HPLC-MS/MS法测定大鼠血浆样品中4-FMA含量

Determination of the Content of 4-FMA in Rat Plasma Samples by HPLC-MS/MS Method

目的建立一种测定大鼠血浆中新精神活性物质4-氟甲基苯丙胺(4-fluoromethamphetamine,4-FMA)含量的高效液相色谱-串联质谱(high performance liquid chromatography-tandem mass spectrometry,HPLC-MS/MS)分析方法,为4-FMA在大鼠体内的毒物代谢动力学研究提供方法基础。方法大鼠血浆样品中加入内标物甲基苯丙胺(methamphetamine,MA),经甲醇沉淀蛋白后采用Poroshell 120 EC-C18色谱柱分离,0.1%甲酸水溶液和0.1%甲酸乙腈溶液为流动相,流速为0.4 mL/min,采用电喷雾离子源在多反应监测模式下进行检测。结果血浆样品中4-FMA在质量浓度5~1 000 ng/mL范围内线性关系良好(r>0.999),检出限为3 ng/mL,定量限为5 ng/mL。准确度以相对误差(relative error,RE)表示,在±5%范围内,日内精密度与日间精密度[以相对标准偏差(relative standard deviation,RSD)表示]<9%,提取回收率>90%。血浆样品的分析检测在2.5 min内完成。结论本研究建立了大鼠血浆样品中4-FMA的HPLC-MS/MS测定方法,该方法准确、快速、简单、灵敏,适用于4-FMA的代谢动力学研究。

Objective To develop a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method for the determination of the content of 4-fluoromethamphetamine(4-FMA)in rat plasma, and to provide a methodological basis for the study of the toxicokinetics of 4-FMA in rats. Methods Rat plasma samples were added into internal standard methamphetamine(MA). Its proteins were precipitated with methanol and then separated with Poroshell 120 EC-C18 chromatographic column. A 0.1% formic acid aqueous solution and a 0.1% formic acid acetonitrile solution were used as the mobile phase at the flow rate of 0.4 mL/min. Electrospray ionization source was used for detection in the multiple reaction monitoring(MRM) mode. Results The linear relationship was good when the mass concentration of 4-FMA in plasma samples was in the range of 5-1 000 ng/mL(r>0.999).The limit of detection(LOD) was 3 ng/mL and the limit of quantification(LOQ) was 5 ng/mL. The accuracy was expressed as relative error(RE), and in the range of ±5%, the intra-day precision and inter-day precision(relative standard deviation, RSD) less than 9%, and the extraction recovery rate was more than 90%. The analysis and detection of plasma samples were completed within 2.5 min.Conclusion This study developed a HPLC-MS/MS method for the determination of 4-FMA in rat plasma samples. This method is accurate, rapid, simple and sensitive and can be applied to the study of toxicokinetics of 4-FMA.