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Optimization and validation of a methodology based on solvent

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JournalofChromatographyA,1154(2007)87–96

Optimizationandvalidationofamethodologybasedonsolventextractionandliquidchromatographyforthesimultaneousdeterminationofseveralpolyphenolicfamiliesinfruitjuices

opez-M´arquez,B.Abad-Garc´ıa,L.A.Berrueta∗,D.M.L´

I.Crespo-Ferrer,B.Gallo,F.Vicente

DepartamentodeQu´ımicaAnal´ıtica,FacultaddeCienciayTecnolog´ıa,UniversidaddelPa´ısVasco/Euskal

HerrikoUnibertsitatea,P.O.Box4,E-48080Bilbao,Spain

Received8November2006;receivedinrevisedform27February2007;accepted5March2007

Availableonline16March2007

Abstract

Asolventextractionprocedureoffreeze-driedaliquotsfollowedbytheanalysisofphenoliccompoundsbyreversed-phasehigh-performanceliquidchromatography(RP-HPLC)withphotodiodearraydetection(DAD)hasbeendevelopedfortheanalysisofpolyphenoliccompoundsinfruitjuices.Thismethodologyisfocussedonthecharacterizationoffruitjuices,mainlyforqualitycontrolpurposes.Theeffectsofexperimentalvariables,suchassolventcompositionandvolumeandtimeandtemperatureonextraction,havebeenstudied.Auniquegradientprogramfortheseparationofseveralphenolicclasses(hydroquinones,hydroxybenzoicacids,ﬂavan-3-oles,hydroxycinnamicacids,coumarins,ﬂavanones,ﬂavones,dihydrochalconesandﬂavonols)hasbeenoptimized,usingstandardsof55commerciallyavailablephenoliccompoundspresentinfruits,aswellasrepresentativerealextractsfromfruitjuices.Allphenoliccompoundsshowedahighrepeatabilitywithin-day(n=5)andbetweendays(n=3)inpeakarea(RSD<8%)andexcellentstabilityoftheirretentiontimes.Highprecisionwasalsoobservedincalibrationslopes(RSD<8%).Detectionlimitsrangedbetween0.005and0.03␮g/mLforthedifferentdetectedpolyphenols.Completerecoveries(98–100%)wereobtainedforthemajorityofthephenolicstructuresofallrepresentativephenolicfamiliespresentinfruits.Themethodwassuccessfullyemployedtomeasurediversephenolicfamiliesinjuicesfrom18differentfruitsandconsequentlycouldbeusedforevaluatethequalityoffruitjuices.©2007ElsevierB.V.Allrightsreserved.

Keywords:HPLC-DAD;Polyphenols;Flavonoids;Phenolicacids;Strawberry;Apple;Tangerine;Fruitanalysis;Solventextraction;Optimization;Validation

1.Introduction

Polyphenols,widelydistributedinfruits,havegreatimpor-tanceinthenutritional,organolepticandcommercialpropertiesofthesefruitsandtheirderivedproducts(juices,jams,jellies...)throughtheircontributionstosensory-attributesofthesefruits(colour,bitternessandastringency)[1,2].Thus,theyplayanimportantroleinjuicequalitysincetheycanbeinvolvedintheenzymaticandnon-enzymaticoxidationsthatleadtothefor-mationofundesirablebrownpigmentsandcanberesponsibleforhazesandsedimentsinfruitjuices[3,4].Moreover,phenoliccompoundsareverysuitableaschemotaxonomicmarkers,andsomeofthemarecharacteristicofsomespeciesorvarieties[5,6],

∗

Correspondingauthor.Tel.:+34946015505;fax:+34946013500.E-mailaddress:luisangel.berrueta@ehu.es(L.A.Berrueta).

whereasquantitativedifferencesmayoccurdependingonfruitvariety,stagesofmaturity,storageconditions[7–10],andthepresenceofthepeelinfruit-basedproducts[11,12].Forcertainfruits,characteristicphenoliccompoundshavebeensuccess-fullyusedforthedeterminationofadulterationoffruitjuices[13,14],nectars[5,15,16]andjams[17–19]withcheaperfruits.Anotherimportantaspectofphenoliccompoundsisthepositivehealthbeneﬁtsonhumans.Amongthese,antioxidant,antiviral,antiallergic,cardioprotective,andanticarcinogeniceffectshavebeeninvestigated[20–25].Epidemiologicalstudieshaveshownaninverserelationshipbetweentheintakeoffruits,vegetablesandtheirproducts,richinpolyphenols,andthesechemoprotec-tiveeffects[26,27].

Knowledgeoftheprecisecompositionoffruitsandfruitvari-etiesandtheirproductssuchasfruitjuices,whoseconsumptionhaveincreasedsigniﬁcantlyinthelastyears,maycontributetoabetterunderstandingoftheirinﬂuenceinthequalityand

88B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–96

biologicalpropertiesoftheseproducts/foods.Forthisreason,

characterizationstudiesbasedonthepolyphenolicproﬁleshavebeencarriedoutwithfruitsandfruitjuices[28–32].However,despitetheextensiveresearchperformedinthisﬁeld,manycom-poundsstillremainunidentiﬁedinfruitsandtheirproductsandthereisnotaconsensusonthemostappropriateexperimen-talconditionsforthedeterminationofphenoliccompoundsoffoods.

Themethodologyusedtoanalyzethesephenoliccompoundsinfruitsandfruitproducts,generallyincludesaseriesofstepsrangingfromexhaustivesolventextraction,clean-upofextractsandpre-concentrationprocedurestosimpleﬁltrationandcen-trifugationinliquidsamples;however,inmanyinstances,simpleﬁltrationisineffectiveinrecoveringabroadrangeofpolyphe-nolsandalternativestrategiesarenecessary[33].

Sampleextractionproceduresareoftenregardedasbot-tlenecksinanalyticalmethods.Moreover,classicalsamplepreparationtechniquesarebothtimeandsolventconsuming,andsamplehandlingcandecreasethequalityoftheanalyti-calresults.Inthissense,thesamplepreparationstepaccountsforatleastone-thirdoftheerrorgeneratedbytheanalyticalmethod[34].Therefore,theimportanceofsamplepreparationinanalyticalmethodsshouldnotbeundervalued.

Themorebroadlyappliedextractionprocedureissolventextractionusingextractantssuchasmethanol,ethanolandace-toneormixturesofthesewithwater[35]fortherecoveryofawiderangeofpolyphenolsofdiversephenolicstructures.Antioxidantssuchastert-butylhydroquinone,2,6-di-tert-butyl-4-methyphenol(BHT),ascorbicacidorsulﬁtesareaddedtoextractionsolventtoprotecttheanalytesfromoxidation[36].Thisextractionprocedureisusuallyfollowedbyclean-upofextractsbysequentialextractionorliquid–liquidextractionand/orsolidphaseextraction(SPE)[33]andpre-concentration.Differentextractiontechniquessuchassupercriticalﬂuidextrac-tion(SFE),whichuseseitherpureormodiﬁedCO2[37],andmicrowave-assisted[38]extractionhavealsobeenused.Themethodsbasedonthesetechniquesofferbettercontroloftheextractionconditionscomparedwithconventionalalternatives,thusallowingtheextractiontobecarriedoutwithinrelativeshorttimesandinaselectiveway.Aftertheextractionproce-dures,thephenoliccompoundsarecharacterizedandquantiﬁed.Variousanalyticalmethodshavebeenpublishedforthedeter-minationofthesecompoundsinfoodsamples.Themostwidelyusedarebasedonreversed-phasehigh-performanceliquidchro-matography(RP-HPLC)coupledwithUV–visdetectionand/ormassortandemmassspectrometry.Theselattermethods,liquidchromatography–massspectrometry(LC–MS)[39–42]andliq-uidchromatography–tandemmassspectrometry(LC–MS/MS),havebecomethebestalternativeforseparation,identiﬁca-tionandquantiﬁcationofthesecompoundsinfruitsandtheirderivedproducts[43,44];however,thenumberofpublishedstudiesdealingwithLC–MSandLC–MS–MStechniquesandthepossibilityofaccesstothesetechnologiesformostlab-oratoriesaresofarlimited.Accordingtothemostrelevantbibliography,DADisanindispensabletoolfortheprovisionalidentiﬁcationofthemainphenolicstructurespresentinfoods[45,46].Generally,inpublicationsaboutnaturalproductsin

plants,suchasthoseaboutdeterminationofpolyphenolsinfruitsandtheirderivedproducts,littleinformationisgivenabouttheoptimizationproceduresoftheanalyticalmethodsused,andfrequently,thesemethodshavenotbeenexhaustivelyvali-dated;moreover,theirapplicabilityislimitedtoafewphenolicstructures.

Inthepresentwork,amethodologytoseparate,identifyandquantifysimultaneouslythemostrepresentativephenoliccompoundsinfruitjuices,suchashydroquinones,hydroxyben-zoicacids,ﬂavan-3-oles,hydroxycinnamicacids,coumarins,ﬂavanones,ﬂavones,dihydrochalconesandﬂavonols,usingasolventextractionassistedbysonicationandfollowedbyRP-HPLCanalysiscoupledwithaphotodiodearraydetector(DAD)isreported.Thismethodwasoptimizedandcarefullyvali-datedbyevaluatingtheselectivity,thelinearrange,thelimitsofdetectionandquantiﬁcation,theaccuracy,therepeatabilitieswithin-dayandbetweendays,therobustnessandthepolyphenolstabilitiesduringanalysis.2.Experimental

2.1.Reagents,solventsandstandardphenolics

Methanol(RomilChemicalLtd,Heidelberg,Germany)wasofHPLCgrade.WaterwaspuriﬁedonaMilli-QsystemobtainedfromMillipore(Bedford,MA,USA).Glacialaceticacid,providedbyMerck(Darmstadt,Germany),ascorbicacid,byPanreac(Barcelona,Spain)andsodiumﬂuoride,byFluka(Steinheim,Germany),wereofanalyticalquality.Allsolventsusedwerepreviouslyﬁlteredthrough0.45␮mnylonmembranes(Lida,Kenosha,WI,USA).

Polyphenolsstandardsweresuppliedasfollows:ellagicacid,procyanidinB1,procyanidinB2,(−)-epicatechingallate,arbutin,eriodictyol-7-O-rutinoside,eriodictyol-7-O-neohesper-idoside,naringenin-7-O-rutinoside,hesperetin-7-O-rutinoside,hesperetin-7-O-neohesperidoside,isosakuranetin-7-O-rutin-oside,ferulicacid,sinapicacid,quercetin-3-O-galactoside,quercetin-3-O-glucoside,quercetin-3-O-rhamnoside,kaempf-erol-3-O-glucoside,kaempferol-3-O-rutinoside,kaempferol-7-O-neohesperidoside,kaempferol-3-O-robinoside-7-O-rhamno-side,isorhamnetin-3-O-glucoside,isorhamnetin-3-O-rutino-side,myricetin,scopoletin,luteolin-7-O-glucoside,luteo-lin-6-C-glucoside,luteolin-8-C-glucoside,luteolin-3󰀇,7-di-O-glucoside,luteolin-4󰀇-O-glucoside,diosmetin-7-O-rutinoside,apigenin-7-O-glucoside,apigenin-6-C-glucoside,apigenin-8-C-glucoside,apigenin-7-O-neohesperidoside,apigenin-7-O-rutinosideandsinensetinbyExtrasynth`ese(Genay,France);whereasgallicacid,(+)-catechin,(−)-epicatechin,phloretin-2󰀇-O-␤-glucoside,naringenin,5󰀇-caffeoylquinicacid,caffeicacid,p-coumaricacidandquercetin-3-O-rutinosidewereprovidedbySigma–AldrichChemie(Steinheim,Germany);caftaricacid,apigenin-8-C-glucoside-4󰀇-O-rhamnoside,quer-cetagetin,quercetin-3-O-glucopyranoside,kaempferol-3-(p-coumaryl)glucoside,tangeretinandnobiletinbyChromadex(SantaAna,CA,USA);andnaringenin-7-O-neohesperidoside,quercetindihydratedandapigeninbyFlukaChemie(Steinheim,Germany).

B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–96

Allstockstandardsolutions(inconcentrationsrangingfrom

250to2500␮g/mLdependingoneachpolyphenol)werepre-paredinmethanol,exceptforhesperitin-7-O-rutinosidethatwasdilutedwithwater–dimethylformamide(80:20,v/v),andallwerestoredat4◦Cindarkness.2.2.Fruitsamples

Samplesofstrawberries(FragariaxananassaDuch.),apples(MalusdomesticaBorkh.)andtangerines(Citrusreticulata)oftheCamarosa,GoldenDeliciousandClemenulevarieties,respectively,werepurchasedfromalocalmarket.Tangerineswerepeeledseparatingtheﬂavedoandthealbedofromthepulp,whereasappleswerecored.1kgoffruit(strawberries,applesortangerines)wassqueezedandthecollectedjuiceaftermeasuringitsvolume,wasmixedwith50mLofanaqueoussolutioncon-tainingascorbicacid0.2g/mLandsodiumﬂuoride0.2g/mL,inordertoinactivatepolyphenoloxidasesandpreventpheno-lic◦degradation[47],andcentrifugedat6000×gfor15minat4C.Aliquotsof1mLweresampled,storedat−20◦Candlyophilizedlater.Thefreeze-driedmaterialwasstoredatroomtemperatureandindarknessinadessiccatoruntilanalysis.Thesethreefruitswereselectedinordertocoverallpolyphenolclassesandwereconsideredasrepresentativesduringtheoptimizationprocess.

2.3.Analyticalprocedures

2.3.1.Solventextractionoffreeze-driedsamples

Aliquotsoffreeze-driedjuice(1mL)weresubmittedtodirectsolventextractionwithanappropriateextractionsolventundernon-oxidativeconditions(inthepresenceofascorbicacid(HAs)at0.2%(w/v)inanultrasonicbath(Selecta,Barcelona,Spain)duringacertainperiodoftime.Thisconventionalextractionpro-cedurewasoptimizedinordertoobtainaquantitativeextraction.Thecriticalparametersunderstudyontheextractionprocedurewerethesolventchoice,thevolumeoftheextractionsolvent,andtheextractiontimeandtemperatureintheultrasonicbath.Inthiscase,thetestedsolventswereaqueousmixturesofmethanolacidiﬁedwithaceticacid(1%,v/v).

Theextractsobtainedineachoneoftheoptimizationexper-imentswereﬁlteredthrough0.45␮mPTFEﬁltersandstoredinglassvialsat4◦C(Waters,Milford,CA,USA)priortotheirinjectionintotheHPLCsystem.

2.3.2.Reversed-phaseHPLCanalysis

ChromatographicanalysiswasperformedonaShimadzu(Kyoto,Japan)liquidchromatograph,equippedwithavacuumdegasserDGU-14A,aquaternarypumpLC-10DVP,ather-mostattedautosamplerSIL-10ADVP,athermostattedcolumncompartmentandaDADdetectorSPD-M10AVP,andcon-trolledbyCLASS-VPsoftware.AreversedphaseLunaC18(150×4.6mmi.d.,3␮m)column(Phenomenex,Torrance,CA,USA)andaNova-PakC18(10×3.9mmi.d.,4␮m)guardcolumn(Waters,Barcelona,Spain)wereused.Solventsthatcon-stitutedthemobilephasewereA(aceticacid–water,0.5:99.5,v/v)andB(methanol).Theappliedelutionconditionswere:

0–2min,0%Bisocratic;2–6min,lineargradientfrom0%to15%B;6–12min,15%Bisocratic;12–17min,lineargradientfrom15%to20%B;17–35min,20%Bisocratic;35–90min,lineargradientfrom20%to35%B;90–136min,35%Biso-cratic;andﬁnally,washingandreconditioningofthecolumnwasdone.Theﬂowratewas0.8mL/minandtheinjectionvol-umewas50␮L.Thecolumnwasoperatedat30◦Candsamplevialsontheinjectorwerepreservedat4◦C.Hydroxybenzoicacidsweremonitoredandquantiﬁedat254nm,ﬂavan-3-ols,dihydrochalcones,ﬂavanonesandhydroquinonesat280nm,hydroxycinnamicacidsat320nm,andﬂavonols,ﬂavonesandcoumarinsat370nm.

2.3.3.Identiﬁcationandquantitationofpolyphenoliccompounds

Theidentiﬁcationofphenoliccompoundsforwhichstan-dardswereavailablewascarriedoutbycomparisonoftheirretentiontimeandtheirUV–visiblespectrawiththoseobtainedbyinjectingstandardsinthesameconditions,whilethepres-enceofothercompoundswasconﬁrmedbycomparisonofUV–visiblespectrawiththoseofstandardsofthesamepolyphe-nolicfamilyandbibliographicdata.

Quantitationwasperformedbyintroducingthemeasuredintegrationareasinthecalibrationequationofstandardsmoresimilartopolyphenolquantiﬁed.Thus,ﬂavan-3-olesandprocyanidinswerequantiﬁedas(+)-catechin;dihy-drochalconesasphloretin-2󰀇-O-␤-glucoside;ﬂavanonesasnaringenin-7-O-rutinoside;theunknownapigeninderivateasapigenin-7-O-glucoside;5󰀇-caffeoylquinicacidwithitself;p-coumaricacidderivateandtheunknownellagicacidderivatesasp-coumaricacidandellagicacid,respectively;andknownandunknownquercetin,isorhamnetin,kaempferolglycosidesasquercetin-3-O-rutenoside,isorhamnetin-3-O-rutenosideandkaempferol-3-O-rutenoside,respectively.3.Resultsanddiscussion

3.1.Optimizationofsolventextractionprocedure

Theoptimizationofthesolventextractionwasperformedusingfreeze-driedstrawberry,appleandtangerinejuices,threedifferentrepresentativematricesthatcontainthemostimportantpolyphenolfamiliesinfruits.Thus,theoptimalconditionsfoundforstrawberry,appleandtangerinejuicecouldbeappliedtojuicesofotherfruits.

Themostinﬂuentialexperimentalvariablesontheextractionprocedure,thepercentageofmethanolinthesolvent(mixturesofmethanol–water–aceticacid,v/v/v,containing1%ofaceticacidandascorbicacid(HAs)0.2%w/v),thesolventvolume,theextractiontimeandthetemperatureintheultrasonicbath,wereevaluatedtoincreasetheextractionefﬁciencyinthethreedifferentmatrices.

Theresultsshowedthatanincreaseinthepercentageofmethanolinﬂuencespositivelyontheextractionefﬁciency(Fig.1),followingasimilartendencyforallthesevenclassesofphenoliccompounds.Thisfactcouldbeattributedtomethanolwhichhasasuitablepolaritytoextractpolyphenolsbesides

90B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–96

Fig.1.Relativeefﬁcienciesofextractionforeachpolyphenolicclassinstraw-berry(A),apple(B)andtangerine(C)juicesasafunctionofthepercentageofmethanolintheextractantsolvent.Polyphenolicclasses:HBZ,hydroxybenzoicacids;FA,ﬂavan-3-oles;HCA,hydroxycinnamicacids;FVL,ﬂavonols;DHC,dihydrochalcones;FVNN,ﬂavanones;andFVN,ﬂavone.

toreducepolyphenoloxidase(PPO)activity.Solventscontain-inglowpercentagesofmethanoldonotinactivatetheenzymecompletely,resultinginlowextractionefﬁciencies[33].How-ever,asigniﬁcantdecreaseontheresolutionofchromatographicpeakswasobservedwhenvolumeshigherthan10␮Lofextractswith99%ofmethanolwereinjected.Incontrast,volumesashighas50␮Lcouldbeinjectedwhenextractswithlowmethanolpercentages,suchas30%,wereused,gettingglobalincreasesinsensitivityinspiteofhavinglowerextractionefﬁ-ciencies.Somelossesofmethodprecisioncanbeexpectedbythischoice,buttheaimoftheproposedmethodwasnotonlythequantitation,butalsotheidentiﬁcationofthehighestnum-berandclassesofpolyphenoliccompounds.Thus,amixtureofmethanol–water–aceticacid(30:69:1,v/v/v)wasselectedastheextractant.

Ontheotherhand,differentextractionvolumesweretestedtocometotheconclusionthatthebestvolumewas2mL,whichallowsextractionofallpolyphenolspresentinstudiedfruitjuiceswithoutsolventsaturation.Onstudyingtheextractiontimeinthethreematrices,itwereobservedthattheamountsofextractedanalyteswerenearlyconstantduringthedifferentperiodsoftimeexamined:10,15,20and30min.However,atimeof15minwaschosentowarranttheruptureofcellmembranesandenhancetheextractionofendocellularmaterialsinfruitjuicesduringthesoniﬁcationprocess.

Whentheextractiontemperaturewasexamined(18,25and

40◦C),

itwasfoundthatthisparameterhadnoinﬂuenceon

theefﬁciencyoftheprocessprovidedthattheextractiontimewaslessthan15min,exceptforhesperetin-7-O-rutinosideandanunknownﬂavanone(XVI)oftangerinejuice,forwhichhigherrecoverieswasobservedat40◦Cbecauseofthesig-niﬁcantincreaseintemperatureofthelowsolubilityoftheselow◦polarpolyphenolsintheextractant.Temperaturesabove40Cproducedanextractionyielddecreaseduetoapossibledegradationofpolyphenoliccompounds,causedbyhydrolysis,internalredoxreactionsandpolymerisations[48].Takingintoaccountthesefacts,25◦Cwasthetemperatureselectedastheoptimum.3.2.Liquidgradientoptimizationstrategy

Thedevelopmentofanuniqueliquidgradientforagreatnumberofpolyphenolicclassescouldconstituteaninterestingandquicksolutionforthestudyoftheproﬁleofphenoliccom-poundsfocussedonthecharacterizationoffruitjuicesmainlyforqualitycontroloftheseproductspriortotheircompletechar-acterization.Consequently,afterreviewingbibliographyaboutthemainpolyphenolspresentinfruit,fourstandardmixturesof55commerciallyavailablepolyphenoliccompoundsrepresenta-tiveofallfruitswereusedforoptimizationofchromatographicmethod,andextractsfromrealstrawberry,appleandtangerinejuiceswereinjectedastheﬁnaltesttoevaluatetheperfor-manceofthechromatographicseparation.Differentgradientsandsolvents(methanolandacetonitrile,andaqueousphos-phoricandaceticacidofdifferentpHvalues)weretestedtocometotheconclusionthatthebestmobilephasewascon-stitutedbymethanolasanorganicmodiﬁer,whichshowedthehighestselectivityandagoodresolutionof55phenoliccompounds(resultswithothermodiﬁersarenotshowed)andaceticacid–water0.5:99.5(v/v),whichwasvolatileandmassspectrometry-compatible.Thus,theoptimizedmobilephasecouldbedirectlyappliedtoLC–MSforfurtherpolyphenoliden-tiﬁcation.Otherparametersstudiedwereﬂowrate,timeandgradientsteps.Theﬁnalchromatographicseparationneededtocombinefourisocraticandthreegradientstepstoachieveaccept-ableresolutionofthehighernumberofphenoliccompoundsofdifferentphenolicfamiliesreportedinfruits.Fig.2showsthechromatogramsobtainedfromthefourmixturesofcommer-ciallyavailablephenoliccompoundsandTable1listsretentiontimes,includingstandarddeviation(SD)forthreeinjectionscar-riedoutondifferentdaysaswellasUVabsorptionmaximaofeachpeak,obtainedbyDADfromthestandardmixturesof55phenoliccompounds.Theseparameterswerelateremployedforprovisionalidentiﬁcationofunknownphenoliccompounds.Theresultsshowedthefollowingelutionorder:hydroquinones,hydroxybenzoicacids,ﬂavan-3-ols≈hydroxycinnamicacids,coumarins,ﬂavanones,dihydrochalcones,ﬂavonols,ﬂavonesandpolymethoxylatedﬂavones,accordingtothatreportedbyRobardsetal.[49].Hydroquinones,coumarins,ﬂavan-3-olsanddihydrochalconeswerenotmentionedbythisauthor.Frombibli-ographicaldata[14,50–53],certaintendencieswithinthesamepolyphenolfamilythatcharacterizetheseparationinreversed

B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–9691

Table1

PhenoliccompoundsandtheirchromatographicandspectroscopicparametersemployedintheoptimizationofthegradientmethodandintheprovisionalidentiﬁcationofphenolicsinthefruitjuicesCompoundno.1234567101112131415161718192021222324252627282930313233343536373839404142434445474849505152535455

Phenoliccompounda

ArbutinGallicacidProcyanidinB1(+)-CatechinCaftaricacidProcyanidinB2

5󰀇-CaffeoylquinicacidCaffeicacid(−)-Epicatechinp-CoumaricacidScopoletinFerulicacid

(−)-EpicatechingallateSinapicacid

Luteolin-8-C-glucosideEriodictyol-7-O-rutinosideLuteolin-6-C-glucoside

Eriodictyol-7-O-neohesperidosideApigenin-8-C-glucosideLuteolin-3󰀇,7-di-O-glucoside

Apigenin-8-C-glucoside-4󰀇-O-rhamnosideApigenin-6-C-glucosideNaringenin-7-O-rutinosideLuteolin-7-O-glucosideQuercetin-3-O-galactosideQuercetagetin

Quercetin-3-O-glucopyranosideQuercetin-3-O-glucoside

Kaempferol-3-O-robinoside-7-O-rhamnosideNaringenin-7-O-neohesperidosideQuercetin-3-O-rutinosideEllagicacid

Hesperetin-7-O-rutinosidePhloretin-2󰀇-O-␤-glucosideMyricetin

Apigenin-7-O-glucoside

Hesperetin-7-O-neohesperidosideApigenin-7-O-rutinoside

Kaempferol-7-O-neohesperidosideQuercetin-3-O-rhamnosideKaempferol-3-O-glucosideLuteolin-4󰀇-O-glucoside

Apigenin-7-O-neohesperidosideDiosmetin-7-O-rutinosideKaempferol-3-O-rutinosideIsorhamnetin-3-O-glucosideIsorhamnetin-3-O-rutinosideQuercetinNaringenin

Isosakuranetin-7-O-rutinoside

Kaempferol-3-(p-coumaryl)glucosideApigeninSinensetinNobiletinTangeretin

Synonyms

PhenolicstructureHydroquinone

HydroxybenzoicacidFlavan-3-olFlavan-3-ol

HydroxycinnamicacidFlavan-3-ol

HydroxycinnamicacidHydroxycinnamicacidFlavan-3-ol

HydroxycinnamicacidCoumarin

HydroxycinnamicacidFlavan-3-ol

HydroxycinnamicacidFlavoneFlavanoneFlavoneFlavanoneFlavoneFlavoneFlavoneFlavoneFlavanoneFlavoneFlavonolFlavoneFlavonolFlavonolFlavonolFlavanoneFlavonol

HydroxybenzoicacidFlavanone

DihydrochalconeFlavonolFlavoneFlavanoneFlavoneFlavonolFlavonolFlavonolFlavoneFlavoneFlavoneFlavonolFlavonolFlavonolFlavonolFlavanoneFlavanoneFlavonolFlavoneFlavoneFlavoneFlavone

tR±SD(min)7.638.8515.2519.2820.6321.7523.3525.7030.0940.6842.4549.4149.9354.0060.1162.2163.9867.7968.5469.1572.8577.4377.2477.7679.0581.8181.7181.6882.6082.9182.9482.9486.9688.9090.01.2491.6393.1093.55.9497.2297.7098.7299.3699.45100.80104.40123.21123.21124.75143.31144.15145.21146.08146.80

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±0.040.060.260.150.690.050.070.130.200.190.250.210.140.230.090.250.020.260.050.090.090.060.370.260.250.050.210.260.060.230.260.260.270.280.250.280.270.080.060.270.330.060.100.360.370.340.440.510.520.110.010.030.010.010.03

UVbands(nm)283271279279328275326323279309

252;344323275323

269;349284

268;337268;340269;340270;338283

268;348255;354257;357256;353256;354266;347283

255;354254;365281285

254;372267;339281

267;340265;3257;353265;348268;337267;338277;335265;348254;353254;354255;367288283

267;314267;338332334

270;325

Chlorogenicacid

OrientinEriocitrinHomoorientinNeoeriocitrinVitexin

Vitexin-4󰀇-rhamnosideIsovitexinNarirutinHyperosideIsoquercetinIsoquercitrinRobininNaringinRutinHesperidinPhloridzinApigetrinNeohesperidinIsorhoifolinQuercitrinAstragalinRhoifolinDiosminNicotiﬂorinNarcisin

DidyminTiliroside

MaximumUVbandindicatedinbold.

aForparticularchemicalstructuresseeforexamplereference[58].

92B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–96

Fig.2.Chromatogramsat280nmobtainedforthestandardphenoliccompoundsemployedintheoptimizationofchromatographicmethod.Foridentiﬁcationof

peaksseeTable1.

phasewereobserved:(a)theretentiontimedecreasethemorehydroxylgroupshasthepolyphenol;(b)ifchemicalstructuresofpolyphenoliccompoundsincludesugars,thepolyphenolselutebeforetheircorrespondingaglycones;(c)compoundswithapo-larsubstituents,suchasmethoxygroups,elutelaterthantheirnoalkylestersequivalentsduetotheirmoreapolarcharacter;(d)inthesamewaytheacylatedphenoliccompounds,whoseretentiontimeishigherthantheirnoacylderivatesand(e)C-glycosideselutebeforeO-glycosidesandwithinC-glycosides8-Cbefore6-Cisomers.

Extractsofstrawberry,appleandtangerinejuicewereana-lyzedaftertheoptimizationprocessinordertoprovetheefﬁciencyofthechromatographicseparation.Theproposedgra-dientelutionmethodallowedagoodseparationofphenoliccompoundspresentinfruitjuicesinallcasesandenabledtheidentiﬁcationandquantiﬁcationofthephenoliccompoundsinallsamplesstudied.Fig.3showsthechromatogramsobtainedfromstrawberry,appleandtangerinejuices.Tables2–4summa-rizethephenolicclassandcompoundsidentiﬁedinthesamples.Inrelationtotheidentiﬁcationofunknownpeaks,thiswasperformedasindicatedinSection2.3.3.ThepeaksI,IIandIVwereassignedtohydroxycinnamicacidstructures,sincetheirspectrawereidenticaltothatofp-coumaricacid.PeaksVandXVIexhibitedspectralcharacteristicsidenticaltothoseofﬂa-vanones.PeakVIwascharacterizedasanapigeninderivate[54]andpeaksXIIIandXIVasellagicacidderivates,sincetheirspectrawereidenticaltothoseofapigenin-7-O-glucosideandellagicacid,respectively.PeakXIIwasidentiﬁedasaphloretinderivate.Thespectrumindicatedthattheaglyconewasphloretin,andtheretentiontimeindicatedamorepolarcompoundthanphloretin-2󰀇-O-glucoside.Thiscompoundhasbeenidentiﬁedasphloretin-2󰀇-xyloglucoside[55–57].PeakXVintheapplejuicewascharacterizedasquercetin-3-O-arabinosideaccordingtoitschromatographicandspectroscopiccharacteristicsandbibliog-raphy[40].Inadittion,tangerinejuiceshowedthepresenceofsixunknownﬂavonolsthatelutedbeforetheirmonoglycosidederivatesandareprobablytri-ordiglycosides.Basedontheirspectracharacteristics,peaksIII,IXandXwereassignedto

Table2

Provisionalidentiﬁcation,accuracyandprecisionofphenoliccompoundsinstrawberryjuicesPeakno.a

PhenolicchemistrystructuresandcompoundsPhenolics

Phenolicclass

ProvisionalidentiﬁcationparameterstR±SD(min)

UV(nm)

AccuracyRecovery(%)

Repeatabilitywithin-dayConc.±SD(␮g/mL)1327693.910.511.66.0

±±±±±±±1240.30.60.70.3

BetweendaysConc.±SD(␮g/mL)1428683.910.111.76.1

±±±±±±±1230.20.60.40.4

34III31XIIIXIVProcyanidinB1(+)-Catechin

p-Coumaricacidderivatep-CoumaricacidderivateQuercetin-3-O-rutenosideEllagicacidderivateEllagicacidderivateFlavan-3-olFlavan-3-ol

HydroxycinnamicacidHydroxycinnamicacidFlavonol

HydroxybenzoicacidHydroxybenzoicacid15.3419.2521.8623.4181.4081.9084.68±±±±±±±0.100.010.060.100.080.030.04278279315312

255;354251;366254;366100

98.4±0.198.7±0.398.6±0.498.7±0.2100100

MaximumUVbandindicatedinbold.

aRomannumbersfornon-availablecommerciallypolyphenolstandards;arabicnumbersforavailablecommerciallypolyphenolstandards.

B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–9693

Fig.3.Chromatogramsofstrawberry(A),apple(B)andtangerine(C)juiceextractsat254,280,320y370nm.Compoundspresentinthestandardmixturesare

numberedasinTable1.UnknownpolyphenolsarelabeledwithRomannumerals.Peaks:(I)p-coumaricacidderivate;(II)p-coumaricacidderivate;(III)quercetinglycoside;(IV)p-coumaricacidderivate;(V)unknownﬂavanone;(VI)apigeninderivate;(VII)kaempferolglycoside;(VIII)isorhamnetinglycoside;(IX)quercetinglycoside;(X)quercetinglycoside;(XI)kaempferolglycoside;(XII)Phloret´ın-2󰀇-O-xyloglucoside;(XIII)ellagicacidderivate;(XIV)ellagicacidderivate;(XV)quercetin-3-O-arabinoside;and(XVI)unknownﬂavanone.

Table3

Provisionalidentiﬁcation,accuracyandprecisionofphenoliccompoundsinapplejuicesPeakno.a

PhenolicchemistrystructuresandcompoundsPhenolics

Phenolicclass

ProvisionalidentiﬁcationparameterstR±SD(min)

UV(nm)

AccuracyRecovery(%)

Repeatabilitywithin-dayConc.±SD(␮g/mL)13.620861705612.218.01.4513.63.268.29

±±±±±±±±±±±0.312520.20.30.040.30.080.09

BetweendaysConc.±SD(␮g/mL)13.220851655311.8181.4313.33.18.2

±±±±±±±±±±±0.7171340.610.070.80.20.5

34679IVXII2834XV40ProcyanidinB1(+)-CatechinProcyanidinB2

5󰀇-Caffeoylquinicacid(−)-Epicatechin

p-CoumaricacidderivatePhloretin-2󰀇-O-xyloglucosideQuercetin-3-O-glucosidePhloretin-2󰀇-O-␤-glucosideQuercetin-3-O-arabinosideQuercetin-3-O-rhamnosideFlavan-3-olFlavan-3-olFlavan-3-ol

HydroxycinnamicacidFlavan-3-ol

HydroxycinnamicacidDihydrochalconeFlavonol

DihydrochalconeFlavonolFlavonol15.2519.2821.23.5930.0940.5680.7181.9888.7793.0396.16±±±±±±±±±±±0.040.070.050.230.231.260.600.350.710.420.44279279279327279311284

255;355284

256;352256;35099.099.199.099.299.199.399.299.099.199.199.1±±±±±±±±±±±0.30.20.20.20.30.30.10.30.20.40.3

MaximumUVbandindicatedinbold.

aRomannumbersfornon-availablecommerciallypolyphenolstandards;arabicnumbersforavailablecommerciallypolyphenolstandards.

94B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–96

Table4

Provisionalidentiﬁcation,accuracyandprecisionofphenoliccompoundsintangerinejuicesPeakno.a

PhenolicchemistrystructuresandcompoundsPhenolics

Phenolicclass

ProvisionalidentiﬁcationparameterstR±SD(min)

UV(nm)

AccuracyRecovery(%)

Repeatabilitywithin-dayConc.±SD(␮g/mL)15.231.25.58.18.34.11.592.718710.13.814119.711.133

±±±±±±±±±±±±±±±0.20.70.10.10.20.20.030.270.10.08820.30.26

BetweendaysConc.±SD(␮g/mL)15.0315.48.08.14.21.612.819010.03.7679.611.038

±±±±±±±±±±±±±±±0.410.30.20.20.20.030.280.10.051100.30.17

IIIVVIVIIVIII16IXX23XI31334547XVIQuercetinglycosideUnknownﬂavanoneApigeninderivateKaempherolglycosideIsorhamnetinglycosideEriodictyol-7-O-rutinosideQuercetinglycosideQuercetinglycoside

Naringenin-7-O-rutinosideKaempferolglycosideQuercetin-3-O-rutenosideHesperetin-7-O-rutinosideKaempferol-3-O-rutinosideIsorhamnetin-3-O-rutinosideUnknownﬂavanoneFlavonolFlavanoneFlavoneFlavonolFlavonolFlavanoneFlavonolFlavonolFlavanoneFlavonolFlavonolFlavanoneFlavonolFlavonolFlavanone34.0747.07.7949.7054.5962.3467.9872.3177.4279.3783.4487.0099.96105.00125.69±±±±±±±±±±±±±±±0.680.680.560.500.480.430.310.370.390.330.310.330.370.360.50255;354283

272;338265;339256;354282

257;357256;366283

265;346256;354284

265;348254;35728310083±797.7±0.510010082±210010078±2100

97.7±0.526±2100

98.5±2.144±3

MaximumUVbandindicatedinbold.

aRomannumbersfornon-availablecommerciallypolyphenolstandards;arabicnumbersforavailablecommerciallypolyphenolstandards.

unknownquercetinglycosides,peaksVIIandXItounknownkaempferolglycosidesandﬁnallypeakVIIItoanunknownisorhamnetinglycoside.3.3.Methodvalidation

Theproposedanalyticalmethodforthedeterminationofpolyphenolsfromfreeze-driedfruitjuiceswascarefullyeval-uatedintermsofselectivity,linearity,precision,accuracy,robutnessandstabilityofphenoliccompoundsfromextractsduringanalysisandfromstockstandardsolutions.

Theselectivityofthemethodwasassessedbycomparingthechromatogramsoftheextractsofrepresentativesamplestothoseofthemethodblank(extractantsolvent)andtoasolutionofstandards.Peakshapes,retentiontimesandspectralpurityofthechromatographicpeakwereconsideredinordertodetectpossibleinterferences.Nointerferingpeakswereobservedintheblankchromatogramsatthequantitationwavelengths(254,280,320and370nm).Inmostcases(peaksinTables2–4),peakpurityanddegreeofmatchwiththestandardspectraweregreaterthan98%,exceptforeriodictyol-7-O-rutinosideinthetangerinejuicewhosepeakpuritywas84%duetoitslowconcentration.

Thelinearitywascheckedbymeansoftheexternalstandardmethod.Thus,standardsolutionswereinjectedinthechromato-graphicsystemfromthelimitsofquantitationuntillevelsofconcentrationofthephenoliccompoundswereusuallypresentinfruitjuices.Severalcalibrationequationsweresetforeachanalyteanddifferentconcentrationintervals.Table5showstheslope,theoriginordinate,thecorrelationcoefﬁcientsandthelimitsofdetectionandquantitationforthepolyphenolsusedforquantiﬁcation.Thehighestslopewasshownforp-coumaricand5󰀇-caffeoylquinicacidat320nmwhile(+)-catechinobtainedthelowestsensitivityat280nm.Thelimitsofdetectionanddeterminationwerecalculatedasthosecorrespondingtosig-naltonoiseratiosof3:1and10:1,respectively.Detectionlimitsrangedbetween0.005and0.03␮g/mLforp-coumaricacid,andapigenin-7-O-glucoside,isorhamnetin-3-O-rutinosideandellagicacid,respectively.Thedeterminationlimitsrangedfrom0.02to0.1␮g/mLfornaringenin-7-O-rutinosideandp-coumaricacid,andkaempferol-7-O-rutinoside,isorhamnetin-3-O-rutinosideandapigenin-7-O-glucoside,respectively.Theresultsobtainedforthequantitationlimitsshowthatthepro-posedmethodissensitiveenoughforthedeterminationofthephenoliccompoundsinfruitjuices.

Theprecisionstudieswerecarriedoutfromtheevaluationoftheretentiontimesstabilityandrepeatabilitywithin-dayandbetweendaysofthepeakareasusingfruitextractssuitablystored(Tables2–4).Therepeatabilitieswithin-dayofpeakareas(ﬁvereplicates),expressedbymeansofthepercentageofrel-ativestandarddeviation(%RSD(n=5)),werelowerthan8,7and5%andtherepeatabilitiesbetweendays(threereplicates)werelessthan8,8,and5%forstrawberry,appleandtangerinejuices,respectively,exceptforhesperetin-7-O-rutinosideandtheunknownﬂavanoneXVI(20%and17%,within-day;and23%and19%betweendays,respectively),foundinthetanger-inejuice,duetothelowpolarityofthesephenoliccompoundsthatproduceslowextractionrecoveries.TheRSD(n=3)valuesobtainedforretentiontimeswerelowerthan1%inallcases.Ontheotherhand,changesintheexperimentalvariables:±5%ofmethanolintheextractionsolvent;±5%ofaceticacidintheextractionsolvent(theseexperimentswerecarriedoutinduplicate)aswellasthevariationofthecalibrationslopesatdifferentdayswereusedasameasureoftherobustnessofthemethod.Theresultsobtainedfromthevariationofpercent-agesofmethanolandaceticacidinextractionsolventwerenotsigniﬁcantlydifferentfromthoseachievedbythevalidatedmethodandtheRSDobtainedforcalibrationslopesondifferent

B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–96

Table5

ParametersofthecalibrationcurvesinstandardsolutionsforpolyphenolsusedforquantitationPolyphenola(+)-Catechin

Linearrange(␮g/mL)0.04–11–1010–2500.04–11–1010–1000.02–11–1010–2500.03–0.10.1–11–1010–1000.02–0.10.1–11–1010–1000.09–11–100.07–11–1010–2500.1–11–1010–1000.1–11–500.1–11–1010–100

Slope±Sb(×10−3)43±0.2±143.0±0.4137±2136±4142±4108±1107±3116±115414315817639020421

±±±±±±±±233341109

Originordinate±Sa(×10−3)−0.7±0.33±7−5±490±1

(1±3)101(−2±2)1020.7±0.79±19

(−3±2)1020.05±0.12±2

(−0.5±2)101(−3±2)1020.02±0.3−0.8±0.8(1±6)101(−2±6)102−5±1−57±280.±0.05(0.5±2)101(−2±1)1021.6±0.53±1

(−2±2)1021.4±0.3−1±40.1±0.30.6±0.8(0.6±1)102

r20.99950.99930.99990.99990.99930.99930.99970.99920.99980.99990.99960.99960.99970.99970.99990.99940.99960.99980.99981.00000.99910.99980.99981.00001.00000.99991.00000.99990.99960.9991

LOD(␮g/mL)0.01

LOQ(␮g/mL)0.04

Phloretin-2󰀇-O-␤-glucoside

0.020.04

Naringenin-7-O-rutinoside0.010.02

5󰀇-Caffeoylquinicacid

0.0080.03

p-Coumaricacid0.0050.02

Ellagicacid

Quercetin-3-O-rutenoside

193±1221±383.8±0.187±392.7±0.997.1±0.995.9±0.2105±473.0±0.573.1±0.269.2±0.666±165±2

0.030.02

0.090.07

Isorhamnetin-3-O-rutinoside0.030.1

Kaempferol-3-O-rutinosideApigenin-7-O-glucoside

0.020.03

0.10.1

Regressionequation:y=a+bx,whereyisthepeakareaandxtheconcentrationin␮g/mL;r2,correlationcoefﬁcient.

days(n=3)werelowerthan8%,exceptforkaempferol-3-O-rutinosidewhichwaslowerthan11%.Theseresultsprovedthattheproposedmethodwasrobust.

Theabsenceofreferencematerialsandthelackofofﬁ-cialmethodsforanalysisofphenoliccompoundsmakethetaskofmeasuringaccuracydifﬁcult.Likewise,thecompleteextractionofphenolicsfromcellwallsisnoteasyand,subse-quently,notmuchinformationdealingwithcompleteextractionofphenoliccompoundscanbefoundintheliterature.Toevaluatetheaccuracyofthemethod,consecutiveliquidextrac-tions(threereplicates)wereperformedonthesamesampleundertheoptimizedconditionsuntilnophenoliccompoundwasdetectedbyHPLCanalysis.Therecoveryofeachextrac-tionwascalculatedastheamountextractedrelativetothetotalquantityextractedfromeachsampleinallthesuccessivesextractions.Thenumberofextractionsneededweredependentonphenolicstructureandindependentofthematrix.Recov-eriesof98–100%wereobtainedintheﬁrststepofextraction(Tables2–4)forﬂavan-3-ols,hydroxybenzoicandhydroxycin-namicacids,dihydrochalcones,ﬂavonolsandﬂavonesinthethreestudiedmatrices,whereasrecoverieslessthan83%werefoundforﬂavanones,beingnecessaryahighernumberofextrac-tionsteps(tenconsecutiveextractions)toobtainquantitativeyield.Hesperitin-7-O-rutinosideandtheunknownﬂavanoneXVIshowedthelowestrecoveriesintheﬁrstextractionstep(26%and44%,respectively),whichwasattributedtotheirlowpolarstructures.Thus,themethodaccuracywasadecuatedforallphenolicclassespresentinfruitsexceptforﬂavanones.Theselesspolarcompoundstendtobemoresolubleinnon-aqueoussolvents.

Thestabilityofphenoliccompoundsoffruitjuicesextractsandstockstandardsolutionswasstudied.Dataobtainedshowedthatextractedphenoliccompoundsfromrealsamplesweresta-bleforatleasttwodaysprovidedthattheywerekeptat4◦Cinthethermostatizedautoinjector,andphenoliccompoundsfromstocksolutionswerestableinmethanolat4◦Candindarknessforatleastsixmonth,exceptfor(+)-catechinand(−)-epicatechin,thatweredegradedabout15%inthattime.Thesepolyphenolswerestableatleastfor1monthunderthementionedconditions.

96B.Abad-Garc´ıaetal./J.Chromatogr.A1154(2007)87–96

4.Conclusion

Theproposedmethodexhibitexcellentprecisionsanddetec-tionlimits,aswellascompleterecoveries(≥98–100%)forthemajorityofthephenolicstructurespresentsinfruits.Therobust-nessofthemethodanditsgoodselectivityallowedthatitssuccessfulapplicationinourlaboratoryformeasurementofphe-noliccompoundsofninedifferentfamiliesinjuicesfrom18dif-ferentfruits(orange,tangerine,lemon,lime,grapefruit,peach,nectarine,plum,apricot,sweetcherry,apple,pear,grape,straw-berry,raspberry,blueberry,redcurrant,blackberry)atthepresenttime.Allthesefactsmadethismethodsuitableforrutineanalysisofphenoliccompoundsforthequalitycontroloffruitjuices.Acknowledgements

ThisresearchwassupportedbytheUniversidaddelPa´ısVasco/EuskalHerrikoUnibertsitatea(projectn◦13468-2001).BeatrizAbadGarc´ıathankstheUniversidaddelPa´ısVasco/EuskalHerrikoUnibertsitateaforaPh.D.grant.References

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