瓜子金药材高效液相色谱指纹图谱研究

瓜子金药材高效液相色谱指纹图谱研究

1风险行为recog：中国,英国,以服务于tcms中国的特征性儿媳（tms）正在等待尝试，而非正统方面。这是一个渐进的教训，证据是相互关联的。这是一个非常相似的系统的结果。线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？线人在哪里？。InTCMtheaerialpartofPolygalajaponicahaslongbeenusedasanexpectorant,anti-inflammatory,antibacterialandantidepressantagent.P.japonicaisaperennialplantharvestedinautumnafterthreeyearsofgrowth.PhytochemicalstudiesonP.japonicahaveledtothediscoveryoftriterpenoidsaponinsandflavonolderivatives.Ourpreliminarytestshaveshownthatethylacetatefraction(richinflavonolderivatives)andn-butanolfraction(richintriterpenoidsaponins)exertedremarkableanti-inflammatoryeffectsTherefore,simultaneousdetectionofbothflavonolderivativesandtriterpenoidsaponinswouldbealogicalapproachforthequalityevaluationofPolygalajaponica.DADisveryconvenientandsensitiveforthedetectionofflavonolderivatives,whileHPLCcoupledwithELSDhasbeenadoptedasanefficienttoolforqualitativeandquantitativeanalysisoftriterpenoidsaponinsofPolygalajaponicainourlaboratory.Inrecentyears,DADconnectedwithELSDhasbeensuccessfullyusedforthesimultaneousdeterminationofmultiplecomponentswithdifferentstructuresinasinglerunforTCMs.DADcanrevealUVabsorbingcomponentssuchasflavonolderivativesinPolygalajaponica,andELSDcanrevealpoorUVabsorptionsuchastriterpenoidsaponins.TheDADandELSDsignalscontaincomplementaryinformationandthecombinationofthemoffersanefficienttoolforthefingerprintingofPolygalajaponica.Inthisstudy,achromatographicfingerprintmethodwasdevelopedtoassessthequalityofPolygalajaponicabyhighperformanceliquidchromatography-photodiodearraydetection-evaporativelightscatteringdetection(HPLC-DAD-ELSD).Principalcomponentanalysis(PCA)wasperformedonthedatageneratedfromDADandELSD.Itwasdemonstratedthatanaccurateclassificationofvarioussamplesfromdifferentlocationswasobtained.2母乳喂养2.1anhi.20samplesoftheherbPolygalajaponicawerecollectedfromprovincesofJiangsu,Anhui,Jiangxi,Guangdong,Guangxi,Yunnan,Hunan,HubeiandHenaninChina.Thevoucherspecimens,identifiedbyDr.XUZeng-Lai(JiangsuZhongshanArboretum,Nanjing,China),havebeenstoredattheHerbariumofChinaPharmaceuticalUniversity,Nanjing,China.2.2相关程序性别合作Thereferencestandardsofrhamnocitrin3-O-β-D-galactopyranosideandpolygalasaponinXXIIwereisolatedpreviouslyfromthemethanolextractofP.japonicainourlaboratory.Theirstructureswereestablishedbasedonspectroscopicanalysescomparedwiththereferencedata.Thepuritiesweredeterminedtobegreaterthan98%bythenormalizationofthepeakareasdetectedbyHPLC-ELSDandconfirmedbyLC-MS,NMRspectroscopy.Methanol(HPLC-grade)andacetonitrile(HPLC-grade)werepurchasedfromHanbonScientific(Jiangsu,China)andMerck(Darmstadt,Germany)respectively.DeionizedwaterwasfromRobust(Guangzhou,China).Trifluoroaceticacid(TFA)waspurchasedfromShanghaiChemicalReagentCompany(Shanghai,China).OthersolventsfromNanjingChemicalFactory(Nanjing,China)wereofanalyticalgrade.2.3pca&alislaskApproximately1.0gofdriedherbwasmilledintopowder,accuratelyweighed,andtransferredintoaround-bottomflaskcontaining50mLmethanolandthemixturewasheatedat80°Cunderrefluxfor6h.Afterevaporatingmethanoltodrynessbyarotaryevaporator,residuewasdissolvedinmethanolina25mLflask,andthenfiltratedthrougha0.45μmmilliporefilter.Analiquotofthesolution(20μL)wasinjectedtotheHPLC-DAD-ELSDsystemforanalysis.2.4raphysaindex,elsd.见表1AnalyseswereperformedonWatersseriesliquidchromatographer,consistingofWaters600pump,996DADcoupledwith2000ESELSD,Millennium32ChemStationsoftware.ThechromatographywascarriedoutonaDiscoveryC18column(5μm,250mm×4.6mm)atacolumntemperatureof30°Candflowrateof1mL·min-1using(A)acetonitrile-methanol(90∶10)and(B)0.05%aqueousTFAasmobilephasewithalineargradient:0-18min(25%A),18-35min(25%A→35%A),35-45min(35%A→80%A),45-55min(80%A).TheevaporatortemperaturefortheELSDwassetat105°Cwiththenebulizinggasflow-rateof2.6L·min-1,andtheDADdetectorwassetat350nmforacquiringchromatogramswithUVspectraintherangeof200-400nmat1nm/step.2.5resp果类.标准实行受精神品牌共同生活的so阶段,以资源整合为原则,见表1.3Methodprecisionandreproducibilitywereevaluatedbytsixconsecutiveinjectionsofthesamesamplesolutionandsixreplicatesofthesamepowdersample,respectively.Thestabilityofsamplesolutionwasevaluatedatthetimepointsof0,2,4,8,24and48h.2.6数据mi类2.6.1chromatograptching,asimmetsim,algorithmTheresultsofmultivariableanalysisweregreatlyinfluencedbythedifferencesinbaseline,suchasslopesandlevelsespeciallywhenusingtheentirechromatographicprofiles.Asasimplebuteffectiveandcompletelyautomaticalgorithm,thecombinationofWhittakersmootherandasymmetricleastsquareswasappliedtoestimatethebaseline.Inthisapproachthesmoothingfactor(generally1e5to1e8),asymmetryfactor(generally0.001)andorderofdifferencesinpenalty(generally2)were1e7,0.001and2,respectively.TheMatlabfunctionsofbaselinecorrectioncanbedownloadedfrom.2.6.2slackspin-definedgage,kn.3.3s-vegesThecorrelationoptimizedwarping,COW,aimstoeliminatetheshiftsalongthetimeaxisinchromatograms.Itdemandsselectionofatleasttwouser-definedvariables:segmentlength(m)andslackparameter(t).IntheprocessofaligningthechromatographicsignalsfromDADorELSD,mandtwere50and5,respectively.2.6.3PrincipalcomponentanalysisPrincipalcomponentanalysis(PCA),seenforexamplefromthereference,hasbeenusedtovisuallyevaluatetheresultsobtainedinthepreprocessingofchromatographicdataPriortoPCA,thechromatographicdatamatrix,X(xij),wasnormalized:wherexijrepresentsthejthvalueoftheithchromatogram,xj,minandxj,maxaretheminimumandmaximumvaluesofeachcolumninthematrix,andzijisthevalueintherange,,afternormalization.ScoreplotsofthefirsttwoprincipalcomponentsshowedtheprocessingresultsofDAD,ELSDandDAD-ELSDdatamatrices.3影响的神圣迪迪斯运营3.1elution&spinthosepe高校elstelatTodevelopanaccurate,validandoptimalchromatographicfingerprint,thedifferentmobilephases(methanolwater,methanol-water-trifluoroaceticacid,acetonitrile-watertrifluoroaceticacidandacetonitrile-methonol-trifluoroaceticacid)wereassessed.AfinaloptimizedHPLCconditionwasidentifiedasacetonitrile-methanol-trifluoroaceticacidasmobilephasewithalineargradientelutionbycomparingtheresolution,baselinequietness,retentiontimesandnumberofcharacteristicpeaksineachchromatogram.MostchemicalcomponentsintheDADchromatogramshowedthebiggestabsorbanceat260nmand350nm.Therefore,thewavelengthof350nmwasselectedforoptimumdetectionwavelengthbyinvestigatingthespectraofallcharacteristicpeaksandanalyzing3D-plotsofthosepeaks.ForELSD,theoperatingconditionssuchasthenebulizinggasflowrateandthedrifttubetemperatureareoptimizedtoobtainthebestS/N.Ingeneral,largedropletsareformedatlowgasflow,whichresultsinspikesandnoisybaseline;ontheotherhand,increasingthegasflowresultsinasubstantialdecreaseoftheresponses.Theoptimumnebulizinggasflowinthiscasewassetat2.6L·min-1.Withrespecttothedrifttubetemperature,solventevaporationisnotcompletedatlowtemperaturewhilethedetectorresponseisdecreasedathightemperature.Therefore,theoptimaldrifttemperaturewasdeterminedtobe105°CaccordingtothedatacomputedwiththeELSDsoftware.TheoptimalHPLC-DAD-ELSDconditionwasshowninSectionpe直利亚联邦lactPrecision,reproducibilityandstabilitywereevaluatedtoensurethevalidityofthisnewlydevelopedHPLC-DAD-ELSDfingerprintingmethod.Sincetoomanypeakswereintegrated,itwasimpossibletotesteachpeak.Peaks1-6inDADand1’-6’inELSDfingerprintwereselectedastherepresentativesofthecomponentsinPolygalajaponicaforvalidation(Fig.1).Allthevalidationswereexpressedbycalculatingtheirrelativestandarddeviations(RSD)oftherelativeareasandrelativeretentiontimesofeachtargetpeakcomparedwiththereferences5(rhamnocitrin3-O-β-D-galactopyranoside)andreferences6’(polygallasaponinXXII).Theprecision,reproducibilityandstabilityoftheproposedmethodarelistedinTable1.AllRSDsarelessthan5%,whichindicatedthatthemethodofHPLC-DAD-ELSDforthefingerprintanalysisisvalidandsatisfactory.3.3revidualnain,elsdfingprin,elsdfingprin,第三,反应物.............3.4和...3.4和3.4工作re原料非负保护.......4.3.4和...4.3.4和.3.4和.....和........................3.TheDADandELSDsignalscontaincomplementaryinformation;theDADsignalrevealsvolatilecompoundsorcompoundswhoselevelsarebelowthedetectionlimitoftheELSD,whilethelatterdetectorrevealsnon-andpoorUVactivecompounds.AsshowninFig.1A,theDADfingerprintrevealspeakswhichcouldnotbedetectedintheELSDfingerprint,andthesepeaksareindicatedwith“a”.ItisalsoseenthattheELSDsignalisratheruniquebetweenRTsat35and45min,whichcontainssomeadditionalpeaksindicatedinFig.1B.Someoftheadditionalpeakssuchas3’,4’,5’,6’wereidentified,asforinstancethepolygalasaponins.Ourpreliminarytesthadshownthattriterpenoidsaponinsandflavonolderivativesarethemainactivecomponents.WhileintheDADchromatogrammostpeaksshowthecharacteristicspectraofflavonolderivatives(260nm,350nm),andintheELSDchromatogrammostpeaksareidentifiedastriterpenoidsaponinsbycomparisonwiththereferencestandards.Inconclusion,thedevelopedHPLC-DAD-ELSDfingerprintshowssuperioritytoindividualDADorELSDfingerprintinthequalitycontrol.3.4whichsone关于lahengeInthisstudy,DADfingerprintandELSDfingerprintwereappliedrespectivelyandcomparedwiththeDAD-ELSDfingerprintforthequalitycontrolofPolygalajaponica.Weintegratedallthedistinguishedpeaksofthesefingerprintsforeachsample.Thecorrespondingpeaksofvariousfingerprintswerematchedanddatanormalizationtransformationwasalsoperformedusingthealignmentmethods.Thenqualityassessmentonthesecorrectedobjectswasperformed.Principalcomponentanalysis(PCA)isawell-knownchemometricmethodforthedecompositionoftwo-dimensionalmatrices,whichdescribesthevariationindatawithminimumlatentvariables.Inourstudy,PCAwasperformedtoallowvisualizationofthevariancebetweenPolygalajaponicafingerprintsfromdifferentorigin,basedondifferencesintheirpeakprofile.WeperformedPCAuponthedataofDADfingerprintsfirstly,andthediscrepanciesofthesamplesareshownclearlyinthescoreplotofPC1versusPC2(inFig.2A).Fromthescatterpoints,thesamplescanbeclassifiedintotwogroups,whicharemarkedasgroupsIandII,respectively.Samples8(Yunnan)and1(Guangdong)werechosenasrepresentativesofthesamplesinthetwogroups.Inthefingerprintofsample8,comparedwiththatofsample1,somepeaksbetweentheretentiontimeof5and40minaremuchlower,oralmostinvisible,whilefourextrapeaksappearinthefingerprintofsample8attheretentiontimeof3-5min(markedbyarectanglefra