内标法及外标法方法、原理、优缺点VIP专享VIP免费

AninternalstandardshouldbeusedwhenperformingMSquantitation.Anappropriateinternalstandardwillcontrolforextraction,HPLCinjectionandionizationvariability.InacomplexmatrixitisnotuncommonfortwodifferentstandardlevelsinSRMintegratedplots,atthelowerendofthestandardcurve,togivenearlyanidenticalresponse.Itisonlywhenaninternalstandardisusedthatthetwopointscanbedifferentiated.Someresearchersattempttopreparestandardcurvesandrunsampleswithoutaninternalstandardandfindmoderatesuccess.Oftenwithoutaninternalstandard%RSDsofreplicatescanbeashighas20%.Usinganinternalstandardthe%RSDscanbebroughtdowntoapproximately2%.Weruntriplicatesateachlevelofourstandardcurve.HowdoIchooseaninternalstandard?Thebestinternalstandardisanisotopicallylabeledversionofthemoleculeyouwanttoquantify.Anisotopicallylabeledinternalstandardwillhaveasimilarextractionrecovery,ionizationresponseinESImassspectrometry,andasimilarchromatographicretentiontime.Ifyouareperformingnon-clinicalPKquantitationitmaybedifficulttojustifysuchastandardsinceaspecialsynthesisofanisotopicallylabeledstandardcanbeexpensiveandtimeconsuming.Oftenifyouareworkingwithmedicinalchemiststheywillhavealibraryofcompoundanalogsthatcanbeusedasinternalstandards.Theseanalogsweremadeintheevolutionofthecompoundtobetestedandwillbesimilartothecompoundtobequantifiedandmoreimportantlywillbeslightlydifferentbyparentmass.Trytoavoidusingde-methylated(-14)orhydroxylated(+16)analogsasinternalstandardssincethesearethemostcommonmassshiftsobservedinnaturallyoccuringmetabolitesoftheparentcompound.Acommoninternalstandardisachlorinatedversionoftheparentmolecule.Achlorinatedversionoftheparentmoleculewillcommonlyhaveasimilarchromatographicretentiontimewhichisanimportantcharacteristicofaninternalstandard.Wehavefoundthatoneofthemostimportantcharacteristicsofaninternalstandardisthatitco-eluteswiththecompoundtobequantified.HowdoIuseaninternalstandard?Firstofallaninternalstandardshouldbeaddedatthebeginningofthesamplework-up,typicallybeforetheplasmacrashorsolidphaseextraction.Theinternalstandardshouldbeaddedatthesamelevelineverysampleincludingthestandards.AninternalstandardshouldgiveareliableMSresponse.Careshouldbetakenthattheamountoftheinternalstandardiswellabovethelimitofquantitationbutnotsohighastosuppresstheionizationoftheanalyte."HowmuchinternalstandardshouldIadd?",thisisanimportantquestion.Itpaystoknowroughlyhowmuchcompoundisinyoursample.Thiscanbeaccomplishedbymakingtrialanalysesofanearly,middleandlatetimepointwithperhapsoneortwostandardpoints.Thisinformationwillbeveryvaluablewhenbuildinganappropriatestandardcurveandinknowinghowmuchinternalstandardtoadd.Ifyouweretryingtoquantifysamplesintherangeof100fgto25pgandthelimitofdetectionwas100fgyoumightadd5to10pgofinternalstandardtoeverysample.Agoodruleofthumbistotargettheinternalstandardtothelower1/3oftheworkingstandardcurve.Thisisarangethatwillgiveacomfortableresponsewithoutinterferingwiththeionizationoftheanalyte.--------------------------------------------------------------------------------中文：什么叫内标法?怎样选择内标物?内标法是一种间接或相对的校准方法。在分析测定样品中某组分含量时，加入一种内标物质以校谁和消除出于操作条件的波动而对分析结果产生的影响，以提高分析结果的准确度。内标法在气相色谱定量分析中是一种重要的技术。使用内标法时，在样品中加入一定量的标准物质，它可被色谱拄所分离，又不受试样中其它组分峰的干扰，只要测定内标物和...