高效液相-质谱法联用测定大鼠体内去甲基斑蝥素血药浓度

HPLC-MS Determination of Norcantharidin in Rat Plasma

目的:建立大鼠体内去甲基斑蝥素的 HPLC-MS 分析方法。方法:选用烟尿酸为内标,血浆样品经乙腈沉淀蛋白处理。色谱条件为 Hypersil SAX 阴离子交换色谱柱(4.6mm×250mm,5μm),甲醇-水(25∶75,含2.5%甲酸)为流动相,流速0.5mL·min^(-1),采用 HPLC-MS检测系统,AP-ESI 负离子模式,质谱检测参数如下:AP-ESI 负离子模式;雾化压力276kPa;温度350℃;保护气流量8 L·min^(-1);毛细管电压3500V;裂解电压为90V(NCTD)和120V(内标);SIM 检测,NCTD m/z185(M+H_2O-H)、烟尿酸m/z 179(M-H)。结果:血浆中去甲基斑蝥素检测方法的线性范围为0.2-20μg·mL^(-1),最低检测限可达2ng·mL^(-1)。血浆中去甲基斑蝥素的平均回收率为96.7%-100.3%,日内、日间 RSD 均小于8%。结论:本法灵敏、准确、选择性高,可用于去甲基斑蝥素的药代动力学研究。

Objective:To establish an HPLC-MS method for the determination of norcantharidin in rat plasma. Method:The plasma was precipitated with acetonitrile and the internal standard was nicotinoylglycine.A SAX col- umn was used to separate norcantharidin in plasma with a mixture of methanol-water(25:75,containing 2.5% for- mic acid)as mobile phase at flow rate of 0.5mL min^(-1).The analysis was detected by the negative electrospray ioni- zation(ESI^-)-MS method under selected-ion monitoring(SIM)mode.The MS detection parameters was set as follows:the nebulizer pressure was 276 kPa,the temperature was 350℃,the drying gas flow was 8L·min^(-1),the capillary voltage was 3500V,the fragmentor was 90V(norcantharidin)and 120V(internal standard),the SIM ions were m/z 185(norcantharidin,M+H_2O-H)and m/z 179(internal standard,M-H).Results:The linear range of the standard curve of norcantharidin was 0.2-20μg·mL^(-1),and the test limit was 2ng·mL^(-1).The recovery was 96.7%-100.3%,intra-day and inter-day RSD were less than 8%.Conclusion:The method had high sensitiv- ity,good selectivity and precision.It is suitable for determination of the norcantharidin in plasma.