超高效液相串联质谱法同时定量检测6个细胞色素P450酶探针代谢产物  

Simultaneous Quantitation of Six Cytochrome P450 Enzyme Probe Metabolites by Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry

作　　者：沈国林[1] 钟玉环[1] 原梅[1] 庄笑梅[1] 李桦[1] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850

出　　处：《分析化学》2013年第4期488-493,共6页Chinese Journal of Analytical Chemistry

基　　金：国家"重大新药创制"科技重大专项(Nos.2008ZXJ09006-001;2012ZX09301003)资助

摘　　要：应用超高效液相色谱-质谱联用技术(LC-MS/MS),建立了同时定量测定6个细胞色素P450酶(CYP)探针代谢产物的方法。用甲醇和乙腈混合溶剂沉淀肝微粒体孵育液中的蛋白,在ZORBAX-C18色谱柱(100 mm×4.6 mm,3.5μm)上,以5 mmol/L甲酸铵和0.1%甲酸-乙腈为流动相,梯度分离待测物。在串联质谱正离子多反应监测模式下定量检测待测物。方法学验证结果表明,6个代谢产物在1.0～1000.0μg/L的范围内均呈良好的线性关系(r2>0.994);定量限为1μg/L;方法的日内和日间精密度(RSD)均小于12%;加标回收率为92.8%～104.4%;不同储存条件下样品稳定性实验的浓度偏差(RSD)小于10%。人肝微粒体活性测定的结果显示CYP1A2和CYP3A4的酶活性最强,分别为(466.1±32.1)和(694.3±11.7)pmole/(mg.min),与最低的CYP2C19酶活性分别相差27.7和41.3倍。本方法简便、快速、灵敏,适用于大量化合物CYP酶诱导和抑制评价的酶活性测定。A quantitative method was established and validated for the simultaneous determination of six cytochrome P450 enzyme（CYP） probe metabolites using ultra-high performance liquid chromatography-tandem mass spectrometry（UHPLC-MS/MS）.Human liver microsomal samples were processed with the mixed solvent of methanol and acetonitrile to precipitate proteins.The separation of the 6 probe metabolites was performed on a ZORBAX-C18 column（100 mm×4.6 mm,3.5 μm） by the gradient elution with the mobile phase consisting of 5mmol/L ammonium formate-0.1% formic acid aqueous solution and acetonitrile.The mass spectrometric quantification was carried out in positive electrospray ionization and multiple reactions monitoring mode,with propranolol as the internal standard.The validation results indicated that the calibration curves of the 6 metabolites were linear（r20.994） over the range of 1.0~1000 μg/L,with the lower limits of quantification of 1 μg/L.The intra-and inter-day precisions（RSD） were all below 12%.The recoveries for the spiked samples were in the range from 92.8% to 104.4%.The concentration variations（RSD） of the stability tests under different experimental conditions were all less than 10%.The results of human liver microsomes activity measurement showed that the CYP1A2 and CYP3A4 had the highest enzymatic activity of 466.1±32.1 and 694.3±11.7 pmole/（mg·min）,which were 27.7 and 41.3 times of CYP2C19 activity,respectively.This method had been applied to the activity determination of the CYP isoforms.It was proved to be simple,rapid,sensitive and suitable for the evaluation of CYP inhibition and induction.

关 键 词：细胞色素P450酶 探针底物 代谢产物 液相色谱-质谱联用 肝微粒体 

分 类 号：R96[医药卫生—药理学]