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[摘要] 目的 建立快速、简便、灵敏可以同时测定大鼠体内外“鸡尾酒”探针药物和代谢产物的LC-MS/MS方法，并用于大鼠肝微粒体外代谢和大鼠体内的药代动力学研究。 方法 所有样品均加入乙腈进行沉淀处理，然后在UPLC CORTECS C18（2.1 mm×50 mm，1.6 μm）色谱柱分离，质谱方法采用多反应监测模式（MRM）。液相方法采用梯度洗脱流动相乙腈-0.1%甲酸水。 结果 6种探针药和代谢产物的体内外样品方法学验证符合生物样品检测要求。该方法成功应用于大鼠体内外“鸡尾酒”实验中。 结论 本实验建立的“鸡尾酒”方法可用于体内外快速评价药物或中草药对相应的CYP450亚型酶活性的影响，可以用于潜在的药物相互作用的预测。
[关键词] 细胞色素P450;大鼠肝微粒体;探针药;UPLC-MS/MS
[Abstract] Objective To establish a rapid， simple and sensitive LC-MS/MS method for simultaneous determination of "cocktail" probe drugs and metabolites in vivo and in vitro of rats， and to apply it to studies of in vitro metabolism of rats′ liver microsomes and in vivo pharmacokinetics of rats. Methods Both in vivo and in vitro samples were treated by acetonitrile precipitation method. Probe drugs and metabolites were detected by UPLC CORTECS C18 （2.1 mm×50 mm， 1.6 μm） chromatographic column combined with UPLC-MS/MS method of multiple reaction monitoring mode（MRM）， in which acetonitrile-0.1% formic acid water was used as gradient elution method of mobile phase. Results Methodological verification of samples in vitro and in vivo of 6 probe drugs and metabolites met the requirements of biological sample detection. The method had been successfully applied to "cocktail" experiments in vivo and in vitro of rats. Conclusion The "cocktail" method established in this experiment could be used to rapidly evaluate the efficacies of drugs or Chinese herbal medicines on the activities of the corresponding CYP450 subtypes enzymes in vivo and in vitro and could be used to predict potential drug interactions.
[Key words] Cytochrome P450; Rat liver microsomes; Probe drugs; UPLC-MS/MS
细胞色素P450酶（Cytochrome P450 proteins，CYP）是体内I相代谢的主要代谢酶超家族，大约能代谢超过90%的药物和内源性物质[1-2]，其中，经CYP1A2、CYP2B6、CYP2D6、CYP2C9，CYP2E1和CYP3A4代谢的药物占所有经CYP代谢药物的80%以上[3-5]。药物、食物和中草药等可通过抑制或诱导药物代谢酶来改变其他药物的代谢和药代动力学特性，从而导致药物之间相互作用。其中，对CYP酶的抑制作用会增加药物不良反应的风险或降低所需的治疗效果。研究者通常使用体外P450酶抑制试验来研究经P450代谢的药物相互作用（Drug-drug interaction，DDI）或中草药药物相互作用（Herb-drug interaction，HDI）的可能机制。目前研究者已开发了几种混合使用P450探针底物的混合物进行肝微粒体温育的鸡尾酒方法，可以高通量地检测经P450酶代谢药物相互作用的预测，但是，目前大多數文献都是单一的体内或体外“鸡尾酒”方法[6-9]，本实验将体内和体外“鸡尾酒”方法相结合，采用UPLC-MS/MS检测方法，为预测药物相互作用提供较全面的数据，可以用于潜在的药物相互作用的预测。
1 材料与方法
1.1 材料来源
非那西汀（Ph，批号：ILTFG-PD）和安非他酮（Bu，批号：31677-93-7）购于Tokyo Chemical Industry公司，右美沙芬（Dex，批号：D299445）、甲苯磺丁脲（Tol，批号：64-77-7）和氯唑沙宗（Chl，批号：95-25-0）购于J
[关键词] 细胞色素P450;大鼠肝微粒体;探针药;UPLC-MS/MS
[Abstract] Objective To establish a rapid， simple and sensitive LC-MS/MS method for simultaneous determination of "cocktail" probe drugs and metabolites in vivo and in vitro of rats， and to apply it to studies of in vitro metabolism of rats′ liver microsomes and in vivo pharmacokinetics of rats. Methods Both in vivo and in vitro samples were treated by acetonitrile precipitation method. Probe drugs and metabolites were detected by UPLC CORTECS C18 （2.1 mm×50 mm， 1.6 μm） chromatographic column combined with UPLC-MS/MS method of multiple reaction monitoring mode（MRM）， in which acetonitrile-0.1% formic acid water was used as gradient elution method of mobile phase. Results Methodological verification of samples in vitro and in vivo of 6 probe drugs and metabolites met the requirements of biological sample detection. The method had been successfully applied to "cocktail" experiments in vivo and in vitro of rats. Conclusion The "cocktail" method established in this experiment could be used to rapidly evaluate the efficacies of drugs or Chinese herbal medicines on the activities of the corresponding CYP450 subtypes enzymes in vivo and in vitro and could be used to predict potential drug interactions.
[Key words] Cytochrome P450; Rat liver microsomes; Probe drugs; UPLC-MS/MS
细胞色素P450酶（Cytochrome P450 proteins，CYP）是体内I相代谢的主要代谢酶超家族，大约能代谢超过90%的药物和内源性物质[1-2]，其中，经CYP1A2、CYP2B6、CYP2D6、CYP2C9，CYP2E1和CYP3A4代谢的药物占所有经CYP代谢药物的80%以上[3-5]。药物、食物和中草药等可通过抑制或诱导药物代谢酶来改变其他药物的代谢和药代动力学特性，从而导致药物之间相互作用。其中，对CYP酶的抑制作用会增加药物不良反应的风险或降低所需的治疗效果。研究者通常使用体外P450酶抑制试验来研究经P450代谢的药物相互作用（Drug-drug interaction，DDI）或中草药药物相互作用（Herb-drug interaction，HDI）的可能机制。目前研究者已开发了几种混合使用P450探针底物的混合物进行肝微粒体温育的鸡尾酒方法，可以高通量地检测经P450酶代谢药物相互作用的预测，但是，目前大多數文献都是单一的体内或体外“鸡尾酒”方法[6-9]，本实验将体内和体外“鸡尾酒”方法相结合，采用UPLC-MS/MS检测方法，为预测药物相互作用提供较全面的数据，可以用于潜在的药物相互作用的预测。
1 材料与方法
1.1 材料来源
非那西汀（Ph，批号：ILTFG-PD）和安非他酮（Bu，批号：31677-93-7）购于Tokyo Chemical Industry公司，右美沙芬（Dex，批号：D299445）、甲苯磺丁脲（Tol，批号：64-77-7）和氯唑沙宗（Chl，批号：95-25-0）购于J