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目的建立同时检测小鼠血浆中4种细胞色素P450(CYP450)探针药物,即咖啡因(CYP1A2)、氨苯砜(CYP3A4)、苯妥英(CYP2C9)和氯唑沙宗(CYP2E1)的反相高效液相色谱方法。方法血浆样品用乙酸乙酯提取处理。色谱柱为Dikma Dia-monsil-C18(4.6 mm×200 mm,5.0μm),保护柱4.6 mm×10 mm(Agilent),流动相为甲醇-磷酸盐缓冲系(含0.02 mol.L-1的磷酸二氢钾,0.02 mol.L-1的三乙胺,磷酸调pH6.5),体积比为48∶52,流速1.0 mL.min-1,柱温25℃,安替比林为内标,检测波长为265 nm。结果咖啡因、氨苯砜、苯妥英和氯唑沙宗的线性范围分别为0.2~1.6,0.075~0.600,0.2~1.6,0.25~2.00 mg.L-1,4种探针药物日内、日间RSD均<5.14%,方法回收率均>95.0%(RSD<6.14%)。结论该反相高效液相色谱法快速、准确、灵敏度高、专属性强,可用于探针药物评价主要CYP450亚型酶的活性。
OBJECTIVE To establish a reverse phase high performance liquid chromatography (HPLC) system for the simultaneous detection of four cytochrome P450 (CYP450) probe drugs in mice plasma, namely, caffeine (CYP1A2), dapsone (CYP3A4), phenytoin (CYP2C9) Liquid chromatography method. Methods Plasma samples were extracted with ethyl acetate. The column was Dikma Dia-monsil-C18 (4.6 mm × 200 mm, 5.0 μm) and the guard column was 4.6 mm × 10 mm (Agilent). The mobile phase consisted of methanol-phosphate buffer Dihydrogenpotassium, 0.02 mol.L-1 triethylamine, phosphoric acid pH6.5), the volume ratio of 48:52, the flow rate of 1.0 mL.min-1, the column temperature of 25 ℃, the internal standard of antipyrine, The detection wavelength is 265 nm. Results The linear range of caffeine, dapsone, phenytoin and chlorzoxazone were 0.2 ~ 1.6,0.075 ~ 0.600,0.2 ~ 1.6,0.25 ~ 2.00 mg.L-1, 4 kinds of probe drugs daily intraday and interday RSD All <5.14%. The recoveries of the methods were all> 95.0% (RSD <6.14%). Conclusion The RP-HPLC method is rapid, accurate, sensitive and specific. It can be used as a probe drug to evaluate the activity of major CYP450 subtype enzymes.