Sensitivity enhancement in LC-MSMS using derivatiz

SensitivityenhancementinLC-MS/MSusingderivatizationKeMa09Jun2010IntroductionDefinition•IntroducefunctionalgroupsintoanalytesPurpose•Modifyphysicalorchemicalproperties•Improveanalysis–detection,quantitation,separation,identificationGC-MSLC-MSVerycommonImprovevolatilityImprovethermalstabilityDerivatizationinGC-MSandLC-MSLesscommonEnhanceionizationefficiencyModifychromatographicbehaviorCompoundscanbefavorablyanalyzedbyLC-MS/MS.Chargeablethroughadductformationingas-phasereaction.IonicforminthesolutionphaseCompoundscanbefavorablyanalyzedbyLC-MS/MS.GenerateanintenseproductionforthesensitiveMS/MSdetectionMusthaveanon-polarregionDerivatizationImproveionizationefficiencyChangefragmentationpatternImproveseparationproperties123WhyderivatizeinLC-MS?SimultaneousdeterminationofgemcitabineanddFdUinhumanplasmagemcitabinecytidinedeaminasedFdUChallenges1.OnlyonemassunitdifferencebetweengemcitabineanddFdU,whichrequiredLCbaselineseparation.2.Poorchromatographicretentionofbothcompoundsonreversedphasecolumns.3.Insufficientsensitivity.BowenCetc.JChromatogrB,15;877(22):2123-9SimultaneousdeterminationofgemcitabineanddFdUinhumanplasmaPlasmasamples(50μL)+50μLaliquotofinternalstandardsolutionMixwellAdd100μLof10mg/mLdansylchlorideinacetoneand100μLsodiumbicarbonate(100mM;pH11)Sealedandmixedincubationat60◦Cfor5min.Add1mLofMTBEwellstoextractthedansyl-derivativesofgemcitabine,dFdU,andtheirinternalstandards.Evaporateunderastreamofnitrogenat45◦CReconstitutein150Lof25/75(v/v)acetonitrile/water,injectionof2μLwasusedm/z171H+BowenCetc.JChromatogrB,15;877(22):2123-9DerivatizationofanalytesR1R2O4.Amines1.Ketonesandaldehydes(carbonylcompounds)2.Alcoholsandphenols3.Carboxylicacids(includingaminoacids)RNH2ROHROHOOtherKetonesandaldehydes(carbonylcompounds)OtherreagentsDNPHCyclohexanedioneGirPGirTDns-HzHMPHPDAABD-MHzAlcoholsandphenolsOtherreagentspocolinicacidpropionylanhydridebenzoylanhydrideCarboxylicacids(includingaminoacids)Classsensitivecompounddetection–fattyacids/organicacids–AminoacidsApplication:newbornscreeningOtherreagentstrimethylamino-ethylalcohol(TAME)4-dimethylamino-benzylamineDAABD-AEAminesReagentsNBD-FNBD-FaceticanhydrideaceticanhydridepentafluorobenzylclorideNITpentafluoropropionicacidanhydridenitrobenzylchloroformDns-ClTHASDisadvantageofderivatizationExtrawork,time,cost,errorratePoorfragmentationpatterns(sometimes)YieldisomerformationandsideproductformationSignalsuppressionLowrecoveryDisadvantageAdvantagesDisadvantagesThanksforlistening!