地非三唑的RP-HPLC法测定及其体外代谢研究

RP-HPLC DETERMINATION OF DIPHENYTRIAZOL IN RAT LIVER MICROSOMAL INCUBATES AND ITS APPLICATION IN IN VITRO METABOLISM

目的 为研究抗早孕新药地非三唑 (DL111 IT)的代谢作用机理和进一步开发利用 ,建立其体外代谢的RP HPLC测定法。方法 以LichrospherODS C1 8为色谱柱 ,甲醇 pH 7 5磷酸盐缓冲液 ( 70∶3 0 )为流动相 ,检测波长 :2 3 5nm ,流速 1 0mL·min- 1 ,地西泮为内标 ,对鼠肝微粒体孵育液中DL111 IT的RP HPLC法进行方法学研究。应用建立的方法对DL111 IT在不同来源的鼠肝微粒体中的体外代谢进行试验。结果 DL111 IT浓度在 1 0 1～ 10 1 0 μg·mL- 1呈良好的线性关系 ,在不同浓度下测得平均绝对回收率和相对回收率分别为 ( 92± 4 ) %和 ( 10 0 3± 1 9) % (n =5 ) ;DL111 IT在鼠肝微粒体中的代谢 ,β 萘黄酮组明显快于其他组。 结论 本法简便、准确 ,可用于DL111

AIM To establish a RP HPLC method for determination of diphenytriazol (DL111 IT) in rat hepatic microsomes. METHODS DL111 IT in rat hepatic microsomal incubates was extracted with chloroform, using diazepam as internal standard. The determination was performed on a Lichrospher ODS C 18 reversed column (25 cm×0 46 cm ID) with mobile phase of methanol pH 7 5 phosphate buffer (70∶30) at a flow rate of 1 0 mL·min -1 . A UV VIS detector was operated at 235 nm.RESULTS The assay was linear from 1 01～101 0 μg·mL -1 for DL111 IT. The limit of detection was 0 15 μg·mL -1 (signal to noise ratio 3) and the limit of quantification was 1 01 μg·mL -1 (RSD<10%, n =4). The method afforded average recoveries of (100 3±1 9)% ( n =5), and intra day and inter day RSD were less than 5 0%( n =5). The method allowed study of the in vitro phase I metabolism of DL111 IT in rat liver microsomal incubates. The microsomes induced by β naphthoflavone showed high enzymatic activity for DL111 IT phase I metabolism. CONCLUSION The method is simple, accurate and can be used to study the metabolism of DL111 IT in rat hepatic microsomes.