地骨皮甲素盐酸盐的体外代谢稳定性和体内药动学研究

目的 建立测定大鼠肝微粒体和血清中地骨皮甲素盐酸盐(kukoamine A hydrochlorid,KuA-H)含量的方法,研究其在肝微粒体中的代谢稳定性和血清中的药动学特征。方法 将含KuA-H的大鼠肝微粒体孵育体系于37℃的水浴下分别孵育多个时间点时加入含76 ng·mL^-1卡马西平的冰乙腈终止反应,再采用乙腈沉淀蛋白对血样预处理,以HPLC-MS/MS测定大鼠肝微粒体和血清中KuA-H的含量。以ACE Excel Super C₁₈为色谱柱,以水(0.1%甲酸)-乙腈(0.1%甲酸)为流动相梯度洗脱,流速为0.2 mL·min^-1,柱温40℃,进样量5 μL;采用电喷雾离子源,以多反应监测模式进行正离子检测,分别以m/z 531.3→222.4(KuA-H)、m/z 237.3→192.2(内标)为定量分析的离子对。以孵育0 min时KuA-H的含量为参照,计算其在大鼠肝微粒体孵育体系中剩余百分率,测定大鼠灌胃给药后不同时间点血样中KuA-H含量,用DAS 2.0软件计算药动学参数。结果 KuA-H在大鼠肝微粒体和血清中检测的线性范围分别为30~900 ng·mL^-1和12.5~2 000 ng·mL^-1,精密度和准确度、提取回收率、基质效应和稳定性均符合生物样品定量分析要求。KuA-H在大鼠肝微粒体中孵育60 min内剩余百分率范围为85.5%~101.5%,药动学参数:C_max为(107.9±29.2)ng·mL^-1,t_1/2为(4.71±1.92)h,AUC_0-t为(252.2±34.7)ng·h·mL^-1,t_max为(0.46±0.10)h。结论 建立的HPLC-MS/MS快速、灵敏,适用于KuA-H体外代谢稳定性和体内药动学研究。

Metabolic Stability In vitro and Pharmacokinetic Studies In vivo of Kukoamine A Hydrochlorid

OBJECTIVE To establish a method for determining the content of kukoamine A hydrochloride(KuA-H) in liver microsomes and plasma of rats, and to study its metabolic stability in liver microsomes and pharmacokinetics characteristics in plasma. METHODS The rat liver microsome incubation system containing KuA-H was incubated in a water bath at 37℃ for multiple time points, and ice acetonitrile containing 76 ng·mL^-1 carbamazepine was added to terminate the reaction. The plasma samples were precipitated with acetonitrile, and the content of KuA-H in rat liver microsomes and plasma was determined by HPLC-MS/MS. The determination was performed on ACE Excel Super C₁₈ column with mobile phase consisted of water(0.1% formic acid)-acetonitrile(0.1% formic acid) for gradient elution at the flow rate of 0.2 mL·min^-1. The column temperature was set at 40℃, and the injection volume was 5 μL. Electrospray ionization source was performed in a positive electrospray ionization mode in the multiple reaction monitoring mode. The ion transitions for quantitative analysis were m/z 531.3→222.4 (KuA-H) and m/z 237.3→192.2(internal standard), respectively. Taking the content of KuA-H at 0 min of incubation as a reference, the remaining percentage of it in the rat liver microsome incubation system was calculated, and the content of KuA-H in plasma samples at different time points was determine after intragastric administration. The pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS The linear ranges of KuA-H in rat liver microsomes and plasma were 30-900 ng·mL^-1 and 12.5-2 000 ng·mL^-1, respectively. The precision and accuracy, extraction recovery rate, matrix effect and stability met the quantitative analysis requirements of biological sample. The remaining percentage of KuA-H within 60 min of incubation in rat liver microsomes ranged from 85.5% to 101.5%. The main pharmacokinetic parameters were as follows:C_max was (107.9±29.2)ng·mL^-1, t_1/2 was (4.71±1.92)h, AUC_0-t was (252.2±34.7)ng·h·mL^-1, and t_max was (0.46±0.10)h. CONCLUSION The HPLC-MS/MS established is rapid and sensitive, which is suitable for in vitro metabolic stability and in vivo pharmacokinetic studies of KuA-H.