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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,Mass spectrometry for the,chromatographer,LC/MS in Drug Development,Identify unknown peaks to support method,development and validation,Characterize degradation products in forced,degradation studies and stability samples,Elucidate drug degradation pathway to support,excipient selection and formulation development,Confirm peak identity for DS and related,substances,Support QA issues and NDA,Support Manufacturing process and finished drug,products,Features of LC/MS,Advantages:,High sensitivity,High selectivity,Speed,Combined power of separation and identification,Molecular weight and structure information,Disadvantages:,Some compounds are not ionizable,Difficult to identify complete unknowns,Limited structure information compared to NMR,Identification Strategy:,When you start LC/MS analysis.,Is the method MS compatible?,Conditions where degradation compounds form,Synthetic scheme(Byproduct?Intermediate?),Structure of drug substance,active sites and functional groups,Number of nitrogen atoms,Isotope ratio,Full MS spectrum of unknown:Which is M+H?,MS/MS spectra of DS and unknown,UV spectra of DS and unknown,LC/MS Analysis,Work Request,LC/MS compatible method,Confirm Chromatogram,Select Ionization Mode,ESI vs APCI,(+)vs(-),LC/MS incompatible method,Modify HPLC conditions,Show Peak Equivalency,Determine MW,MS/MS,(n),Study,Isolation/Purification,Proposed Structure,NMR Confirmation,Report,No,Confirmation?,Yes,In-house Synthesis,LC-MS,LC,Interface,Extraction of,Ion evaporation,Mass,Analyzer,Detector,The analyte from or ionization.,The solvent,Fragmentation,LC Separation of the mixture of analytes,Interface Separation of the analyte from the solvent,MA(mass analyzer)separation of the analyte molecular,ion and fragments according to their mass to charge ratio,Infusion MS,MicroTee,MS,LC,100,l/min,pump,1,l/min,Sample solution,Sample in continuous flow,No sample injection,No LC separation,MS spectrum obtained over an given time,Improved S/N due to the average of multiple spectra,Select LC flow to assist ionization,-4,Basic Electrospray Schematic,N,760 Torr,1 Torr,10,-2,10,-5,Torr,10,-5,Torr,Ion optics,Formation of gas phase ions from solution phase,Richard B.Cole,Electrospray Ionization Mass Spectrometry,Fundamentals Instrumentation and Applications,Wiley Interscience,1997,ELECTROSPRAY,Factors to consider,Ionic strength,Surface tension of the solvent,Volatility of the solvent,Character of the analyte ions in solution:solvated,ion paired,etc,Mobile phase composition and amount of water,pH of the mobile phase,Intensity,Intensity,Confirmation of molecular weight,ESI(+),ESI(-),1.8x107,1.6x107,1.4x107,294.05,7x106,6x106,5x106,292.04,1.2x107,4x106,1.0 x107,8.0 x106,6.0 x106,4.0 x106,2.0 x106,3x106,2x106,1x106,0.0,100.00,200.00,300.00,400.00,500.00,600.00,700.00,800.00,0,100.00,200.00,300.00,400.00,500.00,600.00,700.00,800.00,m/z,m/z,1.Effect of instrumental parameters on ESI response,Mobile phase flowrate*,Capillary*/corona voltage,Fragementor*/cone voltage,All play important roles in analyte ions formation and,ionization efficiency,Effect of flow rate on ESI response,Dependence of mass analyzed ion intensity of the BH,+,ion of,protonated cocaine(10,-5,M)on flow rate.,O,CH,3,O,H,3,C,N,O,O,Increasing the flow rate increases droplet size which,decreases the yield of gas-phase ions from the charged droplets.,-Use shorter columns of same phase and proportionally decrease,flow rate.,P.Kebarle,et.al,Anal.Chem.1993,65,972A,Effect of capillary voltage,G.Valaskovic,J.Murphy,M.Lee,Milestone,development,Formation of small micron sized droplets is not a problem if flow rate,surface tension are low.,An increase,of these may make it difficult to for the electric field to produce the desired charged aerosol.,The electric field,strength can be increased to try to overcome those effects.However if electric field is too high will give,rise to electrical discharge and this is detrimental to ES signal.Nebulizing gas can be used to help focus the#,of ions transported into vacuum envelope of the mass spectrometer.,0,0,Effect of Cone/Fragmentor Voltage on fragmentation,FB-25CV+1,FB 60CV+1,TAE226 FB-1 807(8.225),1:Scan ES+TAE 226 FB-2 805(8.205),1:Scan ES+,100,469.18,4.86e7,100,438.16,5.13e7,25V,Single Quad,3:1,DS,60V,Single Quad,469.18,3:1,3:1,DS,403.22,%,471.08,937.44,%,372.20,471.08,100.19 155.62,214.09,242.19 255.11,315.23,348.40 434.62,506.91,577.06,652.27 696.09,721.16,813.47 831.58,891.09 941.49,976.06,m/z,99.94 117.53,193.08,315.61 359.54,491.09 534.26,635.18,720.40,757.50,870.45 913.51 937.57 959.60,m/z,100,200,300,400,500,600,700,800,900,1000,100,200,300,400,500,600,700,800,900,1000,As potential difference(deltaV)between end of capillary(N)and,first skimmer(S)is increased the ions are accelerated through,the background gas,which leads to more collisions.,Effect of experimental parameters on ESI response,A.Dependence of ion intensities on analyte concentration,Concentration effect of tetracycl
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