UPLC-Orbitrap HRMS法测定小鼠血液中香豆素及其代谢物

Determination of Coumarin and Its Metabolite in Mouse Blood with UPLC-Orbitrap High Resolution Mass Spectrometry

以香豆素为探针底物,建立了测定小鼠血液中香豆素及其代谢物的超高效液相色谱-轨道阱高分辨质谱(UPLC-Orbitrap HRMS)法.以4-甲基伞形酮为内标,样品用乙腈沉淀蛋白后,直接进入XDB-C_(18)色谱柱分离并采用一级全扫描和数据依赖二级扫描(Full MS/dd MS^2)模式进行质谱分析.通过测定7-羟基香豆素的生成量评估5-甲氧基补骨脂素(5-MOP)对小鼠CYP2A5酶活性的影响.结果表明:各目标物的检出限为0.011~0.016 ng/m L,回收率和日内精密度分别为90.2%~110.4%和1.86%~7.08%.方法选择性好,分析效率高,适合于小鼠血液中香豆素及其代谢物的测定.5-MOP组小鼠香豆素的最大血药浓度Cmax为对照组的1.42倍(P<0.01),药时曲线下面积AUC0-∞为1.59倍(P<0.05),半衰期t_(1/2)为1.05倍(P<0.01),5-MOP能够显著抑制小鼠CYP2A5酶活性.

In order to accurately determine coumarin and its metabolite in mouse blood, a UPLC-Orbitrap high resolution mass spectrometry method with coumarin as the probe substrate was established. Using 4-methylumbelliferone as internal standard, immediately after protein precipitating with acetonitrile, the samples were separated by an XDB-Ci8 column in UPLC spectrometer with mass spectrometry under full scan/data-dependent MS2 mode. The effect of 5-Methoxypsoralen （5-MOP） on the activity of cytochrome P450 2A5 （CYP2A5） enzyme in mouse was evaluated by the production of 7-hydroxycoumarin. The results showed that the limits of detection of analytes ranged from 0. Oil to 0. 016 ng/mL, and the recoveries ranged from 90. 2% to 110.4% with the intra-day precision of 1. 86% to 7. 08% respectively. The developed method was suitable for the simul-taneous analysis of coumarin and its metabolite in mouse blood with good selectivity and high efficiency. Compared with the control group, the maximum plasma concentration of 5-MOP-treated group was increased by 1. 42 fold （P〈C0. 01） , the area under the curve was increased by 1. 59 fold （P〈C0. 05） , the elimination half-lie was increased by 1. 05 fold （P〈C0. 01）. The re-sults indicate that the activity of CYP2A5 is markedly inhibited by 5-MOP.