Maktub II - bivir

33 Pages·2004·0.54 MB·Spanish

by  Paulo Coelho

MASS SPECTROMETRY IN GRAPE AND WINE CHEMISTRY.
PART I: POLYPHENOLS
Riccardo Flamini*
Istituto Sperimentale per la Viticoltura, Viale XXVIII Aprile 26, I-31015
Conegliano (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
Massspectrometry,hadandstillhas,averyimportantrolefor complexstructuresofgrapepolyphenols,suchasprocyanidins,
researchandqualitycontrolintheviticultureandenologyfield, proanthocyanidins, prodelphinidins, and tannins, and provides
and its analytical power is relevant for structural studies on experimental evidence for structures that were previously only
aroma and polyphenolic compounds. Polyphenols are respon- hypothesized. The matrix-assisted-laser-desorption-ionization-
sibleforthetasteandcolorofwine,andconferastringencyand time-of-flight(MALDI-TOF)techniqueissuitable todetermine
structure to the beverage. The knowledge of the anthocyanic thepresenceofmoleculesofhighermolecularweightwithhigh
structure is very important to predict the aging attitude of accuracy, and it has been applied with success to study
wine, and to attempt to resolve problems about color stability. procyanidin oligomers up to heptamers in the reflectron mode,
Moreover, polyphenols are the main compounds related to the and up to nonamers in the linear mode. The levels of
benefits of wine consumption in the diet, because of their resveratrolinwine,animportantpolyphenolwell-knownforits
properties in the treatment of circulatory disorders such as beneficialeffects,havebeendeterminedbySPMEandLC-MS,
capillaryfragility,peripheralchronicvenousinsufficiency,and and the former approach led to the best results in terms of
microangiopathy of the retina. Liquid Chromatography-Mass sensitivity. # 2003 Wiley Periodicals, Inc., Mass Spec Rev
Spectrometry (LC-MS) techniques are nowadays the best 22:218–250, 2003; Published online in Wiley InterScience
analytical approach to study polyphenols in grape extracts (www.interscience.wiley.com). DOI 10.1002/mas.10052
and wine, and are the most effective tool in the study of the Keywords: grape and wine polyphenols; anthocyanins;
structure of anthocyanins. The MS/MS approach is a very procyanidins and tannins; resveratrol; mass spectrometry;
powerful tool that permits anthocyanin aglycone and sugar liquid chromatography; MALDI
moiety characterization. LC-MS allows the characterization of
I. INTRODUCTION
————
OnthebasisofdatareportedfromtheOfficeInternational
*Correspondence to: Riccardo Flamini, Istituto Sperimentale per la
De la Vigne et Du Vin (O.I.V.)—The State of Vitivini-
Viticoltura, Viale XXVIII Aprile 26, I-31015 Conegliano(TV), Italy.
E-mail:fl[email protected] cultureintheWorldandStatisticalInformationin1999—
MassSpectrometryReviews,2003,22,218–250
#2003byWileyPeriodicals,Inc.
MSINGRAPEANDWINECHEMISTRY &
in the years 1990–1999 a large increase of production MacDonaldetal.,1999;Wong&Halverson,1999),andthe
and consumption of table grape was registered; that in- study of plant metabolism and diseases (Pe´rez, Viani, &
crease was also encouraged by the amply demonstrated Retamales,2000;Tabacchietal.,2000).
beneficialeffectsofthisfoodonthehumanhealth.Also,the For the improvement of product quality, aroma and
worldwidewineproductionregisteredasensibleincrease, polyphenolic compounds have been widely studied. The
favoredbythesuppressionofmeasuresthatencouragedthe numerous classes of grape polyphenols transferred to the
permanent uprooting implemented in recent years by wineareresponsibleforthetasteandcolorofbeverage.In
European Union, and by recent plantations in certain thisreview,theimportantroleofmassspectrometryinthe
number of non-European countries with potentially high studyofgrapeandwinepolyphenolsisdiscussed—afield
yields(Dutruc-Rosset,1999). where a rapid increase of knowledge has been observed,
Trends of grape and wine worldwide production of due also to the development of the new technologies
theseyearsarereportedthehistogramsofFigure1a,b.The introducedintherecentyears.
particularincreasein1999canbeobserved.
The worldwide market has been influenced by the
repercussions on the media of seminars and scientific
II. QUALITY IMPROVEMENT
symposiainthefieldofresearch,medicine,toxicologyof
foods, and human health. In particular, the beneficial To improve the final product—the wine—the research in
effects of moderate wine consumption on certain cate- viticultureisaddressedtoimprovethequalityofthegrape
gories of diseases, such as cardiovascular diseases, brain through the study of grape-ripening, which involves
degeneration from aging, and certain carcinogenic dis- cultural techniques, the selection of clones and varieties
eases, have been discussed. The report of O.I.V. also of best potentiality (clonal selection), and the study of
indicatesraisinsasaninterestingmeansbywhichtofight environmental influence on the vineyard (zoning). In the
againsthungerintheworld. enology field, main efforts are devoted to optimize
Asaconsequenceofthesetrends,effortsofthelargest industrial processes finalized to obtain products with
grape- and wine-producer countries (Argentina, France, peculiarcharacteristics.Inthisframe,(i)theinoculumof
Italy,SouthAfrica,Spain,UnitedStates)areaddressedto selectedyeastpermitsregularfermentationwithminimum
improve the product quality, rather than to increase pro- secondaryprocessesbyothermicroorganisms,(ii)theuse
duction,soastoremaincompetitiveonemergentcountries of selected enzymes leads to a better extraction of grape
by the growth of systematic positioning in the market components, (iii) the maceration of grape skins is
niches of ‘‘premium’’ and ‘‘super premium’’ wines. The performed in controlled conditions of temperature and
main efforts of researchers and Organisms of Control are atmosphere, (iv) malolactic fermentation is employed to
addressed to develop new methods to detect the product improveorganolepticcharacteristicsandtoconfermicro-
origin (Ogrinc et al., 2001), the detection of adulteration biologicalstabilitytothewine,and,finally,(v)thebarrel-
involving sugar-beet, cane sugar, or ethanol addition and and bottle-aging refine the final product. To reach the
watering(Guillouetal.,2001),theprotectionofconsumer proposedaimsandtobeabletoestimatethepotentialityof
healththroughdeterminationoffoodcontaminantssuchas startingmaterialandhowitcanbetransferredtothefinal
heavymetals,toxins,andpesticides(Szpunaretal.,1998; product,agoodknowledgeofgrapechemistryisessential.
FIGURE1. Trendsoftotalworld-widewine(a)andgrapeproductions(b)fortheperiod1991–1999.
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To define characteristics and identity of product,
the research in viticulture attempts to determine origin
parameters.Forvarietycharacterization(chemotaxonomic
classification), DNA, amphelography, isoenzymes, and
secondary metabolites of plant are studied (Costacurta
etal.,2001).
Secondary metabolites in the grape are compounds
(terpenesandterpenols,methoxypyrazines,volatilesulfur
compounds,benzenoids,norisoprenoids,andpolyphenols)
mainly linked to thevariety but not indispensable for the
plant survivor, also whether environmental and climatic
variables can influence their contents in the fruit (Di
Stefano,1996;Flamini,DallaVedova,&Calo`,2001).
III. GRAPE AND WINE POLYPHENOLS
Inthewinemaking,grapecompoundsaretransferredtothe
mustandtothewine,whichcontainseveralpolyphenolsat
different 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), coniferyl
aldehyde(8),benzoicacidssuchasgentisicacid(9),gallic
acid(10),vanillicacid(11),salicilicacid(12),andsyringic
acid(13).Alsothehydroxycinnamicacids(HCA)caffeic
(14),ferulic(15),andp-coumaric(cis-andtrans-isomers)
FIGURE2. Structuresofmono-,di-,andtri-phenolspresentingrape:
(16)andtheirestersformedbycondensationwithtartaric (1)pyrocatechol,(2)resorcinol,(3)hydroquinone,(4)phloroglucinol,
acid (hydroxycinnamoyltartaric acids HCTA) (17) are (5) vanillin, (6) p-hydroxybenzaldehyde, (7) syringic aldehyde, (8)
presentingrapeandwineinconsiderableamounts.Inorder coniferylaldehyde,(9)gentisicacid,(10)gallicacid,(11)vanillicacid,
(12)salicilicacid,(13)syringicacid,(14)caffeicacid,(15)ferulicacid,
to give to the reader a general view of the chemistry in-
(16)p-coumaricacid,and(17)hydroxycinnamoyltartaricacids.
volvedinthiscontext,thestructureofthesemoleculesare
reportedinFigure2.
More-complexgrapepolyphenolscontaintwoormore
aromatic rings (cumarines, benzopyrones, and flavilium
ions)toformflavanols(18),flavonols(19),andanthocya-
nins(20)(Macheix,Fleuriet,&Billot,1990)(seeFig.3).
Thesemoleculesarepresentinthegrapemainlyinthe
monoglycosideform,withthesugarresiduelinkedtothe
hydroxyl group in position C-3 of the O-containing ring.
Theglycosideflavonolskaempferol(19a),quercetin(19b),
and myricitin (19c) (Fig. 3) form co-pigments with
anthocyanins(inredwines);they,togetherwithoxidation
products of tannins, are in the main responsible for the
colorofwhitegrapesandwines(Cheynier&Rigaud,1986;
Usseglio-Tomasset, 1995). Anthocyanins contain in their
skeletonthebenzopyriliumionasbasemolecule,whichis
responsible for the color of red berry varieties and red
wines. They are present in the grape as mono- or di-
glucosides,dependingonvariety,withthesecondglucose
moleculelinkedtotheC-5hydroxylgroup.Theflavan-3-
ols (þ)-catechin, (þ)-gallocatechin, ((cid:1))-epicatechin, and FIGURE3. Chemical structures of polyphenol aglycones present in
((cid:1))-epigallocatechinarepresentinthegrapeasmonomers, grape:(18)flavanols,(19)flavonols,and(20)anthocyanins.
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MSINGRAPEANDWINECHEMISTRY &
or linked between them to form procyanidins, proantho- proanthocyanidins, gallic acid, catechin, and epicatechin
cyanidinsandtanninsoftypereportedinFigure4. from seeds (Figs. 2 and 4). Moreover, these molecules
With grape pressing, polyphenols are released in the can undergo condensation and polymerization processes
must from the different parts of berry: HCTA, phenolic during the winemaking and wine aging, to produce new
acids,andaldehydesfromjuiceandpulp;HCTA,phenolic structures.
acids,anthocyanins,procyanidinsandproanthocyanidins, Polyphenols play an important role in the organo-
and flavonols from skin; and tannins, procyanidins and lepticcharacteristicsofwine;inparticular,tanninsconfer
FIGURE4. Structuresofcatechins,andprocyanidindimersandtrimersingrapeseeds.(Reprintedfrom
Phytochemistry49,deFreitasetal.,Characterizationofoligomericandpolymericprocyanidinsfromgrape
seedsbyliquidsecondaryionmassspectrometry,p.1436,Copyright1998,withpermissionfromElsevier.)
221
& FLAMINI
astringencyandstructuretothebeveragebyformationof metricmeasurements.TheLCmethodsaremainlyapplied
complexeswiththeproteinsofsaliva.Theirknowledgeis for chemotaxonomic studies by determination of poly-
veryimportanttopredicttheagingattitudeofwine,andto phenolandanthocyaninprofilesofgrapeextracts(Flamini
attempttoresolveproblemsaboutcolorstability—inparti- &Tomasi,2000).Severalmethodsbyspectrophotometric
cularinthecaseofpremiumredwinesthataredestinedto measurements havebeendeveloped todetermineindexes
longagingperiods(e.g.,BrunellodiMontalcino,Chianti, relatedtothedifferentclassofpolyphenolsinthegrapeand
Barolo). wine and to their polymerization state; some of them are
Polyphenols are the principal compounds related to easyandfasttoperformandareusuallyappliedtomonitor
the benefits of wine consuming inthe diet because of the processesinthewinemaking(Paronetto,1977;DiStefano,
propertiesattributedtothem.Procyanidinsandproantho- Cravero,&Gentilini,1989;DiStefanoetal.,2000).
cyanidins from Vitis vinifera seeds are used as active
ingredients in medicinal products for the treatment of
circulatorydisorderssuchascapillaryfragility,peripheral
IV. MASS SPECTROMETRY IN THE STUDY OF
chronicvenousinsufficiency,andmicroangiopathyofthe
POLYPHENOLS AND PROCYANIDINS
retina. Pharmacological properties of selected proantho-
cyanidinsfromgrapeseeds(LeucoselectTM)arerelatedto Gas Chromatography-Mass Spectrometry (GC-MS) has
an increase of tonicity and resistance of capillary walls, beenappliedinthefieldofgrapeandwinearomasincethe
as well as to radical scavenging and inhibition of super- seventies,butbecauseofthelowvolatilityofpolyphenols,
oxide ion formation. Proanthocyanidins supplementation significant papers on their characterization in grape and
intherat,madetheheartlesssusceptibletotheischemia/ winebytheuseofthistechniquehavenotbeenfoundinthe
reperfusion damage, and increased the total antioxidant literature. To increase the volatility of these polar com-
plasmacapacityandtheascorbicacidplasmalevel(Maffei pounds,thesamplederivatizationmustbeperformed,but
Facinoetal.,1996,1998).Additionally,proanthocyanidins oftenstructureofderivativescannotbedeterminedbyGC/
from grape seeds decreased the susceptibility of healthy MS.Theirhighmolecularweight(MW)exceedsthemass
cells to toxic and carcinogenic agents. Phenolic com- rangeavailableforthemostcommonGC/MSsystems,thus
poundsofthegrapehavebeenassociatedwithcardiovas- makingthisapproachineffective.Moreover,derivatization
cular benefits, a reduction of platelet aggregation, and a leads to a more difficult interpretation of fragmentation
modulationofeicosanoidsynthesis.Recently,theantiox- patterns,alsoforsimpleprocyanidinmonomerswithaC
15
idant activity of phenolic compounds from 12 different (C -C -C )skeleton.Consequently,intheearlyinvestiga-
6 3 6
varieties of grape toward human low-density lipoprotein tions structural characterization of grape and wine poly-
(LDL) in vitro has been evaluated (Maffei Facino et al., phenolswasusuallyperformedbyhydrolysisorthiolysis,
1994; Frankel, Waterhouse, & Teissedre, 1995; Bagchi steps and the subsequent identification of hydrolysis pro-
et al., 1997; Meyer et al., 1997; Waterhouse & Walzem, ducts by LC, spectrophotometric analysis, or thin layer
1997; Joshi et al., 1998; Schramm et al., 1998; Maffei chromatography (TLC) methods (Wulf & Nagel, 1978;
Facino et al., 1999). The flavonol quercetin blocked the Hebrero,Santos-Buelga,&Rivas-Gonzalo,1988;Hebrero
aggregationofhumanplateletsbyADPandthrombin,and et al., 1989; Hong & Wrolstad, 1990b; Lee & Jaworski,
thiscompoundhasgainedconsiderableprominenceasan 1990).
inhibitorofcarcinogensandofcancercellgrowthinmany One of the earlier studies by mass spectrometry on
experimentalandhumantumors(Goldbergetal.,1998). grapeunderivatizedpolyphenolswaspublishedin1990.In
Furthermore,theanthocyaninprofileisausefultoolto that research Fast Atom Bombardment (FAB) (De Pauw,
characterize and to determine the origin of products, and 1986;DePauw,Agnello,&Derwa,1991)inthepositive-
intheidentificationofpossibleadulterations.Forexample, and negative-ion modes was used to perform analysis
in some countries the production and commercialization of grape extract samples with glycerol as matrix. The
of wine from not Vitis vinifera grape are prohibited. catechin-gallate(Fig.4)(identificationofion[M(cid:1)H](cid:1)at
Hybrid grapes are characterized by peculiar anthocyanin m/z 441, ions at m/z 151 and 137 as qualifiers), catechin-
3,5-O-diglucosidecontents,whicharepracticallyabsentin catechin-gallate(Fig.4)(ion[M(cid:1)H](cid:1)atm/z729,ionm/z
grapes from Vitis vinifera, and these compounds can be 577correspondingtothelossofgallicacidfragment)and
consequently employed for the identification of possible gallocatechin-gallate(Fig.4)(ionatm/z460)wereidenti-
adulterations. Finally, anthocyanins from grape are also fied in extracts from Niagara Grapes (Lee & Jaworski,
importantinthesyntheticcolorantsmarket,inparticularin 1990).
thefoodindustry(Hong&Wrolstad,1990a). Inthenineties,thedevelopmentandtheavailabilityof
Traditional methods to determine polyphenols and effective Liquid Chromatography-Mass Spectrometry
anthocyaninsinnaturalextractsareusuallyperformedby (LC-MS) and the Multiple Mass Spectrometry (MS/MS
liquid chromatography (LC) analysis and spectrophoto- andMSn)systems(Niessen&Tinke,1995;deHoffmann,
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MSINGRAPEANDWINECHEMISTRY &
1996;Abian,1999)suppliedveryusefultoolstostudythe gallate, ((cid:1))-epicatechin gallate, up to decamers, were
polyphenolstructuresaswellasthemechanismsinwhich identified(m/zvaluesbetween290and3100Da).
they are involved in winemaking and wine aging. In this The authors emphasized the advantages of FAB as a
frame, Cheynier et al. studied tannins (oligomers and rapidtechniquethatrequiredonlylittleamountsofsample
polymersofflavan-3-ols,Fig.4)ingrapeseedextractsby for analysis and without derivatization. In that in-
a simple LC-MS system equipped with an Electrospray vestigation,theMWofoligomericprocyanidinsthatcon-
Ionization(ESI)source(Fennetal.,1990;Gaskell,1997; taineduptosevencatechinunitsweredeterminedforthe
Cole, 2000; Cooks & Caprioli, 2000) operated in the firsttime.
negative-ionmodeandaquadrupolemassanalyzer.They Atmospheric-Pressure-Chemical-Ionization (APCI)
determinedaseriesof peaksattributedtonon-substituted (Wachs et al., 1991) and Electrospray-Ionization (ESI)
procyanidins from trimers [detected as [M(cid:1)H](cid:1)] to techniques were used to study a series of low-molecular
hexadecamers[detectedas[M(cid:1)3H]3(cid:1)]andtheiracylated mass phenols and polyphenols present in wine, such as
derivatives that contained one, two, or three gallic acid vanillin(5),syringicaldehyde(7),gallicacid(10),vanillic
residues (Cheynier et al., 1997). The mass spectrum of a acid (11), caffeic acid (14), ferulic acid (15), p-coumaric
grape-seed extract is reported in Figure 5. They also acid (16), (þ)-catechin, ((cid:1))-epicatechin, ((cid:1))-epigalloca-
proposed the fragmentation of B-type and A-type dimers techin,((cid:1))-epicatechin-3-O-gallate,andepigallocatechin-
and trimers of catechin (structures reported in Fig. 6), 3-O-gallate (see Figs. 2 and 4) (Pe´rez-Magarin˜o et al.,
previouslystudiedbyKarchesyetal.withFAB(Karchesy 1999). The investigation was performed at different cone
etal.,1986). voltages (60, 120, 180, and 210 V) in the positive- and
The analysis of the collision data generated by in- negative-ion modes, and ESI was a particularly effective
creasingtheorificevoltageshowedtwodifferentfragmen- techniquefortheanalysisofflavan-3-olsinbothmodes.In
tation patterns for the two trimeric species that were thenegative-ionmodewithaconevoltageof60V,thelow-
detected.CollisionspectraarereportedinFigure7together molecularmassphenolswereidentifiedbytheproduction
withtherelatedfragmentationpatterns.Theionatm/z863, ofveryabundantdeprotonatedmolecules.Theincreaseof
correspondingtothetrimerA-type,leadstotheformation conevoltageupto120Vcausedareductionofthemole-
of two different ‘‘dimeric’’ ions at m/z 575 and 573, and cularspeciesintensity,andthemostabundantpeakswere
undergoes the Retro-Diels-Alder (RDA) fragmentation duetofragmentsthatoriginatedfromthelossesofcarboxyl
process to produce the ions at m/z 711. Furthermore, the [M(cid:1)H-45](cid:1), hydroxyl [M(cid:1)H-17](cid:1), or/and aldehyde
lossofaneutralfragmentof152Da,correspondingto3,4- [M(cid:1)H-30](cid:1)groups.Ahigherconevoltageleadstoquite
dihydroxy-a-hydroxystyrene, and the formation of two complex mass spectra, with many peaks due to either
fragmentsatm/z285and289,generatedbycleavageofthe fragmentionsorpolymericadducts.Inparticular,thelatter
A-typeinterflavaniclinkage,arealsoobserved. phenomenon was observed for flavan-3-ols, due to their
A study of oligomeric and polymeric procyanidins highself-polymerizationcapability.
(structures formed by linkage of (þ)-catechins and ((cid:1))- On the contrary of what was observed in ESI
epicatechins units) present in grape seed extracts was conditions,theAPCImethodexhibitedalowersensitivity
reportedayearlater.Extractswerepreviouslyfractionated and did not lead to relevant results in the positive- and
bygelchromatography,andthem/zvaluesofdeprotonated negative-ion mode. Only operating in positive-ion mode
molecules[M(cid:1)H](cid:1)weredeterminedbyFAB.Therelated (APIþ)withaconevoltageof60Vandinjectingsolutions
spectra are reported in Figure 8 (de Freitas et al., 1998). thatcontained4.5%formicacid,theprotonatedmolecule
Oligomeric (þ)-catechin, ((cid:1))-epicatechin, (þ)-catechin ion, [MþH]þ, offlavan-3-ols was obtained with a better
FIGURE5. ESI mass spectra (negative-ion mode) of grape seed extract. (Reprinted from Analusis
Magazine25,Cheynieretal.,ESI-MSanalysisofpolyphenolicoligomersandpolymers,p.35,Copyright
1997,withpermissionfromEDPSciences.)
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& FLAMINI
Inthesameyear,usingaLC-ESI-quadrupoleanalyzer
system that operated in the negative-ion mode, Fulcrand
etal.(1999)characterizedtanninsofaCabernetSauvignon
wine(vintage1994).Thedealcoholizedwinesamplewas
fractionated on a Fractogel column by elution with an
ethanol/water/trifluoroaceticacidmixture.Afterthiolysis,
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
FIGURE6. StructuresofB-type(a)andA-type(b)dimerofflavan-3-
highestmassdetected.Theresultsshowedthatcondensed
ols. (Reprinted from Journal of Agricultural and Food Chemistry 47,
Lazarusetal.,High-performanceliquidchromatography/massspectro- tannins present in wine consist of procyanidins, prodel-
metryanalysisofproanthocyanidinsinfoodsandbeverages,p.3693, phinidins, and polymers that contain di- and tri-hydro-
Copyright1999,withpermissionfromAmericanChemicalSociety.) xylatedflavanolunits.
Lazarusetal.(1999)studiedproanthocyanidinsfrom
grapeseedsextracts,ingrapejuice,andinthePinotNoir
wine.Differentlyfromotherauthors,theyperformedLC/
sensitivity than that achieved in the negative-ion mode. MSanalysesinnormalphasechromatographywithasilica
However, because usually acid and non-acid compounds column.Thenormalphasechromatographyhadpreviously
are both present in natural samples, and acid compounds showed to be the better method to obtain a satisfactory
are detected in low yield in the positive-ion mode, the separation of proanthocyanidin oligomers on the basis of
negative-ion modewas proposed as more suitable for the their MW.Analyses were performedinESIconditionsin
analysisofthenaturalextractsofinterest. thenegative-ionmodewithNHþOH(cid:1)asbuffer.[M(cid:1)H](cid:1),
4
FIGURE7. MassspectraofA-typeprocyanidintrimersobtainedbyLC-ESI-MSinthenegative-ionmode
andrelatedfragmentationpatterns.(ReprintedfromAnalusisMagazine25,Cheynieretal.,ESI-MSanalysis
ofpolyphenolicoligomersandpolymers,p.34,Copyright1997,withpermissionfromEDPSciences.)
224
MSINGRAPEANDWINECHEMISTRY &
FIGURE8. FABspectrumofoligomericandpolymericprocyanidinsingrapeseedextracts.(Reprinted
fromPhytochemistry49,deFreitasetal.,Characterizationofoligomericandpolymericprocyanidinsfrom
grapeseedsbyliquidsecondaryionmassspectrometry,p.1438,Copyright1998,withpermissionfrom
Elsevier.)
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& FLAMINI
[M(cid:1)2H]2(cid:1) and [M(cid:1)3H]3(cid:1) ions of the different com- 643) with adsorption maximum at 450 nm were also
poundsweredetected.Anionatm/z881wasalsopresentin identified. The authors suggested that these compounds
the spectrum, and it was suggested to correspond to two couldplayanimportantroleinwhitewinesandingrape-
different isobaric compounds: the dimer of epicatechin- derivedfoodbrowning,andcouldbeinvolvedinred-wine
gallate or the trimer epicatechin-epicatechin-epigalloca- aging.SomestructuresidentifiedbyEs-Safietal.(2000b)
techin. Because a MS/MS system (by which to perform arereportedinFigure9.
collisional experiments) was not available, the authors A study on the characterization of proanthocynidins
assigned this peak to the dimer on the basis of retention containedintheLeuconoselectTMcommercialbatch(from
times. The ion at m/z 325 was identified as [M(cid:1)H](cid:1) of Vitis vinifera seeds) by the use of LC coupled with
feruloyltartaric acid (structure 17 in Fig. 2). Differently thermosprayhasbeenpublishedin2000.Itwasbasedon
1
fromthegrapeseedsextracts,galloylatedoligomerswere thefractionamentoverSephadex LH-20resincolumnof
notobservedintheredwine,soauthorshypothesizedthat LeuconoselectTM, and the analysis of fractions by the
theyarepoorlyreleasedfromthegrapeinthewinemaking. employmentofatriple-quadrupolemassspectrometerthat
Thereactionbetween(þ)-catechinandglyoxylicacid recordedpositiveionsfromm/z160to1200(Gabettaetal.,
in a model solution system was investigated by LC/ESI- 2000). Signals that corresponded to the protonated
MS-quadrupole analyzer system that operated in the molecule ions, [MþH]þ, of catechin (m/z 291), epica-
positive- and negative-ion modes (Es-Safi et al., 2000a). techingallate(m/z443),andflavan-3-oldimers(m/z579),
Glyoxylic acid is formed in the wine by an oxidation of and to cationized molecules [MþNa]þ of flavan-3-ol
tartaricacid,andithasbeendemonstratedthatitreactswith dimers (m/z 601) and flavan-3-ol dimer galloylated (m/z
(þ)-catechin to give colorless compounds that consist of 731)wereidentified(seeFig.10).
oligomeric molecules, where flavanol units are linked InthatstudyanESImethodwasalsodeveloped,andby
betweenthembycarboxymethinegroups(Fulcrandetal., this approach, [MþH]þ ions of dimers, trimers, and
1997; Es-Safi et al., 2000b). Structures with one or two tetramersofcatechin(m/z579,867,1155),theirmono-and
formylgroupsinpositionsC-6,C-8orC-6,andC-8of(þ)- di-galloyl derivatives (m/z 731, 1019, 1307, 883, 1171,
catechin ([M(cid:1)H](cid:1) ions at m/z 317 and 345) and dimers 1459), and the trigalloyl derivatives of trimers and tetra-
formed by a two (þ)-catechin linkage through a methine mers(m/z1323and1611)wereeasilyfound.Identification
group([M(cid:1)H](cid:1)ionsm/zat587)wereidentified.Methyl of[MþH]þionsofflavan-3-olspentamers,hexamers,and
andethylestersofxanthyliumcompounds(m/zat629and heptamers (m/z 1443, 1731, 2019), of their monogalloyl
FIGURE9. Structuresofthexanthyliumsalts(b)andwith(þ)-catechinsubstitutedatpositionsC-6,C-8or
C-6,andC-8byformylgroups(a),identifiedinamodelsolutionbyLC/ESI-MS.Thesecompoundscould
playanimportantroleinwhitewineandgrape-derivedfoodbrowning,andinredwineaging.(Reprinted
fromJournalofAgriculturalandFoodChemistry48,Es-Safietal.,Newphenoliccompoundsformedby
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
ChemicalSociety.)
226
MSINGRAPEANDWINECHEMISTRY &
FIGURE10. Identificationofflavan-3-olsandflavan-3-oldimersintheHPLCthermospray-MSprofileof
grapeseedsextract:D,dimer;C,(þ)-catechin;E,((cid:1))-epicatechin;DG,dimergallate;EG,((cid:1))-epicatechin
3-O-gallate.(ReprintedfromFitoterapia71,Gabettaetal.,Characterization ofproanthocyanidinsfrom
grapeseeds,p.171,Copyright2000,withpermissionfromElsevier.)
derivatives (m/z 1595, 1883, 2171), of pentamers and the basis of accurate mass values; the analysis was per-
hexamers digalloyl derivatives (m/z 1747 and 2035), of formed without any prior separation or purification step
pentamers and hexamers trigalloyl derivatives (m/z 1899 (Cooper & Marshall, 2001). This study was possible
and2187)wasalsoreported(seeFig.11). because of the high mass-resolving power (typically m/
Inarecentstudy,ESIcoupledwithFourierTransform Dm (cid:2)80000) and mass accuracy ((cid:3)1 ppm) typical of
50%
Mass Spectrometry (Amster, 1996) was applied to deter- this technique. The method was ideal for the study of
mine polyphenolic fingerprints of five different wines on complexmixturessuchaswine,becausetheaccuratemass
227