UPLC-MS/MS法检测大鼠血浆中阿法替尼浓度及其药动学研究

Determination and Pharmacokinetic Study of Afatinib in Rat Plasma by UPLC-MS/MS

目的:建立一种检测大鼠血浆中阿法替尼浓度的UPLC-MS/MS方法。方法:选择用乙腈沉淀的蛋白质样品进行样品处理并运用Waters XEVO TQD三重四级杆液质联用仪和CORTECS BEH C18柱(50 mm×2.1 mm,1.6μm)分离分析物。流动相由乙腈和水(0.1%甲酸)组成,流速为0.4 ml·min^(-1),柱温为40℃,内标为来那替尼。采用正离子电喷雾多反应监测(MRM)模式对分析物进行定量,目标碎片离子为阿法替尼m/z 486.19→112.1,来那替尼(IS)m/z 557.3→112.15。结果:阿法替尼在1-200 ng·ml^(-1)范围内线性关系良好(r=0.998 1),定量下限为1 ng·ml^(-1)。日内精密度和日间精密度均≤9.51%,阿法替尼从血浆中回收率高于77.1%。大鼠灌胃给药和静脉给药阿法替尼后,t_(1/2)分别为7.19 h和2.69 h,C_(max)分别为97.78 ng·ml^(-1)和123.37 ng·ml^(-1),AUC_((0-∞))分别为1 505.4 ng·h·ml^(-1)和405.55 ng·h·ml^(-1)。结论:该方法准确可靠,操作简便,重复性好,适用于灌胃和静脉注射10和2 mg·kg^(-1)剂量的阿法替尼的药动学研究。

Objective： To establish an accurate and selective UPLC-MS/MS） method for the determination of afatinib in rat plasma. Methods： Protein precipitating by acetonitrile was used to prepare the samples. A CORTECS BEH C18 column（ 50 mm×2. 1 mm,1. 6 μm） was used to separate the analytes at 40℃. The mobile phase consisted of acetonitrile and water（ 0. 1% formic acid） with the flow rate of 0. 4 ml·min-1. The analytes were quantified by multiple reaction monitoring（ MRM） mode with positive electrospray ionization,while the target fragment ions were m/z 486. 19→112. 1 for afatinib and m/z 557. 3→112. 15 for neratinib（ IS）. Results： The calibration curve obtained good linearity for afatinib within the range of 1 – 200 ng·ml-1（ r=0. 998 1）,and the LLOQ in rat plasma was 1. 0 ng/ml. The intra-and inter-day precisions were both ≤9. 51%. The recovery of afatinib from plasma was above 77. 1%. After intragastric administration and intravenous administration of afatinib in rats,the t1/2 was 7. 19 h and 2. 69 h,Cmax was 97. 78 ng·ml-1 and 123. 37 ng·ml-1,and AUC（ 0-∞）was 1 505. 4 ng·ml-1·h and 405. 55 ng·ml-1·h,respectively. Conclusion： The validated method can be applied in the pharmacokinetic study of afatinib at the intragastric and intravenous dosage of 10 and 2 mg·kg-1,respectively.