内标法及外标法方法、原理、优缺点

《内标法及外标法方法、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,atthelowerendofthestandardcurve,togiven

2、earlyanidenticalresponse.Itisonlywhenaninternalstandardisusedthatthetwopointscanbedifferentiated.Someresearchersattempttopreparestandardcurvesandrunsampleswithoutaninternalstandardandfindmoderatesuccess.Oftenwithoutaninternalstandard%RSDsofreplicatescanbeashighas20%.Usin

3、ganinternalstandardthe%RSDscanbebroughtdowntoapproximately2%.Weruntriplicatesateachlevelofourstandardcurve.HowdoIchooseaninternalstandard?Thebestinternalstandardisanisotopicallylabeledversionofthemoleculeyouwanttoquantify.Anisotopicallylabeledinternalstandardwillhaveasim

4、ilarextractionrecovery,ionizationresponseinESImassspectrometry,andasimilarchromatographicretentiontime.Ifyouareperformingnon-clinicalPKquantitationitmaybedifficulttojustifysuchastandardsinceaspecialsynthesisofanisotopicallylabeledstandardcanbeexpensiveandtimeconsuming.Of

5、tenifyouareworkingwithmedicinalchemiststheywillhavealibraryofcompoundanalogsthatcanbeusedasinternalstandards.Theseanalogsweremadeintheevolutionofthecompoundtobetestedandwillbesimilartothecompoundtobequantifiedandmoreimportantlywillbeslightlydifferentbyparentmass.Trytoavo

6、idusingde-methylated(-14)orhydroxylated(+16)analogsasinternalstandardssincethesearethemostcommonmassshiftsobservedinnaturallyoccuringmetabolitesoftheparentcompound.Acommoninternalstandardisachlorinatedversionoftheparentmolecule.Achlorinatedversionoftheparentmoleculewillc

7、ommonlyhaveasimilarchromatographicretentiontimewhichisanimportantcharacteristicofaninternalstandard.Wehavefoundthatoneofthemostimportantcharacteristicsofaninternalstandardisthatitco-eluteswiththecompoundtobequantified.HowdoIuseaninternalstandard? Firstofallaninternalstan

8、dardshouldbeaddedatthebeginningofthesamplework-up,typicallybeforetheplasmacrashorsolidphaseextraction.T