Ren Xinxin, Wang Zhipeng, Yun Yunlei, Meng Guangyi, Zhang Xialan, Ding Huamin, Xu Ying, Bai Hansheng, Liu Jing, Li Xia, Gao Shouhong, Huang Lifeng, Chen Wansheng
Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Center for Molecular Medicine, Xiangya Hospital, Key Laboratory of Molecular Radiation Oncology of Hunan Province, Central South University, Changsha 410008, China.
Int J Anal Chem. 2019 Feb 3;2019:1536532. doi: 10.1155/2019/1536532. eCollection 2019.
To establish and validate a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of methotrexate (MTX) and its major metabolite 7-hydroxy-methotrexate (7-OH-MTX) in human plasma.
The chromatographic separation was achieved on a Zorbax C column (3.5 m, 2.1 × 100 mm) using a gradient elution with methanol (phase B) and 0.2% formic acid aqueous solution (phase A). The flow rate was 0.3 mL/min with analytical time of 3.5 min. Mass spectrometry detection was performed in a triple-quadruple tandem mass spectrometer under positive ion mode with the following mass transitions: m/z 455.1/308.1 for MTX, 471.0/324.1 for 7-OH-MTX, and 458.2/311.1 for internal standard. The pretreatment procedure was optimized with dilution after one-step protein precipitation.
The calibration range of methotrexate and 7-OH-MTX was 5.0-10000.0 ng/mL. The intraday and interday precision and accuracy were less than 15% and within ±15% for both analytes. The recovery for MTX and 7-OH-MTX was more than 90% and the matrix effect ranged from 97.90% to 117.60%.
The method was successfully developed and applied to the routine therapeutic drug monitoring of MTX and 7-OH-MTX in human plasma.
建立并验证一种简单、灵敏、快速的液相色谱串联质谱法（LC-MS/MS），用于测定人血浆中甲氨蝶呤（MTX）及其主要代谢物7-羟基甲氨蝶呤（7-OH-MTX）。
采用Zorbax C柱（3.5μm，2.1×100mm）进行色谱分离，以甲醇（B相）和0.2%甲酸水溶液（A相）进行梯度洗脱。流速为0.3mL/min，分析时间为3.5分钟。在三重四极杆串联质谱仪上采用正离子模式进行质谱检测，其质荷比跃迁如下：MTX为m/z 455.1/308.1，7-OH-MTX为m/z 471.0/324.1，内标为m/z 458.2/311.1。预处理程序经一步蛋白沉淀后稀释进行优化。
甲氨蝶呤和7-OH-MTX的校准范围为5.0 - 10000.0 ng/mL。两种分析物的日内和日间精密度及准确度均小于15%且在±15%以内。MTX和7-OH-MTX的回收率均超过90%，基质效应范围为97.90%至117.60%。
该方法已成功开发并应用于人血浆中MTX和7-OH-MTX的常规治疗药物监测。
