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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 a high performance liquid chromatography (HPLC) method for the determination of sufentanil concentration in human liver microsomes, and to provide a new method for the further study of sufentanil drug metabolism. Methods: Sufentanil was added into human liver tissue microsomes. The concentration of sufentanil at different time points in the incubation solution was determined by HPLC. The internal standard of fentanyl was treated with n-hexane - absolute ethanol (19 : 1), using a Diamonsil C18 column (4.6 mm × 200 mm, 5 μm) with acetonitrile-0.015 mol·L-1 KH 2 PO 4 buffer (31:69, pH 3.0) as the mobile phase at a flow rate of 1.0 mL · min -1, detection wavelength 205 nm, injection volume 50μL. Results: There was a good linear relationship between sufentanil (0.999 5) and 0.1 mg.L-1 in the range of 0.125 ~ 3 mg.L-1, and the recoveries were 90.66% -100.02%. 24 patients Metabolic rates of sufentanil per mg of protein in liver microsomes were (0.19 ± 0.06) nmol.min-1. Sufentanil was completely separated from the internal standard fentanyl and was not disturbed by endogenous microsomal substances , Retention time was 9.588 min and 4.765 min respectively. Conclusion: Supuntamycin in liver microsomes detected by HPLC in vitro has high sensitivity and specificity, which is suitable for the study of drug metabolism rate in vitro.