ContentslistsavailableatSciVerseScienceDirect
Journal
of
Chromatography
A
j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a te / c h r o m a
Development
of
a
novel
microextraction
by
packed
sorbent-based
approach
followed
by
ultrahigh
pressure
liquid
chromatography
as
a
powerful
technique
for
quantification
phenolic
constituents
of
biological
interest
in
wines
João
Gonc¸
alves,
Berta
Mendes,
Catarina
L.
Silva,
José
S.
Câmara
∗CQM-UMa,CentrodeQuímicadaMadeira,CentrodeCiênciasExactasedaEngenhariadaUniversidadedaMadeira,CampusUniversitáriodaPenteada,9000-390Funchal,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received28September2011
Receivedinrevisedform6January2012 Accepted6January2012
Available online 18 January 2012 Keywords:
Wines
Bioactivecompounds
Microextractionbypackedsorbent(MEPS) Ultrahighpressureliquidchromatography (UHPLC)
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b
s
t
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Anovelanalyticalapproach,basedonaminiaturizedextractiontechnique,themicroextractionbypacked sorbent(MEPS),followedbyultrahighpressureliquidchromatography(UHPLC)separationcombined withaphotodiodearray(PDA)detection,hasbeendevelopedandvalidatedforthequantitative deter-minationofsixteenbiologicallyactivephenolicconstituentsofwine.Inadditiontoperformingroutine experimentstoestablishthevalidityoftheassaytointernationallyacceptedcriteria(linearity, sensi-tivity,selectivity,precision,accuracy),experimentsareincludedtoassesstheeffectoftheimportant experimentalparametersontheMEPSperformancesuchasthetypeofsorbentmaterial(C2,C8,C18, SIL,andM1),numberofextractioncycles(extract-discard),elutionvolume,samplevolume,andethanol content,werestudied.TheoptimalconditionsofMEPSextractionwereobtainedusingC8sorbentand smallsamplevolumes(250L)infiveextractioncycleandinashorttimeperiod(about5minforthe entiresamplepreparationstep).Thewinebioactivephenolicswereelutedby250Lofthemixture con-taining95%methanoland5%water,andtheseparationwascarriedoutonaHSST3analyticalcolumn (100mm×2.1mm,1.8mparticlesize)usingabinarymobilephasecomposedofaqueous0.1%formic acid(eluentA)andmethanol(eluentB)inthegradientelutionmode(10minoftotalanalysis).The methodgavesatisfactoryresultsintermsoflinearitywithr2
-values>0.9986withintheestablished con-centrationrange.TheLODvariedfrom85ngmL−1(ferulicacid)to0.32gmL−1((+)-catechin),whereas theLOQvaluesfrom0.028gmL−1(ferulicacid)to1.08gmL−1((+)-catechin).Typicalrecoveriesranged between81.1and99.6%forredwinesandbetween77.1and99.3%forwhitewines,withrelative stan-darddeviations(RSD)nolargerthan10%.TheextractionyieldsoftheMEPSC8/UHPLC–PDAmethodology werefoundbetween78.1(syringicacid)and99.6%(o-coumaricacid)forredwinesandbetween76.2 and99.1%forwhitewines.Theinter-dayprecision,expressedastherelativestandarddeviation(RSD%), variedbetween0.2%(p-coumaricando-coumaricacids)and7.5%(gentisicacid)whiletheintra-day precisionbetween0.2%(o-coumaricandcinnamicacids)and4.7%(gallicacidand(−)-epicatechin).On thebasisofanalyticalvalidation,itisshownthattheMEPSC8/UHPLC–PDAmethodologyprovestobean improved,reliable,andultra-fastapproachforwinebioactivephenolicsanalysis,becauseofitscapability fordeterminingsimultaneouslyinasinglechromatographicrunseveralbioactivemetaboliteswithhigh sensitivity,selectivityandresolvingpowerwithinonly10min.Preliminarystudieshavebeencarried outon34realwholewinesamples,inordertoassesstheperformanceofthedescribedprocedure.The newapproachoffersdecreasedsamplepreparationandanalysistime,andmoreoverischeaper,more environmentallyfriendlyandeasiertoperformascomparedtotraditionalmethodologies.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Thechemicalcompositionpresentinawine,coveralarge num-berofmetabolitesincludingprimary(e.g.sugars,organicacids, aminoacids)andsecondarymetabolites(e.g.polyphenolssuchas
∗ Correspondingauthor.Tel.:+351291705112;fax:+351291705149. E-mailaddress:jsc@uma.pt(J.S.Câmara).
flavonoids,anthocyaninsandotherpigments).Allofthese com-poundshaveastronginfluenceonthequalityandcharacterofthe wine,andarethereforeimportantnotonlyforthewine character-ization,butalso,reflectsthehistoryofthewineproducingprocess, includingthegrapevariety,theyeaststrain,thecontainersused forfermentationandstorage,andtheenologicpractices.Aswellas beingagoodsourceofvitaminC,dietaryfiber,andminerals,berries containhighlevelsofnaturalpolyphenolcomponentsthatactas potentantioxidants.Grapeandwineextracts,richinpolyphenols,
0021-9673/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2012.01.023
Fig.1.Chemicalstructuresofthebiologicalactivephenolicconstituentsfoundinwines.
havearangeofbiologicaleffectsthatcanhavebeneficialoutcomes onhumanhealth[1,2].
Polyphenols arise biogenetically from either the shiki-mate/phenylpropanoidpathwayor‘polyketide’acetate/malonate pathway,orboth, producingmonomericandpolymericphenols andpolyphenols,whichfulfilaverybroadrangeofphysiological rolesinplants[3].Apparentlyactasdefense(againstherbivores, microbes,virusesorcompetingplants)andsignalcompounds(to attractpollinatingorseeddispersinganimals),aswellas protect-ingtheplantfromultravioletradiationandoxidants[3].Theycan be categorized as (i) flavonoids and (ii)nonflavonoid phenolic compounds[4].Todate,severalhundredsofdifferentflavonoids havebeendescribedandthenumbercontinuestoincrease[5].This groupcomprisesof15carbons,with2aromaticringsconnected bya3-carbonbridge(Fig.1).Accordingtothemodificationsofthe centralC-ring,theycanbedividedintodifferentstructuralclasses includingflavonols(representedmainlybyquercetin,kaempferol, andmyricetin),flavones(representedbyapigeninandluteolin), flavan-3-ols (ranging from the simple monomers (+)-catechin anditsisomer (−)-epicatechintotheoligomericand polymeric proanthocyanidins),flavanones,isoflavones,andanthocyanidins.
Themainnonflavonoidphenoliccompounds(Fig.1)ofdietary significancearetheC6–C1phenolicacids(gallic,p-hydroxybenzoic, protocatechuic, vanillic, and syringic acids),the C6–C3 hydrox-ycinammates (p-coumaric, caffeic, and ferulic acids, frequently accumulateastheirrespectivetartrateesters,coutaric,caftaric,and fertaricacids)andtheirconjugatedderivatives,andthe polyphe-nolic C6–C2–C6 stilbenes (phytoallexins produced by plants in responsetodisease,injury,andstress).
In recent years the role of bioactive phenolic compounds andflavonoidsasprotectivedietaryconstituentshasbecomean increasinglyimportantareaofhumannutritionresearch.Unlike the traditional vitamins, they are not essential for short-term
well-being,butthereisincreasingevidencethatmodestlong-term intakesmayexhibitapotentialformodulatinghumanmetabolism in a manner favorable contributing to the beneficial effects of fruit-andvegetable-richdiets.Indeed,thereisreasonableevidence fromepidemiologicalstudiestosupportthenotionthatdietsrich inphenolics(derivedfromfruitsandvegetables) areassociated withlowerrisksofcancer,osteoporosis,cardiovasculardiseases, cataracts,and diseases associated withbrain andimmune dys-function [6–13]. Furthermore,polyphenols fromred wine have beenreportedtoexertpotentantioxidanteffectsthatprevent low-andvery-lowdensitylipoprotein(LDLandvLDL)oxidation(crucial stepsinatheroscleroticlesionformation)andDNAbases(relevant totheinductionofcancer)byfreeradicals[14–17],inhibitionof plateletaggregation[18–20],inhibitionofcellproliferation, migra-tion,andangiogenesis,anddespitesomereportsoftheabsenceof anassociation[21,22],theyareseriouscandidatestoexplainthe protectiveeffectsofvegetableandfruitconsumptionagainst sev-eraldiseases.Onepossiblereasonforthisprotectiveeffectmaybe thepowerfulantioxidantandfreeradicalscavengingpropertiesof variousclassesofphenoliccompounds[23].
Developedmethodsregardingtheanalysisofbiologicallyactive phenolicsin winesgenerally includesextractions withsolvents (methanol, ethanol, acetone or mixtures of these with water), cleanupandfurtherfractionationbyliquid–liquidextraction(LLE) [24],usuallywithethylacetate[25];columnchromatography(CC) [26]orsolidphaseextraction(SPE)[27,28].However,these proce-duresarequitetime-consumingandneedrelativelyhighvolume ofsolvent/sample,whichisimpracticalfortheroutineanalysesof manyfoodcommodities.Theever-increasingdemandforcontrol analysishascontributedmarkedly totherenewalofinterest in miniaturizedanalyticaltechniques(MAT)whichhastakenplace overthelastyears.TheMAThadgainedattentionduetoitsmany special features over classical approaches. Usage of little or no
Fig.2. SchematicdiagramoftheMEPSsyringeandMEPS-BINfromSGE,Analytical Science.
AdaptedfromWirthetal.[32].
solvent(reducingexposureofanalysttosolvents),increasing sen-sitivityof analysisand user-friendlysystem,should bepointed out.Inthiscontextthemicroextractionbypackedsorbent(MEPS) appearsasanewformatforsolid-phaseextraction(SPE)thathas beenminiaturizedtoworkwithsamplevolumesassmallas10L [29,30].ThecommerciallyavailablepresentationofMEPSusesthe samesorbentsasconventionalSPEcolumnsandsoissuitablefor usewithmostexistingmethodsbyscalingthereagentandsample volumes.UnlikeconventionalSPEcolumns,theMEPSsorbentbed isintegratedintoaliquidhandlingsyringethatallowsforlowvoid volumesamplemanipulationseithermanuallyorincombination withlaboratoryrobotics.Whenthesamplehaspassedthroughthe solidsupport,theanalytesareadsorbedtothesolidphasepacked inabarrelinsertandneedle(BIN)[30–32](Fig.2).
Thecartridgebedcanbepackedorcoatedtoprovideselective andsuitablesamplingconditions.Silicabased(C2,C8,C18),strong cationexchanger(SCX)usingsulfonicacidbondedsilica,restricted accessmaterial(RAM),hilic,carbon,polystyrene–divinylbenzene copolymer(PS–DVB)ormolecularimprintedpolymers(MIPs),can beusedassorbentmaterials.Thisnewtechniqueisverypromising becauseitisfast,simpleanditrequiresverysmallvolumeof sam-plestoproducecomparableresultstoconventionalSPEtechnique. Furthermore, this technique can be easily interfacedto LC–MS andGC–MStoprovideacompletelyautomatedMEPS/LC–MSor MEPS/GC–MSsystem.Thisextractiontechnique(MEPS)couldbeof interestinclinical,forensictoxicologyandenvironmentalanalysis areas[29,33–39].
ThecurrentavailableHPLCmethodsforquantificationof bioac-tivephenolicsinseveralmatrices,includingwines,haveruntimes intherangeof10–50min[24,28,40,41].Recently,ultrahigh pres-sureliquid chromatography(UHPLC)hasbecomewidelyspread techniqueandnewtrendinseparationsciences[33].Higher sep-aration efficiency of sub-2-m particle sorbents allows faster chromatographic separation keeping the same resolution com-paredtoHPLCsorbentswithconventionalparticlesize[33,42,43]. ThisisexpressedbyKnoxcurves,wherelowerheightequivalent oftheoreticalplateindicatesimprovedseparationefficiencyand higheroptimumlinearvelocity,whichmeansdecreaseinanalysis time[33,44].
The current research study describe the development and validation of an ultra-fast, efficient, sensitive, reliable and highthroughputMEPS-basedmethodologyincombination with UHPLC–PDAforthesimultaneousdetermination of16bioactive phenolicconstituentsinwines.TheUHPLCsystemusedincludes a binarysolventmanagerwhich delivers upto15,000psi pres-sure,aphotodiodearray(PDA)detectorwithaspectrainarangeof between200and400nm,a1.8mparticlesizecolumnanda sam-plemanagerwithsmallinjectionvolumeused(2L).Compared
toLLEandSPE,MEPSsavespreparationtimeandreducessolvent wastesignificantly.Spikedsyntheticwinesolution,redwine(Real Lavrador,codedasRL)andwhitewine(GrãoVasco,codedasGV) wereusedtoevaluatetheperformanceofthedevelopedmethod. SomefactorsinfluencingtheMEPSextractionefficiency,suchas type of sorbent material, number of extractioncycles (extract-discard),volumeofeluentandsamplevolume,wereevaluatedand optimized.Themethodwasthenappliedto34winesamplesfrom differentproducersandgeographicorigin.Tothebestofour knowl-edgenopapershasbeenpublishedfortheanalysisofbioactive phenolicconstituentsinwinesbyMEPS.
2. Experimental
2.1. Reagents,standardsandmaterials
All chemicals were of analytical grade. Methanol (99.9% purity, Sigma–Aldrich), formic acid (Merck), and acetic acid (Riedel-de-Haën) of HPLC gradient were purchased through Labodidáctica-EquipamentosdeLaboratórioDidácticos,Lda. (Fun-chal, Portugal). Ethanol absolute (99.5% purity), and sodium hydroxidewereobtainedfromPanreac(Barcelona,Spain). Bioac-tivephenolicstandards,gallicacid,gentisicacid,(−)-epicatechin, m-coumaricacid,o-coumaricacid,cinnamicacid,cinnamicacid, vanillicacid,p-coumaricacid,rutin,ferrulicacidandkaempferol, allfromFlukaBiochemicaAG(Buchs,Switzerland),and protocat-echuic acid, tartaricacidand syringicacid, from Sigma–Aldrich (St. Louis, MO, USA), were supplied through Labodidáctica-EquipamentosdeLaboratórioDidácticos,Lda.(Funchal,Portugal). MyricetinandsyringaldehydewereacquiredfromAcros Organ-ics(Geel,Belgium)and(+)-catechin,CaymanChemicalCompany (Montigny-le-Bretonneux,France).Internalstandard,Trolox,was acquired from Fluka Biochemica AG (Buchs, Switzerland). The purity of all polyphenolicstandards and internal standard was greater than 95%. Ultrapure water (18Mcm at 23◦C) was obtainedbymeansofa Milli-Qwater purificationsystem (Mil-lipore, Milford,MA,USA). Alltheeluates werefilteredthrough 0.22m PTFE membrane filters (Millipore, Milford, MA, USA) suppliedbyViaAthena-GestãodeLaboratórios,Lda.(Sacavém, Lis-bon,Portugal).TheMEPSgas-tightsyringe(250-L)andtheBIN (BarrelInsertand Needle)containingthesorbentmaterial from SGEAnalyticalScience(Melbourne,VIC,Australia)wereprovided by I.L.C.-Instrumentosde LaboratórioeCientíficos,Lda. (Lisbon, Portugal). The Waters Acquity UPLC HSS T3 analytical column (100mm×2.1mm,1.8mparticlesize)waspurchasedfromVia Athena-GestãodeLaboratórios,Lda.(Sacavém,Lisbon,Portugal).A HANNAinstrumentspH209pHmeter(Woonsocket,USA)was sup-pliedbyLabodidáctica-Equipamentos deLaboratórioDidácticos, Lda.(Funchal,Portugal).
2.2. Standardpreparationandwinesamples
Individual stock solutions of each polyphenol tested were prepared in pure methanol at concentration of 1000gmL−1, aliquotedin4mLvials,andstoredat−20◦C,inthedark.Under
theseconditionstheywerestableforatleast4month(asassessed byUHPLC).Workingstandardsolutionscontainingthe16 polyphe-nolswas prepareddaily fromtheindividual stock solutionsby dilutingtheminthesyntheticwine(5gL−1tartaricacid,12%(v/v) ethanol).Thisstandardwasusedbothtospikethewinesinorderto performtheassaysforoptimizationofextractionconditionsandfor calibrationstudyatdifferentconcentrations.Thechemical struc-turesoftheinvestigatedbioactivemetabolitesarereportedinFig.1. Calibrationstandards(1–50gL−1)werepreparedfreshontheday oftheanalysisbydilutingtheappropriateworkingsolutionsin syn-theticwinesolution.Therangesofconcentrationswereselected
Table1
Varietalcomposition,ethanolcontentandproductionyearofinvestigatedwines.
Wine Region Year Alcohol(%,v/v) Varietalcomposition
Redwine
Esteva Douro 2009 13 TintaRoriz,TourigaFranca,TintaBarrocaandTourigaNacional
JPAzeitão Setúbal 13 Castelão,AragonezandSyrah
Terrasd’elRei Alentejo 2010 13 Trincadeira,Castelão,MoretoandAragonez
Reguengos Alentejo 2010 13 Aragonez,TrincadeiraandCastelão
MonteVelho Alentejo 2009 14 Aragonez,TrincadeiraandCastelão
CasadeSantar Dão 2007 13.5 TourigaNacional,AlfrocheiroandTintaRoriz
Periquita Azeitão 2008 13 Castelão,AragonezandTrincadeira
Contemporal Dão 2008 12 TourigaNacional,TourigaFrancesa,TintaRorizandTintaBarroca
FreiBernardo Beiras 2009 12.5 Rufete,MarufoandTintaRoriz
RealLavrador Alentejo 2010 13 Castelão
PorcadaMurc¸a Douro 2009 13 TourigaNacional,TintaRoriz,ToutigaFrancesaandTintoCão
AdegadeBorba Alentejo 2009 14 Aragonez,TincadeiraandAlicanteBouschet
FontedaSerrana Alentejo 2009 14 Aragonez,Trincadeira,CabernetSauvignonandAlicanteBouschet
Pelão Douro 2009 12 BagaandTourigaNacional
TerrasAltas Dão 2007 12 Bastardo,Jean,TourigaNationalandAlfrocheiro
GrãoVasco(GV) Dão 2008 13 Jaen,TintaRorizandTourigaNacional
Torcaz Madeira 2004 12 TintaNegraMole
TMR1 Madeira 2009
TerrasdeLava Azores 2009 13 Merlot,CabernetSauvignon,SaborinhoandSyrah
TerrasdeLava Azores 2010 13 Merlot,CabernetSauvignon,SaborinhoandSyrah
Basalto Azores 2009 12 Periquita,AgronómicaandSaborinho
Basalto Azores 2010 12 Periquita,AgronómicaandSaborinho
Whitewine
RealLavrador(RL) Alentejo 2010 12.5 RoupeiroandRabo-de-ovelha
Vidigueira Alentejo 2009 13.5 AntãoVaz,Manteúdo,PerrumandRoupeiro
Navegante Alentejo 2009 13 AntãoVaz,Manteúdo,PerrumandRoupeiro
Planalto Douro 2009 12.5 MalvasiaFina,Viosinho,GouveioandCódega
Periquita Azeitão 2008 12 MoscatelandArinto
GrãoVasco Dão 2009 13 Encruzado,BicalandMalvasiaFina
Vi ˜natigoGual Canary 2005 13 AlbilloReal
TMW1 Madeira 2010
TMW2 Madeira 2010
TMW3 Madeira 2010
Lajido Ac¸ores 1999 16.5 Arinto,VerdelhoandTerrantez
VG-Verdelho Madeira 2004
infunctionofsensitivityoftheUHPLC–PDAtowardseach bioac-tivephenoliccompound.Physical-chemicalcharacteristicsaffect theanalyticalsignalandforsomecompoundshigher concentra-tionshadtobeusedinordertodetectedthecompound.Trolox wasusedasinternalstandard(IS).
Thirty-fourrepresentative commercial table wines available from different regions of Portugal, and Macaronesia Islands (Madeira,AzoresandCanary),whichwereproducedaccordingto standardproceduresanddefinedvarietalcomposition,were ana-lyzed.The listof the studied wines,production year and their varietalcompositionaresummarizedinTable1.
Allsamplesweretakenfrombottledwinesreadyforsaleand storedat−20◦C.
Syntheticwinesampleswerepreparedbyanethanolsolution at12%(v/v)towhich5gL−1tartaricacidwereadded.SolutionpH wasadjustedto3.4with1Msodiumhydroxide.
Winesamplesandsyntheticwinesweredealcoholizedunder vacuumat40◦C,upto
¼
ofinitialvolume,inordertoavoid inter-ferences of ethanol. The volume of dealcoholized extracts was adjustedtoinitialsamplevolumewithwaterandthepHadjusted to3.4with30% (v/v)acetic acid. Allsampleswereanalyzed in triplicate.2.3. OptimizationofthefactorsaffectingtheperformanceofMEPS TheMEPSprocedurewascarriedoutbymeansofaSGE Ana-lyticalScience(I.L.C.,Lisbon,Portugal)apparatus,consistingofa 250-Lgas-tightsyringewitharemovableneedle.Thesyringewas fittedwithaBIN(BarrelInsertandNeedle)containing4mgofthe sorbentmaterialandwasusedtodrawanddischargesamplesand solutionsthroughtheBIN.Asyntheticwinesamplespikedwith
knownamountsofbioactivephenolicswasusedtooptimizethe MEPSprocedure.SeveralimportantMEPS-influencingextraction factorssuchasthetypeofsorbentmaterial,numberofextraction cycles,elutionvolume,samplevolume,andethanolcontent,were evaluated.
Selectionofsorbentisanimportantfactortoachieveacceptable clean-upandgethighextractionyield[30].Thereforethe perfor-manceof thefive MEPS sorbentmaterials:C2(ethyl-silica), C8 (octyl-silica),C18(octadecyl-silica),SIL(unmodifiedsilica)andM1 (amixedmodesorbentcontaining80%C8and20%strongcationic exchange(SCX))wastestedandcompared,inordertoselectthe bestsorbent for thetarget analytes.C2–C8phases are suitable forlipophilicanalytes(non-polar)andpolymericphasessuchas polystyrene–divinylbenzeneormixedmodephases(anion–cation exchange mode) are suitable for polar analytes such as acidic andbasiccompounds.Toselectthenumberofextractioncycles (extract-discard)andsamplevolume,analiquotof50L,100L and250Lofsyntheticwinewaspumpedupanddownonce,five andtentimes,inordertoobtainthebestextractionefficiency. Dif-ferentelutionvolumes(50,100,250,350and500L)werealso tested.Theflowrateduringaspirationislimitedtoabout20Ls−1 topreventcavitation.Thiswillincrease analyte/sorbentcontact timeandextractionefficiency.Inordertochecktheinfluenceof ethanolcontent,asyntheticwinewith12%(v/v)ofethanolcontent wascomparedtodealcoholizedsyntheticwineextract.The deal-coholizedextractwasadjustedtoinitialvolumeusingsolutions withdifferentalcoholiccontent: (a)100%water;(b) 90% aque-oussolutionofformicacidat0.1%and10%methanol;and(c)80% aqueoussolutionofformicacidat0.1%and20%methanol(mobile phase used at initial step of gradient elution). Since the influ-enceofmethanolonMEPSpolyphenolsextractionefficiencywas
similartoethanol,anditwasusedaseluentintheUHPLCsystem, weselectthemethanoltoevaluatetheeffectofalcoholiccontent onextractionefficiency.Alloptimizationprocedureswerecarried outintriplicate.
2.3.1. MEPSprocedureforbioactivephenolicconstituents
Theprecisionengineeringusedinthedesignandmanufacture ofMEPSallowsthesamefunctionsasSPE,suchastheremovalof interferingmatrixcomponentsandtheselectiveisolationand con-centrationofanalytes.MEPSexperimentswereconductedusing 4mgofsolid-phasematerial(C8sorbentselected,inthe optimiza-tionstep,asthebestsorbenttoisolatethetargetanalytes).Before beingusedforthefirsttime,thesorbentwasmanuallyconditioned firstwith100Lmethanolandthenwith100Lwater(0.1%formic acid). Thisstep activates thesorbent and ensures reproducible retentionoftheanalytes[30].250Lsampleswerepassedthrough theC8sorbentfivetimesata flowrateofabout20Ls−1.The solidphasewasthenwashedwith100Lofwatercontaining0.1% formicacidtoremoveinterferences,ataspeedofabout50Ls−1. Theanalyteswerethenelutedwith250Lof95%methanoland 5%waterdirectlyintoavial.Betweeneveryextraction,the sor-bentwasrinsedwith250Lmethanolfollowedby250Lofthe washingsolution.Thisstepdecreasedmemoryeffects(carry-over), butalsofunctionedasconditioningstepbeforethenextextraction. TheextractswerefilteredthroughMilliporemembranePTFE fil-ters(0.22mparticlesize).Analiquotof2Lofthissolutionwas injectedintriplicateintotheUHPLC–PDAsystem.Thesame pack-ingbedwasusedforabout100extractions;thenitwasdiscarded duetoboththelowanalyteextractionyieldsandcloggingofthe sorbent.AllMEPSstepsincludingactivation,loading,washing,and elutionwerecarriedoutmanually.Inallmeasurements(standards andsamples)Troloxwasaddedastheinternalstandard(IS). 2.4. UPLC–PDAanalysisandoperatingconditions
BesidesmaximumenrichmentperformancebyMEPS,the deter-minationofthetargetanalytes,insmallsamplevolumesrequires asensitivedetectionmethod.Theanalysisofbioactivesecondary metaboliteswascarriedoutonaWatersUltraPerformanceLiquid ChromatographicAcquitysystem(UPLC,AcquityH-Class)(Milford, MA,USA) combined witha Waters Acquity quaternary solvent manager(QSM),anAcquitysamplemanager(SM),acolumnheater, a 2996 PDAdetector, and a degassing system. Thewhole con-figuration was driven by Empower software v2.0 from Waters Corporation. Optimum separation was achieved with a binary mobilephase whichconsisted of(A) waterat 0.1%formic acid, and(B)methanol,withaconstantflowrateof250Lmin−1, giv-inga maximum backpressure of 6000psi,which is withinthe capabilitiesoftheUPLC.2Lofextractswereinjectedintothe WatersAcquityUPLCsystem,equippedwithanAcquityUPLCTM
strength silica HSS T3 analytical column (1.8m particle size, 2.1mm×100mm)andprotectedwithanAcquityUPLCTMHSST3
VanGuardTMPre-column(Waters,Milford,MA,USA);column
tem-peraturewasthermostatedat40◦Candthesampleswerekeptat 4◦C inthesample manager.The5mingradientwasasfollows: 80%A(0min);80–70%A(0.50min);68%A(1min);20%A(8min); and80%A(10min),followedbyare-equilibrationtimeof3min,for bringingthecolumntotheinitialconditionsaftergradientanalysis, givenatotalruntimeof13min.Allsolventsandsampleswere fil-teredthrough0.22mmembranefiltersfromMillipore(Millipore, Milford,MA,USA),beforeuse.ForquantificationpurposesthePDA detectionwasconductedbyusingfourdistinctchannelsthatwere settothemaximumabsorbancewavelengthofeachpolyphenol,as indicatedinTable2.Theywereidentifiedbycomparingthe reten-tiontimeandspectralcharacteristicsoftheirpeakswiththoseof Table
2 Peak number, retention time (RT), UV–vis data, and calibration parameters for the validation of sixteen bioactive phenolic compounds on synthetic wine, by the newly developed methodology (MEPS C8 /UHPLC–PDA). Peak number RT (min) Bioactive phenolics max a(nm) Analytical performance Conc. Range ( g mL − 1) Regression equation br 2 LOD c( g L − 1) LOQ d( g L − 1) % Matrix effect Red wine White wine 1 1.894 Gallic acid 270 1–50 0.0015 x e− 0.0005 0.9989 0.22 0.73 87.3 93.3 2 3.045 Protocatechuic acid 260 1–50 0.057 x + 0.0034 0.9986 0.036 0.12 84.4 87.7 3 3.333 (+)-Catechin 278 2–50 0.0014 x + 0.0017 0.9993 0.32 1.08 95.7 81.4 4 4.030 Gentisic acid 325 1–30 0.0028 x − 0.0012 0.9992 0.085 0.28 90.7 90.7 5 4.256 (− )-Epicatechin 280 1–25 0.002 x + 0.0017 0.9980 0.18 0.59 95.0 90.0 6 4.507 Vanillic acid 260 1–50 0.0193 x + 0.0027 0.9994 0.044 0.15 84.8 88.8 7 4.695 Syringic acid 277 1–25 0.0226 x − 0.0029 0.9998 0.050 0.17 86.6 95.7 8 5.167 Syringaldehyde 308 1–50 0.0334 x + 0.0043 0.9997 0.070 0.23 93.1 87.7 9 5.555 p -Coumaric acid 308 1–40 0.0686 x − 0.0432 0.9990 0.013 0.043 83.5 79.2 10 5.775 Ferulic acid 323 1–25 0.0643 x − 0.009 0.9998 0.0085 0.028 90.0 91.8 11 6.181 m -Coumaric acid 278 1–40 0.0653 x − 0.0427 0.9991 0.028 0.093 89.1 88.4 12 6.360 Rutin 256 1–25 0.0542 x + 0.0464 0.9958 0.15 0.50 87.2 102.6 13 6.781 o -Coumaric acid 276 1–40 0.0659 x − 0.0359 0.9993 0.026 0.088 86.0 90.6 14 7.029 Myricetin 372 1–30 0.0699 x − 0.0084 0.9995 0.078 0.26 112.6 110.4 15 8.112 Cinnamic acid 277 1–40 0.1617 x − 0.0468 0.9995 0.026 0.088 99.6 102.0 16 8.965 Kaempferol 366 1–25 0.1659 x + 0.0049 0.9993 0.022 0.074 97.6 87.6 17 9.178 Trolox 289 50 – – – – – – a Maximum absorbance values obtained in PDA system detection. b Correlation coefficient, give an estimating how well the experimental points fit a straight line. c Limit of detection. d Limit of quantification. Values obtained from ordinary least-squares regression data. e x = analyte concentration.
Table3
Accuracy,extractionyield,intra-andinter-dayperformance,obtainedforbioactivephenoliccompoundsbyusingMEPSC8/UHPLC–PDAmethodology. Bioactivephenolics Fortification
level(gmL−1)
Accuracy(%) Extractionyield(%) RSD(%)
Redwine Whitewine Redwine Whitewine Intra-day(n=7) Inter-day(n=25)
Gallicacid LLa 1 85.7 82.8 84.3 86.4 4.7 5.9 MLa 30 89.9 94.2 89.4 94.5 3.2 4.0 HLa 50 95.1 96.7 80.1 96.8 2.6 1.4 Protocatechuicacid LL 1 83.1 86.6 88.8 82.3 1.1 1.9 ML 20 95.0 87.0 95.2 88.5 1.2 1.6 HL 50 98.9 95.6 98.9 95.8 1.6 0.7 (+)-Catechin LL 2 89.2 80.2 82.4 87.7 3.5 7.5 ML 20 91.3 83.6 79.3 79.1 2.2 2.5 HL 50 88.9 88.7 80.9 89.8 1.9 1.5 Gentisicacid LL 1 87.9 82.0 81.9 85.2 3.2 4.0 ML 10 90.1 89.6 91.0 83.0 1.7 1.0 HL 30 97.1 93.7 97.2 94.1 1.5 1.2 (−)-Epicatechin LL 1 91.2 94.9 83.0 95.2 4.7 5.6 ML 10 87.4 98,0 85.4 98.0 1.3 1.8 HL 25 89.1 99.1 82.7 99.1 1.1 1.6 Vanillicacid LL 1 89.0 82.4 86.3 85.0 0.7 1.4 ML 20 80.7 94.9 83.8 95.1 0.7 0.8 HL 50 93.7 98.1 94.0 98.1 2.0 0.4 Syringicacid LL 1 80.3 78.9 85.8 76.2 1.6 1.8 ML 10 86.9 84.1 78.1 86.3 0.6 0.7 HL 25 91.6 91.7 84.4 92.3 0.6 0.6 Syringaldehyde LL 1 95.8 89.6 80.5 90.6 2.0 1.3 ML 20 95.9 97.8 96.1 97.9 1.6 0.9 HL 50 98.6 99.3 98.6 99.3 2.4 0.6 p-Coumaricacid LL 1 85.8 86.0 90.9 91.0 0.5 0.9 ML 20 88.6 93.7 89.7 94.0 0.4 0.2 HL 40 94.7 97.1 95.0 97.2 0.4 0.3 Ferulicacid LL 1 94.0 85.3 94.4 89.1 0.3 1.3 ML 10 90.3 93.3 91.1 93.7 0.5 0.7 HL 25 95.3 96.6 95.5 96.7 0.3 0.4 m-Coumaricacid LL 1 81.3 92.2 83.0 92.8 1.0 1.0 ML 20 87.8 97.9 81.8 98.0 0.4 0.4 HL 40 89.6 99.0 90.6 99.0 0.2 0.3 Rutin LL 1 82.6 81.4 83.1 89.3 0.5 3.8 ML 10 84.7 93.6 93.4 94.0 0.4 2.9 HL 25 90.1 96.6 88.8 96.7 0.6 0.5 o-Coumaricacid LL 1 93.4 77.1 93.8 81.4 0.8 0.9 ML 20 99.0 94.7 99.0 95.0 0.7 0.2 HL 40 99.6 97.6 99.8 97.7 0.8 0.3 Myricetin LL 1 87.0 91.8 81.4 82.4 0.6 2.6 ML 10 81.1 81.5 84.1 84.4 0.5 1.4 HL 30 92.4 94.8 92.9 95.1 0.6 0.18 Cinnamicacid LL 1 85.1 86.9 84.1 81.2 0.5 0.9 ML 20 90.6 94.7 91.4 95.0 0.2 0.5 HL 40 94.9 97.4 95.2 97.5 0.4 5.7 Kaempferol LL 1 94.2 82.0 94.6 87.5 0.4 0.7 ML 10 99.0 80.4 99.0 83.6 0.4 0.6 HL 25 99.4 91.6 99.4 92.2 0.3 0.5
aConcentrationlevelsusedinMEPS
C8/UHPLC–PDAvalidationstudies:LL-lowlevel;ML-mediumlevel;HL-highlevel.
standardsand theywerequantifiedusingthestandardsofeach one.
2.5. Methodvalidationdesign
ThenewlydevelopedMEPSC8/UHPLC-DADapproachwasfully
validatedintermsofselectivity,linearity,limitsofdetection(LOD) andquantification(LOQ),inter-andintra-dayprecisions,accuracy andextractionefficiency(Table2).Theassayswerecarried out at Laboratory of Analytical Chemistry and Enology (Centro de QuímicadaMadeira)usingtheC8sorbentandaWatersAcquity
H-Class equipped with a HSS T3 analytical column (100mm× 2.1mm,1.8mparticlesize).
Theselectivityofthemethodwasassessedbytheabsenceof interferingpeaksattheelutiontimesofthebioactivemetabolites. Thelinearityoftheanalyticalmethodwasevaluatedbuildingthree calibrationcurves(thepeakareaanalyte/peakareaISratioobtained
wereplottedagainstthecorrespondingstandardconcentration)for eachbioactivephenolicusingstandards,preparedinsyntheticwine fromindividualstocksolutions,at7differentconcentrationslevels, includingthezeropoint(Table3).Thezeropoint(unspiked syn-theticwine)enablestoverifythatnoneofthecompoundsshowed
residuallevelorbackgroundsignal.Eachlevelofconcentrationwas preparedintriplicateandinjectedthreetimes,sotherewereatotal ofninereplicates.
Thesensitivityofthemethodwasassessedbydeterminingthe LOD(thelowestanalyteconcentrationthatproducesaresponse detectableabovethenoiselevelofthesystem)andLOQ(thelowest levelofanalytethatcanbeaccuratelyandpreciselymeasured)for eachcompound.LODandLOQwerecalculatedwiththedata gen-eratedinthelinearitystudies,beingLODdefinedas(a+3Sa/b)and
LOQas(a+10Sa/b),where“a”representsoriginordinate,“Sa”the
originordinatevarianceand“b”theslope[45].Theseparameters werecalculatedforeachanalytefromthestandardsolutionsused toobtainthecorrespondingcalibrationcurves,usingtheUHPLC developedmethod.
Precisionisafunctionofconcentrationanditwascalculated bydividingthestandarddeviation(SD)bythemeansof concen-trationtoobtainthecoefficientof variation(C.V.),which when expressedonapercentagebasisgivestherelativestandard devia-tion(RSD).TheuseoftheRSDvalues,whichdescribesthecloseness ofagreementbetweenseriesofmeasurements,facilitates compar-isonsofvariabilitiesatdifferentconcentration.Methodprecision wasevaluatedbyspikingasyntheticwineatthreedifferent con-centration levels, correspondingto the low level (LL), medium level(ML)andhighestpoint(HL)ofcalibrationcurve(Table3)of eachphenoliccompoundandtreatedbyMEPSC8.Sevenreplicates
(n=7)wasperformedinthesamedaytoobtainrepeatability (intra-dayprecision).Forinter-dayprecision(intermediateprecision—IP) evaluation,thesameprotocolwasfollowedbutsixreplicatesof eachlevelwereanalyzeddailythroughthreedifferentdays(n=25; 7+18).
In order to check the accuracy of the proposed method a recovery study was carried out by spiking a red wine (RL) and a white wine (GV), in triplicate at three concentration levels correspondingto theLL, ML and HL (Table 3), and sub-jected to the MEPSC8 procedure described in Section 2.3. The
RL and GV wines phenolics concentration was previously determined by MEPSC8, and the recovery values were
calcu-lated according to the following formula: Accuracy=100× ([analyte]afterspiking−[analyte]beforespiking)/[analyte]added; where
[analyte]afterspikingistheanalyteconcentrationmeasuredinspiked
wine;[analyte]beforespikingistheanalyteconcentrationmeasured
inunspikedwine,and[analyte]addedisthenominalconcentration
of the analyte added to wine. Extraction efficiency (EE) was determinedbyreplicateanalysis(n=3)ofsyntheticwinespiked withbioactivephenolicsatthreeconcentrationlevels(LL,ML,and HL;seeTable3)andsubjectedtoMEPSC8procedure(CSWMEPS);a
secondsetofdifferentaliquotsofsyntheticwinewassubjectedto MEPSC8andtheextractsspikedwithbioactivephenolicsatLL,ML,
andHLconcentrationlevels(CSW).Thepeakarearatioobtainedfor
spikedsyntheticwinematrixbeforeandafterMEPSC8wasused
tocalculatethecorrespondingconcentrationthroughregression analysis (interpolationof signals in calibrationgraphs). The EE wasthencalculatedasfollow:%EE=(CSWMEPS/CSW)×100,where
CSWMEPS and CSW representstheconcentrationofthebioactive
phenolicsbeforeandafterMEPSC8procedure.
Thematrixeffectwasevaluatedbythepercentageofthe quo-tientbetweentheslopesofthestandardsinsyntheticwineand those obtainedby spikingRLand GV wines (standardaddition method).
3. Resultsanddiscussion
3.1. OptimizationoftheMEPSprocedure
Tooptimizemicroextractionbypackedsorbent,factors affect-ingtherecoverysuchasnatureofsorbent,numberofextraction
cycles,elutionvolume,samplevolume,andethanolcontent,were carefullyinvestigated.Selectionofsorbentisimportanttoachieve acceptable clean-up and extraction yield, therefore the perfor-mance of different kinds of sorbents such as C2, C8, C18, SIL andM1(mixedmodeC8+SCX)wasevaluated.Fig.3ashowsthe UHPLC–PDAresponseforthetargetanalytesusingdifferent sor-bents.
EachMEPSsorbentwasevaluatedintermsofextraction effi-ciency and reproducibility. M1, C2 and C8 sorbents (Fig. 3a) providedthebestextractionyields,howeverC8sorbentwaschosen fortheMEPSprocedurebecauseprovidedbetterresultsfor extrac-tionyieldandprecisioncomparedtobothM1andC2sorbents.In additiontheC8sorbentshowedgoodreproducibilityandstability forrepeateduse.Itwasusedformorethan100extractionswithout lossofextractionpower.Ontheotherhand,theSILsorbent(silica) gavelowextractionyieldsofanalytes.Theinfluenceofalcoholic contentontheMEPSextractionefficiencywasevaluatedbytesting differentsolutionstoadjustthedealcoholizedwineextracttoinitial volumeandcomparingthemwiththeefficiencyobtainedbyusing syntheticwinewith12%(v/v)ofethanol.Theresultsshowedthat thebestefficiencywasobtainedwhenultra-purewaterwasused toadjustthedealcoholizedwineextractvolumetoinitialsample volume.
InMEPStheretentionoftheanalytestothesorbentphaseis affectedby thenumberof extractioncyclesperformedand the speedapplied.Practically,analiquotofthevolumeofthesample can be drawn upand downthrough the syringe,once or sev-eraltimes(cycles)withoutdiscardingit.Themultiplyextraction cyclescanbecarried outfromthesamealiquot (draw–ejectin thesamevial)orbydrawupfromaliquotanddiscardinwaste (extract–discard).Thelastoptionwasselectedinthisstudy.The influenceofthenumberofextractioncyclesandsamplevolume onextractionefficiencyofbioactivephenoliccompoundstudiedis illustratedinFig.3b.Assaysshowedthatthecompetitionforactive adsorptionsitesoftheC8sorbentincreasedslightlyastheapplied extractionnumberandsamplevolumeincreasedFig.3b.
Statistically,nosignificantdifferenceswereobservedbetween once,fiveandtentimesof250Lsamplewinearepassedthough theC8sorbent.Forthisreason5×250Lwasselected,sincethe resultsshowedthatfiveextractioncyclesgaveagoodrecoveryand canextendthelifetimeoftheMEPScartridge.Fig.3cshowsthe UHPLC–PDAresponsefordifferentelutionvolumes,namely50L, 100L,250L,350L,and500L.Theresultsshowedthata vol-umeof50Lisenoughtoobtainthehighestextractionefficiency. 3.2. Methodvalidation
To demonstrate the feasibility of the present approach for determinationofwinebioactivephenolicsandtotestits practi-cability,themethodwasfullyvalidatedconsideringthelinearity, limitofdetection(LOD),limitofquantification(LOQ),extraction yield, accuracy and intra/inter-day precision.These parameters werecalculatedforeachbioactivephenolicusingconcentrations usuallyfoundinwines.Thevalidationparametersareshownin Tables2and3.
For each compound a linear regression of the peak areaanalyte/paekareaISvsconcentration,wascalculatedto
deter-minethelinearityofthemethodusingthreereplicatesatseven levelsofconcentration(Table2).
TheUHPLC–PDA systemgave linearresponseover the stud-iedrangeofconcentrationsandtheleast-squareslinearregression analysisofthedataprovidedexcellentcorrelationcoefficient val-ues for all compoundstested (r2>0.9986). The calibration was
performedbytheuseofsyntheticwine-matchedcalibration stan-dardsprepared asdescribed in Section2.2.The quantitationof the samples was performed using the means of equation of
Fig.3. (a)ComparisonoftheperformanceoffivedifferentMEPSsorbentsandtheinfluenceofthealcoholiccontent;(b)effectofnumberofextractioncycles (extraction-discard)andsamplevolume;and(c)influenceofdifferentelutionvolumes;ontheextractionefficiencyofbioactivephenoliccompoundsinwinesbyMEPS.Valuesexpressed asmean±standarddeviation(n=3).
calibrationcurves,obtainedfromordinaryleast-squaresregression data(Table2).
Thelimitsofdetection(LOD)andlimitsofquantification(LOQ) (correspondingtothebioactive phenolic amountforwhich the areaisequalto3timesand10timesthechosenstandard devi-ation,respectively)werecalculated fromordinaryleastsquares regressiondata[46].Thestandarddeviationchosentocalculate theLODandLOQvaluesistheresidualstandarddeviationofthe regressionlineforallbioactivephenoliccompoundsinthe ana-lyzedmatrix.AsitcanbeseeninTable2,theMEPSC8/UHLPC–PDA
methodologygavein generalvery lowdetectionlimits,ranging between0.0085and0.32gmL−1,whilelimitsofquantification rangedfrom0.028to1.08gmL−1,forferulicacidand(+)-catechin, respectively.
The percentage of matrix effect was evaluated through the percentageofthequotientbetweentheslopesofthestandards obtainedinsyntheticwineandthoseobtainedfromspikedRL(red) andGV(white)wines(standardadditionmethod).Matrixeffect valuesrangedfrom79.2to110.4%forwhitewineandfrom83.5 to112.6%forredwine,thereforenosignificantmatrixeffectwas observed(Table2).
Inordertoevaluatetheaccuracyofthemethod,arecoverystudy wascarriedoutbyspikingRLredwineandGVwhitewinesamples atthreeconcentrationlevels,withaknownamountofeach bioac-tivephenolic(Table3).Theconcentrationofphenolicsaddedtothe wineswaschosentocovertheexpectedvaluesinthewinesamples.
Theaccuracywasdeterminedaccordingtotheequationpresented inSection2.5.
Themeanaccuracy ofthebioactivepolypenols foreach for-tification level is listed in Table 3. At all concentration levels, satisfactory results were found with recovery values ranging from81.1(myricetinat10gmL−1)to99.6%(o-coumaricacidat 40gmL−1)forredwines,andbetween77.1(o-coumaricacidat 1gmL−1)and99.3%(syringaldehydeat50gmL−1).The extrac-tionyields(absoluterecovery)whenusingMEPSsinglepushing andpullingoff250LofsamplethroughtheC8sorbentmodewas examined.Theresultsshowedanaverageextractionyieldofabout 89.2%forredwineand91.3%forwhitewineatallconcentration levelsinvestigated (Table 3).Furthermore, theabsolute extrac-tionyield increase slightlyfrom low concentrationlevel(86.1% forredwines,and86.4%forwhitewines)tomedium(89.2%red wines,and91.3%for whitewines)and highconcentrationlevel (92.1%forredwines,and96.4%forwhitewines).Therefore,low concentrationofbioactivephenoliccompoundsresultsinlower extractionyieldsthantheobtainedtothemiddleandhigh fortifica-tionlevel.Thereasonforthismaybeduetothefactthatthesurface chemistryof sorbentcanbechanged byinterferingcompounds fromwineandthereforesorptionpropertiescanbechangedand maymixedretentionmechanismwasinvolved[31].Thiseffectis morepronouncedatlowconcentrationsoftheanalytewhen ana-lyte/matrixratioisverylow.Thesamebehaviorwasobservedfor accuracy.
J. Gonc ¸ alves et al. / J. Chromatogr. A 1229 (2012) 13– 23 21
Concentrationaofbioactivephenolicconstituentsinstudiedwines(valuesobtainedwerenotcorrectedbytherecoveries).
Concentration
Gal.ac.b Prot.ac.b Cat.b Gent. ac.b
Epicat.b Van.ac.b Syr.ac.b Syr.b p-Coum.ac.b Fer.b m-Coum. ac.b Rut.b o-Coum. ac.b Myr.b Cin. ac.b Kaemp.b Redwines
RealLavrador 12.8±0.1 –c 15.6±0.3 – 1.2±0.1 – 2.6±0.02 – 2.4±0.02 – – 4.3±0.1 – 3.2±0.04 – <LOQd
FontedaSerrana 13.5±0.3 – 11.5±0.8 – 1.4±0.1 – 2.6±0.02 – 2.4±0.02 – – 2.3±0.1 – 5.8±0.2 – 0.4±0.001 Torcaz 10.2±0.2 0.2±0.008 – – – 1.6±0.02 4.2±0.02 – 1.6±0.008 – – <LOQ – – – – Pelão 10.5±0.1 – 8.1±0.1 – 1.3±0.03 – 3.1±0.04 – 1.6±0.01 – – 1.7±0.1 – 3.0±0.03 – 0.3±0.004 JPAzeitão 12.5±0.1 – 15.1±0.2 – 0.9±0.001 – 2.5±0.001 – 1.9±0.004 – – 2.8±0.01 – 4.4±0.03 – 0.2±0.001 PorcadeMurc¸a 9.7±0.1 – 10.5±0.6 – 0.7±0.0001 – 1.9±0.02 – 1.5±0.04 – – 4.4±0.05 – 6.7±0.04 – 0.8±0.003 Periquita 10.5±0.05 – 9.2±0.2 – 2.4±0.04 – 3.8±0.02 – 2.0±0.008 – – 3.3±0.05 – 2.2±0.04 – – Contemporal 9.6±0.2 – 8.4±0.1 – 3.4±0.01 – 1.8±0.01 – 1.7±0.04 – – 4.0±0.002 – 3.8±0.04 – 0.4±0.01 Reguengos 8.7±0.02 – 10.0±0.05 – – – 2.4±0.01 – 1.8±0.003 – – 4.2±0.02 – 1.8±0.01 – 0.2±0.001 FreiBernardo 22.2±0.2 – 19.5±0.1 – 3.5±0.04 – 1.5±0.01 – – – – 3.7±0.06 – 3.8±0.004 – 0.5±0.002
GrãoVasco 6.8±0.1 – 11.7±0.1 – <LOQ – 2.1±0.01 – 2.3±0.002 – – 1.4±0.03 – 8.1±0.01 – <LOQ
CasadeSantar 9.7±0.1 – 10.7±0.02 – 2.2±0.05 – 3.9±0.01 – 1.9±0.002 – – 3.9±0.03 – 2.2±0.004 – 0.6±0.002
AdegadeBorba 10.0±0.1 – 11.8±0.02 – 0.7±0.001 – 2.4±0.01 – 2.5±0.02 – – 5.0±0.04 – 4.5±0.002 – 0.9±0.005
TerrasAltas 12.4±0.4 – 7.2±0.0001 – 2.4±0.04 – 3.1±0.002 – 2.2±0.01 – – 0.8±0.009 – 2.2±0.02 – 0.1±0.001
TMR1 19.1±0.04 – 10.0±0.05 – 0.9±0.0001 – 1.7±0.003 – – – – 1.6±0.02 – 3.2±0.004 – 0.1±0.0002
Esteva 6.2±0.2 – 8.6±0.1 – – – 2.8±0.01 – 1.8±0.001 – – 1.8±0.001 – 3.0±0.001 – 0.2±0.001
MonteVelho 5.6±0.1 – 9.0±0.02 – <LOQ – 2.8±0.001 – 1.7±0.001 – – 4.2±0.001 – 3.8±0.003 – 0.8±0.001
Terrasd‘elRei 8.5±0.1 – 9.7±0.01 – 0.8±0.001 – 3.7±0.01 – 1.9±0.004 – – 3.8±0.005 – 1.2±0.004 – 0.1±0.001
TLT2009 22.4±0.2 0.2±0.001 20.0±0.07 – 8.5±0.1 – 2.7±0.01 – 2.8±0.004 1.2±0.02 – 3.4±0.005 – 6.7±0.03 – <LOQ
TLT2010 29.1±0.4 – 21.3±0.2 – 8.9±0.2 1.1±0.01 1.7±0.01 – – – – 3.1±0.06 – 5.1±0.04 – 0.5±0.01
Basalto2009 23.7±0.5 0.2±0.003 16.6±0.1 – 6.6±0.02 – 1.6±0.005 – 2.6±0.03 – – 2.4±0.02 – 5.2±0.04 – <LOQ
Basalto2010 23.0±0.1 – 31.4±0.2 – 7.3±0.2 2.1±0.01 1.8±0.01 – 2.2±0.03 – – <LOQ – 2.8±0.006 – 0.6±0.01
Whitewines
GrãoVasco 1.9±0.01 – 3.2±0.2 – <LOQ – – – 1.7±0.001 0.7±0.0006 – <LOQ – – – –
Vidigueiro 1.7±0.03 – 1.6±0.1 – <LOQ – – – – 0.3±0.001 – 3.4±0.01 – – – 0.3±0.002
Navegante 2.5±0.01 – 3.8±0.2 – <LOQ – – – – – – 3.1±0.02 – – – –
Planalto 1.5±0.03 – <LOQ – <LOQ – – – 2.1±0.002 0.6±0.002 – <LOQ – – – –
RealLavrador 17.0±0.05 – – – – – – – 1.5±0.002 0.5±0.002 – <LOQ – – – –
Periquita 1.0±0.03 <LOQ – – – – – – – 0.4±0.001 – – – – – –
TMW1 – <LOQ – – – – – – 0.9±0.002 0.5±0.001 – 0.6±0.03 – – – –
TMW2 – <LOQ – – <LOQ – – – 0.8±0.01 0.3±0.0003 – <LOQ – – – –
Vi ˜natigoGual – <LOQ – – – – – – 1.5±0.003 0.4±0.0003 0.7±0.001 – – – – –
TMW3 3.6±0.03 – 5.9±0.02 – <LOQ 0.6±0.01 0.9±0.002 – – – – 0.9±0.02 – 0.4±0.002 – –
Lajido 4.3±0.04 <LOQ – – – – – – 3.0±0.003 0.5±0.004 – – – – – –
VG-Verdelho – <LOQ – – – 0.3±0.002 1.0±0.004 0.8±0.004 1.0±0.001 – – <LOQ – 0.4±0.0004 – –
aThecontentofeachofthe16polyphenolsanalysedinthewinesamplestestedisthemeanofthreereplicates±SDandindicatedasgmL−1.
b Gal.ac.(gallicacid),Prot.ac.(protocatechuicacid),Cat.((+)-catechin),Gent.(gentisicacid),Epicat.((−)-epicatechin),Van.ac.(vanillicacid),Syr.ac.(syringicacid),Syr.(syringicaldehyde),p-coum.ac.(p-coumaricacid),Fer.
(ferulicacid),m-coum.ac.(m-coumaricacid),Rut.(rutin),o-coum.ac.(o-coumaricacid),Myr.(myricetin),Cin.ac.(cinnamicacid)andkaemp.(kaempferol).
c (–)notdetected.
Fig.4.ProfileofMEPSC8/UHPLC–PDAchromatogramof(a)syntheticwineandsyntheticwinespikedwith16bioactivepolyphenols;(b)unspikedredwine(RL);and(c)
unspikedwhitewine(GV)(forpeakidentificationseeTable2).
Methodprecisionwasmeasuredthroughrepeatability inter-day and intra-day studies, expressed by the relative standard deviation(RSD)andcalculatedusingthemeasurementofpeakarea ofeachbioactivephenoliccompoundinthematrix.Theintra-day precisionwasmeasuredbycomparingstandarddeviationofspiked syntheticwine(LL,ML,HL)runinthesameday(n=7).Theinter-day precisionwasdeterminedbyanalyzingspikedsyntheticwine sam-plesforthreealternatedays.TheresultsaresatisfactorywithRSD valueslowerthan8%foreachmeasuredanalyteatallspiking lev-els(Table3).Theintra-dayprecisionvaluesatthethreedifferent levelsrangedfrom0.2(m-coumaricacidat40gmL−1 and cin-namicacidat25gmL−1)to4.7%(gallicacidand(−)-epicatechin at1gmL−1)(n=7),whereastheinter-dayprecisionvariedfrom 0.2(p-coumaricando-coumaricacidat25gmL−1)to7.5% ((+)-catechinat1gmL−1)(n=18+7).
Combination of fast MEPS technique together with quick UHPLC–PDA system proves to be an improved, with excellent recoveries,sensitivity,and repeatability,which makeitpossible touseasaquickapproachtoanalyzetheselectedbiologicalactive constituentsinwines.
3.3. Determinationofbioactivephenolicsinwinesby MEPSC8/UHPLC–PDA
UHPLCcanberegardedasanewdirectionforliquid chromatog-raphy.Usingsub-2mparticlesandmobilephasesathighlinear velocities,andinstrumentationthatoperatesathigherpressures thanthoseusedinHPLC,dramaticincreasesinresolution, sensitiv-ity,andspeedofanalysiscanbeobtained.
Inordertotesttheapplicabilityofthedevelopedmethod,the microextractionbypackedsorbentprocedurewasfirstappliedto amixtureofpolyphenolsstandardsandthento34winesamples (22redand12whitewines)(Table4).AsshowninFig.4a,the sep-arationofthestandardmixtureof16phenolicmetabolitesisvery fast,beingachievedwithinonly10min.Allsampleswereanalyzed intriplicate.
Goodpeakshapeandresolutionwasachievedforallthe com-poundswithnointerferencefromthewinematrix(Fig.4a).The chromatogramsforthewhiteandredwinessamplesshowedquite differentprofiles(Fig.4bandc)andtheircomplexityincreasesor decreasesaccordingtothewavelength.Themaximumabsorbance valuesofeachbioactivephenoliccompoundlistedinTable2was usedforquantificationpurposes.
The content of bioactive phenolic compounds found in the winesamplesassayedis summarizedinTable4.Ascanbe eas-ilyobserved,thebioactivephenolicsanalyzedareaboutsixtimes moreabundantinredwines.Thefactthatpolyphenolscontentis higherinredwineswaswidelydescribedbeforeintheliterature [47].Thephenolicacidsgentisic,o-coumaricandcinnamicalong tosyringaldehyde,werenotfoundinanyofthestudiedwines.On contrast,gallicacid,(+)-catechin,syringicacid,rutinandmyricetin werefoundinallredwinesanalyzed.
RedwinefromAzores,Basalto,wasbyfartheonethatshowed higherpolyphenoliccontentconsideringthesumofthe16 bioac-tivepolyphenolstested(almost71.24gmL−1),followedbyTerras de Lava,with polyphenoliccomposition around70.68gmL−1. In whitewines, thepolyphenoliccontentis significantlylower, varying from about 1.09gmL−1 in the TMW1 wine up to 18.98gmL−1inRLwine.Thisfactmakeswhitewineslessprone tobeeffectiveinhealthprotectionagainstoxidativedamageasthe protectiveeffectsassociatedtomoderatewineconsumptionhave beenattributedtotheircontentinpolyphenols.Infact,Fuhrman etal. [48]have shownthat itwaspossibletohavewhite wine withantioxidantcharacteristicssimilartothoseofredwinejust byincreasingitspolyphenolscontent,whatcouldbeeasily obtain-ingbyincreasingtheextractionofgrapeskinpolyphenolsduring thematurationprocess[48].
Regardingtotheindividualphenolicconstituents,gallic acid and rutin were themost abundantbiologically active foundin allwinesinvestigated,followedbyp-coumaricacid,(+)-catechin and(−)-epicatechin.Theseresultsconfirmedthatwine polyphe-noliccompositionisveryheterogenic,beingdependentofdiverse
factors,namelylocalclimateandvinificationconditionsthatare quitedifferentamongthewinesamplesusedinthiswork. 4. Conclusions
A novel, ultrafast, sensitive and reproducible MEPSC8/UHPLC–PDA-based methodology, using a 100mm
ana-lytical column (Acquity HSS T3) packed with 1.8m particle size, was developed, validated and successfully applied to the simultaneous determination of 16 bioactive phenolics in wine matrices.Theextractionprocedureissimpler,moreefficientand lesstime-consuming,andmoreovercanbeusedforsmallsample volumes(50L)aswellaslargevolumes(>1000L).Aftera care-fulselectionoftheeluentsystems,itwasdemonstratedthatthe chromatographicseparationofthebioactivephenolicmetabolites couldbeachievedinlessthan10minwithhighresolvingpower. Thecombinationoftheshorterrunningtimewithasmallerflow ratealsoreduced drasticallythesolventconsumption and thus ismoreenvironmentalfriendly.Thepackedsyringecanalsobe used several times (100 or more) depending from the matrix nature.Thevalidatedmethodshowedagoodperformance with regard to selectivity, LODs, LOQs, linearity, extraction yields, accuracy and intra/inter-day precisions. The results obtained fromapplicationofthemethodologytowinesamplessuggested thatthismethodcanbepotentiallyusefultoquantitate eachof thesecompoundsandserve aspromising alternativetoexisting methodology for bioactive phenolic constituent determination. Moreover,themethodcouldbeappliedtotheanalysisofbioactive phenolicinotherfoodmatrices(vegetables,fruits),asanattractive andverypromisingapproachfortheanalysisofothergroupsof compoundsduetothepossibilityofautomation,easytouse,rapid and minimumcost ofanalysiswhen compared toconventional SPE.
Acknowledgements
TheauthorsthankthefinancialsupportofFEDER(Transnational Cooperation MAC 2007-2013 Program) through VinSaudeMAC project(MAC/1/M105)andFCTthroughtheMSPortuguese Net-works (REDE/1508/RNEM/2005) and Pluriannual base funding (QUI-Madeira-674).
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