A fast method using a new hydrophilic lipophilic balanced sorbent in combination with ultra performance

(1)

Other uses, including reproduction and distribution, or selling or

licensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of the

article (e.g. in Word or Tex form) to their personal website or

institutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies are

encouraged to visit:

(2)

Talanta

j ourna l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / t a l a n t a

A

fast

method

using

a

new

hydrophilic–lipophilic

balanced

sorbent

in

combination

with

ultra-high

performance

liquid

chromatography

for

quantification

of

significant

bioactive

metabolites

in

wines

Catarina

L. Silva

a

_,

_Jorge

_Pereira

a

_,

_Van

_G.

_Wouter

a,b

_,

_Carme

_Giró

a,c

_,

_José

_S.

_Câmara

a,∗

a_CQM_–_Centro_de_Química_da_Madeira,_Centro_de_Ciências_Exactas_e_da_Engenharia_da_Universidade_da_Madeira,_Campus_{Universitário}_da_Penteada,_9000-390_Funchal,_Portugal b_Katholieke_Hogeschool_Kempen,_{Kleinhoefstraat}_4,₂₄₄₀_Geel,_Belgium

c_Institut_de_l’Aigua,_Universitat_de_Barcelona,_Avinguda_Diagonal,_684,₀₈₀₃₄_Barcelona,_Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received8June2011

Receivedinrevisedform2August2011 Accepted6August2011

Available online 1 September 2011

Keywords: Wines

Bioactivemetabolites Solidphaseextraction Ultra-highperformanceliquid chromatography

a

b

s

t

r

a

c

t

Thismanuscriptdescribesthedevelopmentandvalidationofanultra-fast,efficient,andhigh through-putanalyticalmethodbasedonultra-highperformanceliquidchromatography(UHPLC)equippedwitha photodiodearray(PDA)detectionsystem,forthesimultaneousanalysisoffifteenbioactivemetabolites: gallicacid,protocatechuicacid,(−)-catechin,gentisicacid,(−)-epicatechin,syringicacid,p-coumaricacid, ferulicacid,m-coumaricacid,rutin,trans-resveratrol,myricetin,quercetin,cinnamicacidandkaempferol, inwines.A50-mmcolumnpackedwith1.7-␮mparticlesoperatingatelevatedpressure(UHPLC strat-egy)wasselectedtoattainultra-fastanalysisandhighlyefficientseparations.Inordertoreducethe complexityofwineextractandimprovetherecoveryefficiency,areverse-phasesolid-phaseextraction (SPE)procedureusingassorbentanewmacroporouscopolymermadefromabalancedratiooftwo monomers,thelipophilicdivinylbenzeneandthehydrophilicN-vinylpyrrolidone(OasisTM_HLB),_was performedpriortoUHPLC–PDAanalysis.Thecalibrationcurvesofbioactivemetabolitesshowedgood linearitywithintheestablishedrange.Limitsofdetection(LOD)andquantification(LOQ)rangedfrom 0.006␮gmL−1to0.58␮gmL−1,andfrom0.019␮gmL−1to1.94␮gmL−1,forgallicandgentisicacids, respectively.Theaveragerecoveries±SDforthethreelevelsofconcentrationtested(n=9)inredand whitewineswere,respectively,89±3%and90±2%.Therepeatabilityexpressedasrelativestandard deviation(RSD)wasbelow10%forallthemetabolitesassayed.Thevalidatedmethodwasthenapplied toredandwhitewinesfromdifferentgeographicalorigins(Azores,CanaryandMadeiraIslands).The mostabundantcomponentintheanalysedredwineswas(−)-epicatechinfollowedby(−)-catechinand rutin,whereasinwhitewinessyringicandp-coumaricacidswerefoundthemajorphenolicmetabolites. Themethodwascompletelyvalidated,providingasensitiveanalysisforbioactivephenolicmetabolites detectionandshowingsatisfactorydataforalltheparameterstested.Moreover,wasrevealedasan ultra-fastapproachallowingtheseparationofthefifteenbioactivemetabolitesinvestigatedwithhigh resolutionpowerwithin5min.

1. Introduction

Themoleculespresentinwinescoveralargenumberof metabo-litesincludingprimary (e.g.,sugars,organicacids,amino acids) andsecondarymetabolites(e.g.,polyphenolsincludingflavonoids, anthocyanins, and other pigments). These compounds have a stronginfluence onthequality and character of thewine, and therefore areimportant not only for theircharacterization and differentiation,butalsoforfrauddetection[1–6].Inrecentyears considerableattentionis beingpaid tonatural substances with

∗Correspondingauthor.Tel.:+351291705112;fax:+351291705149. E-mailaddress:jsc@uma.pt(J.S.Câmara).

antioxidant activity,which playa crucialrole intheprevention of cardiovascular,neurodegenerativeand cancerdiseases acting throughdifferentmechanisms,namelydirecttrappingofreactive oxygenspecies(ROS)andreactivenitrogenspecies(RNS,i.e.nitric oxide),inhibitionofenzymesresponsibleforproducing superox-ideanions,chelationoftransitionmetalsinvolvedinprocessesthat originateradicalsandpreventionoftheperoxidationprocessby reducingalkoxylandperoxylradicals.Grapesandgrape-derived products suchas red wineare an abundant source of antioxi-dants(phenoliccompounds)andrepresentanimportantdietary component[1,2].Theirnatureandcontentcanvarysignificantly accordingtodifferentintrinsicandextrinsicfactorssuchasplant genetics and cultivar,soil composition and growingconditions, maturity stateand post-harvest conditions,among others [5,6].

(3)

Fig.1.Chemicalstructuresofdifferentclassesofpolyphenols.

Phenoliccompounds,mainlypolyphenols,areresponsibleforsome oftheorganolepticcharacteristicsofthefruits,foodand drinks

[7],and actas potentantioxidants,reinforcing antioxidant sys-temagainst ROS and RNS. In fact, several studies associate to polyphenolsawiderangeofbiologicaleffects,including antibacte-rial,anti-inflammatory,anti-allergicandantithromboticactivities

[8].Moreover,accumulatingdatafromnumerousepidemiological studiesindicatethatregularintakeofpolyphenol-richbeverages andfoodssuchasredwine,tea,chocolate,fruitandvegetables leadtoa reductionof vascular oxidativestressand consequent increase of endothelial function,thus improvingcardiovascular protection[9].Theroleofpolyphenolsinthisprotectionasinvivo antioxidantsisclearlystrengthenedbytheirabilitytobindtoLDL, preventingits oxidation and consequent atherosclerosis devel-opment[9,10].Thesebioactivemolecules canbecategorizedas flavonoidsandnonflavonoidsphenoliccompounds.Thefirstclass includesflavonols,representedmainlybyquercetin,kaempferol, and myricetin; flavones represented by apigenin and luteolin; flavan-3-ols,rangingfromthesimplemonomers(+)-catechinand itsisomer(−)-epicatechintotheoligomericandpolymeric proan-thocyanidins; flavanones, isoflavones, and anthocyanidins. The nonflavonoids includes C6–C1 phenolic acids such as gallic, p -hydroxybenzoic,protocatechuic,vanillic,andsyringicacids;C6–C3 hydroxycinammates:p-coumaric, caffeic, and ferulic acids (fre-quentlyaccumulate as theirrespectivetartrateesters, coutaric, caftaric, and fertaric acids) and their conjugated derivatives;

andstilbenes[3,11].Thechemical structuresofpolyphenols are reportedinFig.1.

Owingtothecomplexnatureofthewinematrix,thereisa con-sensusonthatefficientsamplepreparationtrace-leveldetection andidentificationareimportantaspectsofanalyticalmethodsto determinepolyphenolsinwines.

Thetechniquemostfrequentlyusedforsampleextractionor clean-upisliquid–liquidextraction(LLE).However,insomecases, suchmethodologyisstillnotselectiveenoughtocomplywiththe needsof food safety and regulations. NowadaysSPEis becom-ingmorepopularbecauseitrequirelessorganicsolvent,iseasy to implement, allow high sample throughputs and, in general, isusedwithgoodresults.In thisway,C18 cartridgeshavebeen

widelyusedinpolyphenoliccompoundsextraction,nevertheless theypresentsomedisadvantages,suchaslowrecoveriesforsome polarcompounds,namelyhydroxybenzoicandhydroxycinnamic acids.

AccordingtoPerez-Magarinoetal.[12]theuseofpolymer car-tridge, mainly theHLB with N-vinylpyrrolidone-divinylbenzene copolymerassorbentseemstobeagoodalternativetoreplace C18cartridges.TheseHLBcartridgespresentstrongerretentionand

(4)

method (time%B) method

Wines

Flavan-3-ols,phenolic acidsandstilbenes

•Filtration(0.45␮m)

•10×dilutioninthemobile phases.

SynergiHydroRP(150mm×4.6mm, 4␮m)

(A)0.1%H3PO4,(B)MeOH;0min10% B,15min22%B,25min50%B,34min 66%B,35min10%B.

HPLC–UV/VIS [19]

(A)0.25%AA+50%ACN,(B)2%AA; 0min70%B,10min70%B,30min0%B, 350%B,40min70%B.

HPLC–MS/MS

•Acidification(pH2.0) •Extraction2×DE(20min) •Organiclayerseparation; evaporation

•Regenerationin50% MeOH

Nova-PakC18column (150mm×3.9mmi.d.,4␮m)

(A)2%AA+10%MeOH,(B)2%AA+90% MeOH;0min0%B,15min15%B, 25min50%B,34min70%B,46min0% B.

RP-HPLC–DAD [20]

Flavan-3-olsand stilbenes

•Filtration(0.45␮m) UPLCHSST3column

(150mm×2.1mm,1.8␮m)

(A)0.1%FA,(B)ACN;0min%B,2.5min 20%B;7min80%B,7.5min80%B; 8min20%B.

UPLC–PDA [21]

Flavonols,flavan-3-ols, anthocyaninsand phenolicacids

•Driedskinsandseeds addedto

CH3OH/H2O/HCOOH (50:48.5:1.5,v/v) •Centrifugation(2500×g, 5◦_C,₁₅_min)

Ace®₅_C18_column₍₂₅₀_mm_×_4.6_mm) _(A)_NH4H2PO4₍₅₀_mM,_pH_2.6),_(B)_20%

mobilephaseA+80%ACN(C)H3PO4 (200mM,pH1.5);0min100%A,2min 100%A,5min92%A8%B,17min0%A 14%B,22min0%A18%B,29.5min0% A21%B,55min0%A33%B,70min0% A50%B,75min0%A50%B,78min20% A80%B,81min20%A80%B,86min 100%

HPLC–DAD-FD [22]

Redwines

Flavan-3-ols,flavonols, flavanoldimmers, phenolicacidsand organicacids

•Freeze-dryingandliquid extraction

•Extractfractionationby semipreparative C18-RP-HPLC.

BEHC18column(100mm×2.1mmi.d., 1.7␮m)

(A)5%FA,(B)ACN;0min10%B,12min 30%B;12.5min50%,13.5min100%B; 13.6min10%B.

UPLC–MS [15]

Flavan-3-ols,flavonols phenolicacidsand stilbenes

•Dilution1:10(10% MeOH+10mMammonium formatebufferpH3.75).

HTC-18reversedphase (50mm×2.1mm,1.8␮m)

(A)0.1%FA+1%ACN,(B)0.1%FA+99% ACN;0min2%B,1.5min2%B, 11.25min45%B,12.75min70%B, 12.82min2%B.

LC–MS/MS [23]

Flavan-3-ols,flavonols andphenolicacids

•Filtration(0.45␮m) OmniSpherC18column

(250mm×4.6mm);ChromSepguard column(100mm×3mm)

(A)0.1%H3PO4,(B)MeOH;0min55% B,30min80%B,33min80%B,35min 5%B. HPLC–PDA [24] Flavanols, anthocyaninsand phenolicacids •Dealcoholizationand acidification(concentrated FApH2.0)

•Fractionationinto aqueousandorganic fractionsusingLLEandSPE

XTerraTM_MS_C18_reverse_phase column(150mm×2.1mm,3.5␮m); XTerraTM_MS_C18_guard_column (10mm×2.1mm,3.5␮m)

Aqueous(anthocyanin)fraction: (A)2%FA+2%ACN,(B)2%FA+80% ACN;0min5%B,11min15%B,26min 30%B,46min60%B,69min80%B, 94min5%B,124min55B.

HPLC–DAD-MS [17]

Organic(non-anthocyanin)fraction: (A)0.1%FA+2%ACN,(B)0.1%FA+80% ACN;0min5%B,30min25%B,35min 30%B,40min55%B,45min55%B, 50min80%B,85min5%B,90min5%B. Flavonolsandflavones •Filtration(0.45␮m) LiChrospher100RP-18ecolumn

(250mm×4.0mmi.d.,5␮m);RP-18

(10mm×4.0mm)guardcolumn

(A)19%ACN+5%MeOH+1%THF(pH 3.0),(B)55%ACN+15%MeOH(pH3.0); 0min2%B,15min2%B,28min28%B, 40min36%B,44min36%B,45min 80%B,52min80%B.

HPLC–UV/VIS [16]

Flavan-3-ols,flavonols, flavones,phenolic acidsandothers phenolics

•Dilutionto90%with Milli-Qwater.

KinetexC18column (100mm×4.6mmi.d.,2.6␮m)

(A)0.1%FA,(B)ACN;0min5%B,4min 5%B,10min23%B,15min23%B, 19min50%B,20min95%B.

HPLC–DAD [25]

Whitewines Glutathione,catechin

andcaffeicacid

•Centrifugation (14,000rpm,5min) •Derivatizationwith p-benzoquinone.

BEHC18column(100mm×1.7mm, 1.7␮m)

(A)0.05%TFA,(B)MeOH;0min10%B; 5min17%B;7.5min30%B;8.5min 35%B;8.8min100%B;9.3min10%B; 11min10%B.

UPLC–PDA [26]

Phenolicacids •Infusion(dryleaves incubationindistilled water10min,80◦_C) •Filtration(3500rpm, 5min,0.22␮m)

BEHC8reversedphasecolumn (150mm×2.1mm,1.7␮m)

(A)7.5mMFA,(B)ACN;0min5%B,5% B0.8min,1.2min10%B,1.9min10%B, 2.4min15%B,3.7min15%B,4min21% B,5.2min21%B,5.7min27%B,8min 50%B,9min100%B,9.5min5%B.

UPLC–MS/MS [27]

Otherwines(Brandies,fruitwinesandmodelwinesolutions) Phenolicacids,

aldehydesandfuran compounds

•Filtration(0.22␮m) LiChrospherRP-C18column

(250mm×4mmi.d.,5␮m)

(A)2%AA+5%MeOH,(B)2%AA+90% MeOH;0min0%B,20min10%B, 60min75%B.

(5)

Table1(Continued)

Analytes Samplepreparation method

Stationaryphase Mobilephases;gradientconditions (time%B)

Detection method

Ref.

UPLCBEHC18column (100mm×2.1mmi.d.,1.7␮m)

(A)2%AA+3%ACN,(B)2%AA+85 ACN;0min0%B,3min10%B,4min 10%B,6.5min75%B,9.5min100%B, 12.5min0%B.

UPLC–PDA

Anthocyaninsand hydroxycinnamic acids

•Referencesolutions preparedinawine-like medium(tartaricacidin 12%EtOHacidifiedtopH 3.5)

BEHC18column(50mm×2.1mmi.d., 1.7␮m)

(A)acidifiedwater(pH2.6),(B)ACN; 0min3%B,2min10%B,6.8min24%B, 10.8min40%B,11min3%B,15min3% B.

UPLC/ESI-TOF MS

[13]

Abbreviations:PDA,photodiodearraydetection;DAD,diodearraydetection;DAD–ESI-MS,diodearraydetectionandelectrosprayionizationmassspectrometry; DAD–FD-ED,diodearray,fluorescenceandelectrochemicaldetectors;FD,fluorescencedetector;LLE,liquid–liquidextraction;SPE,solid-phaseextraction;SS-LLE,solid-supported liquid–liquidextraction;AA,aceticacid;ACN,acetonitrile;DE,diethylether;EA,ethylether;EtOH,ethanol;FA,formicacid;MeOH,methanol.

Liquidchromatographic(LC)methodologiesrepresent,todate, themostwidelyusedapproachtophenolicanalysis.Several meth-odsforthedetectionandquantificationofphenoliccompoundsin winesandothermatrices,usingthisapproach,havealreadybeen developed(Table1).However,insomeofthereportedanalytical methodsthepolyphenolsseparationrequire40,50oreven60min ofrunanalysistimepersample[14–18].

Asanalternative,anultra-fastUHPLCmethodappearsrecently toovercomesomeoftheLCdrawbacksretaining theprinciples ofLCwhileofferingsomeclearbenefitsintermsofanalysistime, resolvingpower,solventconsumptionand,toalesserextent, sen-sitivity[25,13].Uptonow,onlyafewapplicationsusingUHPLC havebeenreportedfortheanalysisofpolyphenols(Table1).Spacil etal.[14]comparedaconventionalHPLCsystemandaUHPLC sys-tem,equippedwithcolumnscontainingsimilarstationaryphases, intheanalysisofwineandteasamples.Whilebothanalytical meth-odsgavegoodresults,theUHPLCsystemappearedtobesuperior. UHPLCmethodsarenotonlyfaster,moresensitiveandmore effi-cient,butarealsomorereliableandecological[14,29].

Hence, the current research study is dedicated towardsthe developmentand validation of an ultra-fast, efficient and high throughputUHPLC–PDAmethodusinga50-mmcolumn(Acquity BEHRP18)packedwith1.7-␮mparticles,fortheseparationand

quantificationofawiderangeofbioactivepolyphenolsinwines. Inordertoreducethecomplexityofwineextractandimprovethe recoveryefficiency,areverse-phaseSPEprocedureusingassorbent anewcopolymermadefromabalancedratiooftwomonomers,the lipophilicdivinylbenzeneandthehydrophilicN-vinylpyrrolidone, wasperformedpriortoUHPLC–PDAanalysis.Thismethodpresents manyadvantagesthatincludehighresolution,speed,an exception-allysmallsamplevolumeandshortrunningtimefortheseparation. Itwasappliedinredandwhitewinesfromdifferentregionsto characterizethephenolicswithanefficientseparation. Neverthe-less,themethodcanbeappliedinseveralotherapplicationssuch asplantextractsanddietarysupplements.

2. Materialsandmethods

2.1. Chemicalsandstandards

For the UHPLC–PDA experiments, HPLC-gradient grade methanol (99.9% purity) was obtained from Sigma–Aldrich (St. Louis, MO, USA). Ethanol and formic acid, both of HPLC-gradientgrade,wereprovidedbyPanreacQuimicaSA(Barcelona, Spain) and ethyl acetate from Lab-Scan (Dublin, Ireland). The eluentswerepreviously filteredwith0.22␮mmembrane filters

(Millipore,Milford,MA, USA).The SPEcartridges used,OasisTM

HLBsorbent(30mg) fromWaters was provided byVia Athena (Lisbon,Portugal).Finally,ultra-purewaterwaspreparedfroma

Milli-Qwaterpurificationsystem(Millipore,Bedford,MA,USA), Polyphenolsstandards gallicacid, gentisicacid, (−)-epicatechin, m-coumaric acid, cinnamic acid,p-coumaric acid, rutin, ferrulic acid and kaempferol, were supplied by Fluka Biochemica AG (Buchs,Switzerland), protocatechuicacid, (−)-catechin, syringic acidandtrans-resveratrol,bySigma–Aldrich(St.Louis,MO,USA), myricetinwasacquiredfromAcrosOrganics(Geel,Belgium)and quercetinfromRiedel-de-Haën(Seelze,Germany). Thepurity of allpolyphenolicstandardswasgreaterthan95%.

Individual stocksolutions of all bioactivemetabolitestested werepreparedinpuremethanolatconcentrationsof1000mgL−1_,

aliquotedin 4mLvials,andstored at−20◦_C._Working_standard solutionsatvariousconcentrationswereprepareddailyfromthe individualstocksolutionsinthemobilephaseusedatinitialstepof gradientelution(waterat0.1%formicacidwitha20%ofmethanol solution).Thesestandardswereusedtospikethewinesinorder toperformtheassaysformethodvalidation.Thechemical struc-tures of the investigated bioactive metabolites are reported in

Fig.1.Calibrationstandardswerepreparedfreshonthedayofthe analysisbydilutingtheappropriateworkingsolutionswithinitial mobilephasesolution.Therangesofconcentrationswereselected infunctionofsensitivityoftheUHPLC–PDAtowardseachbioactive metabolite.Physical–chemicalcharacteristicsaffecttheanalytical signalandforsomecompoundshigherconcentrationshadtobe usedinordertodetectedthecompound.

2.2. Samples

Themethodologywasappliedtoninerepresentative commer-cialtablewinesavailablefromdifferentregions,Madeira(TT,PT,LB, EB,SB,RBB),Azores(TLT)andCanaryIslands(VT,VGB)whichwere producedaccordingtostandardproceduresanddefinedvarietal composition.

2.3. Bioactivemetabolitesextractionprocedure

Thepolyphenolsextractionprocedurewasbasedon reverse-phaseSPEusing OasisTM _HLB _extraction_cartridges. _Briefly, ₁_cc

(30mg)OasisTM_HLB_extraction_cartridges_were_conditioned_with

1mLofethylacetateandmethanolandequilibratedwith1mLof water.Analiquotofsample(900␮L),previouslyacidifiedtopH

2.7withaceticacid30%,waspassedthroughtheSPEcartridges at around 1.5mLmin−1 _using _an _extraction_unit _(Alltech, _SGE).

Thecartridgeswerewashedwithasolutionofmethanol5%and subsequentlythepolyphenolswereelutedwith500␮Lmethanol,

1000␮L ethylacetateandfinally500␮Lmethanol.Theextracts

wereevaporated todrynessusinga nitrogenstream.The dried residuewasredissolvedin1000␮Lofinitialmobilephase(water

(6)

times.

2.4. UHPLC–PDAanalysisandoperatingconditions

The analysisof bioactive metabolites werecarried out on a WatersUltraPerformance LiquidChromatographicAcquity sys-tem(UPLC,AcquityH-Class)(Milford,MA,USA) combinedwith aWatersAcquityquaternarysolventmanager(QSM),anAcquity samplemanager(SM),acolumnheater,a2996PDAdetector,anda degassingsystem.ThewholeconfigurationwasdrivenbyEmpower softwarev2.0fromWatersCorporation.Optimumseparationwas achievedwithabinarymobilephasewhichconsistedof(A)water at0.1%formicacid,and(B)methanol,witha constantflowrate of 250␮Lmin−1,giving a maximum backpressure of 6.000psi,

whichiswithinthecapabilitiesoftheUHPLC.2␮Lofextractswere

injectedintotheWatersAcquityUPLCsystem,equippedwithan AcquityUPLCTM_bridged_ethylene_hybrid_BEH_C

18analytical

col-umn(1.7␮mparticlesize,2.1mm×500mm)andprotectedwith anAcquity UPLCTM _BEH _C

18 Van GuardTM Pre-column(1.7␮m,

2.1mm×5mm)(Waters,Milford,MA,USA);columntemperature wasthermostatedat40◦_C_and_the_samples_were_kept_at₄◦_C_in_the samplemanager.The5mingradientwasasfollows:0min,80%A, 0.25min,70%A,0.5min,68%A,4min,20%A,and5min,80%A, followedbyare-equilibrationtimeof3min,forbringingthe col-umntotheinitialconditionsaftergradientanalysis,givenatotal runtimeof8min.Allsolventsandsampleswerefilteredthrough 0.22␮mmembranefiltersfromMillipore(Millipore,Milford,MA,

USA),beforeuse. Forquantificationpurposes thePDAdetection wasconductedbyusingfourdistinctchannelsthatweresettothe maximumabsorbancewavelengthofeach polyphenol,210,270, 307,and360nm,asindicatedinTable3.Theywereidentifiedby comparingtheretentiontimeandspectralcharacteristicsoftheir peakswiththoseofstandardsandtheywerequantifiedusingthe standardsofeachone.

Low Medium High

(LL) (ML) (HL)

Gallicacid 0.5 5.0 10.0

Protocatechuicacid 2.0 12.0 30.0

(−)-Catechin 2.0 15.0 45.0

Gentisicacid 4.0 16.0 40.0

(−)-Epicatechin 5.0 20.0 60.0

Syringicacid 1.5 5.0 16.0

p-Coumaricacid 2.0 20.0 80.0

Ferulicacid 1.0 3.0 8.0

m-Coumaricacid 2.0 10.0 30.0

Rutin 3.0 15.0 32.0

Trans-resveratrol 2.0 15.0 60.0

Myricetin 3.5 20.0 60.0

Quercetin 2.0 10.0 30.0

Cinnamicacid 3.0 10.0 30.0

Kaempferol 2.0 15.0 40.0

2.5. Methodvalidation

Retentiontimeswerepreviouslydeterminedusingindividual standards.Themethodwasvalidatedintermsofselectivity, lin-earity,limitsofdetection(LOD) andquantification(LOQ), inter-andintra-dayprecisions,accuracyandextractionefficiency.The accuracyandprecisionstudieswerecarriedoutbyspikingred(PT) andwhite(LB)winesatthreedifferentconcentrationlevels, cor-respondingtothelowlevel(LL),mediumlevel(ML)andhighest point(HL)ofcalibrationcurve(Table2).

Theselectivityofthemethodwasassessedbytheabsenceof interferingpeaksattheelutiontimesofthebioactivemetabolites. Thelinearityoftheanalyticalmethodwasevaluatedbuildingthree calibrationcurves(theareasobtainedwereplottedagainstthe cor-respondingstandardconcentrations)foreachbioactivemetabolite usingstandards,preparedinwaterat0.1%formicacidwitha20% ofmethanolsolutionfromindividualstocksolutions,at7 differ-entconcentrationslevels,includingthezeropoint,runatdifferent

Table3

Validationprocessdatashowingtheconcentrationrangeinsidewhichthelinearitywastested,retentiontimes(RT),andresultsofregressionfortotalareaversusconcentration andanalyticalperformanceforthebioactivemetabolites(polyphenols)determinedbySPEOasis®_{HLB/UPLC-PDA.}

Peaknumber RT(min) Bioactivemetabolites maxb(nm) Analyticalperformancea

Conc.range(␮gmL−1) yc Rd LOD

(␮gmL−1)e

LOQ (␮gmL−1)e

1 0.664 Gallicacid 270 0.01–10.00 8.98E+07x–7.82E+03f _0.995 _0.006 _0.019

2 1.032 Protocatechuicacid 270 0.10–30.00 1.85E+07x–1.53E+04 0.985 0.008 0.026

3 1.234 (−)-Catechin 210 2.50–45.00 7.67E+07x+2.09E+03 0.983 0.089 0.297

4 1.595 Gentisicacid 307 2.00–40.00 2.06E+06x+1.58E+03 0.996 0.581 1.938

5 2.044 (−)-Epicatechin 270 1.00–60.00 9.52E+05x+7.03E+02 0.994 0.180 0.599

6 2.210 Syringicacid 270 0.25–16.00 5.90E+06x+4.18E+03 0.989 0.008 0.026

7 2.615 p-Coumaricacid 307 0.05–80.00 6.07E+06x+1.19E+03 0.997 0.011 0.035

8 2.791 Ferulicacid 307 0.25–8.00 122E+07x+4.46E+03 0.989 0.014 0.047

9 2.946 m-Coumaricacid 315 0.25–30.00 1.65E+07x+1.42E+03 0.994 0.076 0.255

10 3.029 Rutin 360 1.00–32.00 1.02E+07x+3.18E+03 0.998 0.011 0.038

11 3.187 Trans-resveratrol 307 0.80–60.00 3.52E+06x–5.57E+03 0.989 0.032 0.108

12 3.393 Myricetin 360 2.50–60.00 3.64E+06x–1.40E+04 0.995 0.038 0.128

13 3.891 Quercetin 360 1.00–30.00 6.14E+06x–1.31E+04 0.982 0.010 0.032

14 4.108 Cinnamicacid 270 0.05–30.00 2.27E+07x+2.70E+03 0.999 0.013 0.044

15 4.388 Kaempferol 360 0.40–40.00 1.09E+06x+5.56E+03 0.987 0.059 0.198

a_RT:_retention_time_(min).

b _max:_maximum_absorbance_wavelength. c _y_:_represents_the_peak_area_of_bioactive_phenolics. d _R_:_correlation_coefficient.

e_LOD:_limit_of_detection,_the_lowest_analyte_{concentration}_that_produces_a_response_detectable_above_the_noise_level_of_the_system_–₍_a₊₃_Sa_)/_b_,_and_LOQ,_{quantification} limit,thelowestlevelofanalytethatcanbeaccuratelyandpreciselymeasured–(a+10Sa)/b.

(7)

days.Thezeropoint(extractionsolvent)enablestoverifythatnone ofthecompoundsshowedresiduallevelorbackgroundsignal.Each levelofconcentrationwaspreparedintriplicateandinjectedthree times,sotherewereatotalofninereplicates.

MethodsensitivitywasassessedbydeterminingtheLOD(the lowestanalyteconcentrationthatproducesaresponsedetectable abovethenoiselevelofthesystem)andLOQ(thelowestlevelof analytethatcanbeaccuratelyand preciselymeasured)foreach compound.LODandLOQwerecalculatedwiththedatagenerated inthelinearitystudies,beingLODdefinedas(a+3Sa)/bandLOQ as(a+10Sa)/b,where“a”representsoriginordinate,“Sa”theorigin ordinatevarianceand“b”theslope.Theseparameterswere calcu-latedforeachanalytefromthestandardsolutionsusedtoobtain thecorrespondingcalibrationcurves,usingtheUHPLCdeveloped method.For methodprecision,PTredwineandLBwhitewine, spiked at three different concentration levels (LL, ML and HL, seeTable2)ofeachpolyphenolandtreatedbySPEOasisHLBwere

measuredin seven replicates(n=7)in the same dayto obtain repeatability(intra-dayprecision),andthreetimesoverfour differ-entdaystoobtaininter-dayprecision(intermediateprecision– IP). Bothprecisionmeasureswereexpressedas%RSDwhichdescribes theclosenessofagreementbetweenseriesofmeasurements.

In order to check the accuracy of the proposed method a recoverystudywascarried outby spikinga redwine(PT) and a white wine (LB), at three concentration levels (Table 2) in triplicate.The mixtures were subjected to the SPEOasisHLB

pro-ceduredescribedinSection2.3.ThePTandLBwinesphenolics concentration was previously determined, and the recovery valueswerecalculatedaccordingtothefollowingformula: Accu-racy=100×([analyte]afterspiking–[analyte]beforespiking)/[analyte

added];where[analyte]afterspikingistheanalyteconcentrationin

spikedwine;[analyte]_before_spiking istheanalyteconcentrationin unspikedwine,and[analyteadded]isthenominalconcentration of the analyte added to wine. Extraction efficiency (EE) was determined by replicate analysis (n=3) of bioactive phenolics standardsolutions at medium level of concentration (ML) and processedasdescribedabove(ASS-SPE);asecondsolutionwiththe

sameconcentrationwasanalysedbutnotsubmittedtoSPEOasisHLB

(ASS).Theobtainedpeakarearatioswerecompared,andextraction

efficiencywasthuscalculated:%EE=(ASS-SPE/ASS)×100.

3. Resultsanddiscussion

Thereisavarietyofpolyphenolcompoundspresentinwines whichhasbeendemonstratedthatpossessratherpotent antioxi-dantpropertiesandasignificantdegreeofbioavailability,resulting in beneficial health effects. For this reason it is important to developefficientanalyticalmethodsabletoassessthesebioactive metabolites.Inthis work,theUHPLCmethoddevelopedforthe quantificationofbioactivephenolicsinwinesextractswasfound tobecapableofgivingafasteranalysiswithgoodresolutionthan thatachievedwithconventionalHPLC.

3.1. Methoddevelopmentandvalidation

Themostabundantbioactivemetabolitesoftenreportedtobe presentinwinecomposition,suchasgallicacid,(−)-catechinand (−)-epicatechin,aswellothers,likequercetin,thatarenotso abun-dant,buthaveahighbioactivepotentialagainstseveraldiseases

[30],werechosen tobeanalysedin this work.Thewavelength thatcorrespondtothemaximumabsorbanceintheUVregionwas determinedforeachoneofthepolyphenolsanalysed(Table3).

Themethodwasvalidatedbydeterminingtheselectivity,the linearity,thelimitsofdetection(LOD)andquantification(LOQ),the inter-andintra-dayprecisions(expressedas%RSD),theextraction

efficiencyandtheaccuracyatdifferentlevelsoffortification.The spikinglevelsusedfortheaccuracyandprecisionstudiesare sum-marizedinTable2.

Selectivitywasassessedbytheabsenceofinterferenceinthe same chromatographic windows as examined in a solution of standardsofbioactivephenolicsandanalysisof“blankmatrices” (extraction solvent). Nointerfering peakswere observedin the blankchromatograms(Fig.2a)atthequantificationwavelengths (210,270,307,315and360nm).

Methodlinearitywasevaluatedforeachoneofthe15bioactive metabolitesusingstandardsolutionsinordertodemonstratethat concentrationislinearlydependentondetectorresponse, while otherparametersinfluencingmethodprecisionandaccuracywere determined usingspikedrealwines treatedbeforeor afterSPE procedure.Todeterminethelinearityofthemethod,calibration plotsof theanalytepeak areaversusnominalstandard concen-trationsusing replicates(n=3)at sevenlevelsof concentration (Table3)wereconstructed.Theseconcentrationlevelscoveredthe concentrationrangesexpectedforthebioactivemetabolitesfound inwines.Table3summarizesthevalidationprocessdatashowing theconcentrationrangeinsidewhichthelinearitywastestedfor eachanalyte,theslope,theoriginordinate,thecorrelation coeffi-cients,andthelimitsofdetectionandquantificationforthetarget metabolites.

Asitcanbeseenthroughcorrelationcoefficients,the linear-itywassatisfactorywhenusingSPEOasisHLBmethod,withR-values

above 0.991 for allbioactive metabolites (Table 3). The lowest value ofR was obtainedfor both quercetinand protocatechuic acid(R>0.991)andthehighestvalueforcinnamicacid(R=0.999). Theslopeoftheregressionmodeldependsontheextraction effi-ciencyand onthedetectorresponsefor each compound.Gallic acid,cinnamic acid,andprotocatechuicacids,showedthe high-estslopevalueswithSPEOasisHLB/UPLC–PDAmethodology,while

(−)-epicatechinobtainedthelowest(Table3).

Thelimitsofdetectionwereestimatedasthepolyphenol con-centration which gave a signal equal to a+3Sa/b(described in Section2.5)[31].AsitcanbeseeninTable3,theSPEOasisHLB

/ULPC-PDAmethodologypresentedingeneralverylowdetectionlimits, rangingbetween0.006and0.58␮gmL−1,forgallicacidand

gen-tisicacid,respectively.Thelimitsofquantification,estimatedasthe polyphenolconcentrationwhichgaveasignalequaltoa+10Sa/b

[31],rangedfrom0.019␮gmL−1to1.94␮gmL−1,againforgallic

andgentisic acids,respectively.ComparingLODandLOQvalues obtainedwiththosereportedinliteratureforthephenolic com-pounds(Table1)thevaluesprovidedbythismethodaregenerally lowerthantheonesfromthecitedliterature.Theverylowvalues forLODandLOQconfirmedthehighsensitivityoftheproposed method for determination of bioactive phenolic metabolitesin wines.

Theprecisionofthemethod(Table4)wasevaluatedbasedonits repeatability,whichwasascertainedbyperformingseven succes-sivesampleextractionsfromspikedredandwhitewinesatthree differentconcentrationlevelsonthesamedayplusthree extrac-tionsperdayfor4days(IP).Theintra-dayrepeatabilityofpeak areas,expressedbymeansofthepercentageofrelativestandard deviation(%RSD,n=7),waslowerthan7%forredwinesand5% forwhitewines,withanaverageprecisionof3±3%,and3±1%, forredandwhitewines,respectively.TheIP(inter-day repeatabil-ity,4days;n=3),waslessthan10%,inbothwinetypes.Forred winesthelowestIPvaluewasobtainedformyricetin(1.4%)spiked at3.5␮gmL−1,andthehighestwasobtainedforgallicacidspikedat

0.5␮gmL−1(10%).ForwhitewinestheIPvaluesrangingfrom1.1%

(myricetin,3.5␮gmL−1)and8.9%forkaempferol(40.0␮gmL−1).

(8)

Intra-day(n=7) Inter-day(n=12)

Redwine Whitewine Redwine Whitewine

LL ML HL LL ML HL LL ML HL LL ML HL

Bioactivemetabolites Gallicacid 2.55 1.57 0.89 5.32 1.41 2.41 10.30 4.50 9.31 3.06 6.88 3.94 Protocatechuicacid 7.30 3.33 1.55 2.51 1.26 3.43 9.66 4.93 3.66 3.16 4.58 1.37 (−)-Catechin 2.85 1.42 0.34 2.42 1.77 2.82 2.02 3.90 4.10 2.00 1.23 4.36 Gentisicacid 4.75 2.30 3.82 1.57 1.61 1.30 9.13 6.93 2.18 1.94 4.02 4.55 (−)-Epicatechin 8.11 1.36 6.51 4.64 2.54 2.17 9.44 6.19 4.45 6.52 8.83 3.83 Syringicacid 3.44 2.89 3.01 5.21 2.44 1.48 7.10 2.36 1.76 3.89 6.39 2.21 p-Coumaricacid 1.62 3.10 2.55 3.20 1.65 4.14 1.54 8.66 8.16 2.86 6.15 2.26 Ferulicacid 5.02 1.52 1.70 4.17 1.27 6.24 2.21 5.02 5.74 3.68 5.39 8.51 m-Coumaricacid 2.51 1.53 2.91 2.80 1.24 1.89 2.69 6.18 1.13 7.25 5.80 4.06 Rutin 1.38 1.61 2.50 1.69 1.74 4.95 2.53 5.81 6.01 7.87 7.02 4.40 Trans-resveratrol 3.21 1.43 1.97 1.29 0.82 3.15 2.55 6.56 1.80 2.97 5.25 3.56 Myricetin 7.80 3.60 2.93 1.29 1.78 0.76 1.37 3.17 2.20 7.06 4.82 3.04 Quercetin 4.58 1.86 1.14 3.09 5.29 2.59 4.56 9.14 2.89 2.53 4.02 6.02 Cinnamicacid 5.55 2.77 2.43 1.13 1.58 5.54 512 5.68 4.36 4.92 5.65 5.92 Kaempferol 3.72 1.67 0.31 2.07 3.19 3.35 4.97 7.56 9.07 6.69 7.99 8.86

amountsofpolyphenolsatthreeconcentrationlevels,LL,MLand HL(Table2).Eachphenolicmetabolitepeakareaversusthe cor-respondingconcentrationwastheninterpolatedfromthelinear regression equation. In general,good recoveries were obtained forthemajorityofpolyphenoliccompounds,exceptforrutin(red wines)and(−)-epicatechin(whitewines)thatshowedthe low-estrecoveries59±39% and 72±3%, respectively,which can be attributedtotheirlowpolarstructures.Nevertheless,themethod accuracywasadequateforalltargetphenolicspresentinstudied wines.Forredwinestherecoveriesrangedfrom57%(rutin,HL –32␮gmL−1)to114%(quercetin,ML–10␮gmL−1),whereasin

whitewinestherecoveriesvariedbetween71%(gallicacid,LL– 0.5␮gmL−1)and12%(cinnamicacid,LL–3␮gmL−1)withan

aver-agerecovery±SD(n=9)of89±3%and90±1%,forredandwhite wines,respectively.Theextractionefficiencywascarriedoutatone concentrationlevel,ML(Table2)analysedintriplicate.Theresults areshowninTable5.Asonecansee,theresultsaresatisfactory, beingtheextractionefficiencieshigherthan77%(rutin),withan average±SD(n=3)of89±7%.

Takingtogether,theresultsobtainedshowthat,besidesbeing simple,fastandstraightforward,thesampleextractionusingthis SPEmethodwithOasisTM _HLB_cartridges_presents_a_good

repro-ducibilityandaccuracy[12].

3.2. Determinationofbioactivemetabolitesby SPEOasisHLB/UHPLC–PDA

OneofthemainadvantagesofUHPLCtechnologyisrelatedto thepossibilityofattainingeitherultra-fastorhighresolvingpower separations.Inordertodemonstratetheapplicabilityofthe devel-opedmethod,theSPEprocedurewasfirstappliedtoamixtureof polyphenolsstandards(Fig.2a)andthentoredandwhitewine samples(Fig.2b).AsshowninFig.2a,theseparationofthestandard mixtureof15polyphenolscompoundsisveryfast,beingachieved within5min.

Thechromatogramsforwhiteandredwines(Fig.2b)showed quitedifferentprofilesandtheircomplexityincreasesordecreases accordingtothewavelength(datanotshown).Thepolyphenolic contentinthewinesamplesanalysedisrepresentedinTable6.As canbeeasilyobserved,thepolyphenolscontentisaboutsixtimes moreabundantinredwinesthaninwhitewines.Thiswaswidely describedbeforeintheliterature[23].

RedwinefromAzores(TLT)wasbyfartheonethatshowed higher polyphenoliccontent consideringthe sumofthe fifteen polyphenols tested (almost 240␮gmL−1),followed by Madeira

and Canary Islands red wines, withpolyphenolic compositions around175(TT)and150␮gmL−1(VT),respectively(seeTable5).In

Table5

ValidationparametersbasedonrecoveriesandextractionefficiencystudiesforSPEOasis®_HLB/UPLC-PDA_of_bioactive_metabolites_in_spiked_red_and_white_wines.

Recovery(%) Extractionefficiency(%)

Redwine Whitewine

LLa _MLa _HLa _LLa _MLa _HLa _MLb

Bioactivemetabolites Gallicacid 80±7 85±5 84±8 71±4 67±5 80±7 87±9

Protocatechuicacid 84±9 103±8 80±6 84±8 94±6 79±6 92±8

(−)-Catechin 89±11 85±7 85±8 113±9 93±10 89±5 84±6

Gentisicacid 81±13 88±11 82±9 76±9 86±7 92±10 85±3

(−)-Epicatechin 95±10 87±9 80±6 70±4 72±5 75±4 80±5

Syringicacid 84±9 106±4 92±8 97±5 95±5 91±7 91±7

p-Coumaricacid 77±8 90±8 83±8 103±9 94±3 97±6 85±6

Ferulicacid 95±10 98±6 82±9 107±3 98±4 96±9 90±3

m-Coumaricacid 80±4 95±9 89±4 103±4 104±3 82±3 96±6

Rutin 59±7 63±5 57±4 74±4 76±8 79±6 77±5

Trans-resveratrol 105±7 101±7 100±5 86.±7 99±6 97±6 93±5

Myricetin 85±8 90±4 111±5 99±5 92±3 89±1 90±7

Quercetin 104±9 114±6 102±6 89±5 84±2 78±4 94±6

Cinnamicacid 98±7 96±6 108±8 116±3 97±8 101±5 96±3

Kaempferol 82±7 81±5 76±8 89±4 99±8 102±3 87±3

a_n₌_3;_average ±RSD%. b _n₌_6;_average

(9)

Fig.2.(a)Chromatogramof(i)extractantsolvent,inordertotestthemethodselectivity;and(ii)astandardmixtureofpolyphenols;(b)UHPLC–PDAchromatogramof(iii) aredwine;and(iv)awhitewine;usingabsorbancedetectionat307nm.IndividualretentiontimesandpeakidentificationofthepolyphenolsarepresentedinTable1.

previousworkswherewinepolyphenolscompositionfromAzores IslandswascomparedwithwinesfromotherregionsofPortugal, thissameresultwasobserved[26].Inwhitewines,the polyphe-noliccontentissignificantlylower,varyingfrom14␮gmL−1inthe

RBB,MadeiraIslandwine,upto45␮gmL−1intheCanaryIsland

VGBwine.Thisfactmakeswhitewineslesspronetobeeffectivein healthprotectionagainstoxidativedamageastheprotectiveeffects

associatedtomoderatewineconsumptionhavebeenattributed totheircontentinpolyphenols[10,27].Infact,Fuhrmanand col-laborators haveshown thatit waspossibletohave whitewine withantioxidantcharacteristicssimilartothoseofredwinejustby increasingitspolyphenolscontent,whatcouldbeeasilyobtained byincreasingtheextractionofgrapeskinpolyphenolsduringthe macerationprocess[28].

Table6

Concentrationa_of_bioactive_metabolites_found_in_red_and_white_wines_assayed_through_proposed_{SPEOasis HLB/UHPLC–PDA}_method.

Redwine Whitewine

Sampleidentification VT TLT TT PT LB EB SB RBB VGB

Sampleorigin CANb _AZOb _MADb _MAD _MAD _MAD _MAD _MAD _CAN

Bioactivemetabolites Gallicacid 2.1±1 2.6±7 4.0±5 2.4±6 0.2±10 0.3±16 0.4±10 0.2±10 0.2±3

Protocatechuicacid 10±0.2 6.9±2.3 4.3±3

(−)-Catechin 7.8±11 51.1±6 30.4±18 377±8 1.2±16 1.9±4 0.9±4

Gentisicacid

(−)-Epicatechin 95.8±2 147.2±3 123.8±7 19.9±2 15.9±2 14.9±2 12.7±3 9.8±5 36.9±0.3

Syringicacid 8.9±0.5 7.9±6 0.8±1 0.7±3 0.8±5 0.3±3 1.7±1

p-Coumaricacid 9.4±0.9 7.1±4 2.9±3 2.2±2 1.8±4. 2.8±2 2.1±1

Ferulicacid

m-Coumaricacid 0.2±4 0.1±3

Rutin 19.4±0.4 8.4±10 11.6±1

Trans-resveratrol 6.5±6 5.1±9 2.4±8

Myricetin 9.7±2

Quercetin 5.4±6 2.1±9 2.2±4

Cinnamicacid 0.8±2

Kaempferol 2.7±9 1.1±0

a_The_content_of_each_of_the_fifteen_polyphenols_analysed_in_the_wine_samples_tested_is_the_mean_of_three_replicates

±RSDandindicatedas␮gmL−1.Thegray-shadow

boxesrefertopolyphenolsthatwerenotdetectedintherespectivewinesample.

(10)

abundantpolyphenolis (−)-catechin, althoughit isnot present inEBandVGBwhitewines.Thesetwo polyphenolshave being extensivelyshowntohaveseveralhealthbenefits,namelyinthe cardiovascular and neurodegenerative protection [32,33]. Gallic acid,alsooftenreportedinwinecomposition[34,35],was identi-fiedinallwinesamples,whilesyringicandp-coumaricacidseems tobemorespecifictowhitewines.Inthesameway,m-coumaric acidwas only identified in white wines, while rutin was only detectedinredwines.Myricetin(inVTredwine)andkaempferol (inVGBwhitewineandVTredwine)weredetectedonlyinCanary Islandwines.Gentisicandferulicacidswerenotdetectedinany ofthewinesamplesanalysedandcinnamicacidwasonly iden-tified at very low amounts in the AzoresIsland red wineTLT. Trans-resveratrol,animportant bioactivepolyphenols shown to haveanti-mutagenandanti-fungalproperties[13],usuallypresent inredwineswasidentifiedinAzores(TLT–6.50±0.06␮gmL−1)

andMadeira(PT– 5.12±0.09␮gmL−1)redwines,butnotinthe

CanaryIslandredwine(VT).Curiouslyitwasidentifiedinoneofthe whitewinestested(VGBwinefromCanaryIsland).Ourdataabout trans-resveratrolareinagreementwiththeresultsfromBaptista etal.[26,35]thatfoundamountsofthiscompoundintherange of0.63–5.21mgL−1_in_other_Portuguese_red_wines,_including_one

fromAzoresIsland.

Theseresultsconfirmedthatwinepolyphenoliccompositionis veryheterogenic,beingdependentofdiversefactors,namelylocal climateandvinificationconditionsthatarequitedifferentamong thewinesamplesusedinthiswork.

4. Conclusions

In the current study an ultra-fast and simple UHPLC–PDA methodology,usinga50-mmcolumn(AcquityBEHC18)packed with1.7-␮mparticles,wasdevelopedandvalidatedforthe

simul-taneousseparationandquantificationof15bioactivepolyphenols inredandwhitewines.Themethodwasvalidatedwithrespectto selectivity,linearity,LOD,LOQ,accuracy,precisionandextraction efficiencyforeachofthefifteencompoundsstudied.Thevalidation hasshownthat themethodislinear,and sensitivewith recov-ery,andprecisionvalueswithintheacceptedlimitsforvalidation offoodanalyticalmethods.Afteracarefulselectionoftheeluent systems,itwasdemonstratedthatthechromatographic separa-tionofthepolyphenolscouldbeachievedinwithinfiveminutes withhigh resolution. This is about tentimes faster separation thanthattraditionalHPLCapproachallowsforthesamepurpose. Thecombinationoftheshorterrunningtimewithasmallerflow ratealsoreduceddrasticallythesolventconsumptionandthusis moreenvironmentalfriendlyandeconomical.Inordertoreduce thecomplexityofwineextractsamplesandoptimizethe recov-eryefficiency,arapidprocedurebasedonSPEusingmethanoland ethyl acetateas extractionsolvents was employedprior to the UHPLC–PDAanalysis.Itspracticalapplicationinselectedwineshas alsobeendemonstrated.

Themethodologyherepresentedissuitableandveryusefulfor anunambiguousdeterminationofbioactivemetabolitesinwines andforroutineuseinlaboratory.Moreover,thevalidatedapproach

Acknowledgements

TheauthorsthankthefinancialsupportofFEDER(Transnational Cooperation MAC 2007-2013 Program) through VinSaudeMAC project (MAC/1/M105) and to Portuguese Foundation for Sci-enceandTechnology(FCT)throughtheMSPortugueseNetworks (REDE/1508/RNEM/2005) and Pluriannual base funding (QUI-Madeira-674).

References

[1]J.S.Câmara,M.A.Alves,J.C.Marques,Anal.Chim.Acta555(2006)191. [2]R. Lopez, M. Aznar, J. Cacho, V. Ferreira, J. Chromatogr. A 966 (2002)

166.

[3]V.Ferreira,R.López,J.Cacho,J.Sci.FoodAgric.80(2000)1659.

[4] J.S.Câmara,P.Herbert,J.C.Marques,M.A.Alves,Anal.Chim.Acta513(2004) 203.

[5]V.Ferreira,N.Ortin,A.Escudero,R.Lopez,J.Cacho,J.Agric.FoodChem.50 (2002)4048.

[6] J.S.Câmara,M.A.Alves,J.C.Marques,Talanta68(2006)1512.

[7]M.Prodanov,I.Garrido,V.Vacas,R.Lebron-Aguilar,M.Duenas,C. Gomez-Cordoves,B.Bartolome,Anal.Chim.Acta609(2008)241.

[8]N. Gheldof, X.H. Wang, N.J. Engeseth, J. Agric. Food Chem. 50 (2002) 5870.

[9]V.B. Schini-Kerth,C. Auger,J.H.Kim, N.Etienne-Selloum,T.Chataigneau, PflugersArch459(2010)853.

[10]M.I.Covas,P.Gambert,M.Fito,R.delaTorre,Atherosclerosis208(2010)297. [11] M.Naczk,F.Shahidi,J.Chromatogr.A1054(2004)95.

[12]S.Perez-Magarino,M.Ortega-Heras,E.Cano-Mozo,J.Agric.FoodChem.56 (2008)11560.

[13]M.Schwarz,M.C.Rodriguez,D.A.Guillen,C.G.Barroso,J.Sep.Sci.32(2009) 1782.

[14]Z.Spacil,L.Novakova,P.Solich,Talanta76(2008)189. [15]L.A.Kartsova,A.V.Alekseeva,J.Anal.Chem.63(2008)1024.

[16]E.Rijke,P.Out,W.M.A.Niessen,F.Ariese,C.Gooijer,U.A.Th.Brinkman,J. Chro-matogr.A1112(2006)31.

[17] H.Wang,G.J.Provan,K.Helliwell,FoodChem.81(2003)307.

[18]M.Pelillo,M.Bonoli,B.Biguzzi,A.Bendini,T.GallinaToschi,G.Lercker,Food Chem.87(2004)465.

[19] V.Atanasova,H.Fulcrand,V.Cheynier,M.Moutounet,Anal.Chim.Acta458 (2002)15–27.

[20]J.X.Castillo-Sánchez,M.S.García-Falcón,J.Garrido,E.Martínez-Carballo,L.R. Martins-Dias,X.C.Mejuto,FoodChem.106(2008)18–26.

[21] Z.Kerem,B.A.Bravdo,O.Shoseyov,Y.Tugendhaft,J.Chromatogr.A1052(2004) 211.

[22]V.Pereira,J.S.Camara,J.Cacho,J.C.Marques,J.Sep.Sci.33(2010)1204. [23]J.Gruz,O.Novák,M.Strnad,FoodChem.111(2008)789.

[24] M.A. Rodríguez-Delgado,G. González-Hernández,J.E. Conde-González, J.P. Pérez-Trujillo,FoodChem.78(2002)523–532.

[25]D.Guillarme,C.Casetta,C.Bicchi,Jean-LucVeuthey,J.Chromatogr.A1217 (2010)6882.

[26]J.A.B.Baptista, J.F.d.P. Tavaresa,R.C.B. Carvalho,Food Res. Int.34 (2001) 345.

[27]Y.Kuroda,Y.Hara,Mutat.Res.436(1999)69.

[28]B.Fuhrman,N.Volkova,A.Suraski,M.Aviram,J.Agric.FoodChem.49(2001) 3164.

[29]L.Nováková,SpacilZdenek,M.Seifrtova,L.Opletal,P.Solich,Talanta80(2010) 1970.

[30]A.A.Bertelli,D.K.Das,J.Cardiovasc.Pharmacol.54(2009)468.

[31]J.N.Miller,J.C.Milller,StatisticsandChemometricsforAnalyticalChemistry, PearsonEducation,Harlow,UK,2000.

[32]H.J.Heo,C.Y.Lee,J.Agric.FoodChem.53(2005)1445.

[33]H.Schroeter,C.Heiss,J.Balzer,P.Kleinbongard,C.L.Keen,N.K.Hollenberg, H.Sies,C.Kwik-Uribe,H.H.Schmitz,M.Kelm,Proc.Natl.Acad.Sci.U.S.A.103 (2006)1024.

[34]J.González-Rodríguez,P.Pérez-Juan,M.D.LuquedeCastro,Talanta56(2002) 53.