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JournalofChromatographyA,1260 (2012) 154–163
ContentslistsavailableatSciVerseScienceDirect
Journal
of
Chromatography
A
jo u rn al h om epa ge :w w w . e l s e v i e r . c o m / l o c a t e / c h r o m a
A
new
and
improved
strategy
combining
a
dispersive-solid
phase
extraction-based
multiclass
method
with
ultra
high
pressure
liquid
chromatography
for
analysis
of
low
molecular
weight
polyphenols
in
vegetables
Catarina
L.
Silva
a,
Nathaly
Haesen
b,
José
S.
Câmara
a,∗aCentrodeQuímicadaMadeira,CentrodeCiênciasExactasedaEngenhariadaUniversidadedaMadeira,9000-390Funchal,Portugal
bKatholiekeHogeschoolKempen,Geel,Belgium
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received5May2012
Receivedinrevisedform21August2012
Accepted24August2012
Available online 30 August 2012 Keywords:
Vegetables dSPE-QuEChERS
Freelowmolecularweightpolyphenols
UHPLC–PDA
a
b
s
t
r
a
c
t
ThispaperreportsonthedevelopmentandoptimizationofamodifiedQuick,Easy,CheapEffective, RuggedandSafe(QuEChERS)basedextractiontechniquecoupledwithaclean-updispersive-solidphase extraction(dSPE)asanew,reliableandpowerfulstrategytoenhancetheextractionefficiencyoffree lowmolecular-weightpolyphenolsinselectedspeciesofdietaryvegetables.Theprocessinvolvestwo simplesteps.First,thehomogenizedsamplesareextractedandpartitionedusinganorganicsolventand saltsolution.Then,thesupernatantisfurtherextractedandcleanedusingadSPEtechnique.Finalclear extractsofvegetableswereconcentratedundervacuumtoneardrynessandtakenupintoinitialmobile phase(0.1%formicacidand20%methanol).Theseparationandquantificationoffreelowmolecular weightpolyphenolsfromthevegetableextractswasachievedbyultrahighpressureliquid chromatogra-phy(UHPLC)equippedwithaphodiodearray(PDA)detectionsystemandaTrifunctionalHighStrength Silicacapillaryanalyticalcolumn(HSST3),speciallydesignedforpolarcompounds.
Theperformanceofthemethodwasassessedbystudyingtheselectivity,lineardynamicrange,the limitofdetection(LOD)andlimitofquantification(LOQ),precision,trueness,andmatrixeffects.The validationparametersofthemethodshowedsatisfactoryfiguresofmerit.Goodlinearity(R2
values>0.954;
(+)-catechinincarrotsamples)wasachievedatthestudiedconcentrationrange.Reproducibilitywas betterthan3%.Consistentrecoveriesofpolyphenolsrangingfrom78.4to99.9%wereobservedwhen alltargetvegetablesampleswerespikedattwoconcentrationlevels,withrelativestandarddeviations (RSDs,n=5)lowerthan2.9%.TheLODsandtheLOQsrangedfrom0.005gmL−1(trans-resveratrol,carrot) to0.62gmL−1(syringicacid,garlic)andfrom0.016gmL−1(trans-resveratrol,carrot)to0.87gmL−1
((+)-catechin,carrot)dependingonthecompound.Themethodwasappliedforstudyingtheoccurrence offreelowmolecularweightpolyphenolsineightselecteddietaryvegetables(broccoli,tomato,carrot, garlic,onion,redpepper,greenpepperandbeetroot),providingavaluableandpromisingtoolforfood qualityevaluation.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Fruitsandvegetablescontainseveralthousandofstructurally diverse phytochemicals, such as polyphenols, carotenoids and tocopherols.Itsconsumptionisencouragedduetothereported multiplebiologicaleffectsnamelyantioxidant,anti-inflammatory, anticarcinogenic, antimutagenic and antiproliferative activities [1,2]. This physiologicalbenefits have been attributed to their potential role on theprevention of low- and very-low density lipoprotein(LDLandvLDL)oxidation(crucialstepsin atheroscle-roticlesionformation)andDNAbases(relevanttotheinductionof
∗ Correspondingauthor.Tel.:+351291705112;fax:+351291705149.
E-mailaddress:jsc@uma.pt(J.S.Câmara).
cancer)byfreeradicalsbyactingasfreeradicalscavengers[3–6],on inhibitionofplateletaggregation[7–10],cellproliferation, migra-tion,andangiogenesis[11].Theyalsocanactastransientmetal ion chelators[9],controlproteinoxidationand advanced glyca-tionendproducts(AGEs)formation[12],andaspotentpancreatic lipaseinhibitorsbeingpotentialcandidatesforobesityprevention, namelyepigallocatechin-3-gallate,kaempferolandquercetin[13]. Infood,phenolicsmaycontributetothebitterness,astringency, color,flavor,odor,and oxidativestabilityof products.However, thereisalimitedamountofinformationonthecontentofphenolic compoundsincommonfoodstuffsofplantoriginandtheir antiox-idantactivities.Inthisrespect,screeningofvariousfoodproducts withbeneficialhealthpropertiesisveryimportant.
Evidenceonthehealthbenefitsofpolyphenolsandtheirimpact on foodquality have stimulatedthe developmentof analytical
0021-9673/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved.
methods for their identification and quantification [14–18]. In recent years, the ability of several extraction techniques, such assolid–liquidextraction(SLE)[19],enzyme-assistedextraction [20],heatextraction[21],solid-phaseextraction(SPE)[22],and solid-phasemicroextraction(SPME)[23],fortheisolationfreelow molecular-weightpolyphenols (LMW-PPs)from vegetables and otherfood matrices, hasbeen proposed and evaluated as reli-ablealternativestoclassicliquid–liquidextraction(LLE)technique. Morerecently,anovelanalyticalapproach,basedonminiaturized microextraction by packed sorbent (MEPS), followed by ultra-high pressure liquid chromatography (UHPLC) separation, has beenproposed by Gonc¸alves et al. [24] for quantitative deter-mination of winebiologically active phenolic constituents and trans-resveratrol[25].Inthelastfewyears,aquick,easy,cheap, effective,ruggedandsafe(QuEChERS)extractiontechniqueforthe multiclass,multiresidueanalysisofpesticides infruitsand veg-etables,wasdescribedbyAnastassiadesetal.[26].Thisprocedure involvesinitialsinglephaseextractionwithacetonitrilefollowed bysalting-outextraction/partitioningbyadditionofMgSO4 plus NaCl,andfinallyusingdispersive-solid-phaseextraction(dSPE)for clean-up.Inadditiontopesticidemultiresidueanalysisin food-stuffs[27–34],QuEChERSconceptshavebeenusedforacrylamide [35,36],clinical[37],veterinarydrugresidue[38,39],foodquality [40],supplementtesting[41],perfluorinatedcompounds[42,43], alkaloids[44],environmentalandmycotoxins[45–47]analytical applications.However,up todatenostudies usingthe QuECh-ERStechniquecoupledtodSPEfortheanalysisoffreeLMW-PPs invegetablesoranotherkindofmatriceshavebeenpublished.
Therefore,inthispaper,wereportforthefirsttimetothebest of ourknowledge, a new and reliable QuEChERS-based extrac-tiontechniquecombinedwithadSPEclean-upprocedureinorder toinvestigateif this methodologyis suitable for extractingthe LMW-PPsfromselectedspeciesofcommonlyconsumed vegeta-bles.Importantparametersthatmayaffectextractionefficiency, namelythepartitioning solvents and thesaltsusedtoenhance thesalting-outeffectontheportioningprocess,wereinvestigated andoptimized.Aneffectiveextraction/clean-upprocedure,using primary-secondaryamine(PSA)andC18sorbents,allows achiev-ingclearextracts,noorlowmatrixeffectandlowLODandLOQ, alsoreducingasmuchasorganicsolvents,fulfillingthepurposes toestablishawideracceptabilityofthemethodology.The QuECh-ERSprocedurewasoptimizedusingcarrotsamplesandevaluated inothersevenselectedvegetables.TheLMW-PPsanalysiswere per-formedonanultrahighpressureliquidchromatography(UHPLC) equippedwithaPDAdetectionsystemandanewanalyticalcolumn speciallydesignedforpolarcompounds.Chromatographic condi-tionswereoptimizedinordertoachieveincreasedsensitivityand highresolutiononthefreelowmolecularweightpolyphenols anal-ysisinadditiontoreducedanalysistime(within11min).Novel aspectsofthepresent studyincomparison withsimilarworks, bothintheenvironmentalorbiologicalfields,areconstitutedby extraction,forthefirsttime,ofseveralLMW-PPsinafoodmatrix byQuEChERS-dSPEtechniqueusingasetofsaltsintheportioning processhighlyselectiverelativelytothegenerallyused.
2. Experimental
2.1. Reagents,materialsandstandards
All chemicals and reagentswere of analytical quality grade. HPLC grade acetonitrile (MeCN) and ethyl acetate (EtAc) were obtainedfromLabScan(Dublin,Ireland),formicacid(FA)from Fis-cherScientific (Loughborough,UK), whereas sodiumhydroxide, methanol(MeOH)andethanol(EtOH)weresuppliedbyPanreac
(Barcelona,Spain)andglacialaceticacidbyFlukaBiochemicaAG (Buchs,Switzerland).
LMW-PP standards, gallic acid monohydrate (98%, purity), ferulicacid(98%),gentisic acid(98%),cinnamic acid(99%),( −)-epicatechin(≥95%),m-coumaricacid(99%),p-coumaricacid(99%), o-coumaricacid(99%)andrutin(>95%)andkaempferol(≥97%)and protocatechuicacid(98%),(+)-catechin(≥95%),syringicacid(98%), andtrans-resveratrol(99%)wereobtainedfromSigma–Aldrich(St. Louis, MO, USA), whereas quercetin(98%) was purchased from Riedel-deHaën(Seelze,Germany)andmyricetin(≥97%)fromAcros Organics (Geel, Belgium). Solvents were filtered with 0.22m membranefiltersusingaSolventFiltrationApparatus58061from Supelco(Bellefonte,PA,USA).UltrapurewaterfromaMilli-Q ultra-purewaterpurificationsystem(Millipore,Bedford,USA)wasused forpreparingtheLCmobilephaseandotheraqueoussolutions. Fil-tersof13mmwith0.22mPTFEmembranewereusedforfiltration ofthefinalextractsbeforeanalysis.
Bulk sorbents (50m particle size) for dSPE, including primary–secondary amine(PSA), trifunctionally-bondedC18 sil-icaandtheQuEChERSextraction/partitioningtubescontainingthe bufferedsaltsandtheclean-uptubes,wereobtainedfromWaters (Milford,MA,USA).
2.2. Preparationofstandardsolutions
Stock solutions of each individual standard (1000gmL−1) werepreparedinmethanol.Thesesolutionswerealiquotedin2mL vials,storedat−20◦Candprotectedfromlight;underthese condi-tionstheyarestableforatleast4months(asassessedbyUHPLC).A standardmulticomponentsolutionwaspreparedbydilutingeach primarystandardsolutionwiththechromatographicmobilephase andwasusedforspikingthetargetvegetablesamples,for prepar-ingcalibrationstandardsandforlineardynamicrangeassessment oftheUHPLC–PDAsystem.ThechoiceoftheLMW-PPswasmainly basedontheirimportanceand/orrelevanceforfoodquality, cov-eringthemajorclasses(flavonoidsandnon-flavonoids).
2.3. Vegetablesamples
Samplesofeightvegetables:carrot(DaucuscarotaL.),tomato (LycopersiconesculentumL.),broccoli(BrassicaoleraceaL.),onion (Allium cepaL.),garlic(AlliumsativumL.),greenandredpepper (CapsicumannuumL.),andbeetroot(Betavulgaris),werepurchased fromalocalmarketinFunchal,Portugal.Foreachvegetable sam-ple,approximately1kgwasrandomlysampledfromthemarket shelves,simulatingconsumershoppingbehavior.Vegetableswere washedintapwaterandallinedibleparts wereremoved man-uallyorusingasteelknife.Bruisedorwoundedvegetableswere discarded.Carrot,beetroot,garlicandonionswerepeeledand ana-lyzedonlyaspulp.Foreachindependentanalysis,atleast500gof vegetable,addedwith100mLMilli-Qwater,wereputina com-mercial juiceextractor (Instantpulp, 200W, Worten,Portugal), obtaininga fluid vegetableextract (FVE)which wasusedin all analyses.TheFVEwerethendividedintofractionsandstoredat −20◦CforlaterQuEChERSprocedure.Alldeterminationswere per-formedusingthreeindependentaliquotsofthesameFVEeachone analyzedintriplicate.
2.4. QuEChERSprocedureforextractionofLMW-PPs 2.4.1. Selectionofextractionsolventandbufferedsalts
InordertogetthehighestextractionefficiencyofLMW-PPs, differentpartitioning solvents namelymethanol(MeOH; 100%), water(H2O;100%),ethylacetate(EtAc;100%),acetonitrile(MeCN; 100%)andMeCN:EtAc(50:50,v/v),antwodifferentsetsofbuffered salts,setS1composedbysodiumacetate(1.5g) andanhydrous
156 C.L.Silvaetal./J.Chromatogr.A1260 (2012) 154–163
magnesium sulfate (6g); and set S2 composed by trisodium citrate dihydrate (1g), disodium hydrogencitrate sesquihydrate (0.5g), sodium chloride (1g), and anhydrous magnesium sul-fate (4g) were tested and compared. For clean-up procedure anhydrousMgSO4 (150mg),PSA(25mg) and C18 (25mg) were used in all assays. Carrot (Daucus carota L.) samples were selectedasmatrixforoptimizationQuEChERSextractionprocedure purposes.
2.4.2. Extractionandclean-upprocedure
Athoroughlyhomogenizedsubsample(10g)oftheselectedFVE wasweightedintoa50mLPTFEcentrifugetubeandadded10mL MeCN:EtAc(50:50,v/v) containing1%FA;thetubewasshaken vigorouslyfor2minwithvortexmixerensuringthatthesolvent interactedwellwiththeentiresample.Bufferedsalts,trisodium citrate dihydrate (1g), disodium hydrogencitrate sesquihydrate (0.5g), sodium chloride (1g) and anhydrous MgSO4 (4g) were addedintothehomogenizedmixtureandtheshakingstep was repeatedfor 1minfollowedbyacentrifugationat5000rpmfor 3min.Analiquot(1mL)fromtheupperpartoftheextract (ace-tonitrilephase)wastransferredintoa 2mLPTFE dSPEclean-up tubescontaining25mgofPSA(removesvariouspolarorganicacids, polarpigments,somesugarsand fattyacids),25mgofC18 sor-bent(removesnon-polar interfering substances likelipids) and 150mg MgSO4 andsubjected toclean-up bydSPE.Themixture wasshakenin a vortexand centrifuged for2minat 3000rpm. Then,700Laliquotoftheextractwereevaporatedunder nitro-gen flow to near dryness and the residue was taken up with 100Lofinitialmobilephase(0.1%FAinMilli-Qwaterand20% of MeOH). All the sample and standard extracts were filtered througha0.22mMilliporePTFEfiltermembranepriortoUPLC analysis.
2.5. UPLC–PDAconditions
TheseparationandquantificationofLMW-PPs[48,49]was per-formedonaWatersUltraPressureLiquidChromatographicAcquity system(UPLC,AcquityH-Class)(Milford,MA,USA)combinedwith aWatersAcquityquaternarysolventmanager(QSM),anAcquity sample manager (SM), a column heater,a 2996 PDA detector, andadegassingsystem.Thewholeconfigurationwasdrivenby Empowersoftwarev2.0fromWatersCorporation.Ahighstrength silicaAcquityHSST3analyticalcolumn(2.1mm×100mm,1.8m particlesize)packedwithatrifunctionalC18alkylphase,keptat 40◦C,wasusedfortheseparationofLMW-PPs.Abinarymobile phase with a gradient program was used, combining solvent A (0.1% FA) and solvent B (MeOH) as follows: 80% A (0min); 80–70% A (0.50min); 68% A (1min); 20% A (8min); and 80% A (11min). The flow ratewas 250Lmin−1, gave a maximum backpressureof6000psi,whichiswithinthecapabilitiesofthe UPLC.Theinjectionvolume,ofboth thestandardsolutions and sample extracts,was 2L.After each injectionthe needle was rinsedinitiallywith400Lof washwater:methanolsolutionat 90:10andafterwith200Lofwater:methanolsolutionat10:90. Thesamples werekeptat 6◦C during theanalysis.The system wasre-equilibratedwiththeinitialcompositionfor3min,prior tonext injection. The target compounds eluted within 11min, while the additional equilibration at the initial mobile phase compositionresultedin atotalanalysistimeof 14min.TheUV detectionwavelengthwassettothemaximumofabsorbancefor the compounds of interest. The identification of the LMW-PPs ininvestigated vegetableswasbased onthecomparison of the retention times(tR)and PDAspectra oftheirpeaks in samples with those previously obtained by the injection of pure stan-dards.
2.6. Analyticalmethodvalidation
Validation of the QuEChERS-dSPE/UHPLC–PDAprocedure for thequantificationoffreeLMW-PPsindietaryvegetablesinvolved the assessment of the selectivity, linear dynamic range (LRD), instrumentLODsandmethodLOQs,intra-day(RSDr)andinter-day (RSDR)precision,trueness(expressedasrecoverypercentage)and matrixeffects.
The selectivity of the method was assessed by the absence ofinterferingpeaksattheretentiontimeoftargetLMW-PPs.To testthelineardynamicrangeofthemethodFVEsamplesofeach vegetable,werespikedatsixconcentrationlevels,obtainedby suc-cessivedilutionsofthestockstandardsolution.Calibrationscurves wereconstructedbyplottingtheLMW-PPssignalobtainedagainst theconcentrationofLMW-PPs.Solvent-basedstandardsolutions werealsoanalyzedtoassessthematrixeffects.Leastsquareslinear regressionanalysiswasusedtointerpolatethedatapairsobtained fromeachcalibrationsolution.Thesloperatios(slopematrix/slope solvent) wereusedasameantoevaluatethematrix effectson theextractionefficiency.Avalueabout1(100%similarity) indi-cates thatmatrixdoesnotsignificantlyinfluence theextraction efficiency.TheLOD(thelowestanalyteconcentrationthatproduces aresponsedetectableabovethenoiselevelofthesystem)andthe LOQ(thelowestlevelofanalytethatcanbeaccuratelyand pre-ciselymeasured)werecalculatedforeachanalyteineachvegetable onthebasisoftheconcentrationthatproducedasignal-to-noise (S/N)ratioequalorhigherthan3and10,respectively.The intra-andinter-dayprecisionoftheassaywasevaluatedbypreparing andanalyzingFVEsamplesofeachvegetablespikedwithknown amountsofLMW-PPsattwodifferentlevelsofconcentrations(low level–lowestlevelofcalibrationcurve;highlevel–highestlevelof calibrationcurve),intherangeofexpectedconcentrations, respec-tively, sixtimesinthesamedayand fivetimesina week,and expressedasrepeatability(RSDr%)andreproducibility(RSDR%), respectively. Trueness,expressedasrecovery,wascalculatedas percentratiobetweentheconcentrationestimatedfromthe cal-ibrationcurveandthespikedconcentration.Alltheexperiments weredoneintriplicate.
2.7. Statistics
StatisticalanalysisoftheresultswascarriedoutusingSPSSfor Windows,version19.0(SPSS,Inc.,Chicago,IL)toapplyone-way ANOVAfollowedbyBonferronitest.Trendswereconsidered sta-tisticallysignificantwhenmeansofcompared setsdifferedatp values<0.05.
3. Resultsanddiscussion
3.1. Selectionofextraction/partitioningsolventsandbuffered salts
Togetthehighestextractionefficiencytowardsthetarget LMW-PPs,differentsolventsand mixtures,H2O(100%),MeOH (100%), EtAc(100%),MeCN(100%),MeCN:EtAc(50:50,v/v),wereassayed andevaluatedbasedontheintensityoftheresponseobserved.A carrotFVEwasselectedasmatrixfortheoptimizationofQuEChERS procedureaccordingtodescribedinSection2.4.2.
Fromthecomparison ofthegraphicpresentedin Fig.1,and onbasisoftheaveragetotaltargetLMW-PPs, itwasfoundthat theMeCN:EtAc(50:50,v/v) extraction/partitioningmixturewas themostefficientsolventfor theextractionoftargetLMW-PPs fromcarrotsamples.Conversely,waterwasfoundthesolventwith thelowestextractionefficiencyforthetargetcompounds.Thus, MeCN:AcEtwasselectedinallexperimentsfortheextractionof
Fig.1. Comparisonofaveragepeakarearesponseobtainedwithdifferentextractive
solvents:MeOH,methanol;H2O,water;MeCN:EtAc,acetonitrileandethylacetate
solution(50:50,v/v);MeCN,acetonitrile;EtAc,ethylacetate.
LMW-PPs.MeCNiseasilyandeffectivelyseparatedfromFVEby addingpolarsubstancesincludingbufferedsalts,namelyNaCland MgSO4.
Furthermore, in order to improve the LMW-PPs extraction efficiency, two different sets of bufferedsalts were compared. BesidestheconcentrationofsometargetLMW-PPs,suchas (+)-catechin,(−)-epicatechin,seringaldehyde,ferulicacid,m-coumaric acid, trans-resveratrol,o-coumaric acid, cinnamic acid, wasnot significantlyaffectedbythenatureof bufferedsalts,asshowed in Fig.2. The averagecontent of thetarget LMW-PPs was sig-nificantlyhighusing theS2set,composedbytrisodium citrate dehydrate,disodiumhydrogencitratesesquihydrate,sodium chlo-rideandanhydrousMgSO4.Thus,S2setwasusedinallfurther experiments.
Inallassays,150mgMgSO4,25mgPSAand25mgC18wereused intheclean-upstep.TheuseofC18associatedtoPSAandMgSO4to removelipidsisofcrucialimportancetomaximizethesensitivityof LMW-PPsandtominimizethepresenceofinterferingcompounds intheextract.
3.2. Methodvalidation
To demonstrate the feasibility of the present approach for thedetermination ofLMW-PPsandtotestitspracticability,the performanceofthemethodwasfullyevaluatedintermsof selec-tivity,linearity,LODs,LOQs,intra/inter-dayprecision,trueness,and matrixeffects.Table1describestheLMW-PPsidentification(coded
Table1
Peaknumber,retentiontime(tR)andmaximumwavelengthforthe15investigated
LMW-PPs.
No. tRa(min) maxb(nm) LMW-PPsc
1 3.027 259 Protocatechuicacid 2 3.345 278 (+)-Catechin 3 3.961 327 Gentisicacid 4 4.292 278 (–)-Epicatechin 5 4.518 260 Vanillicacid 6 4.718 274 Syringicacid 7 5.208 308 Seringaldehyde 8 5.581 309 p-Coumaricacid 9 5.807 322 Ferulicacid 10 6.148 277 m-Coumaricacid 11 6.406 356 Rutin 12 6.629 304 trans-Resveratrol 13 6.813 275 o-Coumaricacid 14 8.126 317 Cinnamicacid 15 8.997 366 Kaempferol at
R,averageretentiontimes(RSD<2%).
b
max,maximumabsorbancevaluesobtainedinPDAsystemdetection.
c LMW-PPs,lowmolecularweightpolyphenols.
asanumber),themaximumwavelengthforeachanalyte,andthe retentiontimeobtainedwiththeinstrumentalconditionsused.
The selectivity was assessed by the analysis of three blank solutions (elution solution) extracted by theoptimized QuECh-ERSmethod.Nointerferencewasdetectedattheanalyteretention time (Fig. 3a). The linear dynamic range (LDR) of the method wasestablished onstandardsolutions and spiked FVEsamples of each vegetable, prepared and analyzed using the described extractionprocedure(QuEChERS-dSPE/UHPLC–PDA)intherange of 0.1–25gmL−1 (sixcalibrators evenlydistributed, five repli-cates).Alongwitheachcalibrationcurve,a zerosample (FVEof eachvegetablewithoutspiking)wasalsoanalyzed.
Calibrationcurveswereobtainedbyplottingtheaverage peak-areaofeachanalyteagainstanalyteconcentration.TheUHPLC–PDA systemgavealinearresponseallthroughouttherespective inves-tigated range of concentrations. Least-squares linear regression analysisofthedataprovidedexcellentcorrelationcoefficient val-uesforallLMW-PPsinvestigated(R2>0.954),andthecalibrators’ residualswereconsideredadequate,beingwithin±10%ofthe nom-inalconcentrationforalllevels.
Toevaluatetheimpactofthematrixontheanalytes,theslopes obtainedinthecalibrationwithmatrix-matchedstandardswere comparedwiththoseobtainedwithsolvent-basedstandards, cal-culating matrix/solvent slope ratios for each of the 15 studied LMW-PPsinallFVEmatrices.Table2summarizestheresults.
Fig.2. Comparisonofaveragepeakarearesponseobtainedbyusingtwodifferentsetsofbufferedsaltsontheextraction/partitioningmechanism:S1– CH3COONa(1.5g),
158 C.L.Silvaetal./J.Chromatogr.A1260 (2012) 154–163
Fig.3. RepresentativeUHPLC–PDAchromatogramsofMeCN:EtAcextractsofthe(a)standardmixtureof15LMW-PPsand(b)blanksolutionforselectivityevaluation,and
fromtheinvestigatedvegetables.Forpeakassignment(seeTable1).
Weconsiderthat,ifthevaluewasintherangeof0.85–1.1,the matrixeffectcouldbeignored;ifthevaluewaslowerthan0.85,it couldshowmatrixsuppressioneffect;ifthevaluewashigherthan 1.1,itcouldshowmatrixenhancement.Forsomeofthe investi-gatedanalytesthematrixeffectvaluesareintothisrange.Asitcan beseeninTable2,thesignalisaffectedforthematrixinmostcases: softmatrixeffectwasobservedfor75%ofLMW-PPswhile25%of LMW-PPSshowedstrongmatrixeffect(equalorupto0.15).
Basedontheobtainedresults,itcanbeobservedthatthematrix effectwasnegligibleforsometargetLMW-PPsinsomematrices, andinthiscase,solventbasedstandardscouldbeusedforaccurate quantificationofthetargetanalytes.HoweverforotherLMW-PPs astrongmatrixeffectofthematrixwasobserved,andinthiscase, matrix-matchedcalibrationsolutionsshouldbeusedforLMW-PPs quantificationpurposes.Therefore,inthisstudymatrix-matched standardswereusedascalibrationmode,toquantifythe15 LMW-PPsinalltarget dietaryvegetables,in ordertocompensate the errorsassociatedwithmatrixinducedsuppressionorenhancement effects.Thus,wecandeducethatthematrixeffectdependsstrongly onthenatureofmatrix,onsomespecificcompounds,andalsoon thechemicalnatureoftheanalytes.
Based on calibration curves, method LODs and LOQs were estimated from the theoretical calculations of the lowest con-centration level, obtaining a S/N ratio equal or higher than 3 and10,respectively,thetheoreticalconcentrationsofwhichwas thenexperimentallytestedbytheinjectionofthecorresponding concentration,followedbyconfirmationoftheexpected/required response.TheQuEChERS-dSPE/UHLPC–PDAmethodologygavein generalverylowLODs(Table3),rangingbetween0.005for trans-resveratrolincarrotFVEand0.62gmL−1forsyringicacidingarlic FVE),whileLOQsrangedfrom0.016gmL−1fortrans-resveratrol to0.87gmL−1 for(+)-catechin,both obtainedwithcarrotFVE, showingthatthemethodisenoughsensitiveforthedetermination ofverylowlevelsofLMW-PPsinvegetables.
Theprecisionwasmeasuredthroughinter-day (reproducibil-ity,RSDR)and intra-day(repeatability,RSDr)studies,calculated usingthemeasurementofrelativepeakareaofeachLMW-PPin thematrix(Table4).Intra-dayprecisionandtrueness(expressed asthe relative error(RE); RE=((spiked concentration− nominal concentration)/nominalconcentration× 100))wereevaluatedby analyzing in the same day 6 replicates of carrot samples spikedwithpolyphenolsattwoconcentrationlevels(lowestand
highest levels ofcalibrationcurve). The obtainedRSDswere in generallowerthan2.9%forallcompoundsatbothtested concen-trations,presentingameanrelativeerrorwithina±5.1%interval. Inter-dayprecisionandtruenesswereevaluatedattwo concen-trationswithina5-day period.ThecalculatedRSDswerelower than3.0%forallcompoundsatallconcentrationlevels,while true-nesswaswithina±16%interval.Table4summarizestheintra-and inter-dayprecisionandtruenessdata.
In ordertoevaluatethetruenessoftheanalytical method,a recoverystudy oftheMeCN:AcEt extractsfromFVEsamplesof eachvegetablewascarriedoutbyaddingLMW-PPsstandardswith known amountsof each LMW-PP (Table 4).Satisfactoryresults werefoundformostofLMW-PPswithrecoveryvaluesrangingfrom 78.4%(trans-resveratrol;tomato)to99.9%(cinnamicacid;carrot), respectively.The%RSDoftheaveragerecoveryislessthan2.9%. Approximately71%(7LMW-PPs)oftheanalyzedLMW-PPsyielded recoveriesof90–99.9%,28%yieldedrecoveriesof80–89%,and0.4% yieldedrecoveriesoflowerthan80%.
IncomparisonwithothermethodstoquantifytheLMW-PPs, theQuEChERSmethodoffersbetterselectivitythantheSPEOasis HLB technique[22] andbettersensitivity tosomeanalytesthan obtainedbyMEPSC8[24].However,regardingthefiguresofmerit ofthedifferenttechniques,similarresultswereobtainedforLODs, LOQs,truenessandprecision.
3.3. ApplicationofQuEChERS-dSPE/UHPLC–PDAfortheanalysis ofLMW-PPsonvegetablesamples
Aftervalidation,thehereindescribedprocedurewasappliedto a setofselected commonlyconsumedvegetables.In particular, carrot,tomato,broccoli,greenandredpepper,onion,garlicand beetroot,purchasedfromalocalmarketandsimulatingconsumer shoppingbehavior,wereincludedinthisstudy.Theresulting chro-matograms ofinvestigated vegetables,obtainedby UHPLC–PDA analysis,areshowninFig.3.Goodpeakshapeandresolutionwere achievedfor allthecompoundswithlowinterferencefromthe vegetablematrix.Thechromatogramsforthedifferenttested veg-etablesshowedquitedifferentprofiles(Fig.3)andtheircomplexity increasesordecreasesaccordingtothewavelength.Themaximum absorbancevalueofeachLMW-PPlistedinTable1wasusedfor quantificationpurposes.
Fig.4.ConcentrationoftheLMW-PPsininvestigatedvegetablesasheatmaprepresentation.
Onlytheareasofthecompoundsthatwereclearlyrecognized bytheirPDAspectrumwereextractedtoeliminatefalsepositives. Ascanbeseen,theseparationofthetargetLMW-PPsisveryfast, beingachievedwithinonly11min.
ThenumberofLMW-PPsdetectedatconcentrationabovethe LOQvariedinthedifferentmatrices:itwaspossibletoquantify11
LMW-PPsinbroccoli,10ingreenpepper,9ingarlicandtomato, 8incarrotandbeetroot,7inredpepperandonion.Furthermore someLMW-PPsweredetectedinthesesamplesatconcentrations lowerthantheLOQand couldnotbequantified(Table1S).The resultsoftheanalysisareshownintheformofheatmapinwhich theconcentrationvaluesarewithinagrayscale(Fig.4).
Fig.5. (a)TotalcontentofLMW-PPsintheinvestigatedvegetablesobtainedusingtheoptimizedconditions(extractionsolvent:MeCN:EtAc(50:50)inthepresenceofbuffered
salts:Na3C6H5O7,C6H8Na2O8,NaClandMgSO4;clean-upwith:150mgMgSO4,25mgPSAand25mgC18);(b)distributionoftheconcentrationprofile(Conc.)oftheassayed
160 C.L.Silvaetal./J.Chromatogr.A1260 (2012) 154–163 Table 2 Linear dynamic range (LDR), matrix effects (ME) and determination coefficients (R 2) obtained for the 15 investigated LMW-PPs in studied vegetables by the newly QuEChERS-d SPE/UHPLC–PDA methodology. No. LDR a ( g mL − 1) Matrix: Solvent Carrot Tomato Broccoli Red pepper Green pepper Onion Garlic Beetroot Equation R 2 b ME c( ) R 2 ME ( ) R 2 ME ( ) R 2 ME ( ) R 2 ME ( ) R 2 ME ( ) R 2 ME ( ) R 2 ME ( ) R 2 1 1–25 2.30 x − 0.021 d 0.992 0.92 (0.07) 0.994 0.96 (0.04) 0.990 0.88 (0.12) 0.995 0.92 (0.08) 0.998 1.02 (− 0.02) 0.998 0.93 (0.07) 0.995 0.94 (0.06) 0.973 0.96 (00.4) 0.998 2 1–25 1.42 x + 1.22 0.989 0.74 (0.26) 0.954 0.87 (0.13) 0.980 1.03 (− 0.03) 1 0.98 (0.02) 0.977 0.92 (0.08) 0.999 0.90 (0.10) 0.984 0.92 (0.08) 0.990 0.95 (0.05) 0.986 3 1–25 1.94 x − 5.02 0.990 0.93 (0.07) 0.952 1.06 (− 0.06) 0.990 0.85 (0.15) 0.932 0.87 (0.13) 0.990 0.88 (− 0.12) 0.963 0.84 (0.16) 0.995 0.85 (0.15) 0.990 0.92 (0.08) 0.995 4 1–25 1.21 x + 4.61 0.992 0.86 (0.14) 0.932 0.77 (0.23) 0.990 1.04 (− 0.04) 0.990 0.74 (0.26) 0.997 0.79 (0.21) 0.971 0.89 (0.11) 0.980 0.87 (0.13) 0.985 1.08 (− 0.08) 0.997 5 1–25 6.14 x + 1.04 0.999 0.81 (0.19) 0.988 0.83 (0.17) 0.980 0.88 (0.12) 0.959 0.85 (0.15) 0.958 0.81 (0.19) 0.997 0.86 (0.14) 0.947 0.81 (0.19) 0.995 0.74 (0.26) 0.983 6 1–25 3.15 x + 3.52 0.997 0.82 (0.18) 0.991 0.88 (0.12) 0.980 0.86 (0.14) 0.986 0.93 (0.07) 0.999 0.92 (0.08) 0.997 0.93 (0.07) 0.989 0.89 (0.11) 0.975 0.90 (0.10) 0.996 7 0.5–20 3.06 x + 2.51 0.997 0.88 (0.12) 0.979 0.98 (0.02) 1 1.07 (− 0.07) 0.992 0.92 (0.08) 0.990 0.91 (0.09) 0.998 1.03 (− 0.03) 0.986 0.95 (0.05) 0.949 0.88 (0.12) 0.999 8 1–20 1.45 x − 5.27 0.994 0.77 (0.23) 0.998 0.85 (0.15) 0.999 1.13 (− 0.13) 0.991 0.77 (0.23) 0.990 1.08 (− 0.08) 0.998 1.06 (− 0.06) 0.972 0.97 (0.03) 0.980 0.93 (0.07) 0.989 9 0.5–15 9.50 x − 1.40 0.996 0.91 (0.09) 0.983 0.87 (0.13) 0.990 1.04 (− 0.04) 0.958 0.63 (0.37) 0.987 0.77 (0.23) 0.999 0.88 (0.12) 0.990 0.95 (0.05) 0.980 1.20 (− 0.20) 0.934 10 0.1–7 1.13 x + 7.43 0.998 0.95 (0.05) 0.984 0.84 (0.16) 0.995 0.83 (0.17) 0.999 0.80 (0.20) 0.996 0.91 (0.09) 0.999 0.86 (0.14) 0.990 0.97 (0.03) 0.999 0.83 (0.17) 0.999 11 1–25 1.74 x − 3.31 0.996 0.90 (0.10) 0.991 0.94 (0.06) 0.992 0.81 (0.19) 0.972 0.89 (0.11) 0.962 0.89 (0.11) 0.999 0.94 (0.06) 0.952 0.91 (0.09) 0.983 0.78 (0.22) 0.999 12 0.1–10 1.35 x − 1.29 0.998 0.92 (0.08) 0.970 0.90 (0.10) 1 0.94 (0.06) 0.990 0.77 (0.23) 0.996 0.82 (0.18) 0.999 0.89 (0.11) 0.988 0.88 (0.12) 0.961 0.81 (0.19) 0.999 13 0.1–10 1.17 x − 3.93 0.998 0.93 (0.07) 0.992 0.91 (0.09) 0.999 0.86 (0.14) 0.999 0.88 (0.12) 0.999 0.94 (0.06) 0.999 0.79 (0.21) 0.989 1.03 (− 0.03) 0.997 0.84 (0.16) 0.995 14 0.1–10 1.62 x + 1.45 0.999 0.80 (0.20) 0.987 0.84 (0.16) 0.996 0.88 (0.12) 0.934 0.85 (0.15) 0.999 0.85 (0.15) 0.999 0.73 (0.27) 0.988 1.21 (− 0.21) 0.998 0.90 (0.10) 0.998 15 1–20 7.23 x − 1.81 0.991 0.85 (0.15) 0.989 0.88 (0.12) 0.982 0.82 (0.18) 0.991 0.75 (0.25) 0.978 0.90 (0.10) 0.995 0.92 (0.08) 0.937 0.87 (0.13) 0.911 1.13 (− 0.13) 0.999 a LDR, Linear dynamic range. b R 2, Determination coefficient gives an estimating how well the experimental points fit a straight line. cME, Matrix effect are expressed as the ratio between the calibration curve slopes of standards addition method and solvent-based standards. d x , LMW-PP concentration. Table 3 LODs and LOQs of the 15 LMW-PP in different target vegetables. LMW-PP LOD a( g mL − 1) LOQ b( g mL − 1) Carrot Tomato Broccoli Red pepper Green pepper Onion Garlic Beetroot Carrot Tomato Broccoli Red pepper Green pepper Onion Garlic Beetroot Protocatechuic acid 0.013 0.18 0.43 0.12 0.13 0.21 0.102 0.086 0.021 0.61 1.42 0.041 0.043 0.69 0.034 0.029 (+)-Catechin 0.26 0.15 0.051 0.045 0.57 0.17 0.091 0.024 0.87 0.56 0.17 0.15 0.19 0.55 0.30 0.079 Gentisic acid 0.016 0.17 0.27 0.13 0.018 0.044 0.16 0.047 0.052 0.58 0.89 0.35 0.058 0.16 0.54 0.16 (− )-Epicatechin 0.065 0.15 0.036 0.042 0.025 0.022 0.049 0.12 0.22 0.08 0.091 0.13 0.082 0.073 0.16 0.40 Vanillic acid 0.076 0.026 0.039 0.016 0.018 0.062 0.024 0.015 0.025 0.087 0.095 0.17 0.055 0.28 0.079 0.054 Syringic acid 0.097 0.021 0.28 0.014 0.15 0.084 0.62 0.16 0.13 0.070 0.74 0.55 0.51 0.12 0.26 0.53 Seringaldehyde 0.014 0.029 0.079 0.028 0.039 0.066 0.071 0.083 0.046 0.69 0.21 0.084 0.13 0.32 0.14 0.28 p -Coumaric acid 0.025 0.017 0.013 0.071 0.016 0.14 0.055 0.014 0.083 0.057 0.093 0.25 0.08 0.45 0.10 0.047 Ferulic acid 0.014 0.081 0.080 0.018 0.017 0.023 0.012 0.075 0.048 0.27 0.26 0.060 0.040 0.08 0.02 0.15 m -Coumaric acid 0.0130 0.071 0.019 0.024 0.015 0.033 0.019 0.086 0.043 0.13 0.064 0.080 0.050 0.11 0.064 0.18 Rutin 0.010 0.073 0.044 0.023 0.011 0.042 0.024 0.039 0.34 0.12 0.14 0.076 0.052 0.14 0.081 0.13 trans -Resveratrol 0.005 0.017 0.010 0.006 0.024 0.019 0.010 0.015 0.016 0.06 0.032 0.021 0.080 0.16 0.032 0.049 o -Coumaric acid 0.009 0.038 0.017 0.025 0.023 0.066 0.054 0.019 0.018 0.18 0.055 0.083 0.078 0.12 0.18 0.065 Cinnamic acid 0.016 0.086 0.022 0.021 0.014 0.053 0.015 0.013 0.053 0.28 0.072 0.069 0.039 0.11 0.049 0.043 Kaempferol 0.023 0.017 0.018 0.058 0.045 0.027 0.026 0.058 0.076 0.08 0.09 0.19 0.14 0.088 0.081 0.19 a LOD, Limit of detection. b LOQ, Limit of quantification; values obtained from ordinary least-square regression data.
Table 4 Precision of QuEChERS-d SPE/UHPLC–PDA assays expressed as repeatability intra-day precision (RSD r , %) and reproducibility inter-day precision (RSD R , %), relative error (RE, %) and average trueness (%) obtained for the target LMW-PPs at two spiked levels (lowest and highest points of calibration curve) in different vegetables. LMW-PPs Spiked level ( g mL − 1) Precision RE % a Trueness (RSD%) RSD R (n = 30) RSD r (n = 6) RSD R (n = 30) RSD r (n = 6) Tomato Broccoli Red pepper Green pepper Carrot Onion Garlic Beetroot Protocatechuic acid 1 b 0.70–1.52 0.25–0.76 4.89 − 6.56 84.5 (1.4) 98.8 (2.5) 96.8 (0.9) 90.8 (0.8) 97.6 (0.2) 98.5 (1.2) 90.8 (0.8) 95.1 (0.6) 25 c 0.39–0.15 0.81–0.77 4.94 4.95 90.0 (0.8) 89.8 (0.2) 99.0 (0.8) 99.4 (0.4) 98.8 (0.4) 99.1 (0.6) 91.5 (0.3) 89.0 (0.5) (+)-Catechin 1 0.40–0.30 0.57–0.75 4.76 5.05 81.2 (1.6) 93.3 (1.4) 97.1 (1.0) 92.5 (1.1) 93.0 (0.5) 96.6 (1.1) 91.5 (1.2) 84.5 (1.3) 25 0.54–0.80 0.45–1.02 7.54 5.05 80.3 (0.9) 98.5 (1.1) 99.2 (1.1) 93.6 (1.4) 95.5 (0.7) 97.5 (0.9) 92.8 (1.3) 95.1 (1.6) Gentisic acid 1 0.30–0.40 0.53–0.80 6.48 4.74 90.2 (1.3) 96.6 (2.3) 87.4 (1.2) 92.3 (0.6) 93.0 (0.5) 82.2 (1.8) 92.4 (1.9) 92.5 (1.4) 25 0.57–0.77 0.68–0.70 − 10.13 3.36 98.2 (0.6) 97.5 (1.9) 90.8 (1.6) 98.5 (0.4) 90.2 (1.9) 83.1 (1.2) 99.8 (0.7) 96.8 (0.5) (− )-Epicatechin 1 1.20–1.25 1.00–1.05 − 8.37 − 8.75 84.2 (0.9) 82.2 (0.7) 86.4 (1.5) 93.3 (1.7) 92.6 (1.0) 90.2 (1.0) 90.4 (1.6) 90.5 (0.9) 25 1.25–1.30 1.00–1.99 5.16 0.31 80.4 (0.7) 83.1 (1.2) 90.5 (1.1) 97.1 (1.1) 91.2 (0.5) 91.8 (1.0) 98.5 (1.2) 92.6 (1.5) Vanillic acid 1 0.20–0.60 0.14–0.30 8.47 − 1.43 85.6 (1.2) 90.2 (1.3) 88.5 (0.9) 92.5 (0.9) 90.3 (0.4) 87.5 (0.4) 92.4 (1.7) 95.3 (0.4) 25 0.24–0.42 0.52–0.55 − 1.98 1.01 89.0 (0.4) 91.8 (0.8) 89.6 (0.1) 96.8 (0.7) 89.8 (0.1) 95.5 (0.5) 98.5 (0.7) 94.1 (1.6) Syringic acid 1 0.32–0.50 0.40–0.80 − 6.72 − 1.02 92.3 (1.1) 95.3 (1.1) 85.8 (1.3) 90.5 (1.1) 93.3 (0.4) 90.3 (0.3) 90.4 (1.1) 90.5 (1.4) 25 0.33–0.57 0.12–0.40 − 1.19 2.2 90.5 (0.4) 94.1 (1.6) 88.2 (0.9) 92.6 (0.5) 98.1 (0.3) 88.2 (0.5) 98.4 (0.3) 87.4 (0.6) Seringaldehyde 0.5 0.30–3.10 0.60–2.90 − 1.51 3.67 93.3 (1.2) 90.5 (1.9) 92.5 (1.3) 93.0 (0.9) 90.2 (2.9) 95.4 (1.4) 96.7 (1.8) 85.2 (1.4) 20 1.00–2.97 0.50–3.17 9.04 1.24 99.8 (0.5) 87.4 (0.5) 96.5 (1.1) 94.2 (1.1) 89.1 (0.8) 93.3 (0.6) 99.7 (0.9) 87.1 (1.2) p -Coumaric acid 1 0.30–1.00 0.12–0.53 6.75 − 7.68 95.5 (1.1) 85.2 (0.6) 88.3 (0.6) 90.8 (0.5) 97.3 (0.5) 84.2 (0.3) 85.2 (0.6) 88.3 (0.2) 20 0.18–0.69 0.20–1.18 − 3.94 3.01 98.5 (0.2) 87.1 (0.4) 89.4 (0.4) 91.5 (0.5) 99.2 (1.2) 80.4 (0.4) 87.1 (0.5) 89.4 (0.2) Ferulic acid 0.5 0.28–0.84 0.50–0.74 1.61 9.57 90.3 (1.3) 90.8 (1.8) 80.3 (0.3) 91.5 (0.6) 84.5 (0.7) 85.6 (0.8) 90.8 (0.8) 88.3 (1.4) 15 0.29–0.79 0.57–0.94 − 4.37 − 4.48 92.0 (0.5) 92.4 (0.7) 90.5 (0.4) 92.8 (0.3) 98.5 (0.9) 89.0 (0.5) 95.8 (0.5) 90.5 (0.2) m -Coumaric acid 0.1 0.25–1.25 0.17–0.82 − 0.62 − 4.42 87.5 (1.6) 95.5 (1.3) 90.3 (1.5) 92.4 (1.5) 96.6 (0.7) 92.3 (1.9) 96.6 (0.8) 87.4 (1.2) 7 0.22–1.20 0.36–1.30 − 5.63 1.26 92.4 (0.3) 98.5 (0.8) 90.4 (0.8) 99.8 (0.7) 99.7 (0.6) 90.5 (0.9) 92.4 (0.7) 95.9 (0.6) Rutin 1 0.34–0.80 0.57–1.22 5.98 5.44 85.2 (1.4) 84.4 (1.6) 89.1 (2.0) 90.4 (1.3) 91.3 (0.7) 89.2 (1.7) 91.5 (1.3) 94.7 (1.1) 25 0.50–0.77 0.28–0.55 3.3 3.22 90.4 (1.1) 87.3 (1.7) 89.4 (0.3) 85.0 (0.8) 90.2 (0.8) 85.7 (1.1) 86.7 (0.5) 88.1 (0.9) trans -Resveratrol 0.1 0.44–0.88 0.32–0.89 7.4 4.73 88.2 (0.9) 95.4 (0.6) 93.3 (0.5) 93.8 (1.6) 95.0 (0.3) 95.4 (1.7) 84.4 (0.2) 89.1 (1.4) 10 0.33–0.99 0.36–1.24 − 0.82 1.51 78.4 (0.3) 96.7 (2.7) 99.7 (0.4) 96.4 (1.7) 94.2 (0.6) 96.8 (0.8) 87.3 (1.8) 89.4 (0.7) o -Coumaric acid 0.1 0.27–1.70 0.45–0.98 5.41 2.14 85.6 (2.2) 91.4 (0.9) 87.6 (1.8) 97.5 (1.5) 97.8 (0.5) 96.4 (1.4) 96.7 (0.6) 89.1 (1.6) 10 0.35–1.30 0.05–0.70 − 5.35 6.69 90.8 (0.7) 92.5 (0.4) 88.7 (0.9) 98.1 (0.3) 99.8 (0.5) 97.1 (0.6) 97.5 (0.4) 89.4 (0.8) Cinnamic acid 0.1 0.90–1.30 0.63–0.84 − 7.35 − 2.41 88.4 (0.5) 92.6 (0.1) 96.4 (1.6) 98.5 (1.6) 98.9 (0.6) 98.4 (1.3) 89.7 (0.5) 99.2 (0.6) 10 0.74–0.90 0.50–1.07 − 2.31 1.05 85.6 (0.3) 97.5 (1.2) 97.4 (0.7) 99.8 (0.4) 99.9 (1.7) 98.5 (1.2) 99.8 (1.4) 98.5 (0.6) Kaempferol 1 0.30–0.90 0.38–0.62 6.91 5.90 87.5 (1.3) 89.6 (0.5) 91.4 (0.9) 95.8 (0.7) 96.1 (0.8) 99.1 (1.7) 91.4 (0.6) 87.6 (0.4) 20 0.31–0.78 0.10–0.50 4.54 6.66 85.7 (0.2) 97.6 (1.8) 95.6 (0.3) 96.8 (0.6) 95.4 (0.1) 99.5 (0.5) 92.5 (0.4) 98.7 (0.8) a RE – Relative error = [((spiked concentration − nominal concentration)/nominal concentration) × 100]. b Lowest concentration of calibration curve ( Table 2 ). cHighest concentration of calibration curve ( Table 2 ).
162 C.L.Silvaetal./J.Chromatogr.A1260 (2012) 154–163
Asshown in Fig.4 not all LMW-PPswere detected in each vegetable orthe LOQwas toolow to ensurethat theLMW-PP was quantified. The results concerning the quantitative deter-mination ofthe LMW-PPsfoundin theselected vegetablesare summarized in Table 1S. The total concentration of the target LMW-PPs (Fig. 5a) significantly differed between the investi-gatedvegetablesbeinghigher in thebroccolisamplesthan the other investigated vegetables (Table 1S). The total content of thetargetLMW-PPswasapproximately38-foldhigherfor broc-coli than for carrot, the vegetable with the lowest LMW-PPs content.
Kaempferoland(+)-catechinfollowedbyprotocatechuicacid were the major constituents of all identified LMW-PPs in the target vegetables. (+)-Catechin was detected and quantified in allthevegetablesunlikem-coumaricacidwhich wasnotfound inanyoftheinvestigatedsample.Syringicacidwasonlyfound in broccoli. Kaempferol was detected and quantified in broc-coli, garlic and beetroot, however, wasfound themajor target LMW-PPs in these vegetables. The content of rutin and (+)-catechindeterminedinbroccoliissignificantlyhigherthanthat foundin theother samples. In red pepper the majorLMW-PP isprotocatechuicacidwhereas(+)-catechinwasthemost abun-dant in tomato, green pepper, carrot, onion and beetroot. The main LMW-PP determined in garlic was kaempferol followed by (+)-catechin. The highest content of trans-resveratrol was foundin garlic,whereas carrotand redpepperexhibitedminor amounts.
4. Conclusions
Thispaperdescribesforthefirsttimeaquick,simpleand sen-sitiveanalyticalmethodbasedonQuEChERS-dSPE/UHPLC–PDAfor thesimultaneousdeterminationof15LMW-PPsincommondietary vegetables.
The extraction and clean-up procedures of the described methodareverysimpleandrequiredlittlesamplepreparationor pre-treatment,providingadequateclean-uptotheFVE.Moreover, gradientelutionbythemobilephaseacetonitrile–wateryieldsgood separationandresolutionandtheanalysistimerequiredforthe chromatographicdeterminationofthe15LMW-PPsisveryshort (around11minforachromatographicrun).
Satisfactoryvalidationparameters,suchaslinearity,recovery, precision and LODs and LOQs, were obtained. The effective-ness of different extraction/partitioning conditions was sys-tematically investigated, and the joint use of MeCN:EtAc in presenceoftrisodium citratedihydrate,disodiumhydrogen cit-rate sesquihydrate, NaCl and MgSO4 as buffered salts on the extraction/partitioning, and MgSO4, PSA and C18 as clean-up reagents, was recommended in our final method. For all tar-get LMW-PPs the sensitivity of the method was good enough to ensure reliable determination at levels commonly found in dietary vegetables. These data suggests that the analytical method represents an attractive, reliable and promising high-throughput approach for the quantification of LMW-PPs in a wide range of dietary vegetable samples, and theirapplication couldbesuccessfullyappliedtotheanalysisofarange ofthese health-relatedsecondarymetabolitesinfruitsandotherfood com-modities.
Acknowledgments
TheauthorsthankthesupportofPortugueseFoundationfor Sci-enceandTechnology(FCT)throughthePluriannualBaseFunding
(QUI-Madeira-674) andPortuguese NationalMassSpectrometry Network(REDE/1508/RNEM/2005).
AppendixA. Supplementarydata
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.chroma.2012.08.082.
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