RP-HPLC法测定人血清中天门冬氨酸、谷氨酸、甘氨酸和γ-氨基丁酸含量

Simultaneous determination of glutamate, aspartate, γ-aminobutyric acid and glycine in human serum by RP-HPLC

关键词:  氨基酸  γ-氨基丁酸  反相高效液相色谱法  血清

Key words:  glutamate  aspartate  γ-aminobutyric acid  glycine  RP-HPLC  serum

摘要:

［目的］建立RP-HPLC法测定人体血清中天门冬氨酸、谷氨酸、甘氨酸和γ-氨基丁酸含量的方法。［方法］用邻苯二甲醛（OPA）柱前衍生；氨基酸的衍生物用Nova-Pak C18 (3.9 mm×150 mm，4 μm)分离；流动相A为30%甲醇、2%四氢呋喃、68%磷酸缓冲液（0.1 mol/L，pH 6.8），流动相B为100%甲醇，用二元线性梯度程序洗脱；荧光检测器检测，激发光波长280 nm，发射光波长340 nm。［结果］ 在(4~400) nmol/mL范围内，各组分线性关系良好，各氨基酸的平均回收率为90.0%~93.1%。［结论］本方法操作简便、准确，适用于人体血清中天门冬氨酸、谷氨酸、甘氨酸和γ-氨基丁酸浓度的测定。

Abstract:

［Objective］To develop a high performance liquid chromatography (HPLC) for simultaneous separation and rapid determination of four amino acid [ glutamate(Glu), aspartate(Asp), γ-aminobutyric acid (GABA), glycine (Gly) ] in human serum.［Methods］The precolumn derivazation reagent was OPA。Derivatives were separated on a Nova-Pak C18 (3.9 mm×150 mm，4 μm).Phosphate buffer solution, tetrahydrofuran and methanol as mobile phases, and gradient elution lasted 20 min with fluorescence detection.［Results］The range of calibration curve was （4~400）nmol/mL. The calibrations were characterized by a coefficient of correlation higher than 0.997. The average extracted recovery was 90.0%~93.1%,RSD%＜9.87%.［Conclusions］Our results showed that the method was rapid, accurate and simple.