Department of Clinical Pharmacy, The First People's Hospital of Jiashan, Jiaxing, 314100, China.
Department of Pharmacy, Wenzhou Central Hospital, Wenzhou, 325027, China.
Curr Med Chem. 2024;31(22):3426-3435. doi: 10.2174/0109298673256258231219060950.
Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well.
Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: 313.12 → 148.97 for tofacitinib, 329.10 → 165.00 for M9 and 455.12 → 308.05 for methotrexate.
The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects.
The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.
托法替布是一种用于治疗类风湿关节炎（RA）的口服 JAK 抑制剂。已评估了给予托法替布（单药或与传统合成疾病修饰抗风湿药物，主要是甲氨蝶呤（MTX）联合使用）的临床疗效和安全性。高血浆浓度和药物清除延迟可能会影响肝和/或肾功能。在这项研究中，开发了一种用于检测 Sprague Dawley（SD）大鼠血浆中甲氨蝶呤、托法替布和代谢物 M9 的超高效液相色谱-串联质谱（UPLC-MS/MS）方法，并对其进行了验证。
甲氨蝶呤、托法替布、M9 和 fedratinib（内标）通过梯度洗脱进行分离。色谱在 Acquity BEH C18（2.1mm×50mm，1.7μm）柱上进行，流动相为不同比例的乙腈和 0.1%甲酸水溶液，流速为 0.30mL/min。在正离子模式下，使用 Xevo TQ-S 三重四极杆串联质谱仪进行分析，托法替布的质量转移对为 313.12→148.97，M9 为 329.10→165.00，甲氨蝶呤为 455.12→308.05。
托法替布在 0.1-100ng/mL 范围内、M9 在 0.05-100ng/mL 范围内、甲氨蝶呤在 0.05-100ng/mL 范围内均得到良好的校准线性。甲氨蝶呤、托法替布和 M9 的定量下限（LLOQ）分别为 0.05ng/mL、0.1ng/mL 和 0.05ng/mL。日内和日间准确度值在-6.3%至 12.7%范围内，而日内和日间精密度值均≤14.4%。此外，各化合物的回收率均大于 86.5%，且无明显基质效应。
目前建立的分析方法具有同时评估甲氨蝶呤、托法替布和 M9 血浆浓度的潜力，大大缩短了检测时间，在制定剂量决策以实现个体化治疗、识别引起不良反应的药物以及最终评估药物相互作用方面发挥补充作用。
