目的建立检测大鼠血浆中3种蟾蜍甾烯类化合物蟾毒灵、华蟾酥毒基及酯蟾毒配基的HPLC法,并用于蟾酥在大鼠体内的药动学研究。方法分别于大鼠尾iv给予蟾酥提取物0.8 mg/kg后2、5、10、15、20、30、45、60、90 min,眼眶取血,采用乙腈沉淀蛋白与液液萃取相结合方法进行血浆样品预处理,以HPLC法测定大鼠血浆中蟾毒灵、华蟾酥毒基及酯蟾毒配基的质量浓度,以Kinetica软件拟合药动学参数。结果蟾毒灵、华蟾酥毒基及酯蟾毒配基均得到很好的分离,重现性、精密度、线性关系良好,达到体内分析要求;经非房室模型拟合,得到蟾毒灵、华蟾酥毒基及酯蟾毒配基在大鼠体内主要药动学参数;iv给药30 min时,血药浓度均降至C_max的1/5以下。结论建立的蟾毒灵、华蟾酥毒基及酯蟾毒配基血药浓度测定方法操作简单、结果准确可靠;蟾蜍甾烯类成分在大鼠体内代谢较为迅速,所得数据可为蟾酥提取物的药动学研究提供参考。

Objective To establish an HPLC method for the assay of bufalin (BL), cinobufagin (CBG), amd resibufogenin (RBG),and for study on their pharmacokinetics in plasma of rats. Methods The rat tail was iv injected with 0.8 mg/kg Bufonis Venenum extract, then the blood was obtained form the orbit of rats after 2, 5, 10, 15, 20, 30, 45, 60, and 90 min. The combination of protein precipitation with acetonitrile and liquid-liquid extraction was applied to purifying plasma samples and HPLC was used to determine the concentration of BL, CBG, and RBG in them. The pharmacokinetic parameters were assessed by Kinetica software. Results The BL, CBG, and RBG separation, precision, accuracy and calibration curves were in line with the requirements of methodology in vivo.The main pharmacokinetic parameters were fitted by non-compartment models. After 30 min of administration in vivo, the plasma concentrations of BL, CBG, and RBG were reduced blow 20% C_maxrespectively in rats. Conclusion The established method is simple, rapid, accurate, and precise. BL, CBG, and RBG metabolism are quickly in rats, and the main pharmacokinetic parameters could be referenced in pharmacokinetic study of Bufonis Venenum extract.

“十一五”重大新药创制项目(2009ZX09502-008);“中药生产技术及过程控制技术标准平台”(2009ZX09308-003);教育部博士点基金(20090013110007);北京中医药大学中药复方制药创新团队(2011-CXTD-13)