高效液相色谱法测定大鼠血浆中新型抗纤维化药物美氟尼酮的浓度及其药物动力学研究

摘要

Mefunidone (MFD),a pirfenidone analogue,has been suggested as a novel anti-fibrotic agent in preclinical research stage.In this work,we developed a sensitive and specified HPLC-UV method and validated it for the determination of MFD in rat plasma.A cost-effective protein precipitation method using methanol was used to process the plasma samples,and pirfenidone was employed as the internal standard (IS).Chromatographic separation was performed on an Agilent ZORBAX SB-Aq column (4.6 mm×250 mm,5 μm) with a mobile phase consisting of 10 rnM ammonium formate solution (pH 3.0,adjusted by 1.5‰o formic acid)-acetonitrile-methanol (60:23:17,v/v/v) at a flow rate of 1.0 mL/min,and the samples were monitored at an ultraviolet wavelength of 245 nm.The retention times of MFD and IS were 5.5 and 7.8 min,respectively.The calibration curve was linear (r2=0.9997) between 0.1 and 20 μg/mL.The intra-and inter-day precisions were within 8.6％,and the bias of intra-and inter-accuracies of the method was between-4.2％ and 6.5％.The method was successfully applied to pharmacokinetic study of MFD after i.g.and i.v.administration in rats.The elimination half-life was (3.41±0.81) h for i.g.administration and (2.26±0.87) h for i.v.administration.The absolute bioavailability of MFD in rat was 79.1％.%作为吡非尼酮的类似物,美氟尼酮在临床前研究中显示了良好的抗纤维化活性,具有开发成为新型抗纤维化药物的潜力.本研究建立并验证了测定大鼠血浆中美氟尼酮浓度的高效液相色谱方法.该方法选择吡非尼酮作为内标,采用甲醇沉淀蛋白制备血浆样本.色谱条件:Agilent ZORBAX SB-Aq柱(4.6 min×250 mm,5μm),流动相10mM的甲酸铵(添加1.5‰的甲酸调节pH至3.0)-乙腈-甲醇(60:23:17,v/v/v),流速为1.0 mL/min,紫外检测波长为245 nm.美氟尼酮和吡非尼酮色谱峰的保留时间分别为5.5min和7.8min.美氟尼酮在0.1-20 μg/mL范围内具有良好的线性关系(r2=0.9997).本方法的批间和批内准确度偏差为-4.2％～6.5％,批间和批内的精密度均小于8.6％.该方法成功应用于美氟尼酮的大鼠单剂量灌胃和单剂量尾静脉注射给药的药代动力学研究.灌胃给药和尾静脉注射美氟尼酮后,药物在大鼠体内的消除半衰期分别为(3.41±0.81)h和(2.26±0.87)h,其绝对生物利用度为79.1％.