HPLC法测定CYP450不同亚型探针药物浓度

目的: 建立同时测定细胞色素P450(cytochrome P450,CYP450)亚型CYP1A2、2D6、2E1、2C19、2C9和3A4活性的Cocktail探针药物溶液的高效液相色谱(HPLC)检测方法。方法: 分别选择咖啡因、美托洛尔、氯唑沙宗、奥美拉唑、甲苯磺丁脲和咪达唑仑作为CYP1A2、2D6、2E1、2C19、2C9和3A4的特异性探针药物,根据其各自理化特点配制成Cocktail组合探针溶液。使用HPLC-UV梯度洗脱法同时测定该6种探针药物的血药浓度,并对其进行专属性、精密度、准确度、稳定性的验证。结果: 本研究建立的HPLC检测方法,可以同时测定大鼠血浆中6种探针药物的浓度,各探针药物的吸收峰分离完全,无杂质峰干扰。线性关系良好,线性范围为0.2~50 μg·mL^-1,最低检测限为0.2 μg·mL^-1。日内、日间精密度均小于9%,绝对回收率均大于75%,相对回收率在91%~107%之间,稳定性高,冷冻30 d内的RSD小于10%,室温条件下稳定性RSD小于9%,反复冻融条件下稳定性RSD小于10%。将此方法应用于同时研究大鼠体内该6种酶的活性,证明其具有较好的灵敏度。结论: 经方法学验证,本法可以同时测定6种探针药物咖啡因、美托洛尔、氯唑沙宗、奥美拉唑、甲苯磺丁脲和咪达唑仑的血浆浓度。

Objective: To establish an HPLC method for simultaneously determination of the metabolic activities of cytochrome P450(CYP450)isoforms CYP1A2, 2D6, 2E1, 2C19, 2C9 and 3A4. Methods: Cocktail probe solution was prepared by mixing 6 specific probe drugs of CYP 1A2, 2D6, 2E1, 2C19, 2C9 and 3A4, namely caffeine, metoprolol, chlorzoxazone, omeprazole, tolbutamide and midazolam, respectively.An HPLC-UV method for simultaneous determination of the above 6 probes was established and validated by specificity, precision, recovery and stability. Results: An HPLC method for simultaneous determination of the plasma concentrations of 6 probe drugs was established.The absorption peak of each probe was separated without interference of impurity peak.For each probe, the linear range was 0.2-50 μg·mL^-1, the minimum detection limit was 0.2 μg·mL^-1, the intra-day and inter-day precisions were less than 9%, the absolute recoveries were more than 75%, the relative recoveries were between 91% and 107%, the RSD of stability after frozen 30 d was less than 10%, the RSD of room temperature stability was less than 9%, and the RSD of freeze-thaw stability was less than 10%.The method showed good sensitivity for pharmacokinetic study in vivo. Conclusion: The established HPLC method is efficient and reliable for simultaneous determination of caffeine, metoprolol, chlorzoxazone, omeprazole, tolbutamide and midazolam, thus can be applied to activity evaluation of CYP1A2, 2D6, 2E1, 2C19, 2C9 and 3A4 with a cocktail approach.