radical scavenging and inhibition of super-oxide ion formation. Proanthocyanidins supplementationin the rat, made the heart less susceptible to the ischemia/reperfusion damage, and increased the total antioxidantplasma capacity and the ascorbic acid plasma level (MaffeiFacino et al., 1996, 1998). Additionally, proanthocyanidinsfrom grape seeds decreased the susceptibility of healthycells to toxic and carcinogenic agents. Phenolic com-pounds of the grape have been associated with cardiovas-cular benefits, a reduction of platelet aggregation, and amodulation of eicosanoid synthesis. Recently, the antiox-idant activity of phenolic compounds from 12 differentvarieties of grape toward human low-density lipoprotein(LDL) in vitro has been evaluated (Maffei Facino et al.,1994; Frankel, Waterhouse, & Teissedre, 1995; Bagchiet al., 1997; Meyer et al., 1997; Waterhouse & Walzem,1997; Joshi et al., 1998; Schramm et al., 1998; MaffeiFacino et al., 1999). The flavonol quercetin blocked theaggregation of human platelets by ADP and thrombin, andthis compound has gained considerable prominence as aninhibitor of carcinogens and of cancer cell growth in manyexperimental and human tumors (Goldberg et al., 1998).

Furthermore, the anthocyanin profile is a useful tool tocharacterize and to determine the origin of products, andin the identification of possible adulterations. For example,in some countries the production and commercializationof wine from not Vitis vinifera grape are prohibited.Hybrid grapes are characterized by peculiar anthocyanin3,5-O-diglucoside contents, which are practically absent ingrapes from Vitis vinifera, and these compounds can beconsequently employed for the identification of possibleadulterations. Finally, anthocyanins from grape are alsoimportant in the synthetic colorants market, in particular inthe food industry (Hong & Wrolstad, 1990a).

Traditional methods to determine polyphenols andanthocyanins in natural extracts are usually performed byliquid chromatography (LC) analysis and spectrophoto-

metric measurements. The LC methods are mainly appliedfor chemotaxonomic studies by determination of poly-phenol and anthocyanin profiles of grape extracts (Flamini& Tomasi, 2000). Several methods by spectrophotometricmeasurements have been developed to determine indexesrelated to the different class of polyphenols in the grape andwine and to their polymerization state; some of them areeasy and fast to perform and are usually applied to monitorprocesses in the winemaking (Paronetto, 1977; Di Stefano,Cravero, & Gentilini, 1989; Di Stefano et al., 2000).

IV. MASS SPECTROMETRY IN THE STUDY OFPOLYPHENOLS AND PROCYANIDINS

Gas Chromatography-Mass Spectrometry (GC-MS) hasbeen applied in the field of grape and wine aroma since theseventies, but because of the low volatility of polyphenols,significant papers on their characterization in grape andwine by the use of this technique have not been found in theliterature. To increase the volatility of these polar com-pounds, the sample derivatization must be performed, butoften structure of derivatives can not be determined by GC/MS. Their high molecular weight (MW) exceeds the massrange available for the most common GC/MS systems, thusmaking this approach ineffective. Moreover, derivatizationleads to a more difficult interpretation of fragmentationpatterns, also for simple procyanidin monomers with a C15

(C6-C3-C6) skeleton. Consequently, in the early investiga-tions structural characterization of grape and wine poly-phenols was usually performed by hydrolysis or thiolysis,steps and the subsequent identification of hydrolysis pro-ducts by LC, spectrophotometric analysis, or thin layerchromatography (TLC) methods (Wulf & Nagel, 1978;Hebrero, Santos-Buelga, & Rivas-Gonzalo, 1988; Hebreroet al., 1989; Hong & Wrolstad, 1990b; Lee & Jaworski,1990).

One of the earlier studies by mass spectrometry ongrape underivatized polyphenols was published in 1990. Inthat research Fast Atom Bombardment (FAB) (De Pauw,1986; De Pauw, Agnello, & Derwa, 1991) in the positive-and negative-ion modes was used to perform analysisof grape extract samples with glycerol as matrix. Thecatechin-gallate (Fig. 4) (identification of ion [M�H]� atm/z 441, ions at m/z 151 and 137 as qualifiers), catechin-catechin-gallate (Fig. 4) (ion [M�H]� at m/z 729, ion m/z577 corresponding to the loss of gallic acid fragment) andgallocatechin-gallate (Fig. 4) (ion at m/z 460) were identi-fied in extracts from Niagara Grapes (Lee & Jaworski,1990).

In the nineties, the development and the availability ofeffective Liquid Chromatography-Mass Spectrometry(LC-MS) and the Multiple Mass Spectrometry (MS/MSand MSn) systems (Niessen & Tinke, 1995; de Hoffmann,

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1996; Abian, 1999) supplied very useful tools to study thepolyphenol structures as well as the mechanisms in whichthey are involved in winemaking and wine aging. In thisframe, Cheynier et al. studied tannins (oligomers andpolymers of flavan-3-ols, Fig. 4) in grape seed extracts bya simple LC-MS system equipped with an ElectrosprayIonization (ESI) source (Fenn et al., 1990; Gaskell, 1997;Cole, 2000; Cooks & Caprioli, 2000) operated in thenegative-ion mode and a quadrupole mass analyzer. Theydetermined a series of peaks attributed to non-substitutedprocyanidins from trimers [detected as [M�H]�] tohexadecamers [detected as [M� 3H]3�] and their acylatedderivatives that contained one, two, or three gallic acidresidues (Cheynier et al., 1997). The mass spectrum of agrape-seed extract is reported in Figure 5. They alsoproposed the fragmentation of B-type and A-type dimersand trimers of catechin (structures reported in Fig. 6),previously studied by Karchesy et al. with FAB (Karchesyet al., 1986).

The analysis of the collision data generated by in-creasing the orifice voltage showed two different fragmen-tation patterns for the two trimeric species that weredetected. Collision spectra are reported in Figure 7 togetherwith the related fragmentation patterns. The ion at m/z 863,corresponding to the trimer A-type, leads to the formationof two different ‘‘dimeric’’ ions at m/z 575 and 573, andundergoes the Retro-Diels-Alder (RDA) fragmentationprocess to produce the ions at m/z 711. Furthermore, theloss of a neutral fragment of 152 Da, corresponding to 3,4-dihydroxy-a-hydroxystyrene, and the formation of twofragments at m/z 285 and 289, generated by cleavage of theA-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 wasreported a year later. Extracts were previously fractionatedby gel chromatography, and them/z values of deprotonatedmolecules [M�H]� were determined by FAB. The relatedspectra are reported in Figure 8 (de Freitas et al., 1998).Oligomeric (þ)-catechin, (�)-epicatechin, (þ)-catechin

gallate, (�)-epicatechin gallate, up to decamers, wereidentified (m/z values between 290 and 3100 Da).

The authors emphasized the advantages of FAB as arapid technique that required only little amounts of samplefor analysis and without derivatization. In that in-vestigation, the MW of oligomeric procyanidins that con-tained up to seven catechin units were determined for thefirst time.

Atmospheric-Pressure-Chemical-Ionization (APCI)(Wachs et al., 1991) and Electrospray-Ionization (ESI)techniques were used to study a series of low-molecularmass phenols and polyphenols present in wine, such asvanillin (5), syringic aldehyde (7), gallic acid (10), vanillicacid (11), caffeic acid (14), ferulic acid (15), p-coumaricacid (16), (þ)-catechin, (�)-epicatechin, (�)-epigalloca-techin, (�)-epicatechin-3-O-gallate, and epigallocatechin-3-O-gallate (see Figs. 2 and 4) (Perez-Magarino et al.,1999). The investigation was performed at different conevoltages (60, 120, 180, and 210 V) in the positive- andnegative-ion modes, and ESI was a particularly effectivetechnique for the analysis of flavan-3-ols in both modes. Inthe negative-ion mode with a cone voltage of 60 V, the low-molecular mass phenols were identified by the productionof very abundant deprotonated molecules. The increase ofcone voltage up to 120 V caused a reduction of the mole-cular species intensity, and the most abundant peaks weredue 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 quitecomplex mass spectra, with many peaks due to eitherfragment ions or polymeric adducts. In particular, the latterphenomenon was observed for flavan-3-ols, due to theirhigh self-polymerization capability.

On the contrary of what was observed in ESIconditions, the APCI method exhibited a lower sensitivityand did not lead to relevant results in the positive- andnegative-ion mode. Only operating in positive-ion mode(APIþ) with a cone voltage of 60 Vand injecting solutionsthat contained 4.5% formic acid, the protonated moleculeion, [MþH]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.)


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sensitivity than that achieved in the negative-ion mode.However, because usually acid and non-acid compoundsare both present in natural samples, and acid compoundsare detected in low yield in the positive-ion mode, thenegative-ion mode was proposed as more suitable for theanalysis of the natural extracts of interest.

In the same year, using a LC-ESI-quadrupole analyzersystem that operated in the negative-ion mode, Fulcrandet al. (1999) characterized tannins of a Cabernet Sauvignonwine (vintage 1994). The dealcoholized wine sample wasfractionated on a Fractogel column by elution with anethanol/water/trifluoroacetic acid mixture. After thiolysis,fractions were analyzed, and deprotonated molecules ofoligomers up to pentamers (based on flavanol units withtrihydroxylated ß-ring, prodelphinidins), were identified;pentamers and larger oligomers were detected as doublycharged anions. Heptamer species corresponded to thehighest mass detected. The results showed that condensedtannins present in wine consist of procyanidins, prodel-phinidins, and polymers that contain di- and tri-hydro-xylated flavanol units.

Lazarus et al. (1999) studied proanthocyanidins fromgrape seeds extracts, in grape juice, and in the Pinot Noirwine. Differently from other authors, they performed LC/MS analyses in normal phase chromatography with a silicacolumn. The normal phase chromatography had previouslyshowed to be the better method to obtain a satisfactoryseparation of proanthocyanidin oligomers on the basis oftheir MW. Analyses were performed in ESI conditions inthe negative-ion mode with NH4

þOH� 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.)

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 spectro-

metry analysis of proanthocyanidins in foods and beverages, p. 3693,

Copyright 1999, with permission from American Chemical Society.)

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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.)


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[M� 2H]2� and [M� 3H]3� ions of the different com-pounds were detected. An ion atm/z 881 was also present inthe spectrum, and it was suggested to correspond to twodifferent isobaric compounds: the dimer of epicatechin-gallate or the trimer epicatechin-epicatechin-epigalloca-techin. Because a MS/MS system (by which to performcollisional experiments) was not available, the authorsassigned this peak to the dimer on the basis of retentiontimes. The ion at m/z 325 was identified as [M�H]� offeruloyltartaric acid (structure 17 in Fig. 2). Differentlyfrom the grape seeds extracts, galloylated oligomers werenot observed in the red wine, so authors hypothesized thatthey are poorly released from the grape in the winemaking.

The reaction between (þ)-catechin and glyoxylic acidin a model solution system was investigated by LC/ESI-MS-quadrupole analyzer system that operated in thepositive- and negative-ion modes (Es-Safi et al., 2000a).Glyoxylic acid is formed in the wine by an oxidation oftartaric acid, and it has been demonstrated that it reacts with(þ)-catechin to give colorless compounds that consist ofoligomeric molecules, where flavanol units are linkedbetween them by carboxymethine groups (Fulcrand et al.,1997; Es-Safi et al., 2000b). Structures with one or twoformyl 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 dimersformed by a two (þ)-catechin linkage through a methinegroup ([M�H]� ions m/z at 587) were identified. Methyland ethyl esters of xanthylium compounds (m/z at 629 and

643) with adsorption maximum at 450 nm were alsoidentified. The authors suggested that these compoundscould play an important role in white wines and in grape-derived food browning, and could be involved in red-wineaging. Some structures identified by Es-Safi et al. (2000b)are reported in Figure 9.

A study on the characterization of proanthocynidinscontained in the LeuconoselectTM commercial batch (fromVitis vinifera seeds) by the use of LC coupled withthermospray has been published in 2000. It was based onthe fractionament over Sephadex1 LH-20 resin column ofLeuconoselectTM, and the analysis of fractions by theemployment of a triple-quadrupole mass spectrometer thatrecorded positive ions fromm/z 160 to 1200 (Gabetta et al.,2000). Signals that corresponded to the protonatedmolecule ions, [MþH]þ, of catechin (m/z 291), epica-techin gallate (m/z 443), and flavan-3-ol dimers (m/z 579),and to cationized molecules [MþNa]þ of flavan-3-oldimers (m/z 601) and flavan-3-ol dimer galloylated (m/z731) were identified (see Fig. 10).

In that study an ESI method was also developed, and bythis approach, [MþH]þ ions of dimers, trimers, andtetramers of catechin (m/z 579, 867, 1155), their mono- anddi-galloyl derivatives (m/z 731, 1019, 1307, 883, 1171,1459), and the trigalloyl derivatives of trimers and tetra-mers (m/z 1323 and 1611) were easily found. Identificationof [MþH]þ ions of flavan-3-ols pentamers, hexamers, andheptamers (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. 4235 and 4236, Copyright 2000, with permission from American

Chemical Society.)

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derivatives (m/z 1595, 1883, 2171), of pentamers andhexamers digalloyl derivatives (m/z 1747 and 2035), ofpentamers and hexamers trigalloyl derivatives (m/z 1899and 2187) was also reported (see Fig. 11).

In a recent study, ESI coupled with Fourier TransformMass Spectrometry (Amster, 1996) was applied to deter-mine polyphenolic fingerprints of five different wines on

the basis of accurate mass values; the analysis was per-formed without any prior separation or purification step(Cooper & Marshall, 2001). This study was possiblebecause of the high mass-resolving power (typically m/Dm50%� 80000) and mass accuracy (�1 ppm) typical ofthis technique. The method was ideal for the study ofcomplex mixtures such as wine, because the accurate mass

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.)


227

of the different components are simultaneously determinedto allow an immediate elemental composition assignment.The study was performed on California Red, Corbiere,Zinfandel, Beaujolais, and Sauvignon Blanc wines; theinstrument was operated in the positive- and negative-ionmodes. The positive-ion mass spectra were dominated bysucrose and anthocyanin signals, but more than 30compounds such as anthocyanins, tannins, procyanidins,flavonols and flavanonols, HCTA, and carbohydrates wereidentified. Peaks that corresponded to protonated andcationized (Kþ) homodimers of catechin or epicatechin(theoretical mass of 579.1498 and 617.1057 Da, structure21 in Fig. 12) and an ion at m/z 621.1967 (proposed to be aprotonated heterodimer that contained one monohydrox-ydimethoxylated flavan-3-ol group and one trimethoxy-lated flavan-3-ol group) were determined in the positive-ion mass spectra of red wines. Peaks that corresponded toprotonated and cationized (Kþ) heterodimers that con-sisted of one dihydroxylated and one trihydroxylatedflavan-3-ol units [M1þM2þH]þ and [M1þM2þK]þ

(theoretical mass of 595.1446 and 633.1005 Da, respec-tively), and a protonated heterodimer that contained twohydroxyl and three methoxyl constituents (theoretical massof 637.1916 Da), were identified in the Corbiere spectrum.One must emphasize that these species have been found inwine samples for the first time. Peaks of esculin sodiate

(theoretical mass of 363.0687 Da, structure 22 in Fig. 12)and a series of glucosyl-p-coumaric acids that containedhydroxyl and methoxyl substituents at aromatic ring werealso identified. As flavanonols and flavanols, peaks thatcorresponded to cationized (Kþ) 1-hydroxy flavanonol(taxifolin) glucoside ([MþK]þ, theoretical mass of

FIGURE 11. Positive ESI mass spectrum of a fraction of LeuconoselectTM 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.)

FIGURE 12. Structures of cationized (Kþ) homodimers of catechin or

epicatechin (21), esculin sodiate (22), and cationized (Kþ) 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 spectro-

metric analysis of wine, pp. 5710–5718, Copyright 2001, with

permission from American Chemical Society.)

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489.0794 Da, structure 23 in Fig. 12), cationized (Kþ) 1-methoxy-2-hydroxy flavanonol glucoside (theoretical massof 535.0849 Da), and cationized (Kþ) 2-methoxy-1-hydroxy flavonol glucoside (theoretical mass of 547.0849Da) were evidenced.

The negative-ion spectra showed peaks that corre-sponded to a large number of compounds, and showed fargreater differences among different wines, with respect toeither different components or the relative abundance ofcommon ones. Consequently, the authors proposed that, forclassification of wines by ESI FT-ICR, the negative-ionmode is preferable than the positive-ion one, as was well-evidenced by the results shown in Figure 13.

V. MASS SPECTROMETRY IN THE STUDY OFANTHOCYANINS AND THEIR DERIVATIVES

The five common anthocyanins in the grape from Vitisvinifera are delphinidin (Dp), cyanidin (Cy), petunidin(Pt), peonidin (Pn), and malvidin (Mv)—present as 3-O-

monoglucosides, 3-O-acetylmonoglucosides, and 3-O-(6-O-p-coumaroyl)monoglucosides. In the case of Mv, the3-O-(6-O-caffeoyl)monoglucoside is also present (seeFig. 14). In the not Vitis vinifera grape, anthocyanins witha second glucose linked to the C-5 hydroxyl group may bepresent.

Reversed-phase LC and detection at wavelength520 nm is usually employed in the study of grape antho-cyanin profile, and leads to results analogous to thosereported in Figure 15 for a not Vitis vinifera extract. Ofcourse, the peak assignment is based only on the retentiontime and the chromatographic sequence of the differentcomponents. To achieve more confident data, mass spectro-metry seemed to be highly attractive in this theme.

One of the earlier studies on grape anthocyanins bymass spectrometry was performed by Bakker & Timber-lake (1985). In that research, skin extracts of 16 grapecultivars grown in the Douro Valley in Northern Portugalused for Port Wine production were analyzed by FAB-MS,and confirmed the compound identification obtainedwith 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. 5710–5718, Copyright 2001, with

permission from American Chemical Society.)


229

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 andcontinuous-flow fast atom bombardment (CF-FAB) toseparate and identify anthocyanins in the Japanese grapeMuscat Bailey A. For the analysis of grape extracts,reversed-phase chromatography was used to performgradient elution by a water/acetonitrile/trifluoroacetic acidsolvent mixed with a methanolic solution to which glyceroland dimethyl sulfoxide were added. The positive molecularions of five Mv derivatives were detected, together with

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.

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.)

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signals of several fragments and the Mþ of aglycone(m/z 331).

Until a few years ago, FAB was the most common massspectrometry technique used to achieve structural informa-tion on aglycone and sugar residue of anthocyanins. Adisadvantage of this technique is that analyses must neces-sarily be preceded by purification and dissolution of samplein a polar matrix. In recent years, ESI has been shown to besuitable for the analysis of polar compounds in aqueoussolution without any previous sample derivatization, andthe relatively soft API method (atmospheric pressure,room temperature) provides information on the com-pounds without any interference of signals due to theformation of thermal degradation compounds. As conse-quence, several studies on grape and wine anthocyaninshave been performed by these approaches.

Baldi et al. (1995) used the LC/MS-API method tostudy grape anthocyanins from Vitis vinifera Sangioveseand Colorino varieties. In that study Dp, Cy, Pt, Pn, andMv 3-O-monoglucosides, 3-O-acetylmonoglucosides, andp-coumaroylmonoglucosides, and Mv and Pn caffeoylmo-noglucosides, were identified. Operating in the positive-ionmode and in acid medium (with 7% v/v formic acid),anthocyanins were detected as flavylium cations, Mþ, toprovide signals of high intensity. Spectra of 3-O-mono-glucosides were characterized by Mþ and [M� 162]þ [lossof sugar residue by a rearrangement reaction, the ionlabeled Y0

þ by nomenclature of glycoconjugates intro-duced from Domon and Costello (Domon & Costello,1988)] ions, those of 3-O-acetylmonoglucosides by Mþ

and [M-204]þ (loss of acetylglucose residue) ions, andthose of p-coumaroylmonoglucosides by Mþ and [M-308]þ (loss of p-coumaroylglucose residue) ions. Peaksat m/z 655 and 625 were also present and were identifiedas the Mþ ion of Mv and Pn caffeoylmonoglucosides,respectively (the latter co-eluting from the HPLC columnwith Mv 3-O-acetylmonoglucoside). After background-noise suppression, the mass spectrum appeared to be well-defined, and the authors confirmed the Mv and Pncaffeoylmonoglucosides identification on the basis of iso-topic peaks (13C and 12C) ratio. Ions [MþH]þ of antho-cyanins Dp and Pt 3,5-O-diglucosides (m/z at 627 and 641)were determined, and confirmed that, differently from thebelief until that time, low levels of diglucoside antho-cyanins may be present also in Vitis vinifera grapes.

In 1999, a study on LC-MS methods for the detectionand identification of anthocyanins in methanolic extracts ofTinto Fino and Cabernet Sauvignon (Vitis vinifera) grapeskins and in Cabernet Sauvignon, Graciano, Garnacha, andTinto Fino (young and 2-year bottle-aged) red wines, waspublished (Revilla et al., 1999). By an analysis of standardcompounds in the positive- and negative-ion modes, thepower of APCI and ESI was investigated. The formertechnique showed poor sensitivity, and the best conditions

for analysis were achieved in the ESI positive mode, inacidified medium (10% v/v formic acid) with a conevoltageof 60 V. The authors emphasized that the behavior undernegative- and positive-ion modes was similar, but in theformer conditions a lower sensitivity was observed. In theLC-MS chromatogram of wines, other than the five antho-cyanin monoglucosides and their acetyl and p-coumaroylderivatives, a number of pigments formed by the agingreactions undergone by monomer anthocyanin were identi-fied. They were vitisin A and vitisin B (the structures arereported in Fig. 19), a series of compounds that the authorsproposed formed by a condensation between Dp, Pt, and Pnand pyruvic acid (pyruvic derivatives), and five otherunrecognized pigments. For two components, the authorsproposed structures that consisted of two dimers of Mvmonoglucoside and catechin linked by an ethyl bridge.

In the same year, anthocyanins in Concord (Vitislabrusca) grape juice extracts were characterized by ESI inthe positive-ion mode and tandem mass spectrometry (MS-MS) (Giusti et al., 1999). Collision-induced dissociationexperiments were performed on a triple quadrupole, usingAr as the target gas. Analyses were performed by the directinjection of samples into the mass spectrometer in either anaqueous or methanolic solution. With this approach, thefive anthocyanins Dp, Cy, Pt, Pn, and Mv as 3-O-mono-glucosides, p-coumaroylmonoglucosides, 3,5-O-digluco-sides, and 3-(6-O-p-coumaroyl),5-O-diglucosides wereeasily identified. The authors emphasized that the collisionenergy strongly affected the relative abundance of diagno-stic fragments, and that, for the acylated derivatives, anycleavage mechanism that involved the cleavage of an estergroup was not observed.

Favretto and Flamini developed an ESI-MS/MSmethod to gain structural and semi-quantitative informa-tion on grape anthocyanin contents. The anthocyanincomposition of the two hybrid grape Clinton (Vitislabrusca�Vitis riparia) and Isabella (Vitis vinifera�VitisVitis labrusca) varieties (both characterized by the pre-sence of a large number of different anthocyanins) and ofthe Vitis vinifera Cabernet Franc variety were studied(Favretto & Flamini, 2000). To develop a fast and low-solvent consuming method, analyses were performed bydirect injection in the ESI source of crude extract, pre-viously purified by solid-phase-extraction (SPE), withoutperforming LC separation. First, to obtain a valid MSn

database to allow the unequivocal identification of thedifferent compounds, the anthocyanin extract was fractio-nated by off-line LC semipreparative chromatography, andthe structural characterization of each compound was ob-tained with multiple-step mass spectrometric (MSn) analy-sis by ion trap. The crude extract was directly injected intothe ESI source, to produce the spectrum shown in Figure 16.With this approach, the identification of the componentsreported in Table 1 was easily achieved.


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Isobaric compounds are present, but MSn was ingeneral highly effective for their differentiation. The detec-tion of fragment ions [M-162]þ (Y0

þ), [M-324]þ (dueto consecutive losses of two sugar residues), [M-204]þ,[M-308]þ, and [M-470]þ (due to consecutive losses ofp-coumaroylglucose and sugar residues) allowed thecharacterization of the five monoglucoside and diglucosideanthocyanins. However, an MSn experiment was not effec-tive in the differentiation between two isobaric com-pounds Mv diglucoside and Mv caffeoylmonoglucoside,because of their identical molecular mass and the identi-cal aglycone moiety. To achieve their characterization,deuterium-exchange experiments were successfully em-ployed: samples were dissolved in deuterated water, anddifferent mass shifts were observed in agreement with thedifferent number of exchangeable, acidic protons presentin the molecules (see Fig. 17).

Finally, a semi-quantitative procedure to estimate theamounts and percentage of anthocyanin monoglucosidesand 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 calibrationcurves were calculated by the loop injection of standardsolutions at different concentrations. The areas obtained byrecording the time versus ion current for the molecular ionatm/z 655 and 493 in the SIM mode were measured, and therelative abundance of the Mþ species in the ESI spectrum

were used in order to obtain plots that represented thequalitative and semi-quantitative anthocyanin profile ofcultivar.

Asenstorfer et al. studied oligomeric wine pigments ingrape marc extracts and 4-year-old wine fromVitis viniferacv. Shiraz by ESI in the positive- and negative-ion modes.Analyses were performed by direct injection of sample into the mass spectrometer and by HPLC separation(Asenstorfer, Hayasaka, & Jones, 2001). Before analysis,the isolation of compounds by cation-exchange chromato-graphy, in the absence and presence of a bisulfite buffer,was performed. Bisulfite excess was used to take advantageof the resistance of the 4-substituted anthocyanins to forman anionic bisulfite adduct, and to allow their separationfrom other compounds. Structures of anthocyanidin C-4substituted with vinyl group linked to the hydroxyl group atC-5 such as vitisin B (24), Mv-3-O-glucoside 4-vinyl-guaiacol (35), Mv 4-vinylphenol (31), also with acetyl or p-coumaroyl group linked to glucose, and a series of struc-tures with vinyl malvidin linked to monomer, dimer andtrimer of catechin (28), were proposed (see Fig. 18). Themass spectral data of the proposed pigments isolated fromgrape marc and wine are reported in Table 2.

Recently, Hayasaka and Asenstorfer studied the evolu-tion 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-

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.)

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meter (Hayasaka & Asenstorfer, 2002). The structures ofanthocyanidin C-4 substituted with vinyl linkage pre-viously proposed were confirmed, and new structures werealso identified. The authors emphasized the advantages ofthe nano-ESI-MS/MS technique over conventional ESImass spectrometry, due to a higher sensitivity and smallersample-size requirement (typically 1 mL). Sample solutionis directly infused into the ESI source (without LC separa-tion) and loaded into a glass capillary coated with gold usedas nano-ESI needle at a voltage of 700 V. Fragmentationpatterns of aglycone cations were studied by in-sourcecollisions. Aglycone cations of Mv always follow the samefragmentation pattern, regardless of the modification ofthe Mv moiety; a fingerprint of the five anthocyanins andtheir derivatives could be determined on the basis of neutralfragments. Structures of 10 vinyl aglycones of Mv, two ofPn 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) andvinylformic (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 viniferavarieties (Benabdeljalil et al., 2000). Also, structures withMv linked to 4-vinylcathecol (34) and to 4-vinylsyringol(36), and with Mv, Pt, and Pn 3-O-monoglucosides linkedto 4-vinylphenol (31,32,33) were proposed. Mv-3-O-monoglucoside 4-vinylphenol, formed by condensationof Mv with the 4-vinylphenol produced by enzymaticdecarboxylation of p-coumaric acid, was previously identi-fied by Fulcrand et al., 1996a in wines from Carignanegrape. Structures of anthocyanin derivatives identified arereported in Figure 18.

The characterization and fragmentation patterns ofvitisin A, vitisin B, and their 3-O-acetylglucoside deriva-tives were studied by Bakker and Timberlake by FAB-MS,and the authors proposed the structures reported inFigure 19 (Bakker & Timberlake, 1997; Bakker et al.,1997).

Analyses were performed in the positive-ion modewith a glycerol matrix. The molecular ion (Mþ) of twovitisins at m/z 517 and 561 and of their acetyl derivatives(m/z 559 and 603, respectively) were identified, and wereaccompanied by ions due to the loss of a neutral fragment

TABLE 1. Fragmentation of the Mþ of anthocyanin compounds

Compounds present in the direct infusion ESI mass spectrum of Clinton extract obtained by multiple step mass

spectrometric (MSn) 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 2002.


233

of mass 162 and 204, respectively. The two vitisins aredistinguished by a higher resistance to color losses whenadding SO2, a greater color expression at higher pH values,and a stability higher than those exhibited by anthocyaninglucosides.

The year after Fulcrand et al. (1998) isolated andcharacterized, in a model solution prepared with pigmentsextracted from V. vinifera Carignane grape, a pigmentsimilar to vitisin A; data suggested that the condensationof Mv with pyruvic acid probably proceeding with themechanism postulated from the authors reported inFigure 20.

The Mv vinylformic adduct was studied in an LC/ESI-MS system operated in the negative-ion mode; thepseudomolecular ion [Mþ� 2Hþ]� at m/z 559.2 wasidentified.

VI. MASS SPECTROMETRY IN THE STUDY OFSTRUCTURES FORMED BY POLYMERIZATION OFANTHOCYANINS AND FLAVAN-3-OLS

The changes in the wine color that occur during aging werehypothesized to be related to the formation of products of

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 D2O. (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


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condensation between anthocyanins and flavan-3-ols. Inthe seventies, two different structural models were hypo-thesized: in the first, proposed by Somers, anthocyanin islinked by the C-4 position directly to the C-8 of flavan-3-ol(Somers, 1971); in the second, anthocyanin is linked to aflavan-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 acetalde-hyde to mixtures of anthocyanins and flavan-3-ols caused arapid color augmentation with a shift toward the violet, andconsequently the authors suggested the mechanism of

polymerization between anthocyanins and flavan-3-olspromoted by acetaldehyde.

Bakker, Picinelli, & Bridle (1993) studied, the antho-cyanins present in the Touriga Nacional grape-skin extractwith FAB mass spectrometry. They observed a peak at m/z809, which could correspond to the molecular ion of adimeric species formed by a linkage of Mv 3-O-mono-glucoside with catechin through an acetaldehyde bridge ofthe type proposed by Timberlake & Bridle.

In the study of the loss of astringency that wasobserved during the wine aging, and suspected as a result of

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,


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

the insolubilization of tannins by a reaction with acetalde-hyde, the mechanism of polymerization between flavan-3-ols was evaluated (Fulcrand et al., 1996b). Ion-spraymass spectrometry in the negative-ion mode (LC-ISP-MS)was applied to study reactions in model wine solutions thatcontained (þ)-catechin or (�)-epicatechin and acetalde-hyde, and the mechanism of polymerization reported inFigure 22 was proposed. In that study, ion spray (other thanto take advantage of the soft sample ionization) permittedone to determine oligomers up than hexamers as single-charged ions, and to characterize oligomers with similarchromophores that co-eluted from the LC column and thatwere 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 ofpolymerization and the instability of products.

The year after the results obtained in the study of theevolution of polyphenols during aging of the CabernetSauvignon wine were published, evidence was reported ofproducts formed by polymerization of two (þ)-catechinsby an ethyl bridge (Saucier, Little, & Glories, 1997). Thestructure was characterized by ESI in the positive-ionmode by an application of rapid cone-voltage switchingfrom 70 to 25 V to obtain alternate mass scans offragmented (70 V) and non-fragmented (25 V) ions. Inthe low cone-voltage mode, only the molecular ion (m/z607) was detected, whereas, at high cone-voltage,characteristic fragmentations were activated, with theformation of ions at m/z 317 and 291, which corresponded

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,


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.)

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to vinyl-catechin and catechin fragments, respectively (seeFig. 23).

The same year Cheynier et al. (1997), by ESI in thepositive- and negative-ion modes, confirmed the identifica-tion of the signal at m/z 809 as a product of polymerizationbetween epicatechin and Mv 3-O-monoglucoside by anethyl bridge. Signals at m/z 605 and 921 of the [M�H]�

ions extracted from the total ion chromatogram (obtainedin the negative-ion mode) of a red wine sample wereattributed to ethyl-linked catechin dimers and trimers—even on the basis of their retention times.

In another paper Francia-Aricha et al. (1997) studiedwith LC/MS in the positive-ion mode the pigments formedduring aging in model wine solutions that contained Mvmonoglucoside, acetaldehyde, and procyanidin B2 (dimerof (�)-epicatechin) isolated from grape seeds. A peak atm/z 1097, corresponding to the two different enantiomersformed by an epicatechin dimer linked to Mv monogluco-side by an ethyl bridge, and a peak at m/z 1093, assigned topigment B2-III (Mv-3-O-glucoside-vinylcatechin-epica-techin), were determined. Similar pigments were found insolutions that contained (þ)-catechin, (�)-epicatechin, orprocyanidin B3 (dimer of (þ)-catechin).

With LC-MS, a study on copigmentation (hydrophobicassociation of an anthocyanin chromophore with the planarelectronically unsatured part of a copigment) in the winewas also performed. The transformations that occurred inhydroalcoholic synthetic solutions that contained Mv-3-O-glucoside as pigment and (�)-epicatechin as copigmentwere investigated, and the influence of acetaldehyde on theformation of covalent compounds was evaluated (Mirabelet al., 1999). Operating in the positive-ion mode, peaks atm/z 809,m/z 783 (probably corresponding to the molecularion Mþ of compound formed by direct linkage of Mv-3-O-glucoside with flavan-3-ol), and m/z 621 and 469 (cor-responding to fragments formed by glucose residue lossand the consecutive RDA) were detected. The fragmenta-tion pattern of molecular ion m/z 783 and the structureproposed for the molecular ion at m/z 809 are reported inFigure 24. Also, a peak at m/z 453 was present, and theauthors hypothesized that it corresponded to either a pos-sible yellow-orange product formed by Mv oxidation or acopigment flavonol-anthocyanin complex. A peak at m/z809 was found also in non-acetaldehyde solutions—confirming that acetaldehyde is formed by ethanoloxidation.

Remy et al. (2000) studied the molecular speciesformed by anthocyanin-tannin condensation in solutionsfrom 2-year-old Cabernet Sauvignon wine obtained afterfractionament on a Toypearl TSK gel HW-50 (F) column.Thiolysis of the polymeric fractions was performed, andproducts were analyzed by LC-API in the negative-ionmode. Compounds were detected as their [M�H]�

deprotonated molecules, or [Mþ� 2H]� in the case of

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.)


237

flavylium. Thiolysis experiments confirmed the presenceof a structure with position C-6 or C-8 of Mv-3-O-monoglucoside linked to C-4 of flavan-3-ol to form a dimerof sequence T-A type (A, anthocyanin; T, tannin) (seeFig. 25a). The formation of this compound follows amechanism that involves the nucleophilic attack of anaromatic ring of Mv to the carbonium ion of flavan-3-olliberated by acidic cleavage of a proanthocyanidininterflavanic bond.

Also, the structure previously hypothesized fromSomers was confirmed by this study, and the results in-dicated that acetaldehyde-mediated condensation is not aprevailing mechanism involved in the transformation oftannin in wine (Somers, 1971). LC/MS Analyses alsorevealed the presence of an [M�H]� species at m/z 781that corresponded to two different chromatographic peaksthat were tentatively attributed to a flavene or a bicyclic

structure that originated from the direct reaction of flavonolposition C-6 or C-8 with the C-4 of Mv-3-O-monogluco-side with the formation of a A-T type dimer (see Fig. 25b).The presence of those structures implies a reactionmechanism, where the flavilium ion is subjected to anucleophilic attack from the aromatic ring of flavan-3-ol;however, these structures were not confirmed by thiolysisexperiments. Finally, the authors reported that structures T-A and A-T appeared to be predominantly associated withlower-MW flavanols.

In 4-year-old Shiraz wines, Asenstorfer et al. identifieda series of structures, where Mv-3-O-monoglucoside, Mv-3-O-acetylglucoside and Mv-3-O-p-coumaroylglucosideare linked to monomer, dimer and trimer of catechin by avinyl group, to produce the compounds that are reported inTable 2. The presence of these types of structures in Shirazwines was also confirmed 1 year later by Hayasaka and

FIGURE 23. ESI positive-ion mode mass spectrum of the compound reported in Figure 22. In low cone

voltage mode (25 V) molecular ion atm/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.)

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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


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 atm/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.)


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Asenstorfer, with the identification of Mv-3-O-glucosidevinylcatechin (Asenstorfer, Hayasaka, & Jones, 2001;Hayasaka & Asenstorfer, 2002).

VII. MASS SPECTROMETRY AND GRAPE ANDWINE RESVERATROL

Resveratrol (3,5,40-trihydroxystilbene) is a polyphenol thatis mostly present in red grapes and wines as cis-and trans-isomers in the free or glycosidically bonded form (Fig. 26).

In the last 10 years, it has become an important quali-tative parameter because of the several beneficial effects onhuman health that were revealed by recent studies: anti-cancer activity, cardioprotection, antioxidant activity, in-hibition of platelet aggregation, and anti-inflammatoryactivity (Frankel, Waterhouse, & Kinsella, 1993; Bertelliet 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 than30 mg/L (Paronetto & Mattivi, 1999). As a consequence,the interest for analytical applications to determineresveratrol in grape and wine was increased, and severalstudies have been done to develop suitable analyticalmethods, some of which are based on mass spectrometry.

One of the first applications of mass spectrometry todetermine resveratrol in grape juice and wine was based onGC-MS, and the method permitted one to determinesimultaneously cis- and trans-isomers (Soleas et al., 1995).Preparation of samples by SPE with C18 cartridges, andderivatization by bis-[trimethylsilyl]-trifluoroacetamide(BSTFA), was performed. For analyses, a DB-5HT (5%phenyl, 95% methyl) column was used. The electronionization (EI) 70 eV fragmentation spectra of tris-trimethylsilyl trans-resveratrol derivative is reported inFigure 27. For quantitative evaluation, the signal atm/z 444was employed for SIM analysis, and under those condi-tions, a limit of detection of ca. 10 mg/L was obtained. Forcomparative evaluation, it is to be emphasized that LCmethods, which require sample preparation by multiplesolvent extractions, exhibit a limit of detection between1 and 50 mg/L. Wines from different zones of Canada wereanalyzed, and samples from Ontario showed higher cont-ents of cis- and trans-isomers. Because the cis-resveratrol

was not detected in grape skins or juices, the formation ofthis isomer by isomerization of the trans, or by a break-down of resveratrol polymers during fermentation, hasbeen hypothesized.

The hydroxylated stilbene contents of a number ofcommercial American red wines were determined by GC-MS by performing sample preparation and analyses underconditions that were similar to those reported in theprevious paper (Lamikanra et al., 1996). Six stilbenols (cis-and trans-resveratrol and others mono-, di-, tri-, and tetra-hydroxystilbenes) in wines from different grape varietieswere determined as trimethylsilyl derivatives. Tris-[tri-methylsilyl] stilbene was still quantified on the basis ofthe ion at m/z 444. Standards of mono-, di-, and tetra-hydroxystilbene were not available, and their structuralcharacterization was based on their molecular ions andfragmentation patterns. Ions at m/z 268 (tentatively identi-fied as monohydroxystilbene), m/z 356 (tentatively identi-fied as dihydroxystilbene), and m/z 532 (tentativelyidentified as tetrahydroxystilbene) were employed forSIM. Retention times were compared to those of cis- andtrans-resveratrol, and compounds proposed as dihydroxyand tetrahydroxy stilbenes were eluted from the columnbefore 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 isomerwas absent in some samples. The concentrations of re-sveratrol in wine fromVitis rotundifolia (muscardina) grapewere higher than those present in wine from Vitis viniferaand Vitis labruscana (Concord) grape.

A year later, four different methods, two based on LCand two on GC-MS, to assay cis- and trans-resveratrol inwine samples were compared in an analysis of 169 differentwines (Soleas et al., 1997). The two LC methods used,respectively, a normal phase (isocratic elution) and areversed-phase (gradient elution) chromatography withspectrophotometric detection at 306 nm. The two GC-MSmethods (flow charts of Fig. 28) both required a previoussample preparation with SPE on a C18 cartridge: the firstanalysis was direct injection of sample on a DB-17 HT(diphenyl dimethyl polysiloxane) column, and recordingsignals in the SIM mode at m/z 228 (Mþ), m/z 227([M�H]þ), and m/z 229 (13C-isotope containing species)as qualifiers; the second was the derivatization of samplewith BSTFA, and recording signals at m/z 444 of themolecular ion, and m/z 445 and m/z 446 (30Si-isotopecontaining species) as qualifiers.

By direct injection (i.e., without derivatization), higherlevels of trans-resveratrol were found, this result was, atleast in part, attributed to the thermal decomposition ofresveratrol glucosides. On the contrary, the method thatimplyed the derivatization procedure showed a tendencyto overestimate the cis-resveratrol and underestimatethe trans, possibly as a consequence of the trans-to-cisFIGURE 26. Trans- (a) and cis- (b) isomers of resveratrol.

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isomerization that occurred during the derivatization.Results of analysis of 90 different wines with and withoutprevious sample derivatization with BSTFA are comparedin Figure 29. The greater advantage of GC methods was thevery shorter analysis time (ca. 10 min), whereas the LCmethods required 48 min in the normal phase and 33 min inthe reversed phase.

A method based on solid-phase-micro-extraction(SPME) for the trace analysis of trans-resveratrol in winehas been recently developed in order to eliminate the labo-rious and time-consuming SPE procedure for the samplepretreatment (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 27. GC-MS electron ionization (70 eV) mass spectrum of tris-trimethylsilyl trans-resveratrol.

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.


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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.)

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polyacrilate, 85 mm thickness) directly into the wine, thederivatization step was performed in gas phase by exposingthe fiber in the head space of silylating agent, so as to avoidits hydrolysis. Analyses were performed in the SIM mode,by recording the signal at m/z 444; a linear response over aconcentration range of 10 ng/L to 1 mg/L with a detectionlimit of 5 ng/L (about 2000-times lower than that reportedby Soleas et al. for the SPE method), were obtained. Resultsconfirmed the wider presence of the trans-isomer in grapewith respect the cis-isomer, where their concentrationswere similar in wines.

In 1999, a method to determine trans-resveratrol inwine with ESI LC-MS in the negative-ion mode (capillaryvoltage 4 kV) was developed (Gamoh & Nakashima,1999). The chromatography was performed on a C18

column with a CH3OH/ammonium acetate 20 mM (pH 5.5)(55:45 v/v) eluent. For quantitative analysis, the SIMmethod 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), diagnosticfragment ions are observed (see Fig. 30).

Sample preparation was performed by reversed-phaseSPE, and trihydroxyflavone was used as the internalstandard due to its high ionization efficiency in ESI condi-tions and its hydrophobic properties comparable with thoseof resveratrol. The best condition of cone voltage foranalysis was 35 V, and the SIM detection limit was 200 pginjected on-column (volume injected 10 mL of a 20 mg/Lsolution, signal/noise ratio 3).

Recently, Wang et al. (2002) studied by LC-MS the cis-and trans-resveratrol contents of grape juice and winesfrom Vitis vinifera Negroamaro. The glucoside resveratrol

fraction was determined in the grape by an extraction withmethanol, and in the wine by an extraction with ethylacetate. Enzymatic hydrolysis of extracts was performed toliberate aglycones, and APCI in the positive-ion mode wasused. The reversed-phase chromatography was performedwith CH3OH/HCOOH as eluent; the formic acid was addedto the mobile phase to suppress an on-column ionic dis-sociation of resveratrol due to the acidic character of thehydroxyl groups. SIM of the [MþH]þ ion (m/z 229) wasused for quantitative determination.Cis- and trans-isomerswere determined with a limit of detection of 70 pg injectedon-column with a signal/noise ratio of 10.

VIII. APPLICATION OF MALDI INTHE STUDY OF POLYPHENOLS

The first application of MALDI in wine chemistry was bySzilagyi et al. (1996) and was devoted to the study of wineprotein. Only more recently, some studies on grape poly-phenols with matrix-assisted-laser-desorption-ionization-time-of-flight (MALDI-TOF) (Karas & Bahr, 1991;Sundqvist, 1992; Busch, 1995; Guilhaus, 1995; Gluck-mann & Karas, 1999; Karas, Gluckmann, & Shafer, 2000)mass spectrometry have been published. The MALDI-TOFtechnique has advantages over other mass spectrometricsystems, such as FAB, in sensitivity and mass range, andproduces only a single ionization event (Krueger et al.,2000). Furthermore, it is highly tolerant toward contami-nants, and is thus particularly suitable for the directanalysis of complex mixtures (Yang & Chien, 2000).

Sugui et al. (1999) studied by MALDI-TOF theanthocyanic profile of methanol extracts from five different

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.)


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French hybrid grape varieties: Chancellor, Marechal Foch,MN 1095, Cynthiana and Concord. A system with anitrogen laser that operated at 337 nm, and an acceleratingvoltage 28 kV, was used, and a-cyano-4-hydroxycinnamicacid (CCA) was employed as the matrix. The five antho-

cyanins Dp, Cy, Pt, Pn, and Mv as 3-O-monoglucosides,acetylmonoglucosides, p-coumaroylmonoglucosides, 3,5-O-diglucosides, and p-coumaroyldiglucosides were easilyidentified. The MALDI spectra of five grape extracts arereported 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


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For the considerable laser shot-to-laser-shot variabil-ity, the average value of relative ion abundance wascalculated for three different MALDI analyses. SimpleMALDI-MS cannot differentiate isobaric compounds; inparticular, Mv-3-O-caffeoylmonoglucoside and Mv diglu-coside could not be distinguished, and only after HPLCanalysis could the absence of the former be verified.

A qualitative and quantitative MALDI-TOF study ofanthocyanins in red wine and fruit juice was reported in thesame year (Wang & Sporns, 1999). Analyses of Merlot,Pinot Noir, Zinfandel, and Cabernet Sauvignon wines andof Concord grape juice anthocyanins were performed bythe co-crystallization of extracts with 2,4,6-trihydroxya-cetophenone (THAP) in acetone. The THAP matrixproduced the best spot-to-spot reproducibility. The systemwas operated in the linear-mode and positive-ion modes.With this approach, the five anthocyanin 3-O-monogluco-sides and their derivatives were identified, and quantifica-tion was performed with cyanidin 3-rutinoside as internalstandard. In the quantitative analysis of monoglucosideanthocyanins, the responses were only slightly different;however, for diglucosides and anthocyanin with a secondcarbohydrate moiety to a 3-glucoside (e.g., cyanidin 3-rutinoside), the relative molar response was only ca. one-fourth of that noted for monoglucosides. MALDI-MSresponses were linear for groups of chemically similaranthocyanins, and experiments showed that the internalstandard addition had no effect on the relative responses ofthe other anthocyanins.

The MALDI-TOF technique has also been applied tocharacterize polygalloyl polyflavan-3-ols (PGPF) in grapeseed extracts dissolved in a trans-3-indolacrylic acid (IAA)matrix (Krueger et al., 2000). Ions that corresponded to aseries of PGPF units up to nonamers were observed in thepositive-ion reflectron mode, and ions up to undecamerswere observed in the positive-ion linear mode, also withlower resolution. The highest galloylation degree foundwas six, and sodium adducts [MþNa]þ were revealed inthe reflectron and linear modes. The positive-ion reflectronmode permitted one to identify a series of compounds withMWs two mass units lower than those of the above-described compounds, and that corresponded to polyca-techins A-type (see Fig. 6). On the basis of the galloylatedstructures, an equation was developed to predict themass distribution of PGPF in grape seed extracts: 290þ288cþ 152gþ 23, where 290 is the MW of the terminalcatechin/epicatechin unit, c is the degree of polymeriza-tion, g is the number of galloyl esters, and 23 is the atomicmass of sodium. With this equation, an easy description ofthe MS data can be achieved.

In the same year, Yang and Chien published a study,where LC/MS and MALDI-TOF (operating in the re-flectron and linear modes) were applied to characterizeprocyanidins in grape seeds extracts, with a critical com-

parison of the results achieved by two techniques (Yang &Chien, 2000). LC/MS analyses were performed withnormal-phase and reversed-phase columns, with APCI inthe negative-ion mode. MALDI-TOF analyses were per-formed with 2,5-dihydroxybenzoic acid (DHB) or trans-3-indoleacrylic acid (IAA) as the matrix. LC-MS methodsdid not permit the separation and identification ofoligomers higher than pentamers because, as the MWincreases, the number of diastereomers becomes so largethat separation of individual isomers becomes impossible.On the other hand, MALDI-TOF in the positive-ion re-flectron mode was a rapid method of analysis that allowedone to determine oligomers that contained (þ)-catechin,(�)-epicatechin, and their galloylated derivatives up toheptamers with resolution higher than 3000. This resolu-tion allowed the separation of individual ions of differentisotopic composition: for example, the ion at m/z 1177.46was further resolved into a group of four peaks, as shown inthe expanded view of the spectrum in Figure 32.

The application of MALDI-TOF MS in the linearmode permitted one to detect oligomers up to nonamers assodium adducts. The lower sensitivity of the reflectronmode for the large ions is reasonably due to their colli-sionally induced decompositions that occur in the flightpath. The authors reported that DHB as a matrix leads to thebest analytical conditions for the detection of procyanidinsin reflectron mode to provide the broadest mass range withthe least background noise.

IX. MASS SPECTROMETRY APPLIED TO THESTUDY OF WINE POLYPHENOLS FROM CORKBOTTLE STOPPERS AND OAK BARRELS

The several compounds released in to the wine from thewood materials used in the wine storage and aging providean important contribution to the organoleptic charac-teristics of the final product. Between them, a number ofsimple phenols and polyphenols were identified, in parti-cular by GC-MS, and some of these studies are discussedhere.

The volatile compounds released in to the wine fromthe different types of oak woods that are used in the con-struction of barrels that are used for the aging of wine andbrandy have been studied by a number of researchers. GC-MS analysis of alcoholic wood extracts identified differentclasses of compounds such as ketones, aldehydes, estersand lactones, alcohols, furanic compounds, fat acids, andphenols. Phenols such as vanillin, syringol and syringalde-hyde, 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 wereidentified. In particular, vanillin provides an important


245

contribution to the aroma of barrel-aged wine. Structures ofthose compounds are reported in Figure 33. Those com-pounds are released in different amounts, depending on theheat treatment of the wood (Cutzach et al., 1997; Moioet al., 1999; Perez-Coello, Sanz, & Cabezudo, 1999;Cerdan, Goni, & Azpilicueta, 2002).

Galletti, Bocchini, & Antonelli (1996) characterizedthe structural polymers of cork bottle stoppers by pyrolysis/GC/MS. Pyrolysis at 6008C for 5 s in an inert atmosphereinduced the thermal fission of cork, and the phenolic

macromolecules released from lignin and suberin wereanalyzed by GC-MS.

X. CONCLUSIONS

Mass spectrometry has a very important role for researchand quality control in the viticulture and enology field.After the first applications by GC-MS on grape and winearoma and FAB for polyphenol characterization, LC-MS

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.)

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) isoeu-

genol; (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.)

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methods are the most common techniques to performstructural studies on anthocyanins and polyphenols. Thesoft ionization conditions and the minor sample purifica-tion required, make LC-MS techniques more suitable toperform studies on grape extracts and wine. Different fromthe LC-UV methods that require hydrolysis or thiolysissteps for the compound identification, LC-MS is reason-ably the most effective tool for the study of structures thatcorrelate to the color changing of red wines during aging,which involve reactions of polymerization of anthocyaninswith other compounds, and it has permitted one to con-firm structures that were previously only proposed. Thistechnique has also permitted the characterization of grapeprocyanidins, proanthocyanidins, prodelphinidins, andtannins, to provide experimental evidence for structuresthat until a few years ago were only hypothesized. Also, thecomplex structure of polyphenols often does not permit theavailability of a standard, and, as a consequence, structuresare based on spectra evaluation. On the other hand, theimportant contribution of LC-MS in the structural studiesof polyphenols is also confirmed by the number of appli-cations in different fields of food and agricultural chemistrythat have appeared in the worldwide literature of the lastyears. Furthermore, the application of different massspectrometric techniques show that their complementarityis highly precious to study the large number of grapeand wine compounds. As an example, in the study ofprocyanidins LC-MS determined isomers at a low poly-merization degree, but not for the separation and charac-terization of oligomers up to pentamers. On the other hand,MALDI-TOF is suitable to determine the presence ofmolecules of higher MW with high accuracy, and it hasbeen successively applied to study procyanidin oligomersup to heptamer in reflectron mode, and up to nonamers inthe linear mode. The MS/MS approach is a very powerfultool in the study of anthocyanins, and permitts aglyconeand sugar moiety characterization.

To determine resveratrol in the wine, commonmethods are based on LC, spectrophotometric detection,electrochemical, and fluorescence methods. However,those methods require a long analysis time, and accuratechromatographic separation is necessary for satisfactorysensitivity. On the other hand, GC-MS with SPME showedan excellent sensitivity, but requires a previous derivati-zation of sample to improve the volatility and thermalstability of the resveratrol derivative. Moreover, a hightemperature promoted the thermal isomerization and de-gradation of compounds. The use of LC-MS overcomesthese problems, and yields analyses with a detection limitof ca. 1–10 mg/L.

On the basis of the important results obtained by massspectrometry in the last years, the next step could be, in myopinion, the application of these techniques to study theproteins and pathogenesis-related proteins of grape and

wine, fields where only few studies have been done withthis tool. This application would be a relevant contributionfor the study of plant metabolism and industrial process, inparticular, for sparkling wines.

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