生物样品中5种CYP450酶底物分析方法的建立
Establishment of a method for the analysis of five CYP450 enzyme substrates in biological samples
母磊鑫 1刘湉 1柯彩依 1白延婷 1马群 1陈昭2
作者信息
- 1. 北京中医药大学中药学院,北京 102488
- 2. 中国中医科学院中医临床基础医学研究所,北京 100700
- 折叠
摘要
目的 采用HPLC法建立生物样品中测定CYP450酶底物的分析方法.方法 通过钙沉淀法制备肝微粒体,采用探针底物与大鼠肝微粒体体外温孵法进行孵育反应,以氢溴酸右美沙芬、奥美拉唑、非那西丁、硝苯地平、甲苯磺丁脲分别作为CYP2D6、CYP2C19、CYP1A2、CYP3A4、CYP2C9的探针药物,并以大鼠肝微粒体为基质,通过系统考察建立了 HPLC法测定5种探针药物的方法.结果 该方法专属性好,空白肝微粒体及内标无干扰,右美沙芬线性回归方程为Y=0.001 6X+0.003 7(r=0.994 8),奥美拉唑回归方程为Y=0.005 3 X+0.016 2(r=0.996 3),非那西丁的线性回归方程为Y=0.008 1X+0.0199(r=0.995 0),硝苯地平的回归方程为Y=0.009 3X+0.011 8(r=0.991 7),甲苯磺丁脲的回归方程为Y=0.008 1X+0.022 0(r=0.995 1).日内精密度和日间精密度良好,RSD≤8.75％,样本在4、12、24h稳定性良好,RSD≤14.31％,提取回收率均＞71.36％,且RSD≤6.98％,以上结果均符合样本含量测定要求.结论 本论文建立的探针方法可用于体外快速评价药物对CYP450亚型酶活性的影响,为临床药物-药物相互作用研究提供可靠的检测方法.
Abstract
Objective To establish a method to determine CYP450 enzyme substrates in biological samples using HPLC.Methods Liver microsomes were prepared by calcium precipitation method,the probe substrates were incubated with rat liver microsomes in vitro using the probe substrate approach,Dextromethamine hydrobromide,omeprazole,phenacetin,nifedipine,and tolbutamide were used as probe drugs for CYP2D6,CYP2C19,CYP1A2,CYP3A4,and CYP2C9,respectively,using rat liver microsomes as the matrix.and the detection method for the five probe drugs was established using HPLC.Results Methodological investigations showed that the method had good specificity and was not affected by blank liver microsomes or internal standards.The linear regression equations were as follows:Dextromethorphan(Y=0.001 6X+0.003 7,r=0.994 8),Omeprazole(Y=0.005 3X+0.016 2,r=0.996 3),Phenacetin(Y=0.008 1X+0.019 9,r=0.995 0),Nifedipine(Y=0.009 3X+0.011 8,r=0.991 7),and Tolbutamide(Y=0.008 1X+0.022 0,r=0.995 1).Intra-day and inter-day precision were good with RSD ≤8.75％,and sample stability at 4,12,and 24 hours was good with RSD ≤ 14.31％.The extraction recovery rate was greater than 71.36％,with RSD ≤6.98％.All results met the requirements for sample content determination.Conclusion The probe substrate method established in this experiment can be used for rapid in vitro evaluation of the effects of drugs on CYP450 isozyme activities,providing a reliable detection method for clinical drug-drug interaction studies.
关键词
高效液相色谱法/细胞色素P450/方法学考察/药物-药物相互作用Key words
High-performance liquid chromatography/Cytochrome P450/Methodological examination/Drug drug interactions引用本文复制引用
基金项目
国家自然科学基金青年科学基金资助项目(82104694)
出版年
2024
NETL
NSTL
万方数据