HPLC法测定大鼠血浆中CYP450不同亚型探针药物浓度

Determination of probe concentration of CYP450 isoforms in plasma of rats by HPLC

目的:建立同时测定细胞色素P450 (cytochrome P450,CYP450)亚型CYP1A2、CYP2C9和CYP2E1活性的Cocktail探针药物溶液的高效液相色谱(HPLC)检测方法.方法:分别选择茶碱、甲苯磺丁脲和氯唑沙宗作为CYP1A2、CYP2C9和CYP2E1的特异性探针药物,根据其理化特点配制成Cocktail组合探针溶液.使用HPLC梯度洗脱法同时测定3种探针药物的血药浓度,并对其方法学进行验证.结果:本研究建立的HPLC法可以同时测定大鼠血浆中3种探针药物的浓度,各探针药物的吸收峰分离完全,无杂峰干扰;线性关系良好,线性范围为0.02~50μg/mL,最低检测限为0.02μg/mL.日内、日间精密度均小于10%,回收率在87.74%~ 112.39%之间,稳定性高,室温放置、-20℃冷冻1周和3周的RSD均小于10%.将该方法应用于研究大鼠体内此3种酶的活性,证明其具有较好的灵敏度.结论:经方法学验证,此法可同时测定3种探针药物茶碱、甲苯磺丁脲和氯唑沙宗的血浆浓度.

Objective:To establish an HPLC method for simultaneously determination of the metabolic activities of cytochrome P450 (CYP450) isoforms CYP1A2,CYP2C9和CYP2E1.Methods:Cocktail probe solution was prepared by mixing 3 specific probe drugs of CYP1A2,CYP2C9 and CYP2E1,namely theophylline,tolbutamide and chlorzoxazone respectively.An HPLC method for simultaneous determination of the above 3 probes was established and validated.Results:An HPLC method for simultaneous determination of the plasma concentrations of 3 probe drugs was established.The absorption peak of each probe was separated without interference of impurity peak.For each probe,the linear range was 0.02~50μg/mL,the minimum detection limit was 0.02μg/mL,the intraday and interday precisions were less than 10%,the recovery was between87.74%~112.39%,the RSD of stability after frozen 1 and 3 weeks 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 theophylline,tolbutamide and chlorzoxazone,thus can be applied to activity evaluation of CYP1A2,CYP2C9 and CYP2E1 with a cocktail approach.