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目的:建立LC-MS/MS法测定大鼠肝微粒体酶孵育体系中伊立替康活性代谢物(SN-38)及其次级代谢产物(SN-38G)的浓度,并对体外孵育条件进行优化。方法:采用蛋白沉淀法,用甲醇(含0.1%的甲酸)溶液处理孵育样本后,直接采用LC-MS/MS法测定分析。色谱柱采用ZORBAX Eclipse XDB-C_(18)柱(2.1 mm×50 mm,3.5μm),柱温35℃,流动相由乙腈-0.1%甲酸水(23∶77)组成,流速0.3 ml·min~(-1);采用电喷雾离子源(ESI),以多反应监测方式(MRM)进行定量分析。通过单因素法对各孵育条件进行优化。结果:SN-38、SN-38G分别在2.3~920 ng·ml~(-1)、2.5~1 000 ng·ml~(-1)内线性关系良好(r≥0.997 2);日内、日间精密度RSD均小于14.6%(n=6);回收率均在74.1%~123.4%之间,RSD均小于13.5%(n=6)。最佳的孵育体系为:肝微粒体蛋白浓度为0.3 mg·ml-1,孵育时间为30 min。结论:本研究所建立的LC–MS/MS法分析快速、灵敏、准确,适于体外孵育体系中SN-38与SN-38G浓度的测定,并为葡萄糖醛酸转移酶1A1(UGT1A1)体外活性测定提供方法学基础。
OBJECTIVE: To establish a LC-MS / MS method for the determination of the concentration of irinotecan active metabolite (SN-38) and its secondary metabolite (SN-38G) in rat liver microsomal enzyme incubation system and to optimize the in vitro incubation conditions . Methods: The protein was precipitated and treated with methanol (containing 0.1% formic acid). The samples were directly analyzed by LC-MS / MS. The column was operated on a ZORBAX Eclipse XDB-C 18 column (2.1 mm × 50 mm, 3.5 μm) with a column temperature of 35 ° C. The mobile phase consisted of acetonitrile-0.1% formic acid water (23:77) (-1). The electrospray ionization source (ESI) was used to carry out quantitative analysis by multiple reaction monitoring (MRM). Each incubation condition was optimized by the single factor method. Results: The linear relationship between SN-38 and SN-38G was 2.3 ~ 920 ng · ml ~ (-1) and 2.5 ~ 1 000 ng · ml ~ (-1) respectively (r≥0.997 2) The RSDs were less than 14.6% (n = 6). The recoveries ranged from 74.1% to 123.4% with RSDs less than 13.5% (n = 6). The best incubation system: liver microsomal protein concentration of 0.3 mg · ml-1, incubation time of 30 min. Conclusion: The LC-MS / MS method established in this study is rapid, sensitive and accurate and suitable for the determination of SN-38 and SN-38G concentrations in in vitro incubation system. It is also suitable for in vitro activity of glucuronosyltransferase 1A1 (UGT1A1) Measurement provides the basis of methodology.