LC-MS/MS测定大鼠血浆中辛伐他汀、辛伐他汀酸和利伐沙班浓度的方法建立

Simultaneous determination of simvastatin,simvastatin acid and rivaroxaban in rat plasma by LC-MS/MS

目的建立液相色谱-串联质谱法(LC-MS/MS)同时测定大鼠血浆中辛伐他汀、辛伐他汀酸和利伐沙班的浓度。方法用乙酸乙酯对血浆样品进行二次提取处理后进样测定,分别以洛伐他汀和替格瑞洛为内标。色谱柱为Waters C18柱(150. 0 mm×4. 6 mm,3. 5μm),流动相为乙腈-醋酸铵溶液(0. 1%甲酸调至p H=4. 5)=75∶25。用电喷雾离子源,负-正离子切换模式及多反应监测扫描方式进行检测,用于定量分析的离子反应分别为m/z 419. 3→199. 1(辛伐他汀),m/z 435. 2→319. 3 (辛伐他汀酸),m/z 434. 1→390. 1 (利伐沙班),m/z521. 1→361. 2(替格瑞洛)和m/z 405. 3→199. 0 (洛伐他汀)。考察该方法的专属性、标准曲线与定量下限、精密度与回收率、稳定性和基质效应。结果血浆中的辛伐他汀、辛伐他汀酸和利伐沙班分别在1～100,5～400和1～100ng·m L^(-1)内线性关系良好,标准曲线方程分别为y=0. 78x+7. 00×10-4(r=0. 992 8),y=0. 59x+2. 57×10-2(r=0. 997 2)和y=1. 09x+1. 79×10-2(r=0. 996 4),最低定量下限分别为1,5和1 ng·m L^(-1),日内标准偏差与日间标准偏差均小于15%,提取回收率可达70%以上。血浆样品冻融3次,-80℃放置5d,4℃放置24 h稳定性良好,且不存在明显的基质效应。结论本方法是一种简便、快捷的测定大鼠血浆中辛伐他汀、辛伐他汀酸和利伐沙班3种物质浓度的LC-MS/MS法,适合于药代动力学研究中大量生物样本的测定。

Objective To establish a HPLC-MS/MS method for simultaneous determination of the concentration of simvastatin,simvastatin acid and rivaroxaban in rats plasma.Methods The plasma samples were determined after secondary extraction with ethyl acetate.Ticagrelor and lovastatin were used as internal standards.Simvastatin,simvastatin acid and rivaroxaban in rats plasma were separated by Waters C18(150.0 mm×4.6 mm,3.5μm)with mobile phase consisting of acetonitrile?ammonium(75∶25,pH=4.5).Electrospray ionization source was applied and operated in positive multiple reaction monitoring mode.Polarlity switch(negative-positive mode)was performed.The detector was operated in multiple reaction-monitoring mode at m/z 436.0→144.9 for simvastatin,m/z 452.9→162.3 for simvastatin acid,m/z 324.1→127.2 for rivaroxaban,m/z 521.1→361.2 for Ticagrelor and m/z 405.3→199.0 for lovastatin.The specificity,standard curve and lower limit ofquantification,precision and recovery,stability and matrix effect of the method were investigated.Results The linear ranges of simvastatin,simvastatin acid and rivastatin in plasma were 1-100,5-400 and 1-100 ng·m L-1,standard curve were y=0.78 x+7.00×10-4(r=0.992 8),y=0.59 x+2.57×10-2(r=0.997 2)and y=1.09 x+1.79×10-2(r=0.996 4),and the lower limit of quantitation were 1,5 and 1 ng·m L-1,respectively.The intra-day and inter-day standard deviation were less than 15%,and the recovery rate reached more than 70%.The plasma samples had a good stability with freezing-thawing for three times,-80℃for 5 days and 4℃for 24 h.And there was no obvious matrix effect.Conclusion The method was simple,specific and sensitive,suitable to detect a number of samples for pharmacokinetic study.