Purpose: Precise and timely detection of methotrexate (MTX) concentration played a key role in high-dose MTX individualization therapy in acute lymphoblastic leukemia (ALL) children to avoid serious adverse effects or nonresponse. This report described a sensibility and validation of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for therapeutic drug monitoring (TDM) of methotrexate concentration in children’s plasma.
Methods: One-step protein precipitation of samples was accomplished by adding 200 μL of acetonitrile to 100 μL of plasma sample. The separation of plasma samples was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution HD column with gradient elution using a mobile phase constituted of acetonitrile and 1% formic acid. The detection was executed by electrospray ionization (ESI) of triple quadrupole tandem mass spectrometer (TQMS) in the multiple reaction monitoring (MRM) mode with the transitions m/z 455.2 → 307.9 for methotrexate and m/z 458.2 → 311.2 for IS, separately. Linear concentration range of the calibration curve was 44– 11,000 nmol/L and 44 nmol/L was the lower limit of quantification.
Results: The methotrexate elution time was at 1.577 min, and the overall running time was only 3.3 min. The intra- and interday precision for all the analysis results was within 11.24%, and mean recoveries rate of methotrexate exceeded 87.98%.
Conclusion: The described and fully validated UHPLC-MS/MS method was successfully applied in clinical TDM after infusion of high-dose methotrexate 1– 5 g/m² to 41 childpatients.

Keywords: high-dose methotrexate, UHPLC-MS/MS, therapeutic drug monitoring, children, acute lymphoblastic leukemia


中文翻译：

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UHPLC-MS/MS 分析的开发和验证，用于监测儿童急性淋巴细胞白血病的大剂量甲氨蝶呤治疗药物

目的：准确及时地检测甲氨蝶呤（MTX）浓度在急性淋巴细胞白血病（ALL）儿童大剂量甲氨蝶呤个体化治疗中发挥关键作用，以避免严重的不良反应或无反应。本报告描述了超高效液相色谱串联质谱 (UHPLC-MS/MS) 方法用于儿童血浆中甲氨蝶呤浓度的治疗药物监测 (TDM) 的敏感性和验证。
方法：通过向 100 μL 血浆样品中加入 200 μL 乙腈来完成样品的一步蛋白质沉淀。血浆样品的分离在 ZORBAX Eclipse Plus C18 Rapid Resolution HD 色谱柱上进行，采用梯度洗脱，流动相由乙腈和 1% 甲酸组成。检测通过三重四极杆串联质谱仪 (TQMS) 在多反应监测 (MRM) 模式下的电喷雾电离 (ESI) 进行，甲氨蝶呤的离子对 m/z 455.2 → 307.9 和 IS 的 m/z 458.2 → 311.2，分别. 校准曲线的线性浓度范围为 44–11,000 nmol/L，44 nmol/L 为定量下限。
结果：甲氨蝶呤洗脱时间为1.577 min，总运行时间仅为3.3 min。所有分析结果的日内和日间精密度均在11.24%以内，甲氨蝶呤平均回收率超过87.98%。
结论：在向 41 名儿童患者输注大剂量甲氨蝶呤 1–5 g/m ^{2后，所描述且经过充分验证的 UHPLC-MS/MS 方法成功应用于临床 TDM。}

关键词：大剂量甲氨蝶呤，UHPLC-MS/MS，治疗药物监测，儿童，急性淋巴细胞白血病