【摘要】 基于流动注射-串联质谱(FI-MS/MS)技术,综合运用微孔板低温孵育、低温样品相分离、"六合一"进样和多探针同时定量分析,建立了体外快速筛选复杂中草药组分抑制细胞色素P450同工酶活性的新方法。FI-MS/MS新技术不依赖色谱分离,也无需复杂的仪器设备和样品前处理,分析通量大幅提高。采用基质匹配标样和内标法定量,方法的准确度、精密度、灵敏度和线性范围均满足孵育体系中代谢物分析的需要。优化条件下测得各酶动力学参数和对照抑制剂的半抑制率(IC50)均与参考值一致;仅用两抑制剂浓度点即可获得满意的IC50近似值,两点IC50的测试重现性良好。结合两点IC50程序,FI-MS/MS方法成功应用于中药粗提液对人体主要P450酶抑制效应的快速筛选与分类,为多组分体系复杂药代相互作用的研究和评估提供了一种有效手段。更多还原

【Abstract】 A novel flow injection-tandem mass spectrometric(FI-MS/MS) multiplex enzyme assay was developed and validated for rapid screening of inhibitory potency of herb extracts on activities of five major human drug-metabolizing cytochrome P450 isoenzymes(CYP 1A2,2C9,2C19,2D6 and 3A4 /5) toward six probe substrates in pooled human liver microsomes.The fast FI-MS/MS-based method,without chromatographic separation,complicated instrumentation or labor-intensive sample preparation,enables at least a 20-fold increase in throughput,achieved by use of microplate incubation,low-temperature phase separation,6-in-1 injection and direct simultaneous multi-metabolite quantification for the marker reactions.Adequate accuracy(83%-112%),precision(RSDs < 17%),sensitivity(LODs < 10 nmol/L) and linearity(over a 300-fold range,r > 0.99) were obtained using matrix-matched calibrations and non-isotope labeled internal standard.The P450 enzyme kinetic parameters(Km and Vmax) and half maximal inhibitory concentration(IC50) of the known positive inhibitor for each probe reaction under the optimized conditions were consistent with the literature values.The IC50data derived from just two inhibitory concentrations(2-point IC50) provided comparable values by using the typical 7-point procedure,with only a slight deviation but acceptable reproducibility observed.After validation,the integrated use of the FI-MS/MS-based approach and the quick 2-point IC50 procedure allows rapid screening and ranking the inhibitory potency of 10 herbal extracts in a complex herbal mixture without sample purification or fractionation,and demonstrates its potential applicability for interpreting multiple effects of herbal recipes on P450 system in complex herbal-drug interaction scenarios.更多还原