超高效液相色谱法检测6种探针底物代谢产物并评价人细胞色素P450同工酶活性  

Simultaneous Determination of 6 Kinds of Probe Substrate Metabolites and Rapid Evaluation of Human Cytochrome P450 Isoenzyme Activity by UPLC-MS/MS

作　　者：陆苑[1,2] 谢玉敏[1] 潘洁[1] 黄勇[1] 王永林[1,2] 

机构地区：[1]贵州医科大学贵州省药物制剂重点实验室,贵州贵阳550004 [2]贵州医科大学药学院民族药与中药开发应用教育部工程研究中心,贵州贵阳550004

出　　处：《贵阳医学院学报》2015年第6期560-565,共6页Journal of Guiyang Medical College

基　　金：国家自然科学基金项目(81260688);贵州省中药现代化科技产业研究开发专项[黔科合重G字(2013)4001];贵州省中药现代化专项项目[黔科ZY字(2013)3020];贵阳市科技计划项目[筑科合同(20141001)09号]

摘　　要：目的：建立同时测定非那西丁（CYP1A2）、氯唑沙宗（CYP2E1）、甲苯磺丁脲（CYP2C9）、奥美拉唑（CYP2C19）、睾酮和咪达唑仑（CYP3A4）6种探针底物代谢产物的Cocktail体外方法,评价药物对人肝微粒体CYP450同工酶活性的影响。方法：人肝微粒体与6种探针底物在还原系统NADPH的作用下,37℃共同孵育;以蛋白沉淀法处理样品,采用超高效液相色谱系统（UPLC-MS/MS）,建立Cocktail混合探针,对药物的6种代谢物进行定量分析,并分析药物对人肝微粒体CYP450同工酶活性的影响。结果：6种探针底物物代谢产物乙酰氨基酚（0.26~8.35μmol/L）、6-羟基氯唑沙宗（3.02~96.63μmol/L）、5-羟基奥美拉唑（0.10~3.09μmol/L）、羟基甲苯磺丁脲（0.15~4.88μmol/L）、6β-羟基睾酮（3.67~117.37μmol/L）和α-羟基咪达唑仑（1.64~52.37μmol/L）线性关系良好,各成分相关系数（R2）〉0.996,准确度为95.96%~103.10%,日内和日间精密度RSD（%）〈15%,提取回收率、基质效应RSD（%）〈11%,稳定性好。结论：UPLC-MS/MS准确、灵敏度高,可作为研究受试药物对CYP450酶影响及相关研究的分析测定方法。Objective： To establish cocktail method for simultaneous determination of 6 kinds of probe substrate metabolites： phenacetin and chlorzoxazone（ CYP1A2）, CYP2E1, tolbutamide（ CYP2C9）,omeprazole（ CYP2C19）,testosterone and midazolam（ CYP3A4） in vitro,evaluating the effects of medicine on human liver microsomes＇ CYP450 isozyme activity. Methods： Under the function of NADPH,human liver microsomes interacted with 6 kinds of probe substrates at 37℃. Using protein precipitation to handle the samples. Adopting UPLC / MS / MS and establishing Cocktail mixed probes to carry out quantitative analysis on the 6 kinds of metabolites,analyzing the effect of medicine on human liver microsomes＇ CYP450 isozyme activity. Results： A good linear relationship was found in acetaminophen（ 0. 26 ~ 8. 35 μmol·L- 1）,6-hydroxy chlorzoxazone（ 3. 02 ~ 96. 63 μmol·L- 1）,5-hydroxy omeprazole（ 0. 10 ~ 3. 09 μmol·L- 1）,hydroxytolbutamide（ 0. 15 ~ 4. 88 μmol·L- 1）,6-β-hydroxy testosterone（ 3. 67 ~117. 37 μmol·L- 1） and alpha-hydroxy midazolam（ 1. 64 ~52. 37 μmol·L- 1）. The correlation coefficient（ R2） were greater than 0. 996. The data showed that accuracy range was 95. 96% ~ 103. 10%,RSD（ %） of intra-day and inter-day precision were less than 15%,RSD（ %） of extraction recovery and matrix effect were less than 11%,as well as excellent stability. Conclusion： The method is accurate and sensitive,which can serve as a good analysis mean for effect of researching drugs on cytochrome P450 enzymes and related research.

关 键 词：色谱法 液相 细胞色素P450酶系统 肝微粒体 COCKTAIL探针药物法 

分 类 号：R963[医药卫生—微生物与生化药学]