Development, validation and evaluation of an analytical method forthe determination of monomeric and oligomeric procyanidins inapple extracts

ContentslistsavailableatScienceDirect

Journal of Chromatography A

j ou rn a l h om ep a 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

Development, validation and evaluation of an analytical method for the determination of monomeric and oligomeric procyanidins in apple extracts

Wendy J. Hollands

, Stefan Voorspoels

, Griet Jacobs

, Kjersti Aaby

, Ane Meisland

, Rocio Garcia-Villalba

, Francisco Tomas-Barberan

, Mariusz K. Piskula

,

Deborah Mawson

, Irena Vovk

, Paul W. Needs

, Paul A. Kroon

aInstituteofFoodResearch,NorwichResearchPark,Norwich,UK

bVITONV,FlemishInstituteforTechnologicalResearch,Boeretang200,2400Mol,Belgium

cNofima,NorwegianInstituteofFood,FisheriesandAquacultureResearch,Ås,Norway

dResearchGrouponQuality,SafetyandBioactivityofPlantFoods,CEBAS-CSIC,CampusdeEspinardo,Murcia,Spain

eInstituteofAnimalReproductionandFoodResearch,PolishAcademyofSciences,Olsztyn,Poland

fLGC,Fordham,Cambridge,UK

gNationalInstituteofChemistry,Ljubljana,Slovenia

a r t i c l e i n f o

Articlehistory:

Received28October2016

Receivedinrevisedform14March2017 Accepted16March2017

Availableonline18March2017

Keywords:

Flavanolsflavan-3-ols Tannins

Polyphenols Phenolics HPLC

Inter-laboratoryevaluation

a b s t r a c t

Thereisalackofdataforindividualoligomericprocyanidinsinapplesandappleextracts.Ouraimwasto develop,validateandevaluateananalyticalmethodfortheseparation,identificationandquantification ofmonomericandoligomericflavanolsinappleextracts.Toachievethis,wepreparedtwotypesof flavanolextractsfromfreeze-driedapples;onewasanepicatechin-richextractcontaining∼30%(w/w) monomeric(−)-epicatechinwhichalsocontainedoligomericprocyanidins(ExtractA),thesecondwasan oligomericprocyanidin-richextractdepletedofepicatechin(ExtractB).Theparametersconsideredfor methodoptimisationwereHPLCcolumnsandconditions,sampleheating,massofextractanddilution volumes.Theperformancecharacteristicsconsideredformethodvalidationincludedstandardlinearity, methodsensitivity,precisionandtrueness.Eightlaboratoriesparticipatedinthemethodevaluation.

ChromatographicseparationoftheanalyteswasbestachievedutilizingaHiliccolumnwithabinary mobilephaseconsistingofacidicacetonitrileandacidicaqueousmethanol.Thefinalmethodshowed linearityforepicatechinintherange5–100␮g/mLwithacorrelationco-efficient>0.999.Intra-dayand inter-dayprecisionoftheanalytesrangedfrom2to6%and2to13%respectively.Uptodp3,truenessof themethodwas>95%butdecreasedwithincreasingdp.WithinlaboratoryprecisionshowedRSDvalues

<5and10%formonomersandoligomers,respectively.Betweenlaboratoryprecisionwas4and15%

(ExtractA)and7and30%(ExtractB)formonomersandoligomers,respectively.Ananalyticalmethod fortheseparation,identificationandquantificationofprocyanidinsinanappleextractwasdeveloped, validatedandassessed.Theresultsoftheinter-laboratoryevaluationindicatethatthemethodisreliable andreproducible.

©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Flavanolsareasub-classofflavonoidscomposedofmonomeric

‘catechins’ (e.g. catechin, epicatechin, epigallocatechin) and

∗ Correspondingauthorat:InstituteofFoodResearch,NorwichResearchPark, ColneyLane,Norwich,NR47UA,UK.

E-mailaddress:authorspaul.kroon@ifr.ac.uk(W.J.Hollands).

1 Equalcontributionfirst.

oligomeric/polymericproanthocyanidinswhicharecomprisedof twoormorecatechinsubunits(Fig.1).Flavanolsarepotentially importantdietarycomponentsbecausecardiovascularhealthben- efitshavebeenreportedinnumeroushumaninterventiontrials afteringestionofflavanol-richfoodsandbeverages[1–4].Under- pinningtheestablishmentofacausaleffectforthehealthbenefits observed in thesetrials is thedevelopmentof validated meth- odsfortheaccurateandprecisequantificationofthemonomeric andoligomericflavanolspresentinthesefoods.Indeed,whether thebeneficialeffectsobservedinhumanscausedbysupplement- http://dx.doi.org/10.1016/j.chroma.2017.03.030

0021-9673/©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

Fig.1. Structuresofmonomericandoligomericflavan-3-ols.A,(−)-epicatechin(flavan-3-olmonomer);B,aprocyanidindimer(dp2);C,atetramericprocyanidin(dp4);D, (+)-catechin(monomer);E,(+)-catechingallate;F,(+)-epigallocatechin.ThedimericprocyanidinshownisprocyanidinB2(anepicatechindimerlinked8-4).

ingdietswithcocoaorapplepolyphenolextractsareduetothe monomericflavan-3-ols(epicatechin,catechin)ortheoligomeric procyanidinsisanongoingdebate.Ontheonehand,thedatasup- porting(−)-epicatechinas themajorcauseof improvementsin flow mediateddilatation (FMD) and blood pressureafter cocoa consumptionaremixed[5,6]whereastheresponsestococoathat containsbothepicatechinandprocyanidinoligomersisstronger andmoreconsistent[7].Theauthorsarenotawareofanystud- ies that have assessed the effects of a procyanidin-only (i.e.

monomer-free)dietaryinterventionsonFMDorbloodpressure.

Procyanidinoligomershavebeenshowntohavepotentbiologi- calactivityinvitro[8,9],buttheirbioavailabilityhasbeenshown tobeextremelylimitedwithonlydimersandoccasionallytrimers reportedtohavereachedhumanplasmainmeasurablebutvery lowconcentrations.ThereportbyGarcia-Conesaetal.[8]ispar- ticularlyinterestingbecausetheyshowthatwhereastreatmentof humanumbilical veinendothelialcells(HUVEC)withamixture ofprocyanidinoligomersofaveragesize4units(8.9␮M)caused significantchangesintheexpressionof>1000genes,thetreat- mentswith(−)-epicatechin(25␮M)ofprocyanidindp2caused theexpressionofonlyafewgenestobealteredsignificantlyand thesewereconsideredtolargelybeaccountedforbythefalsedis- coveryrate.Sincethereisevidencethatboththemonomericand oligomericflavan-3-olshavebiologicalactivity,andthecontribu- tionofeachtypeisnotclear,itisimportanttoquantifyindividual flavan-3-ololigomersinadditiontothemonomersinfoodsand extractsusedindietaryinterventions.

Anumberofapproaches havebeenusedforquantifyingand determining the structural nature of flavanols. Monomeric fla- vanolsareroutinelyquantifiedwithwidelyusedtechniquessuch asHPLCwithUV,fluorescence,massspectrometryorcoulometric arraydetection.Theanalysisofoligomericflavanolsarefarmore challenging fora numberof reasonsincludingthepotentialfor largemolecularweightrangeofoligomersthroughpolymersthat maybepresentinsinglesamplesandeacholigomerwithapar- ticulardegreeofpolymerisation(dp)beingcomprisedofmultiple isomericstructures[10].Further,theincreasinglypoorsensitivity ofmostdetectortypesasthedpoftheproanthocyanidinsincreases, anddifficultiesinseparatingtheindividualisomers,especiallyas thedpincreases,offeradditionalchallenges.Methodsthatseekto labeltheterminalcatechin unit ofeach proanthocyanidinprior tohydrolysingtheoligomers/polymersintosingleunitsaresuit- ablefortheaccuratequantificationoftotalflavanolsinasample andprovideanestimateoftheaveragedp[11,12].However,they do not provide information of theconcentrations of individual procyanidinoligomers.Morerecently,chromatographicmethods thatseektoseparatetheindividualdpfractionshavebeenestab- lished and come into wider use. For example, the USDA have publishedfoodcompositiontablesforprocyanidinsinfoods[13]

onthebasisofasilicacolumn-basedseparationofproanthocyani- dinsaqueousalcoholextracts[14].Morerecently,a methodfor theaccuratequantificationofindividualcocoaprocyanidinsupto dp10wasreported,andthiswasbasedonseparationoftheindi- vidualoligomersusingabondeddiolstationaryphase-basedHPLC

column.Importantly,accuratequantificationofprocyanidinsfrom dp2through10wasachievedbyisolatingeachoftheoligomers andusingtheseasindividualreferencestandards[15].TheRobbins etal.report[15]highlights animportantchallengeintheanal- ysisofoligomericprocyanidins,thatisthelackofcommercially availableanalytical referencestandards,withtheexceptionof a fewdimers(e.g.A2,B1andB2)andtrimers(e.g.C1).Thefactthat eachplantfoodcontainingproanthocyanidinswillcontaindiffer- enttypesof‘catechins’asoligomericunitsanddifferentratiosof isomericstructureswithinasingledpnecessitatestheisolationof proanthocyanidinoligomersforuseasreferencestandardsforeach plantfoodproduct,inordertoensurerelevantrelativeresponse factors(RRFs)areestablishedforthedifferentoligomerichomo- logues.OncesuchRRFsareestablishedandvalidated,epicatechin calibrationwouldsufficetoassesstheoligomericprocyanidincon- tent.

Therearemultiplereportsdescribing thecontentofflavanol monomers(−)-epicatechin,(+)-catechin andprocyanidindimers and occasionally trimers in apple extracts [16,17] and reports providing averagedpvalues for procyanidins fromapples [12].

However,apartfromtheUSDAdatabasewhichprovidevaluesfor monomers,dimers,trimers,4–6mers,7–10mersandpolymers[13], wearenotawareofanyreportsprovidingaccuratequantification ofindividualprocyanidinoligomersforapples/appleproducts.

Theobjectivesofthisstudywere:(i)todeveloparobustand reliableanalyticalmethodfortheextraction,separationandiden- tificationofprocyanidinsinappleextracts;(ii)todetermineRRF betweenprocyanidinoligomersandepicatechinforthepurposeof accuratequantificationusingprocyanidinsisolatedin-housefrom anappleextract;(iii)tovalidatetheobtainedmethodandestimate theassociatedmeasurementuncertainty;and(iv)toperforman inter-laboratoryassessmentexercisetodeterminereproducibility oftheestablishedmethodology.

2. Materialsandmethods

2.1. Chemicals/reagents

Epicatechin,dimethylsulfoxide (DMSO),andaceticacidwere purchasedfromSigma-Aldrich(Poole,UK).HPLCgrademethanol, acetonitrile and hexane were purchased from Fisher Scientific (Loughborough,UK).Analyticalstandardsdp2-10weresynthesised in-housefromanappleextractandtheprocedureisdescribedin Section2.3.

2.2. Productionoftheappleextracts

TheappleextractswereprovidedbyCoressenceLtd.Extracts were produced through supercritical fluid extractionof apples that had been freeze-dried and then processed to prepare i) an epicatechin-rich extract containing around 30% (w/w) of monomeric(−)-epicatechinwhilstretainingoligomericprocyani- dins(ExtractA)andii)anoligomericprocyanidinrichextractthat wasdepletedofepicatechin(ExtractB).

2.3. Isolationofindividualoligomersfromanappleextractby preparativeHPLC

Pre-purification of the apple extract was performed as fol- lows:MNPolyamide(28g) waspackedintoa Biotagecartridge (15×3.7cm;idsinglefitreservoir)toadepthof9cm.Appleextract (4g)wasdissolvedinmethanol(50mL)andmixedwithpolyamide (6g). Themixture was evaporated to dryness and loaded onto thepre-wettedpolyamidecolumn.Thecolumnwasconnectedto aGilsonpreparativesystemcomprisingtwo306pumps,an806 manometricmodule,an816bdynamicmixerandaUVdetector.The

columnwaselutedisocraticallyat45mL/minwithwater,acetoni- trile/water(30:70;v/v)andacetone/water(75:25;v/v).Fractions weremonitoredat290nm.Theacetone/waterfractionwasthen evaporated(50^◦C)underreducedpressure togivea mixtureof procyanidins.

Preparative HPLC was performed using an Agilent system (HP1260),equippedwithtwoinfinitypreparativepumps,adual loop autosampler, a diode array detector and a fraction col- lector. Samples were injected from high recovery vials (screw thread;5mL;Agilent)andfractionscollectedintotubes(14mL;

16×100mm). Both the injector and collector were cooled to 4^◦C. Apple extract (100–200mg) was dissolved in methanol (1mL)andloadedontoasemi-preparativecolumn(Luna HILIC 250×21.2mm;5␮m),whichwaselutedat10mL/min.Samples wereelutedwithacetonitrile(A)and97%methanoland3%water (B) as follows: 0–3min, isocratic 7% B; 3–12min, linear gradi- ent7–10% B;12–20min, linear gradient10–22% B; 20–70min, lineargradient22–65%B;70–80min,lineargradient65–100B;

80–85min;lineargradient100–7%B;85–90min,isocratic7%B.

Postcolumn,theeluent passedthroughafluorescencedetector usingwavelengths230nmforexcitationand321nmforemission anda diodearraydetectorusingwavelength280nm.Appropri- atefractionswerepooledandevaporatedat50^◦Cunderreduced pressure.

2.4. RemovaloflipidfractionfromNISTSRM2384

TheNISTreferencebakingchocolatewasdefattedpriortoanal- ysis.Removalofthelipidfractioninvolvedgrindingaportionofthe NISTchocolatebarintoapowderusingahouseholdcoffeegrinder.

Thechocolatepowderwasthenweighedintoapre-weighedcon- ical flask before the addition of hexane. Samples were mixed thoroughlyandthenallowedtosettle.Thehexanelayerwassub- sequentlydecanted.Theprocesswasrepeatedthreemoretimes, filteringandcombiningthehexanefractionseachtime.Residual hexanewasevaporatedfromthechocolatepowdertocomplete dryness in an air ovenat 60^◦C. At theend of the process,the weightof defattedchocolatewasrecorded.The combinedhex- aneextractswereevaporatedtocompletedryness.Theremaining fatwasweighed.Themassbalancebetweentheextractedfatand defattedchocolatewaswithintheexpecteduncertainty(lessthan 1.1%).Basedontheremovedfatfraction,referencevaluesforepi- catechinwereadjustedaccordingly.

2.5. Preparationofanalyticalstandards

Epicatechinpowderwasdriedinanairoven(60^◦C;2h)and thencooledatroomtemperatureinadesiccator.Thepowderwas weighed(0.04g)intoavolumetricflask(10mL)andfilledtovolume withDMSOtoyielda4mg/mLstocksolution.Priortoanalysisof theextracts,thestocksolutionofstandardwasfurtherdilutedwith 70%methanolovertherange5–100␮g/mLsuchthattheworking concentrationscontainthesamequantityofDMSO.

2.6. Methodoptimisation

Ourfinalizedmethodfortheextractionandchromatographic separation of monomeric and oligomeric procyanidins froman appleextractisdescribedfurtheroninSection2.7.Theparameters thatwereoptimisedaredescribedbelow.

2.6.1. AlternativeHPLCcolumnsandconditions

Apple extracts were analysed by HPLC (Agilent HP1100) equippedwitha quaternarypump,cooledautosampler,column ovenandphotodiodeandfluorescencedetectors.Thecolumnsand HPLCconditionstestedwere:

i)LunaSilicacolumn(Phenomenex;250×4.6mm;5␮m)anda mobilephaseconsistingofdichloromethane(A),methanol(B) 50%aceticacid(C).Sampleswereelutedwithanincreasinggra- dientof(B),0min,14%;30min,28.4%;45min,39.2%;50min, 86% ata flow rate of1mL/min.Fluorescence detectionwas achievedusingwavelengths276nmforexcitationand316nm foremission.

ii)Develosildiolcolumn(Phenomenex;250×2mm;5␮m)anda binarymobilephaseconsistingof98%acetonitrileand2%acetic acid(A) and95%methanol,3%waterand2%aceticacid(B).

Sampleswereelutedwithanincreasinggradientof(B),0min, 7%;3min,7%;15min,23%;70min,65%;85min,100%ataflow rateof0.5mL/min.Fluorescencedetectionwasachievedusing wavelengths230nmforexcitationand321nmforemission.

iii)Luna Hilic column (Phenomenex; 250×4.6mm; 5␮m) and HPLCconditionsasdescribedfor(ii)above.

iv)LunaHiliccolumn(Phenomenex150×2.0mm;3␮m)withthe HPLCconditionsdescribedinSection2.7.

2.6.2. Optimisationofextraction

Extractionspeed canbetemperature dependant.To testthe effectsofincreasingtemperatureonextractionefficiency,aliquots ofappleextract(40mg;n=3)wereextractedwith70%methanol atroomtemperatureandat60^◦C.

To assess whetherfiltration has an effect on the analytical process,anepicatechinstandardsolutionwaspreparedattwodif- ferentconcentrationsandappliedtotheHPLCcolumnwithand withoutfiltration.

2.6.3. Samplemassanddilution

To assess the effect of sample mass on monomeric and oligomericprocyanidinconc.,appleextract (20,40 and100mg;

n=3/mass) weredissolvedin 70% methanolsuchthat thefinal dilutionvolumeremainedthesame(100mL).

Toassess theeffects of thedilution procedure, two dilution methodswereinvestigated.MethodA;Appleextract(∼100mg;

n=3)wasweighedintoavolumetricflask(25mL)andfilledtovol- umewith70%methanol.Theextractwasthendiluteda further 2,4and8-fold.Thefinaldilutionvolumeswere25(originalstock solution),50,100and200mL.MethodB;Appleextract(∼100mg) wasweigheddirectlyinto25,50,100and200mLvolumetricflasks (n=3/conc.)andfilledtovolumewith70%methanol.Nofurther dilutionwasapplied.Forbothmethods,extractswereprocessedas describedinSection2.7andthensub-samplesofeachconcentra- tionofextractcentrifugedbeforeapplyingtoHPLC.

2.7. Optimizedmethodfortheanalysisoftheappleextract

The final in-house method developed for the extraction of monomericand oligomericprocyanidinsfromtheappleextract involvedtheweighingofasolidsample(∼100mg)intoavolumet- ricflask(50mL)beforepartiallyfillingwithpre-warmed(60^◦C) aqueousmethanol(70%).Thedissolutionwassonicatedinawater bath(60^◦C;10min)todispersethesamplebeforeincubatingfor 30minat60^◦C.Postincubation,flaskswerecooledandthenfilled tovolumewith70%aqueousmethanol.Sub-samplesofthedisso- lutionwerecentrifuged(2500g;5min)beforeapplyingtoHPLC.

Appleextract A(epicatechinrich extract)wasdiluteda further 10-foldtoavoidasaturationeffectofepicatechinontheHPLCflo- rescencedetectorandthereforeensureitremainedwithintheHPLC detectionlimits.Appleextractswerepreparedconcurrentlywitha certifiedreferencematerial(NISTSRM2384–bakingchocolate;Sec- tion2.4)withaknownepicatechinconc.asameasureofanalytical accuracy.

For chromatographic separation of the monomers and oligomers, extracts were analysed by HPLC (Agilent HP1100)

equipped with a quaternary pump, cooled autosampler, col- umnovenand photodiodeandfluorescencedetectors.Thefinal selectedcolumnwasaLunaHiliccolumn(150×2.0mm; 3␮m) (Phenomenex).Themobilephaseconsistedof98%acetonitrileand 2%aceticacid(A)and95%methanol,3%waterand2%aceticacid (B).Sampleswereelutedwithanincreasinggradientof(B):0min, 7%;3min,7%;15min,30%;40min,49%;40.1min,7%and45min 7%ataflowrateof0.350mL/min.Thetotalruntimewas45min, themid-peakRTofdp10being38min.Theinjectionvolumewas 2␮L.Thecolumntemperaturewasheldat35^◦C.Thefluorescence detectorwavelengthswere230nmforexcitationand321nmfor emission.Toensurethatallanalyteswerewithinthelinearrange ofthedetector,thephotomultipliertubegain (PMT)wassetat 9.Monomericcontentoftheextractswascalculatedrelativetoa response factorderived fromanauthentic epicatechinstandard curve over the range 0–100␮g/mL. Oligomeric procyanidins (dp2-10)werecalculatedagainsttheresponsefactorobtainedfor epicatechin and additional relative fluorescence response data determinedfromprocyanidinsisolatedin-house(Section2.3)for useasanalyticalstandards.

2.8. Methodvalidation

The performance characteristicsconsidered for validation of theoptimizedmethodwere:selectivity,linearity,workingrange, RRFs,limitofquantification(LOQ),precision,truenessandmethod uncertainty. A procyanidin rich appleextract was used for the experiments.

2.8.1. Selectivity

A high degree of selectivitywas achieved by using fluores- cencedetection withexcitationand emission wavelengthsthat arespecificforflavan-3-ols[18].Aschromatographicresolutionis insufficienttoseparateindividualcompoundswithineachdp-class, theresponseperdp-classwassummed.Nofurtherassessmentof specificitywasdone.

2.8.2. Linearityandworkingrange

Linearityoftheepicatechincalibrationwasassessedvisually andbymeansofalack-of-fittest[19].

Theworking rangewas definedas theinterval betweenthe upperandthelowerlevelsoftheanalytewithinthecalibration curve.

2.8.3. DeterminationofRRFs

Authentic reference standards for apple oligomeric pro- cyanidins are not commercially available. During the method developmentandvalidation,RRFsfordp2-10vsepicatechinwere established.To this extentthepuredp’s that wereisolated in- housewereused.RRFsweredeterminedintriplicateandattwo independent laboratories. The RRFs wereincorporated into the finalanalyticalmethodforquantificationofprocyanidinsandwas furthervalidatedthroughaninter-laboratoryevaluationexercise.

Calibrationcurvesforepicatechinandeacholigomer(dp2-10)were measuredovertheconc.range5–100␮g/mL(n=3/conc.)onthree differentdays.

2.8.4. Limitofquantification(LOQ)

Themethodwasappliedtosampleswithrelativelyhighepi- catechinandprocyanidinlevels,thereforepracticaltestsonLOQ werenotperformed.Whenthemethodcalibrationcouldsensibly beforcedthroughzero,LOQwasdeterminedbysystemperfor-

mance.Inothercases,theoreticalestimatesoftheLOQbasedon calibrationcurveswereobtainedusingthefollowingformula:

LOQ= 10

S (1)

where␴isthestandarddeviationoftheresponseandSistheslope ofthecalibrationcurve

2.8.5. Methodprecision

Todeterminetheintra-day(repeatability)andinter-day(inter- mediate)precisionofthemethodology,replicatesamplesofapple extract(n=3) wereanalysed three timeson thesameday and overthreeseparatedays.One-wayANOVAwasusedtocalculate repeatabilityandintermediateprecision.

ReproducibilityoftheRRF-determinationwasassessedsepa- rately.Tothisextent,theRRFsfortheindividualoligomerswere determinedattwo independentlocations,bydifferentanalysts, usingdifferentinstrumentation.VariationoftheRRFscanbeused forreproducibilityassessment.VariationwithintheRRFsofdp2-10 vsepicatechinaddstothemethoduncertaintyandwasconsidered inthefinaluncertaintyestimation.

Reproducibilityoftheentiremethodwasassessedinaninter- laboratoryexercise.Eight laboratoriesin eight countriesacross Europeparticipatedintheevaluationofthemethodforthequan- tification of procyanidins in an apple extract. Each laboratory receivedthe following: 1) the method protocol describing the preparationofthecalibrationstandards,appleextractsandcon- trolsample;therequiredHPLCconditionsandsamplesequence setup;examplechromatogramsandguidanceonpeakintegration procedure;guidanceoncalculationoftheprocyanidincontentof thetestmaterialsusingacommerciallyavailable(−)-epicatechin standardandRRFsfordp2–10asestablishedearlier2)anexcel calculationspreadsheetandreportingtemplateand3)twodiffer- entappleextractslabelledAandB,andacontrolsample(defatted chocolate).Eachparticipantconductedsixindependentmeasure- mentsof the two appleextracts over two different days (n=3 replicates/extract/day)andtwoindependentmeasurementsofthe controlsample(n=1replicate/day).

2.8.6. Methodtrueness

The determination of trueness (the closeness of agreement betweentheaveragevalueobtainedfromasetoftestresultsandan acceptedreference/“true”value)canonlybeestablishedbymeans ofacertifiedreferencematerial(CRM).However,since noCRM wasavailable,therecoveryof analytesin fortifiedsampleswas investigated.

Aspikingexperimentwassetupfortheestimationandevalua- tionoftherecovery.Theextractwasfortifiedwithprocyanidinsto obtainfortifiedsamplescontainingapproximately100%and200%

oftheexpectedcontent.Threereplicatesperspikinglevelwere preparedandanalysed,aswellasthreereplicatesofanunfortified sample.Themeananalyteconcentrationsobtainedforthespiked samples(in␮g/mg)werereferredtotheamountaddedtothesam- plesinordertofindanestimateoftheresponseoriginatedbythe analytesspikedinthesamples.Theexpectedvalueswereplotted againsttheconcentrationsthatwereactuallyfound.

2.8.7. Stabilityoftheextracts

Epicatechinanditsoligomersarecompoundswellknownto bestablein fruitjuices[20]and duringgastro-intestinaltransit [21].Therefore noextensivestability testingwasexecuteddur- ingmethodvalidation.Totestthestabilityoftheanalytesinthe appleextracts,asmallstabilitystudywasperformed.Sampleswere measuredat0handafter48,96and168h.

2.8.8. Methoduncertainty

The maximum expanded uncertainty (U) is based on the combineduncertaintyresultingfromthedifferentuncertaintycon- tributions(u)throughoutthemethod.InthiscaseUwasestimated takingintoaccountthedifferentcontributionsspecifiedinthefol- lowingexpression:

U=k∗

urep(RRF)²+u_il(RRF)²+ur2

n1 +uip2

n2 +u²_bias+u²_rec (2) Where:

U–expandeduncertainty;

k– coverage factor (k=2)resulting in a confidence level of approximately95%;

urep (RRF) – uncertainty resulting fromrepeatability of RRF withinlab,

u_il(RRF)–uncertaintyresultingfromvariationofRRFbetween labs,

ur–uncertaintyresultingfromrepeatability, u_ip–uncertaintyfromtheintermediateprecision, n₁–totalnumberofanalyses,

n₂–numberofdaysmeasured,

u_bias–uresultingfromthemeasurementbias, urec–uresultingfromtherecovery.

3. Resultsanddiscussion

3.1. Methodoptimisation

3.1.1. AlternativeHPLCcolumnsandconditions

Optimization of the chromatographic separation of the oligomerswas based ondegreeof polymerisation(i.e. molecu- larweight)andemployingseveralHPLCcolumnsandappropriate mobilephasetechniques.Analytepeakidentificationwasbased uponretentiontimematchwiththereferencestandards(dp2-10) isolatedin-housecombinedwithexcitationandemissionwave- lengths.

Initially,ourchromatographicseparationofprocyanidinsused anormalphasesilicacolumn.However,becauseofthecomplex- ityinthestructuraldiversityofoligomersintheappleextract,this methodwasnotsuitableforquantificationpurposes,particularly atthehigherdp.Thus,wemodifiedourchromatographicmethod touseadiolcolumnasthiswasreportedtogiveimproveddetec- tionofoligomersatthehigher dp,whilstavoidingfluorescence responsesuppressionissuesinherentintheuseofchlorinatedsol- vents.ThemethodwasadaptedfromthatofRobbinsetal.[15]and isdescribedinSection2.6.1.Whilstchromatographicresolutionof eachdppeakwasachieved,thestructuralisomerswithineachdp werenotapparent.ItwasexpectedthataHILICcolumnmightgive evenbetterresolutionthantheDiolcolumnwiththesameeluent mixture.Therefore,wecomparedtheDevelosildiolcolumnwith theLunaHILICcolumn,whichconfirmedthehypothesisofHILIC superiorityforthisapplication.TheLunaHiliccolumnbearscross- linkeddiolgroups,whichaddsbothfunctionalityandrobustnessto thecolumncomparedtotheDevelosildiolcolumn.Substructure, attributabletoamixtureofstructuralisomers,wasnowapparentin eachdppeakandresolutionofthehigherdp’swasalsoimproved.

However,adrawbackofthesecolumndimensionswasthelengthy 90minchromatographic runtime. Toincrease samplethrough- putwe investigatedanalternativeHILICcolumn,withmodified HPLCconditionstoreducetheruntimeto45min.Fig.2shows thedifferencesinseparationandresolutionofthemonomericand oligomericprocyanidinsbetweenthediolandtwoHILICcolumns.

Oneofthelimitationsofourfinalchromatographicmethodisthe lackofseparationofthemonomericcatechins((−)-epicatechinand (+)-catechin)intheappleextract.

Fig.2.Comparisonbetweencolumnsinthechromatographicseparationofmonomericandoligomericprocyanidinsinanappleextract.

Table1

Theeffectsofsamplemassonvariationinprocyanidinconc.inanappleextract.

%RSDpermassextract

Analyte 20mg 40mg 100mg

Monomer 13.9 1.5 0.6

dp2 14.5 4.6 0.7

dp3 24.9 5.7 1.4

dp4 9.4 3.9 1.2

dp5 36.3 5.3 1.6

dp6 36.2 5.8 1.9

dp7 43.1 5.2 0.7

Finaldilutionvolume=100mLpermassofextract;RSDbasedon3replicatesper massofextract.

3.1.2. Influenceofextractionatelevatedtemperatureand filtration

Themonomericandoligomericconc.oftheappleextractwas higherintheheatedsamples(60^◦C)comparedwiththenon-heated samples(17and13%;p=0.01and0.14;monomersandoligomers respectively)indicatingthatheatingappleextractsmayimprove theextractionefficiency.

Forbothconcentrationstested,filteringsamplesresultedinan increaseinepicatechinconc.(>5.5%).Sincesamplesofappleextract wereroutinelycentrifugedpriortofiltering,andwassufficientto achievesampleclarification,thefiltrationstepwasnotretained.

3.1.3. Samplemassanddilution

As expected, the largest sample mass (100mg) resulted in a lower co-efficientof variation betweenreplicatesin thepro- cyanidinconc.oftheappleextract(Table1).Whilstthevariation

betweenreplicateswasacceptableat40mgofextract,wechoseto incorporatethelargersamplemassintoourfinalmethodology.

Havingdeterminedanappropriatesamplemass,wenextinves- tigateddifferentdilutionvolumestodeterminetheoptimalconc.

of appleextractrequired for identifyingeachof theoligomeric peaks in a singlechromatographic run. Our initial approach to dilutetheappleextractwasmethodA(Section2.6.3).However, withthisapproachweobservedamarkedstep-wisereductionin oligomericprocyanidinconc.oftheextractasthefinaldilutionvol- umeincreased(Fig.3;panelA).Consequently,wetriedadifferent approachtodilutetheappleextracts(MethodB, directdissolu- tioninfinalvolumeinsteadofstep-wisedilution).Whilstwestill observedaninversecorrelationbetweenthedegreeofdilutionand oligomericprocyanidinconc.,theeffectsweremuchlesspromi- nent compared withmethod A(Fig.3; panel B).Theexpected procyanidincontentofthematerialusedfor theseexperiments was∼20%. On this basis, we calculated thatinjecting less than 0.8␮goligomericprocyanidinsontothechromatographiccolumn resultsinsignificantunderquantificationofthesecompoundsin theappleextract(Fig.4)usingourmethod.

Onelikelyexplanationfortheseobservationsisadsorptionof theprocyanidinsontothesurfaceoftheglassvolumetricflasks.

PolarInteractionsinflavanoladsorptionontosolidsurfaceshave beenreportedpreviously[22].Therefore, theinner surfacearea oftheflasksused,werecalculated.Asignificantcorrelationwas observedbetweentheconcentrationsandglasssurfacethesolu- tionhadbeenincontactwith.Cumulativesurfaceareawasusedfor thesubsequentlydilutedsolutions.Hereby,theslopeforthesub- sequentdilutionswashigher,confirmingthehypothesisofglass

Fig.3. %reductioninmonomericandoligomericprocyanidincontentofanappleextractbytwodilutionmethods.n=3replicatesperdilutionvolume.Oligomersarethe summationoftotalprocyanidins(dp2-10).

Fig.4.Oligomeric content(%)ofan appleextract bymass injectedontothe chromatographiccolumn.Hiliccolumn150×2mm;3␮minternaldiameter;2uL injectionvolume*Equivalentto100mgappleextractin50mL70%methanol.

walladsorption. WerepeatedmethodBsubstitutingglasswith polypropylenevolumetricflasksbuttheeffectsremainedthesame (datanotshown).Thiseffectcannotbeovercome.Itsimpactcan bereducedby usingflasksof thesamevolume throughoutthe procedure.

3.2. Methodvalidation

Theperformancecharacteristicsasspecifiedaboveweredeter- minedandthemethodwassuccessfullyvalidated.Resultsofthe variousexperimentsaredescribedbelow.

3.2.1. Linearityandworkingrange

A calibrationcurve wasconstructed for epicatechin by ran- dom injectionof standard solutions. The fluorescence detector responsetoepicatechinwasplotted againsta seriesofconcen-

Table2

Retentiontimesandrelativeresponsefactorsfordp2-10.

Analyte RT MeanofmeansRRF RSDRFF(%)

Epicatechin 2.1 1 4

dp2 4.8 0.587 4

dp3 12.0 0.442 5

dp4 15.9 0.265 12

dp5 18.7 0.238 10

dp6 21.0 0.172 9

dp7 23.6 0.148 9

dp8 26.5 0.113 19

dp9 29.5 0.080 34

dp10 32.4 0.062 4

n=3replicatesperanalyte.

trations(5–100␮g/mL)andlinearitydeterminedvisuallyandby meansofa lack-of-fit-test.Forcing thecalibrationthroughzero wasstatisticallysupportedasbislargerthanthestandarderror ofcorrelation(sey).ThecalculatedF-valuewassmallerthanthe criticalF-valueforallcongeners,indicatingcompliancewiththe linear model. To support the choicefor forcing the calibration throughzero,errorforbothapproacheswerecalculated.Byforc- ingthecurvethroughzero,erroratthelowerendwassignificantly reduced.

Thecalibrationcurveforepicatechinwaspreparedoverthree separatedaystoassesslinearityand acorrelationcoefficientof

>0.999achieved.

3.2.2. DeterminationofRRFs

RRFfordp2-10weredeterminedfromtheepicatechinresponse factorandindividualcalibrationcurvesfordp2-10overthesame rangeandonthreeseparatedays.TheRRFfordp2-10arelisted inTable 2.Becauseof thenatureoffluorescencedetection and itssensitivitytothecompositionoftheHPLCmobilephase sol- vents,theseresponsefactorsarespecificfortheestablishedmethod describedinSection2.7.TheprecisionofRRFdeterminationisfur- therdescribedinSection3.2.4.

Table3 Methodprecision.

Analyte Repeatability(intra-day) Intermediateprecision(inter-day)

Epicatechin 4% 2%

dp2 6% 2%

dp3 6% 3%

dp4 5% 2%

dp5 3% 4%

dp6 2% 7%

dp7 2% 8%

dp8 3% 10%

dp9 4% 13%

dp10 5% 13%

N=3replicatesperanalyte.

3.2.3. LOQ

AtheoreticalLOQwascalculatedat0.5␮g/mLbasedonsystem performanceandequation1.AlthoughlowerLOQsweretechni- callypossibleseeingthecalibrationthroughtheoriginwasallowed, therewasnoneedtodosoforthesamplesunderstudy.Therefore, theLOQwassetatthelowestpointofthecalibrationpointsusedto determinetheRRFs.Inpractice,thisresultedinthefollowingLOQs:

5␮g/mLforepicatechin&dp2-5,10␮g/mLfordp6,25␮g/mLfor dp7-9and50␮g/mLfordp10.

3.2.4. Methodprecision

Table3summarisestheprecisionoftheanalyticalmethodol- ogy.MethodprecisionisexpressedastheRSDbetweenreplicate measurements.Repeatabilityrangedfrom2to6%dependingonthe analyte.Intermediateprecisionwasgooduptodp7(rangingfrom 2to8%)andincreasingto10–13%forthehigherdp’s.Toassess theinter-dayprecisionatthehigher dp,participatinglaborato- riesoftheinter-laboratoryexercisewereinstructedtoperformthe analysesondifferentdays.

ReproducibilityofRRFdeterminationwasassessed.TheRRFfor theindividualoligomerswasdeterminedattwolocations,bydif- ferentanalysts,usingdifferentinstrumentation.TheRRFsforall analytesisgiveninTable4.ThecorrelationoftheRRFswiththe polymerchainlengthisvisualisedinFig.5.Variationwithinthe RRFsofdp2-10vsepicatechinaddstothemethoduncertaintyand wasconsideredinthefinaluncertaintyestimation.

3.2.5. Methodtrueness

Estimationoftruenesswasdeterminedbyfortifyinganapple extract with known amounts of procyanidins as previously described.Truenessisexpressedas%recoveryoftheanalytes.Up todp3thetruenesswas>95%.However,truenessofthesample decreasedasthedpincreased.

Themeananalyteconcentrationsobtainedforthespikedsam- ples(in␮g/mg)werereferredtotheamountaddedtothesamples ofappleextractsinordertofindanestimateoftheresponseorig- inatedbytheanalytesspikedinthesamples.Theexpectedvalues wereplottedagainsttheconcentrationsthatwereactuallyfound.

AnoverviewoftherecoveriesforallanalytesisgiveninTable5.

3.2.6. Methoduncertainty

Theexpandedmethoduncertaintyliesbetween12%and103%, dependingupontheanalyte.Uncertaintyincreaseswithincreasing degreeofpolymerisation.Thisisnotunexpectedasitisanindirect

Table5

Averagerecoveriesandtheirestimateduncertaintybasedonspikingexperiments.

rec,% urec,%

epicatechin 96% 7%

dp2 95% 8%

dp3 97% 6%

dp4 83% 7%

dp5 77% 7%

dp6 69% 11%

dp7 70% 9%

dp8 60% 11%

dp9 63% 15%

dp10 49% 34%

Table6

Estimatedexpandeduncertainty.

Analyte Uopt,% Umax,%

epicatechin 12% 19%

dp2 17% 41%

dp3 16% 48%

dp4 42% 50%

dp5 51% 54%

dp6 65% 70%

dp7 64% 69%

dp8 88% 94%

dp9 101% 122%

dp10 103% 148%

Table7

Stabilityassessment(mg/ganalyte).

Compound T0 48h 96h 168h ip DeltaT0-168h acceptable range(±) Epicatechin 12.9 12.4 12.4 12.4 4% −0.5 0.50

dp2 40.6 39.9 40.7 40.8 6% 0.2 2.43

dp3 45.1 45.4 45.9 45.7 6% 0.6 2.73

dp4 82.0 78.6 81.4 79.6 5% −2.4 4.02

dp5 77.1 72.7 71.8 74.0 3% −3.0 2.22

dp6 93.5 91.0 91.1 91.6 2% −1.9 1.84

dp7 78.6 81.9 77.7 73.3 2% −5.3 1.56

dp8 71.5 69.7 66.7 67.9 3% −3.6 2.07

dp9 61.6 61.5 64.7 64.4 4% 2.8 2.52

dp10 54.9 52.2 52.5 53.2 5% −1.7 2.66

methodthatmakesuseoftheRRFstocalculateresultsforhigher dp’s.AnoverviewofUforeachdpisgiveninTable6

3.2.7. Stabilityoftheextracts

ResultsofthestabilityexperimentsaresummarizedinTable7.

Notrendcouldbeobservedandtheextractswereconsideredstable withinthestudiedtimeframe.Itishoweveradvisabletocomplete theLC-measurementswithinoneweekfromthemomentofsample preparationtopreventdryingoftheextracts.

3.3. Inter-laboratoryvalidationoftheanalyticalmethod

Eightlaboratoriesreceivedthematerialsrequiredtoparticipate intheevaluationofthemethodforextractingandquantifyingpro- cyanidinsfromanappleextract.Alllaboratorieswereinstructedto reportallquantifiableanalytesuptodp10.Itwasexpectedthatall laboratorieswouldbeabletoreportprocyanidinsuptodp6or7 (extractA)anddp10(extractB)usingthemethodologydescribed.

Table4

RelativeResponseFactors(RRFs;mean-of-means)forthedifferentanalytesatthe2testlocations.

Epicatechin dp2 dp3 dp4 dp5 dp6 dp7 dp8 dp9 dp10

Lab1 1 0.489 0.347 0.295 0.244 0.177 0.154 0.103 0.058 0.038

Lab2 1 0.587 0.442 0.265 0.238 0.172 0.148 0.113 0.080 0.062

Fig.5. RRFcorrelationofdifferentpolymerchainlengthsdeterminedin2differentlaboratories.

Table8

Monomericandtotaloligomericprocyanidinconc.(mg/g)determinedbysevenlaboratories.

ExtractA ExtractB Control

Lab Monomers Oligomers Monomers Oligomers Monomers Oligomers

1 337.3 (3.4) 324.5 (2.5) 14.3 (2.7) 495.5 (2.0) 2.9 (2.7) 8.0 (2.6)

2 344.2 (0.9) 381.6 (2.4) 12.5 (3.0) 601.7 (1.0) 2.8 (0.9) 10.7 (10.0)

3 323.8 (2.1) 317.9 (1.8) 12.3 (4.9) 445.7 (6.3) 2.7 (9.0) 7.4 (5.6)

4 332.0 (13.9) 296.6 (12.2) 13.1 (14.8) 439.5 (9.7) 3.1 (1.4) 4.7 (17.6)

5 323.2 (2.3) 233.4 (2.7) 12.5 (3.0) 187.9 (4.8) 3.0 (0.7) 5.7 (6.9)

6 306.2 (0.1) 275.4 (0.1) 12.6 (0.3) 373.0 (2.2) 3.0 (0.6) 7.5 (1.3)

7 312.0 (0.8) 277.1 (0.7) 11.4 (1.6) 408.8 (5.4) 2.6 (1.9) 4.5 (12.1)

Mean 325.5 300.9 12.7 421.7 2.9 6.9

SD 13.5 46.8 0.9 126.4 0.2 2.2

%RSD 4.1 15.6 7.0 30.0 6.6 31.6

Mean(RSD)ofsixindependentmeasurementsoftwoappleextractsanalysedontwodifferentdays(i.e.n=3replicates/extract/day)andacontrolsample(n=1/day).

Alleightlaboratoriesreturnedresults.Sevenoftheeightlaborato- rieswereabletomeasureandquantifyalltheexpectedanalytes andreturnedfulldatasets.Onelaboratoryreturnedaverylimited dataset.Theinitialstepintheevaluationoftheresultswastoassess thequalityofthereturneddatabymeansofthecontrolsample.As aconsequenceofthisassessment,onelaboratorydidnotproduce datainaccordancewiththedesiredquality(faroutsideconfidence intervalofconsensusvalue)andwasthereforenotusedforfurther evaluation.Sevenfulldatasetswereretainedforevaluation.Results fromthesevenlaboratoriesarepresentedinTable8.Thereported dataarethemeanresultsofsixindependentmeasurementsover twodays(extractAandB)andoneindependentmeasurementper dayforthecontrolsample.Thevaluesfortheoligomericprocyani- dinsinTable8arethesummationofallthereportedindividual analytesforeachextract.

3.3.1. Within-laboratoryprecisionofthemethod

For monomericprocyanidins inter-day variance in measure- mentperformancewasexceptionallygood,withsixoftheseven participantsreporting%RSDvaluesbelow5%forbothextractsand controlsample.Inter-dayvariation inthemeasurementoftotal oligomericcontentforeachlaboratoryisalsogood,withmostlab- oratoriesreportingRSDbelow10%.Onanindividualanalytebasis, measurementprecisiondecreasedwiththehigherdp’swhichwas nottotallyunexpected(datanotshown).

3.3.2. Between-laboratoryprecisionofthemethod

For monomericprocyanidins, betweenlaboratoriesprecision ofthemethodwasverygood,withreportedRSDvaluesranging between4and7%dependingupontheextract(Table8).Similarly, precisionwasgood foroligomeric procyanidinquantificationin extractA. Forextract B,%RSDwassomewhathigher. Thiswas largelytheconsequenceofoneparticipant(numberfive)under- estimating total oligomer content quitesignificantly compared withtheothersix,despiteidentifyingandquantifyingallofthe expectedanalytes inthis material. Furtherscrutinyof thedata revealedthattheunder-estimationwasgreatertowardsthehigher endofthechromatogram(dp4–5upwards).

3.3.3. Inter-laboratoryperformance

The quantitative criteria for the evaluation of the inter- laboratoryperformance, wasdetermined usingthe z-score and calculatedusingthefollowingequation:

Z−SCORE= X−

wherexisthelaboratoryresult;␮istheaverageofallparticipants andisthestandarddeviationofallresults.Theclassificationused todescribe method performance is a z-score: ≤2=satisfactory;

<3=doubtful;>3=unsatisfactory.

Allcalculatedzscoreswerebelowtwo(Table9),fromwhich canbeconcludedthatresultsweresatisfactory.Closeinspectionof thez-scoresandtheirstatisticaldescriptors,andthusthelabs’per- formance,showthatbetweenlabbiasispredominantoverwithin labvariation.Averageandmedianz-scoresperlabarecloselyclus-

Table9

Z-scoresofallanalytesperparticipantforbothtestsamples.

Lab 1 2 3 4 5 6 7

ExtractA epicatechin −0.10 −1.46 0.86 0.47 −0.14 −1.00 1.37

dp2 −1.19 −1.10 0.38 0.63 0.52 −0.68 1.45

dp3 −1.47 −0.53 0.51 −0.24 0.81 −0.57 1.50

dp4 −1.50 −0.31 0.63 −0.29 −0.24 −0.05 1.76

dp5 −1.44 −0.05 0.67 −0.83 −0.27 0.31 1.62

avg −1.14 −0.50 0.55 −0.18 0.21 −0.25 1.58

med −1.44 −0.42 0.57 −0.26 0.14 −0.31 1.56

min −1.50 −1.46 0.38 −0.83 −0.27 −1.00 1.37

max −0.10 −0.05 0.86 0.63 0.81 0.31 1.76

ExtractB epicatechin −0.19 −0.13 1.85 0.50 −0.40 −1.45 −0.18

dp2 −1.62 −0.63 0.08 0.90 0.72 −0.60 1.14

dp3 −1.43 −0.97 0.65 0.44 0.41 −0.50 1.40

dp4 −1.59 −0.75 0.64 0.42 0.00 −0.23 1.51

dp5 −1.92 −0.52 0.55 0.63 0.15 −0.03 1.15

dp6 −1.96 −0.30 0.41 0.49 −0.02 0.09 1.29

dp7 −1.92 −0.13 0.46 −0.11 0.01 0.27 1.41

dp8 −1.87 −0.19 0.49 −0.34 0.18 0.32 1.41

dp9 −1.86 −0.38 0.59 −0.17 0.26 0.18 1.38

dp10 −1.69 −0.21 0.75 −0.80 0.27 0.39 1.29

avg −1.61 −0.42 0.65 0.20 0.16 −0.16 1.18

med −1.78 −0.34 0.57 0.43 0.16 0.03 1.33

min −1.96 −0.97 0.08 −0.80 −0.40 −1.45 −0.18

max −0.19 −0.13 1.85 0.90 0.72 0.39 1.51

teredtogether,whichresultsincertainlabsreportingonthehigh endforallanalytes,whileothersaremoreboundtobeonthelower endforallanalytes.Thisbiashoweveriswithinexpectationsasno z-scoreisbiggerthantwoforthisdataset.Wecanthusconclude thatthemethodperformanceisundercontrol.

3.4. Methodperformancecomparedtostate-of-the-art

Themethodpreviouslydeveloped byRobbinset al.[15] has proventobeveryuseful,robustandhasbeenappliedtoquantify cocoaprocyanidinsinanumberofpublishedstudies.Neverthe- less the currentlydeveloped and appliedmethod proves tobe superiorincertainaspects.Thismethodissignificantlyfasterwith thefinaldetectableoligomerelutingaround38mincomparedto 56minfortheRobbinsmethod(a30%improvement).Inaddition, whereasRobbinsetal.[15]havevalidatedfortotalcontent(sumof monomersandprocyanidins),thepresentmethodprovidesperfor- mancecharacteristicsforindividualanalytes.Thisisalsothecase forthetotalrecovery,whereRobbinsetal.[15]reportedaround 93%fortheentiretestmatrix,whereasherewehaveestimatedan in-depthrecoveryofeachindividualoligomer.Further,ourrecov- eryestimateswereaveragedoverdifferentspikinglevelswhich canbeconsideredamoreconservativeapproach.Nevertheless,re- calculationoftherecoveryaswasreportedbyRobbinsetal.[15](i.e.

1pointspikevsnon-spikedmatrix),showsatotalrecoveryof95%

forthesumofanalytes,whichisthesameasreportedpreviously byRobbinsetal.Regardingprecisionandrecovery,bothmethods appeartobecomparable.However,itisimportanttonotethatthe resultsofthecurrentstudyhaveshownthatusingageneralised precisionandrecoveryassessment(i.e.forthesumofanalytes)is notideal,asbothvalidationparametersarecompounddependent (athigherpolymerisation degreeboth anincreasingintermedi- ateprecisionanddecreasingrecoveryisobserved).Thisjustifies theconservativeapproachthatwasadoptedinthecurrentstudy, leadingtowhatweconsiderarealisticmeasurementuncertainty.

Themethod reportedby Robbins et al.[15].was developed forcocoapowdersandchocolateproducts,whereasthemethod describedherewasforapplesandappleextracts.Sincethereare differencesbetweenapplesandchocolateintermsofthematrices (thesugarspresentintheappleextractareofadifferentnatureto thosepresentinchocolate,therearelikelytobedifferencesinthe

natureoftheprocyanidins,thenatureoftheothernon-flavan-3-ol polyphenolsaredifferent,andtherearenumerousnon-phenolic compoundsthat arevery differentbetweenthetwomaterials), it wasimportanttodevelop and validatea methodspecificfor appleflavan-3-olsthatallowedaccuratequantificationofindivid- ualoligomers.Insummary,both methodsperformequallywell, withtheonepresentedhereoutperformingthatofRobinsetal.

[15].intermsofspeedandresolution.Further,thecurrentmethod ismoreextensivelyvalidatedandhasbeenascribedameasurement uncertainty.

4. Conclusion

Ananalyticalmethodfortheextraction,separation,identifica- tionand quantificationof procyanidins inanappleextract was developed,validatedandassessed.Theestablishedmethodutilizes a HILICstationaryphase withabinary mobilephaseconsisting of acidic acetonitrile and acidic aqueous methanol. The devel- opedmethodwasvalidatedforstandardlinearity,RRF,sensitivity, methodprecisionandtrueness.Evaluationofthevalidatedmethod wasconductedbysevenparticipatinglaboratoriesinsevencoun- triesacrossEurope.Theresultsoftheevaluationindicatethatthe established analytical method is reliable and reproducible. The methodisthefirstreportedforaccuratequantificationofindividual procyanidinsinappleextracts,andanimprovementonanexisting methodforquantifyingprocyanidinsincocoaintermsofruntime, resolutionofanalytes,theextentofvalidationandtheinclusionof anestimateofmeasurementuncertainty.

Conflictofinterest

Theauthorsdeclarenoconflictofinterest.

Acknowledgements

ThisresearchhasreceivedfundingfromtheEuropeanCommu- nity’sSeventhFrameworkProgramme(FP7underagreementno.

312090,projectBACCHUS)andtheBiotechnologyandBiological SciencesResearchCouncil(UK)throughanInstituteStrategicPro- grammeGrant(‘FoodandHealth’;GrantNo:BB/J004545/1)tothe InstituteofFoodResearch.