2010QTRAP用户会之二-MRM3定量.ppt

QTRAP实战指南之二----MRM3定量  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MRM3 分辨率与灵敏度关系图解 分辨率/传输率 MRM on 4000 Q TRAP (Optimal CE)Pamaquin (10pg) in presence of Oxycodone (50ng) and Clonazepam (2.5ng) MRM on 4000 Q TRAP (CE 45eV)Pamaquin (100pg) in presence of Oxycodone (50ng) and Clonazepam (2.5ng) 为什么复合型四极杆-线性离子阱是不同的? 使用4000QTRAP的MRM3进行定量，其单位分辨率的选择特异性，相当于FWHH=0.03amu（MS2），干扰概率只有0.25%，而其灵敏度相对于MS/MS定量, 只降低2.5倍 MRM3 Quantitation using the QTRAP? 5500 system 10μg/kg in Matrix (MRM3: %CV5, accuracy 90-110% ) Calcitriol MRM vs MRM3 实例-克伦特罗 Clenbuterol analysis from Urine sample Calibration curves prepared from pooled matrix Subject 1 to 18 pooled to prepare standard calibration curve This mimics approach used in typical bioanalysis Total of 34 subject samples (including the 18 pooled together for calibration curve) were analyzed as individual samples at 3 levels of clenbuterol 0 pg/uL of clenbuterol (unspiked – blank) 0.5 pg/uL of clenbuterol (apporximate) Previous sample diluted 2 fold (0.25pg/uL) All Subject samples injected randomly Calibration curve injected in 4 replicates MRM – monitored 277-203 / 277-168 / 277-132 MRM3 – 277?203?168 / 277?203?132 Recommendations for MRM3 Quant Acquire using DFT mode Min = 0.05 msec Max = 150 msec Target = 1e8 Acquire at 10,000 amu/sec Acquiring faster will not significantly improve the cycle time as the bulk of the time will be spend on Fill Time and Ion Processing (isol., excit. and scan over head) Acquire mass range that encompasses fragment ion of interest Start 10-20amu lower than mass of fragment and several amu above. Optimize Excitation Energy for desired Fragmentation Time Exc.Time can be set to 15-20msec (minimize impact on cycle time) Typical expected Exc.Energy would range from 0.08 to 0.14. MS3 Quant Optimization can be used to find conditions (Fragments and Exc.Energy) Recommendations for MRM3 Quant Best to loop MRM3 and MRM experiment MRM3 will capture low level concentration and elimi