液质联用法测定0期试验中血浆替莫唑胺浓度的方法学研究

Determination of temozolomide concentration in human plasma in phase 0 by liquid chromatography-tandem mass spectrometry

目的建立替莫唑胺血药浓度测定的液质联用方法,检测0期试验中抗肿瘤药替莫唑胺的血药浓度。方法将血浆样本用甲醇和乙腈(1∶2,含1%甲酸)作为沉淀剂进行蛋白沉淀后,涡旋离心,取上清100μL进样分析,用Phenomene C18100A柱(3.0 mm×50 mm,2.6μm),流动相为0.1%甲酸水溶液-0.1%甲酸甲醇溶液,梯度洗脱,流速为0.3 m L·min^(-1),柱温为室温,检测器采用API5500 QTrap质谱检测器,正离子模式,质谱多反应监测(MRM)扫描方式,检测样品中药物的质量浓度。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、基质效应和稳定性。结果该方法的标准曲线范围是1~200ng·m L^(-1)(r=0.995 5),最低定量下限为1 ng·m L^(-1)。血样日内、日间RSD为0.89%~10.20%和3.45%~7.32%。批内批间精密度分别为89.70%~110.00%和95.00%~101.00%,样本回收率为(96.90±3.90)%,(96.30±1.60)%,(93.20±1.40)%,稳定性较好。结论本方法快速、灵敏和专属性好,适用于替莫唑胺0期实验的血浆浓度检测(正常给药剂量的1/100)。

Objective A rapid, sensitive and specific analytical method based on highperformance liquid chromatography - tandem mass spec- trometry （LC -MS/MS） has been developed for the determination of te- mozolomide concentration in human plasma in phase 0 clinical trial. Methods After protein was precipitated by methanol and acetonitrile （1： 2,0. 1% FA）, the analyte and internal standard were separated on a KinetexC18 column using a gradient elution consisting of 0. 1% FA water solution 0. 1% FA methanol as the mobile phase at a flow rate of 0. 3 mL .min-t Thedeteetion was performed using an API 5500 QTrap mass spectrometer in the positive electrospray ionization （ESI） mode. The specificity, standard curve and lower limitation of quantitation, precision and recovery rate and stability as well as the matrix effect were investiga- ted. Results The calibration curve exhibited a linear dynamic range of 1 - 200 ng .mL- 1 （ r = 0. 995 5 ）. The intra - and inter - day precisions （ as relative standard deviation; RSD ） were 0. 89% - 10. 20% and 3.45% -7.32%. The intra- and inter- day accuracy were 89.70% - 110. 00% and 95.00% - 101.00% . The recoveries were （96.90 ± 3.90）%, （96.30 ± 1.60）%, （93.20 ±1.40）%, withsatisfied consistency. Conclusion This method could successfully used for rapid, sensitive and specific detection of plasma temozolomide in phase 0 clinical study （1/100 normal dosage）.