舒芬太尼在体外人肝微粒体中代谢速率的测定

Measurement of metabolic rate of sufentanil in vitro of human hepatic microsome

目的:建立高效液相色谱法(HPLC)测定人肝脏微粒体中舒芬太尼的药物浓度,为进一步研究舒芬太尼的药物代谢提供新方法。方法:于人体肝组织微粒体孵育液中加入舒芬太尼,用HPLC测定孵育液中不同时点的舒芬太尼浓度,以芬太尼为内标,用正己烷-无水乙醇(19∶1)进行萃取,采用Diamonsil C18色谱柱(4.6 mm×200 mm,5μm),以乙腈-0.015 mol.L-1KH2PO4缓冲液(31∶69,pH3.0)为流动相,流速1.0mL.min-1,检测波长205 nm,进样量50μL。结果:在0.125～3 mg.L-1范围内,舒芬太尼线性关系良好(r=0.999 5),最低检测为0.1 mg.L-1,回收率为90.66%～100.02%,24例人肝脏微粒体中平均每毫克蛋白的舒芬太尼的代谢速率为(0.19±0.06)nmol.min-1;舒芬太尼与内标芬太尼分离完全,不受微粒体内源性物质的干扰,保留时间分别为9.588 min和4.765 min。结论:HPLC检测体外孵育液中肝微粒体中的舒芬太尼灵敏度高,专一性强,适用于药物体外代谢速率的研究。

OBJECTIVE To establish an HPLC method for determining the concentrations of sufentanil in human liver microsomes,and to provide a new way for investigating the metabolism of sufentanil.METHODS Sufentanil in an incubation mixture with human liver microsomes was determined by high performance liquid chromatography（HPLC） at the different time points,to calculate the metabolic rate of sufentanil.Fentanyl was used as an internal standard.The samples were extracted by liquid-liquid extraction method using n-hexane and ethanol（19∶1）.Sufentanil was determined by HPLC using of 0.015 mol·L-1 potassium,acetonitrile（31∶69,pH 3.0）was pumped at 1.0 mL·min-1 and the wavelength of detection was set at 205 nm,50 μL of sample was injected.RESULTS The assay was linear over the range of 0.125-3 mg·L-1 for sufentanil（r=0.999 5）,the limit of detection was 0.1 mg·L-1.The method afforded recoveries was 90.66%-100.02%.The metabolic rate of sufentanil in the twenty four cases of microsomes was（0.19±0.06） nmol·min-1.On the condition of this study sufentanil was totally separated with internal standard fentanyl and suffered no endogenous material intervention.Retention time of sufentanil and internal standard fentanyl was 9.588 min and 4.765 min respectively.CONCLUSION HPLC method is sensitive and specific to detect the metabolic rate of sufentanil in human hepatic microsome and helpful to investigate the metabolism of drugs in vitro.