HPLC-MS/MS法测定比格犬血浆中PA-824的浓度及其药动学研究

Determination and Pharmacokinetics of PA-824 in the Plasma of Beagle Dogs by HPLC-MS/MS

目的:建立高效液相色谱-质谱连用(HPLC-MS/MS)测定比格犬血浆中PA-824的浓度,以及进行PA-824在比格犬体内的药动学研究。方法:以卡马西平为内标,生物样品预处理采用乙酸乙酯萃取。色谱柱为Eclipse Plus C18柱(100 mm×2.1 mm,3.5μm),流动相为甲醇-水(90∶10),流速为0.6 ml·min^(-1),柱温为30℃,进样量为5μl,样品分析时间为5 min;电喷雾电离源(ESI),正离子多反应监测扫描分析,PA-824离子对为m/z 360.1→m/z 175.0(碰撞能量:35,解簇电压:65),卡马西平离子对为m/z 237.2→m/z 194.0(碰撞能量:28,解簇电压:83)。比格犬口服给药之后,在不同的时间点取血测定血浆中PA-824的含量,然后再用DAS 2.0软件进行药代动力学参数的计算。结果:比格犬口服给药之后,PA-824在50~10 000 ng·ml^(-1)(r=0.999 1)范围内呈线性关系,回收率为97.7%~105.1%,日内、日间精密度RSD均<5.0%。PA-824 3个不同给药量(100,200,500 mg)的AUC0-t分别为(5 735.18±1 918.76),(11 548.47±1 838.04),(21 987.88±4 587.58)ng·min·ml^(-1),t1/2分别为(14.17±5.97),(11.11±4.39),(13.13±5.46)h,Cmax分别为(626.66±188.48),(2 399.13±516.51),(4 861.33±2 253.61)ng·ml^(-1)。结论:该方法简单、准确、快速、重复性好,适用于比格犬血浆PA-824的药代动力学研究。

Objective： To establish a high performance liquid chromatography-tandem mass spectrometric method （HPLC-MS/ MS） method for the determination of PA-824 in the plasma of Beagle dogs, and study the pharmacokinetics of PA-824 in Beagle dogs. Methods： Carbamazepine was used as the internal standard, and the plasma samples were pretreated with ethyl acetate for the liquid- liquid extraction of PA-824. An Eclipse Plus Clscolumn （100 mm 2.1mm,3.5μm） was used with the mobile phase consisting of methanol-water （90 ： 10）. The flow rate was 0.6 ml min-1 and the column temperature was 30 ℃. The injection volume was 5 μl and the sample analysis time was 5 min. The determination was performed with an electrospray ionization （ESI）source in the positive multiple reaction monitoring （MRM） mode. The ion pairs were m/z 360.1--m/z 175.0 （collision energy of 35, solution cluster volt- age of 65 ） for PA-824 and m/z 237.2--m/z 194.0 （ collision energy of 28, solution cluster voltage of 83 ） for carbamazepine. After the oral administration, PA-824 in plasma was measured at different time points, and then the pharmacokinetic parameters were calcu- lated by DAS 2.0 software. Results： PA-824 showed a good linear relationship within the range of 50-10 000 ng m1-1 （r = 0. 999 1 ）. The recovery was 97.7% -105.1%, and the RSDs of intra-day and inter-day were less than 5.0%. At three different dosa- ges （100,200 and 500 mg） of PA-824, AUCo.,were （5 735.18 ± 1 918.76） ,（11 548.47 ± 1 838.04） and （21 987.88 ±4587.58） ng min ml -1, t1/2 were （ 14.17 ± 5.97 ）, （ 11.11± 4.39 ） and （ 13.13 ± 5.46 ） h, and C max were （ 626.66 ± 188.48 ）, （ 2 399.13 ± 516.51 ） and （4 861.33 ±2 253.61 ）ng ml^-1, respectively. Conclusion： The method is simple, accurate, rapid and reproducible, and suitable for the pharmacokinetic study of PA-824 in the plasma of Beagle dogs.