LC-MS/MS法测定肝微粒体孵育体系中伊立替康活性代谢物及其次级代谢产物浓度  

Determination of Active Metabolite and Secondary Metabolite of Irinotecan in Rat Liver Microsomes Incubation System by LC-MS/MS

作　　者：张新林[1] 汪难喜 朱超然[1] 翟学佳[1] 吕永宁[1] Zhang Xinlin Wang Nanxi Zhu Chaoran Zhai Xuejia Lv Yongning(Department of Pharmacy, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Chin)

机构地区：[1]华中科技大学同济医学院附属协和医院药剂科,武汉430022

出　　处：《中国药师》2017年第2期238-241,共4页China Pharmacist

基　　金：国家自然科学基金项目(编号:81473287)

摘　　要：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 an LC - MS/MS method for the determination of the active metabolite（SN-38） and secondary metabolite （SN-38G） of irinoteean in rat liver microsomes incubation system, and optimize the incubation conditions. Methods： Meth- anol was selected to precipitate protein in the samples, and then the concentrations were analyzed by LC - MS/MS. All the separation was carried out on a ZORBAX Eclipse XDB-C18 column（2.1 mm × 50 mm, 3.5 μm） with the mobile phase of acetonitrile - water （containing 0.1% formic acid） （23 ： 77） at a flow rate of 0.3 ml min-1. The mass spectrometer was operated with muhiple reac- tions monitoring （MRM） using electrospray ionization （ESI）. The incubation conditions were optimized by single factor design. Resuits： SN-38 and SN-38 G showed a good linearity （ r ≥ 0.997 2） respectively within the range of 2.3-920 ng ml- 1 and 2.5-1 000 ng ml-1. The intra- and inter-day RSD was below 14.6% （n = 6）. The average recovery was within the range of 74.1%-123.4% with RSD below 13.5% （n = 6）. The optimal incubation conditions were as follows： the concentration of liver mierosomal protein was 0.3 mg m1-1 and the incubation time was 30 min. Conclusion： The method is rapid, sensitive and accurate in the quantification of SN-38 and SN-38G in the incubation system,which provides methodological basis for the activity determination of UGT1 A1 enzyme in vitro.

关 键 词：7-乙基-10羟基喜树碱 孵育体系 代谢产物 尿苷二磷酸葡萄糖醛酸转移酶1A1 高效液相色谱-质谱联用法 

分 类 号：R968[医药卫生—药理学]