内标法和外标法方法、原理、优缺点

《内标法和外标法方法、原理、优缺点》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、.AninternalstandardshouldbeusedwhenperformingMSquantitation.Anappropriateinternalstandardwillcontrolforextraction,HPLCinjectionandionizationvariability.InacomplexmatrixitisnotuncommonfortwodifferentstandardlevelsinSRMintegratedplots,atthelowerendofthesta

2、ndardcurve,togivenearlyanidenticalresponse.Itisonlywhenaninternalstandardisusedthatthetwopointscanbedifferentiated.Someresearchersattempttopreparestandardcurvesandrunsampleswithoutaninternalstandardandfindmoderatesuccess.Oftenwithoutaninternalstandard%RS

3、Dsofreplicatescanbeashighas20%.Usinganinternalstandardthe%RSDscanbebroughtdowntoapproximately2%.Weruntriplicatesateachlevelofourstandardcurve.HowdoIchooseaninternalstandard?Thebestinternalstandardisanisotopicallylabeledversionofthemoleculeyouwanttoquanti

4、fy.Anisotopicallylabeledinternalstandardwillhaveasimilarextractionrecovery,ionizationresponseinESImassspectrometry,andasimilarchromatographicretentiontime.Ifyouareperformingnon-clinicalPKquantitationitmaybedifficulttojustifysuchastandardsinceaspecialsynt

5、hesisofanisotopicallylabeledstandardcanbeexpensiveandtimeconsuming.Oftenifyouareworkingwithmedicinalchemiststheywillhavealibraryofcompoundanalogsthatcanbeusedasinternalstandards.Theseanalogsweremadeintheevolutionofthecompoundtobetestedandwillbesimilartot

6、hecompoundtobequantifiedandmoreimportantlywillbeslightlydifferentbyparentmass.Trytoavoidusingde-methylated(-14)orhydroxylated(+16)analogsasinternalstandardssincethesearethemostcommonmassshiftsobservedinnaturallyoccuringmetabolitesoftheparentcompound.Acom

7、moninternalstandardisachlorinatedversionoftheparentmolecule.Achlorinatedversionoftheparentmoleculewillcommonlyhaveasimilarchromatographicretentiontimewhichisanimportant...characteristicofaninternalstandard.Wehavefoundthatoneofthemostimportantcharacterist

8、icsofaninternalstandardisthatitco-eluteswiththecompoundtobequantified.HowdoIuseaninternalstandard? Firstofallaninternalstandardshouldbeaddedatthebeginningofthesamplework-up,typicallybeforetheplasmacrashorsolidphaseextracti