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目的建立同时测定大鼠离体肝微粒体中5种细胞色素P-450特异性底物含量的方法。方法选择非那西丁、甲苯磺丁脲、右美沙芬、氯唑沙宗和咪达唑仑分别作为CYP1A、CYP2C、CYP2D、CYP2E、CYP3A的特异性底物。采用高效液相色谱法,以艾司唑仑为内标,色谱柱为Welchrom C18,流动相为乙腈-0.01 mol.L-1乙酸胺(pH 5.25,含0.03%三乙胺)=40∶60,流速为1 ml.min-1,检测波长分别为202,247,280,220 nm,柱温为30℃,进样量为40μl。结果在线性范围内线性关系良好,相关系数r>0.999 0;日内RSD均小于14.4%,日间RSD均小于14.7%;方法回收率在为91.1%～114.3%之间,提取回收率在84.6%～94.9%之间。结论该方法灵敏、简便、准确,可用于大鼠肝微粒体中细胞色素P-450特异性底物的含量测定。
Objective To establish a method for the simultaneous determination of five cytochrome P-450 specific substrates in isolated rat liver microsomes. Methods Phenacetin, tolbutamide, dextromethorphan, chlorzoxazone and midazolam were selected as specific substrates for CYP1A, CYP2C, CYP2D, CYP2E and CYP3A, respectively. High performance liquid chromatography with estazolam as internal standard, the column was Welchrom C18, the mobile phase was acetonitrile-0.01 mol·L-1 acetic acid amine (pH 5.25, containing 0.03% triethylamine) = 40:60 , The flow rate was 1 ml.min-1, the detection wavelength was 202,247,280,220 nm, the column temperature was 30 ℃, the injection volume was 40μl. The results showed good linearity in the linear range with correlation coefficient r> 0.999 0. The intra-day RSD was less than 14.4% and the intra-day RSD was less than 14.7%. The recoveries ranged from 91.1% to 114.3% and the recovery rates were 84.6% ~ 94.9% between. Conclusion The method is sensitive, simple and accurate and can be used for the determination of cytochrome P-450-specific substrate in rat liver microsomes.