LC-MS/MS法定量测定人全血中替西罗莫司及西罗莫司的药物浓度

LC-MS/MS determination of temsirolimus and sirolimus in human whole blood

目的:为临床研究替西罗莫司及西罗莫司人体药物代谢动力学建立一种特异、灵敏、快速、重现性好的液质联用的定量分析方法。方法:使用100μL含待测药物的全血,加入内标D_7-替西罗莫司和32-去甲氧基西罗莫司及萃取剂混匀,采用液液萃取的方法进行样品前处理。HPLC色谱柱为Alltima C_(18)(2.1 mm×50 mm,3.0μm);柱温51℃;流动相为甲醇-10 mmol·L^(-1)醋酸铵(pH 5.1)(80:20);进样量20μL;室温下测定。质谱检测方式为ESI^+,MRM扫描,监测替西罗莫司m/z 1047.5→.980.4,内标D_7-替西罗莫司m/z 1054.6→987.6,西罗莫司m/z 931.5→864.5,内标32-去甲氧基西罗莫司m/z 901.5→834.4。分析时间4.0 min。结果:替西罗莫司在2.5～2500 ng·mL^(-1),西罗莫司在2.5～250 ng·mL^(-1)的范围内,本法线性良好(r>0.99),最低定量限为2.5 ng·mL^(-1),以RSD表示的日内与日间精密度替西罗莫司<7.7%,西罗莫司<14.5%,以相对偏差RE表示的准确度替西罗莫司<±13.6%,西罗莫司<±11.2%。所有稳定性考察项目结果均符合要求。结论:本法在国内首次采用液液萃取的样品前处理方法以及液质联用的定量测定方法同时定量测定替西罗莫司及西罗莫司,建立了一种可以应用于临床试验分析需求的生物样品定量分析方法。

Objective：To develop a specific,sensitive,efficient and robust method using LC - MS/MS technique for the quantitative determination of temsirolimus and sirolimus in human whole blood.Method：After mixed with IS （D7- CCI -779 and 32 - desmethoxyrapamycin） and extraction agent,100μL of human whole blood containing the test drugs was extracted through liquid - liquid extraction.On HPLC,a reversed - phase analytical column of Alltima C18（2.1 mm×50 mm,3.0μm,） was adopted and the column temperature was 51℃,the mobile phase was methanol -10 mmol·L^-1 ammonium acetate（pH 5.1）（80：20）.20μL of analyte was injected onto the column. Positive electro - spray ionization and multiply reaction monitoring（MRM） mode was employed.The transition of m/z was 1047.5→980.4 for temsirolimus,1054.6→987.6 for D7 -CCI -779,931.5→864.5 for sirolimus and 901.5→834.4 for 32 - desmethoxyrapamycin.Each run was 4.0 min.Results：The linear range of the calibration curve（2.5 -2500 ng·mL^-1 for temsirolimus and 2.5 -250 ng·mL^-1 for sirolimus） with a good correlation coefficient （r〉0.99） was obtained.The lower limit of quantification was 2.5 ng·mL^-1 and the intra - and inter - day precisions represented by RSD were〈7.7%for temsirolimus and〈14.5%for sirolimus.The accuracies represented by RE were±13.6%for temsirolimus and±11.2%for sirolimus.Results for all stability tests met the acceptance criteria.Conclusion：It is the first time to set up a liquid - liquid extraction method combined with LC-MS/MS detection to quantify the concentration of temsirolimus and sirolimus at the same time.The results indicate that the present quantitative method is satisfied for the analysis of temsirolimus and sirolimus in human whole blood.