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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0.2~50μg·m L-1,最低检测限为0.2μg·m L-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 a HPLC method for the simultaneous determination of the activity of the Cocktail probe of CYP1A2, 2D6, 2E1, 2C19, 2C9 and 3A4 cytochrome P450 (CYP450) subtypes. Methods: Caffeine, metoprolol, chlorzoxazone, omeprazole, tolbutamide and midazolam were selected as specific probes for CYP1A2, 2D6, 2E1, 2C19, 2C9 and 3A4, According to their own physical and chemical characteristics of the cocktail probe solution was prepared. The HPLC-UV gradient elution method was used to simultaneously determine the plasma concentrations of the six probe drugs and to verify their specificity, precision, accuracy and stability. Results: The HPLC assay established in this study can determine the concentration of 6 kinds of probe drugs in the plasma of rats at the same time. The absorption peak of each probe drug is completely separated without the interference of impurity peak. The linear range was 0.2 ~ 50μg · m L-1 with the lowest detection limit of 0.2μg · m L-1. The intra-day and inter-day precision were less than 9%, the absolute recoveries were more than 75%, the relative recoveries were between 91% and 107%, the stability was high, the RSD within 30 days of freezing was less than 10%, and the stability at room temperature RSD is less than 9%, RSD less than 10% under repeated freezing and thawing conditions. This method was applied to the simultaneous study of the activity of the six kinds of enzymes in rats, which proved to have good sensitivity. CONCLUSION: This method can be used to determine the plasma concentrations of six probe drugs caffeine, metoprolol, chlorzoxazone, omeprazole, tolbutamide and midazolam simultaneously.