(原文)超临界流体色谱对异黄酮的分离

JournalofPharmaceuticalandBiomedicalAnalysis107(2015)364–369ContentslistsavailableatScienceDirectJournalofPharmaceuticalandBiomedicalAnalysisjournalhomepage:∗InstituteofPharmacy,Pharmacognosy,UniversityofInnsbruck,CCB,Innrain80-82,6020Innsbruck,AustriaarticleinfoArticlehistory:Received30October2014Receivedinrevisedform23December2014Accepted6January2015Availableonline14January2015Keywords:IsoflavonesSFCSoyRedgloverKudzuabstractThefirstprotocolfortheanalysisofisoflavonesbysupercriticalfluidchromatographyisreported.OptimumresultswereobtainedonanAcquityUPC2BEH1.7mcolumn,usingasolventgradientofsupercriticalcarbondioxideandmethanol(withphosphoricacidasadditive)forelution.Themethodenablesthebaselineseparationofnineisoflavones(aglycaandglycosides)in8min,andissuitablefortheirquantitativedeterminationindietarysupplementscontainingsoy(Glycinemax),redglover(Tri-foliumpratense)andkudzu(Puerarialobata).Methodvalidationconfirmedthattheassayisselective,linear(R2≥0.9994),accurate(recoveryratesfrom97.6to102.4%),aswellaspreciseontheshort-andlong-termlevel(intra-dayprecision≤2.1%),andshowsanon-columndetectionlimitof0.2ngandbelow.This,togetherwithanexcellentperformanceshownintheanalysisofrealsamples,indicatesthatSFCiswellsuitedforthefastandaccuratedeterminationofisoflavonesincomplexmatrices.Disadvantagescomparedtotheestablishedapproacheswerenotobserved,sothatSFChastobeconsideredinthiscaseasan(atleast)equivalentanalyticalalternative.©2015ElsevierB.V.Allrightsreserved.1.IntroductionIsoflavonesbelongtoarathersmall,butstructurallyandphar-macologicallyuniquegroupofnaturalcompounds.Incontrasttoregularflavonoidstheyderivefrom3-phenylchromen-4-one,whereaphenylmoietyispositionedonC-3ofthechromene-systeminsteadoftheusuallysubstitutedC-2(Fig.1).Thisstructuralmodificationresultsinhormone-likeactivities(theyareassignedtothegroupofphytoestrogens),whichexplaintheiruseinreme-diestoreducemenopausalandpost-menopausalsymptomslikehotflushes[1],boneloss[2]ormoodswings[3].Theconsumptionoffoodrichinisoflavones(e.g.soy)isevenassociatedwithagener-allyloweroccurrenceofbreastcancerinAsia[4]andtheretardedprogressionofAlzheimer’sdisease[5].Fromliteraturedataitisnotpossibletoconfirmtheseindicationswithoutanydoubt,becauseanumberofpharmacologicalstudiesobservednosucheffects[6,7].IsoflavonesarefoundprimarilyinplantsoftheFabaceaefam-ily,andexampleswithmedicalrelevanceareredclover(Trifoliumpratense),kudzu(Puerarialobata)orsoy(Glycinemax).Manyanalyticaltechniqueshavesuccessfullybeenutilizedfortheseparationofisoflavonesindiversematrices(e.g.plantmate-rial,nutraceuticalproducts,plasma,urine),includingTLC[8],GC[9],capillaryelectrophoresis(CE)[10]andHPLC[11–13].What∗Tel.:[phone];fax:[phone].E-mailaddress:[e-mail]©2015ElsevierB.V.Allrightsreserved.neverhasbeenevaluatedbeforeissupercriticalfluidchromatogra-phy(SFC),atechniquethatincreasinglybecomespopularinroutineanalysisduetomanydesirablecharacteristics(seeSection3).HereasupercriticalfluidisusedasmobilephaseforchromatographicseparationsinHPLClikeinstrumentation.Thisneedstobedifferen-tiatedfromSFE(supercriticalfluidextraction),wheresupercriticalfluids,mostlycarbondioxide,areusedforextractionpurposesonly.Thelatterhasbeendescribedfortheenrichmentoftotalisoflavonesfromsoy[14]andothermatrices[15],yettheanalysisofindividualisoflavonesbySFCneverhasbeenattempted.2.Materialandmethods2.1.Samples,standardsandreagentsSamplesIF2014-1toIF2014-6,capsulesortablets(dietarysup-plements)containingsoy-,redclover-orkudzu-extractsortherespectiveplantmaterial,werepurchasedin2014fromdrugstoresinInnsbruck/AustriaorfromtheInternet.Thestandardcompounds(BiochaninA(1),Formononetin(2),Genistein(3),Glycitein(4),Daidzein(5),Genistin(6),Glycitin(7),Daidzin(8)andPuerarin(9);Fig.1)camefromPhytoLab(Vestenbergsgreuth,Germany),andhadapurityof98.3%orhigher.ForSFCanalysis4.5gradecar-bondioxide(purity≥99.995%;Messer,Gumpoldskirchen,Austria)andmethanolwithphosphoricacidwereusedasmobilephase.ThelattertwowereofanalyticalgradeandpurchasedfromMerck(Darmstadt,Germany).M.Ganzera/JournalofPharmaceuticalandBiomedicalAnalysis107(2015)364–369365CompoundR1R2R3R4R5BiochaninA(1)HOHHOHOCH3Formononetin(2)HOHHHOCH3Genistein(3)HOHHOHOHGlycitein(4)HOHOCH3HOHDaidzein(5)HOHHHOHGenistin(6)Hß-D-glcHOHOHGlycitin(7)Hß-D-glcOCH3HOHDaidzin(8)Hß-D-glcHHOHPuerarin(9)ß-D-glcOHHHOHFig.1.Chemicalstructureoftheinvestigatedisoflavones.2.2.SamplepreparationThecontentofonecapsuleorone,inamortarfinelygrindedtabletwasextractedwith15mlofamethanol/water(8/2,v/v)mixturebysonicationfor10minatambienttemperature.Then,thesamplewascentrifuged(3000rpm,10min)andthesuper-natantwasdecantedina50mlvolumetricflask.Thisprocedurewasrepeatedtwomoretimesandtheflaskwasfinallyfilleduptovolumewiththeextractionsolvent.Theextractionprotocolisidenticaltoanalreadyvalidatedandpublishedone,whichensurestheexhaustiveextractionofisoflavonesfr