目的 建立EZ:faast^TM氨基酸分析试剂盒联合高效液相色谱串联质谱法(HPLC-MS/MS)测定大鼠血浆中谷氨酰胺的浓度,并用此方法对谷氨酰胺在大鼠体内的药动学进行研究。方法 大鼠血浆用EZ:faast^TM氨基酸分析试剂盒进行样品处理,而后采用API 3000 HPLC-MS/MS进行分析。色谱柱采用Phenomenex, EZ:faast 4 μ AAA-MS(3.0 mm× 250 mm, 4 μm),柱温为20 ℃,流动相由0.2%甲酸水溶液(含5 mmol·L^-1乙酸铵)-甲醇进行梯度洗脱,流速0.4 mL·min^-1。质谱采用多反应离子监测(MRM)的扫描模式,以电喷雾离子源(ESI)在正离子电离模式下进行测定。结果 本方法在3.003~150.2 μg·mL^-1内线性良好(r=0.996 6),最低定量限为3.003 μg·mL^-1,日内、日间精密度均小于15%,准确度在85%～115%之间,萃取回收率约90%,稳定性考察结果表明,谷氨酰胺在室温放置、处理后放置、反复冻融及长期冷冻条件下稳定性良好。结论 本方法专属性强、灵敏、准确,样品处理操作简便、快速,适用于谷氨酰胺在大鼠体内的药动学研究。

OBJECTIVE To develop an HPLC-MS/MS method coupled with EZ:faast^TM amino acid analysis kits for the determination of glutamine in rat plasma for the pharmacokinetic study in rats.METHODS The plasma samples were prepared by EZ:faast^TM amino acid analysis kits and then analyzed with API 3000 HPLC-MS/MS system. The analytical column was Phenomenex EZ:faast 4 μ AAA-MS(4 micron, 3.00 mm×250 mm) and the column temperature was 20 ℃. The mobile phase was composed of 0.2% formic acid aqueous solution(containing 5 mmol·L^-1 ammonium acetate)-methanol and eluted gradiently at a flow rate of 0.4 mL·min^-1. The detection was performed with multiple reactions monitoring(MRM) using positive electrospray ionization(ESI). RESULTS The calibration curve for glutamine was linear over the concentration range of 3.003-150.2 μg·mL^-1(r=0.996 6). The lower limit of quantification was 3.003 μg·mL^-1. The inter- and intra-day RSDs were less than 15% and the accuracy was within 85%-115%. The extraction recoveries were around 90%, and glutamine was proved to be stable under different circumstances.CONCLUSION This method is specific, sensitive, and accurate, and the sample preparation procedure is simple, rapid, and suitable for the pharmacokinetic study of glutamine in rats.