A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib, cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for the application in daily clinical routine has been developed and validated according to the US Food and Drug Administration and European Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples with acetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5 μm (2.1 × 50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A and methanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400 μL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stable isotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min per run. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2–500 ng/mL for afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6–1500 ng/mL for cabozantinib, dabrafenib, nilotinib, and osimertinib (coefficients of correlation ≥ 0.99). Validation assays for accuracy and precision, matrix effect, recovery, carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughput and was successfully applied to monitor concentrations of kinase inhibitors in patients.

一种用于分析人血清和血浆中十种激酶抑制剂（阿法替尼、阿西替尼、博舒替尼、卡博替尼、达拉非尼、乐伐替尼、尼罗替尼、奥希替尼、鲁索替尼和曲美替尼）的液相色谱串联质谱法，用于日常临床常规应用根据美国食品和药物管理局和欧洲药品管理局的生物分析方法验证指南开发和验证。用乙腈对血浆样品进行蛋白质沉淀后，使用 Waters XBridge® Phenyl 3.5 μm (2.1 × 50 mm) 色谱柱在环境温度下进行色谱分离。流动相由以 10 mM 碳酸氢铵为 A 相的水-甲醇 (9:1, v/v) 和以 10 mM 碳酸氢铵为 B 相的甲醇-水 (9:1, v/v) 组成。以 400 μL/min 的流速应用梯度洗脱。在电喷雾电离正离子模式下使用多反应监测对分析物进行检测和定量。每种激酶抑制剂的稳定同位素标记化合物用作内标。每次运行的采集时间为 7.0 分钟。所有分析物和内标均在 3.0 分钟内洗脱。阿法替尼、阿西替尼、博舒替尼、乐伐替尼、鲁索替尼和曲美替尼的校准曲线在 2–500 ng/mL 范围内呈线性，卡博替尼、达拉非尼、尼罗替尼和奥希替尼的校准曲线在 6–1500 ng/mL 范围内呈线性（相关系数≥ 0.99）。根据监管机构的要求，准确度和精密度、基质效应、回收率、残留和稳定性的验证分析是适当的。