在线固相萃取LC-MS/MS法测定健康人体全血中依维莫司浓度及在药代动力学研究中的应用

目的：为临床研究依维莫司人体药动学建立一种特异、灵敏、快速、重现性好的在线固相萃取（onlineSPE）-液相色谱-串联质谱（LC-MS/MS）方法，并将其应用于依维莫司在中国健康人群药动学研究。方法：200 μL含依维莫司全血样本与20 μL内标混合后经0.4 mol·L^-1硫酸锌80%甲醇水溶液处理，取上清液经在线固相萃取，以含0.1%甲酸5 mmol·L^-1甲酸铵水溶液-含0.1%甲酸甲醇溶液（10：90）为流动相，Waters SunFire^TM C₁₈色谱柱（4.6 mm×150 mm，3.5 μm）为分析柱，进样量50 μL，室温下测定；采用正离子模式，MRM扫描，依维莫司m/z 975.8→908.6，内标m/z 809.6→756.7，分析时间为5 min。12例健康受试者口服1.5 mg依维莫司片，于不同时间点分别采集静脉血进行药代动力学分析。结果：依维莫司质量浓度在0.201~100.7 ng·mL^-1的范围内线性良好（r=0.999 8±0.000 2），最低定量限为0.201 ng·mL^-1，回收率在99.2%~105.4%之间，以RSD表示的日内与日间精密度均小于8%，过程效率良好，所有稳定性考察项目结果均符合要求。药动学结果显示，健康受试者口服依维莫司片1.5 mg后，依维莫司在人体内平均达峰时间t_max为0.69 h，平均药峰浓度C_max为10.56 ng·mL^-1，0~96 h时间段药时曲线下面积AUC为AUC_{0-96 h} 65.70 ng·mL^-1·h，平均半衰期t_1/2 36.50 h。结论：本法经方法学验证，可用于依维莫司的药动学研究和血药浓度监测。

Objective: To offer a specific,sensitive,efficient and robust method for the quantitative determination of everolimus in the whole blood using the online solid phase extraction (SPE)-high performance liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS)system, and to investigate the pharmacokinetics profile in Chinese healthy volunteers. Methods: After mixed with internal standard(IS), 200 μL of the whole blood samples were precipitated by 0.4 mol·L^-1 zinc sulphate solution(containing 80% methanol), and the supernatant was analyzed by the online SPE LC-MS/MS system. The analyte was separated on a Waters SunFire^TM C₁₈(4.6 mm×150 mm, 3.5 μm)column with mobile phase consisted of 0.1% formic acid, 5 mmol·L^-1 ammonium formatemethanol and 0.1% formic acid(10:90). 50 μL of analyte was injected into the column. Positive electrospray ionization and multiple reaction monitoring(MRM)mode was employed. The transition of m/z 975.8→908.6 for everolimus and m/z 809.6→756.7 for the IS were monitored. Each run was 5 min. Venous blood samples for pharmacokinetic measurements were taken at different time points from 12 healthy volunteers after receiving 1.5 mg single dose of everolimus. Results: The linear range of the calibration curve(0.201-100.7 ng·mL^-1) was obtained with a good correlation coefficient(r=0.999 8±0.000 2). The lower limit of quantification was 0.201 ng·mL^-1, the average recovery was 99.2%-105.4% and the intra- and inter-day precision represented by RSD were <8%. Results for process efficiency test and all stability tests met the acceptance criteria. After oral medication,the mean peak time(t_max) of everolimus in healthy volunteers was 0.69 h, and the mean maximum concentration(C_max) was 10.56 ng·mL^-1. The area under the curve(AUC_{0-96 h}) was 65.70 ng·mL^-1·h. This drug's mean half-life was 36.50 h, Conclusion: The method is proved by methodology validation that it can be applied to analyze clinical samples and determine the pharmacokinetics of everolimus.