QTRAP用户会之二MRM定量

1©2010ABSCIEXMRM3定量:•三级四极杆传统定量方法是MRM•典型的是单位质量分辨(0.7amuFWHH)(通常特异性足够)•不幸的是，仍然会有干扰•如果能早期发现，还能修改方法，如果已经确认，将花费大量的时间和资源克服这个问题2©2010ABSCIEX−MRM3,在定量上增加选择性消除干扰;建立置信度!−区别于3D离子阱的MS3,专用在定量方面,−故称为MRM33©2010ABSCIEX如何解决干扰问题消除干扰可以…1.色谱2.变换碎片离子forMRM3.改变CE4.提高母离子的选择性优点操作容易LC时间不受影响LC时间不受影响提高Q1分辨率容易做到缺点延长分析时间影响灵敏度影响灵敏度降低灵敏度4©2010ABSCIEX0.00.20.40.60.81.01.200.10.20.30.40.50.60.70.8FWHH(amu)NormalizedIntensity(max=6.99e5)当FWHH0.7降到0.1时，分辨率升高，但是灵敏度会损失50%以上分辨率与灵敏度关系图解Finnigan高分辨率的定量分析(H-SRM)，FWHH=0.2，此时灵敏度损失20%20%Loss70-75%Loss0.06amuFWHH5©2010ABSCIEX0.00.20.40.60.81.01.200.10.20.30.40.50.60.70.8FWHH(amu)NormalizedIntensity(max=6.99e5)FinniganpromotesHi-ResQuantitationat0.2FWHH使用4000QTRAP进行定量分析，单位分辨率时MS3的特异性，相当于MS2FWHH=0.03分辨率/传输率~90%LossForH-SRM0.03amuFWHHMS2的前体离子选择的分辨率达到FWHH0.03amu时，灵敏度损失超过90%6©2010ABSCIEXFromLibrary-m/z315(181Compound,27containingCland10containingBr)0.000010.00010.0010.010.1100.10.20.30.40.50.60.70.8FWHH(amu)ProbabilityofInterferencem/z315.2192m/z315.2192MSMSm/z315.2192MS^3干扰概率:MS,MS/MSandMS3vs.Resolution100%0.25%0.03amu…要得到与单位分辨率时MS3相同水平的定量分析特异选择性，MS2的前体离子选择的分辨率需达到FWHH0.03amu7©2010ABSCIEXMS3实验:•Q1分析•Q2裂解•LIT分离/裂解/贮存/扫描•特异的定量通路•消除干扰•比提高Q1分辨率更通用定量由…Q0Q1q2Q3LIT8©2010ABSCIEXMRM3定量:实验条件:3种化合物有相同的整数质量和碎片离子在快速色谱条件下可分析CompoundExactMassMassDifference(mmu)Pamaquine315.23110Oxycodone315.1471-84Clonazepam315.0411-1905-500pg50ng2.5ngOncolumn流动相:A)95:5H2O:ACN5mMNH4OAc0.1%FormicB)95:5ACN:H2O5mMNH4OAc0.1%FormicGradientelutionover7minutes色谱柱:LunaC18(2)5(2.1x50mm)流速：350L/minMRMon4000QTRAP(OptimalCE)Pamaquin(10pg)inpresenceofOxycodone(50ng)andClonazepam(2.5ng)OxycodonePamaquinClonazepamClonazepamClonazepamPamaquin(ABSENT)Pamaquin(ABSENT)MRM316/243MRM316/241MRM316/187OxycodonePamaquinClonazepamClonazepamClonazepamPamaquinPamaquinOxycodoneMRM316/243MRM316/241MRM316/187MRMon4000QTRAP(CE45eV)Pamaquin(100pg)inpresenceofOxycodone(50ng)andClonazepam(2.5ng)SampleName:Pamaquin1.0pg/uL_MS3(4000amu/sec)_WITHInterferenceSampleID:File:QTPamaquin_MS3(4000amusec)withI...PeakName:Pamaquin_187Mass(es):186.8-187.5amuComment:Annotation:SampleIndex:7SampleType:StandardConcentration:1.00ng/mLCalculatedConc:1.08ng/mLAcq.Date:2004/02/29Acq.Time:02:05:20PMModified:NoProc.Algorithm:IntelliQuan-MQIINoisePercentage:50Base.Sub.Window:0.20minPeak-Split.Factor:3ReportLargestPeak:NoMin.PeakHeight:0.00cpsMin.PeakWidth:0.00secSmoothingWidth:5pointsRTWindow:30.0secExpectedRT:0.635minUseRelativeRT:NoInt.Type:BaseToBaseRetentionTime:0.597minArea:2.09e+004countsHeight:6.92e+003cpsStartTime:0.561minEndTime:0.707min0.51.01.52.0Time,min-200-10001002003004005006007008009001000110012001300140015001600170018001900200021002200230024002500260027002800290030003100320033003400350036003700380039004000410042004300440045004600470048004900500051005200530054005500560057005800590060006100620063006400650066006700Intensity,cps0.601.620.391.231.900.171.02SampleName:Pamaquin1.0pg/uL_MS3(4000amu/sec)_WITHInterferenceSampleID:File:QTPamaquin_MS3(4000amusec)withI...PeakName:Pamaquin_174-187Mass(es):174.8-175.5amu,186.8-187.5amuComment:Annotation:SampleIndex:7SampleType:StandardConcentration:1.00ng/mLCalculatedConc:1.11ng/mLAcq.Date:2004/02/29Acq.Time:02:05:20PMModified:NoProc.Algorithm:IntelliQuan-MQIINoisePercentage:50Base.Sub.Window:0.20minPeak-Split.Factor:3ReportLargestPeak:NoMin.PeakHeight:0.00cpsMin.PeakWidth:0.00secSmoothingWidth:5pointsRTWindow:30.0secExpectedRT:0.635minUseRelativeRT:NoInt.Type:BaseToBaseRetentionTime:0.609minArea:3.40e+004countsHeight:1.02e+004cpsStartTime:0.573minEndTime:0.768min0.51.01.52.0Time,min-2000200400600800100012001400160018002000220024002600280030003200340036003800400042004400460048005000520054005600580060006200640066006800700072007400760078008000820084008600880090009200940096009800Intensity,cpsMS3316/243/187Summedionsof187and174MRM3onthe4000QTRAPPamaquin(5pg)inpresenceofOxycodone(50ng)andClonazepam(2.5ng)SampleName:Pamaquin2.0pg/uL_MS3(4000amu/sec)_WITHInterferenceSampleID:...PeakName:Pamaquin_241Mass(es):240.5-240.8amuComment:Annotation:0.51.01.52.0Time,min0.01000.02000.03000.04000.05000.06000.07000.08000.09000.01.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e41.8e41.9e42.0e42.1e42.2e42.3e42.4e42.5e42.6e42.7e42.8e42.9e43.0e43.1e43.2e43.3e43.4e43.5e43.6e43.7e43.8e43.9e44.0e44.1e44.2e44.3e44.4e44.5e44.6e44.7e44.8e4Intensity,cpsSampleName:Pamaquin2.0pg/uL_MS3(4000amu/sec)_WITHInterferenceSampleID:...PeakName:Pamaquin_187Mass(es):186.8-187.5amuComment:Annotation:0.51.01.52.0Time,min0.0200.0400.0600.0800.01000.01200.01400.01600.01800.02000.02200.02400.02600.02800.03000.03200.03400.03600.03800.04000.04200.04400.04600.04800.05000.05200.05400.05600.05800.06000.06200.06400.06600.06800.07000.07200.07400.07600.07800.08000.08200.08400.08600.08800.09000.09200.09400.09600.09800.01.0e41.0e41.0e41.1e41.1e41.1e41.1e41.1e41.2e41.2e41.2e41.2e41.2e41.3e41.3e41.3e41.3e41.3e41.4e41.4e4Intensity,cpsSampleName:Pamaquin2.0pg/uL_MS3(4000amu/sec)_WITHInterferenceSampleID:...PeakName:Pamaquin_174-187Mass(es):174.8-175.5amu,186.8-187.5amuComment:Annotation:0.51.01.52.0Time,min0.