液质联用测定血浆替莫唑胺的浓度

Determination of temozolomide concentration in human plasma by liquid chromatographytandem mass spectrometry

目的建立一个快速、灵敏和专属性好的液质联用方法,来检测等效性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,使用Phenomene C_(18) 100A柱(3.0 mm×50 mm),采用等度洗脱,流动相为0.1%甲酸水溶液-0.1%甲酸甲醇溶液(9∶1),流速为0.3 m L·min^(-1),检测器采用API5500 QTrap质谱检测器,正离子模式,MRM扫描方式,待测物和内标的离子对分别是是m/z 195.1→m/z 138.0(替莫唑胺)和m/z 198.1→m/z 138.0(替莫唑胺-d3,内标)。结果该方法的标准曲线范围是0.05~30μg·m L^(-1)(r>0.9973),最低定量下限为0.05μg·m L^(-1)。血样日内、日间RSD为1.06%~7.13%和1.80%~5.19%,批内批间准确度分别为:91.02%~109.33%和95.14%~101.22%,样本回收率、基质效应和专属性都满足检测要求。结论该方法快速、灵敏和专属性好,成功用于替莫唑胺等效性实验的血浆浓度检测。

Objective To develop a rapid,sensitive and specific analytical method based on liquid chromatographytandem mass spectrometry（ LC-MS/MS） for the determination of temozolomide concentration in human plasma in equivalence trial clinical trial. Methods After protein sediment by methanol and acetonitrile（ 1 ∶ 2,0. 1% formic acid）,the analyte and internal standard were separated on a Kinetex C（18） column（ 3. 0 mm × 50 mm） using a isocratic elution consisting of 0. 1% formic acid water solution 0. 1% formic acid and methanol（ 9∶ 1） as the mobile phase at a flow rate of 0. 3 m L · min^-1. The detection was performed using an API 5500 QTrap mass spectrometer in the positive electrospray ionization（ ESI） mode. The multiple reaction monitoring（ MRM） transitions were m/z 195. 1→m/z 138. 0 for the analyte and m/z 198. 1→m/z138. 0 for temozolomide-d3. Results The calibration curve exhibited a linear dynamic range of 0. 05-30 μg·m L^-1（ r 〉0. 997 3）. The intra-and inter-day precisions（ relative standard deviation, RSD） were1. 06%-7. 13% and 1. 80%-5. 19%. The intraand interday precisions were 91. 02%-109. 33% and 95. 14%-101. 22%. The recoveries and matrix effects were satisfactory in all the biological matrices examined.Conclusion This method was successfully used for the determination of temozolomide concentration in human plasma in equivalence trial.