头孢妥仑测定方法的建立及胆汁排泄机制研究

Construction of determination of cefditoren and the investigation of mechanism of biliary excretion

目的:建立高效液相色谱内标法测定大鼠血浆、胆汁、尿液中头孢妥仑含量,阐明多药耐药相关蛋白2(Mrp2)是否与头孢妥仑胆汁排泄有关。方法:用4-二甲氨基安替比林作内标。色谱柱为CapcellpakC18UG120(4.6mm×250mm,5μm),流动相为0.1%醋酸铵-甲醇(67:33,V/V),流速为0.8mL/min,进样量为25μL;用肝灌流法探讨头孢妥仑是否经Mrp2转运。设立对照组(头孢妥仑1μmol/L)和实验组(头孢妥仑1μmol/L加Mrp2抑制剂丙磺舒20μmol/L),在设定时间点于灌流的大鼠肝脏收集出口灌流液和胆汁样品,高效液相色谱法测定样品中头孢妥仑含量,考察实验组和对照组中肝摄取率和胆汁累积排泄率的差异。结果:血浆样品中头孢妥仑在0.1～4μg/mL范围内、胆汁样品中头孢妥仑在0.1～5μg/mL范围内、尿液样品中头孢妥仑在0.1～5μg/mL范围内线性关系良好。回收率为85%～115%,日内、日间RSD均小于13.5%。实验组和对照组相比,肝摄取率变化无统计学意义上的差别;实验组中,肝灌流25min后,头孢妥仑胆汁累积排泄率减少至对照组的40.0%。结论:本方法经济、简单、灵敏、快速,可用于头孢妥仑血浆、胆汁、尿液样品中药物浓度检测和药物代谢动力学研究;头孢妥仑是Mrp2的底物,Mrp2与头孢妥仑的胆汁排泄有关。

AIM： To develop a high-perfonnance liquid chromatography internal standard method for determining of cefditoren in the rat plasma, bile and urine, and to clarify the relation between multidrug resistance-associated protein 2 （Mrp2） and biliary excre- tion of cefditoren using perfused rat livers. METH- Oil： 4-dimethylaminoantipyrine was used as the internal standard. Chromatographic separation was performed on a C18 UG 120 column （4.6 mm ×250 mm, 5μm） and mobile phase was composed of 0.1% ammonium acetate and methyl alcohol （67： 33, V/V）. The flow rate was 0.8 mL/min and 25 μL of mixture was injected. Perfused rat livers were performed to investigate whether cefditoren was transported via Mrp2. We established the control （cefditoren 1μmol/L） and experimental group （cefditoren 1 /nnol/L added the in- hibitor of Mrp2-probenecid, 20 μmol/L）. Perfusate samples and bile were collected at setting times. Cefditoren was determined by HPLC. We compared the hepatic extraction ratio and cumulative biliary excretion rates in control and experimental groups. RESULTS： The concentration range of cefditoren was 0.1 - 4 μg/mL in plasma, 0.1 - 5μg/mL in bile and in urine, with good linearity. The relative recovery was 85 % - 110%. The intra- and inter-day RSD were 〈 13.5%. The hepatic extraction ratio showed no statistically sig- nificant differences, whereas cumulative biliary excretion rates were significantly reduced to 40.0% compared to control over 25 min in experimental group. CONCLUSION： It can be used for the determination of eefditoren concentration in plasma, bilary and uri- nary samples and for pharmacokinetic study since it is economic, simple, sensitive and fast method. Cefditoren is the substrate of Mrp2, which is related with biliary excretion of eefditoren.