LC-MS/MS法测定SD大鼠血浆中紫杉醇前药含量及其在临床前药代动力学研究中的应用

Determination of paclitaxel prodrug in SD rat plasma by LC-MS/MS and its application in preclinical pharmacokinetic studies

本实验建立了一种液相色谱-串联质谱(LC-MS/MS)法快速、灵敏测定Sprague-Dawley(SD)大鼠血浆中紫杉醇前药(Pro-PTX)及紫杉醇(PTX)的浓度。血浆样本以乙腈(0.1%甲酸)沉淀蛋白后,选用Ultimate AQ-C18色谱柱(50 mm×3.0 mm,3μm),以乙腈-1 mmol·L^(-1)甲酸铵(含0.1%甲酸)为流动相,选用三重四极杆串联质谱仪的多重反应监测(MRM)扫描方式进行监测,选择监测离子反应分别为m/z 983.4→415.2(Pro-PTX)、m/z 854.4→286.1(PTX)和m/z 808.3→527.2(多西他赛,内标)。方法验证结果表明,血浆中Pro-PTX和PTX的线性范围分别为2.00~500 ng·mL^(-1)(r>0.99)和4.00~1000 ng·mL^(-1)(r>0.99),定量下限分别为2.00和4.00 ng·mL^(-1);Pro-PTX和PTX低、中、高三浓度的质控样本批内、批间相对标准差(RSD)均小于9%;相对偏差(RE)均在±9%的范围以内;稳定性实验中,血浆中Pro-PTX和PTX在各种贮存条件下均稳定。本方法灵敏度高,专属性、重现性好,成功用于Pro-PTX在大鼠中的药代动力学研究。本研究中实验动物的使用已获得中国医学科学院药物研究所实验动物管理与动物福利伦理委员会批准(批号:00003552)。

A fast and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of prodrug of paclitaxel(Pro-PTX)and paclitaxel(PTX)in rat plasma was developed.The plasma samples were subjected to protein precipitation with acetonitrile(0.1%formic acid),and then separated by LC with an Ultimate AQ-C18 column(50 mm×3.0 mm,3μm)and acetonitrile-1 mmol·L^(-1)ammonium formate(containing 0.1%formic acid)as the mobile phase.Multiple reaction monitoring(MRM)scanning mode was used to detect the ion responses m/z 983.4→415.2(Pro-PTX),m/z 854.4→286.1(PTX)and m/z 808.3→527.2(Docetaxel,internal standard)by using a triple quadrupole tandem mass spectrometer with electrospray ionization source and positive ion mode.The method validation results show that the linear ranges of Pro-PTX and PTX in plasma were 2.00-500 ng·mL^(-1)(r>0.99)and 4.00-1000 ng·mL^(-1)(r>0.99),the lower limit of quantification was 2.00 ng·mL^(-1)and 4.00 ng·mL^(-1),respectively;the quality control samples with low,medium and high concentrations of Pro-PTX and PTX were within the batch,the relative standard deviation(RSD)between batches were all less than 9%;the relative deviation(RE)was within the range of±9%;In the stability test,both Pro-PTX and PTX in plasma were stable under various storage conditions.The method was sensitive,specific,and reproducible,and was suitable for the pharmacokinetic study of Pro-PTX in rats.Animal experiments were approved by the Ethics Committee of Laboratory Animal Management and Animal Welfare,Institute of Materia Medica,Chinese Academy of Medical Sciences(No.:00003552).