基于液质联用技术的抗化疗抗体大鼠体内定量分析方法研究

摘要

Objective To establish a sensitive,accurate and reliable LC-MS method to quantify the monoclonal antibody in rat blood and preclinical pharmacokinetics study. Methods The method of selective reaction monitoring(SRM)was established by using on-the-fly orthogonal array optimization(OAO)with anti-chemotherapeutic antibody-06(ACA-06)as a model drug,and the pharmacokinetics of ACA-06 in rats was studied. The optimal signature peptide selection and mass spectrometry conditions were opti?mized by single LC-MS run. Two highly specific and sensitive peptides,FSGVPDR and VNSAAFPAPIEK,were identified as the sig?nature peptides of quantification based on the OAO optimization result,which were located in the ACA-06 light chain and heavy chain complementary determining region(CDR)respectively. Results The method had good linearity in the range of 12.9~32250 ng/ml (r20.9819-0.9946). The lower limit of quantification was 12.9 ng/ml. The intra-day precision(RSD)was less than 11.5%,the inter-day RSD was less than 6%,and the accuracy was between-4.0%and 4.3%. Conclusion In this study,LC/SRM-MS method is es?tablished for the determination of ACA-06 in rat plasma. The method is specific,accurate and sensitive and enables an accurate phar?macokinetics investigation of ACA-06 in rat plasma.%目的 建立准确、灵敏、可靠的液质分析方法对以单克隆抗体为代表的蛋白药物进行血药浓度测定和临床前药代动力学研究.方法 以抗化疗抗体(ACA-06)为模型药物,采取即时正交优化(OAO)策略建立选择反应监测(SRM)技术的定向质谱分析方法,并在此基础上对ACA-06大鼠体内药代动力学进行研究.结果 通过单次进样同时完成最优信号肽段选择和质谱条件的优化,根据OAO优化结果选定分别定位于ACA-06轻链和重链互补决定区(CDR)的2个高特异性、高灵敏度的肽段FSGVPDR和VNSAAFPAPIEK作为质谱检测的信号肽段.方法确证结果表明,本法在12.9~32250 ng/ml浓度范围内线性良好(r2:0.9819~0.9946),在血浆用量仅为2μl的情况下,定量下限为12.9 ng/ml.日内精密度(RSD)<11.5%,日间RSD<6%,准确度在-4.0%~4.3%之间.结论 本研究建立了LC/SRM-MS方法测定大鼠血浆中ACA-06,本方法特异、准确、灵敏,适用于ACA-06在大鼠体内的药代动力学研究.