HPLC测定大鼠海马中谷氨酸、γ-氨基丁酸的含量

Study on the Determination of Glutamic Acid and γ-Aminobutyric Acid in Hippocampus of Rat by HPLC

目的建立了邻苯二甲醛(OPA)柱前衍生高效液相荧光色谱法检测大鼠海马中谷氨酸(GLU)、γ-氨基丁酸(GABA)含量的方法。方法采用AlltimaC18色谱柱(4.6mm×250mm,5μm),流动相为50mmol·L-1乙酸钠(pH6.8)、甲醇、四氢呋喃梯度洗脱,流速1.0mL·min-1。激发波长(λEx)338nm,发射波长(λEm)425nm。结果GLU、GABA线性范围分别为1.03～20.6μg·mL-1(r=0.9992)和1.13～22.6μg·mL-1(r=0.9997);当信噪比为3时,GLU和GABA的检出限分别为5.27×10-4μg·mL-1和7.35×10-4μg·mL-1;测得精密度RSD分别为3.4%和3.5%;加样回收率分别为90%～96%,85%～91%。结论本方法简便、准确、灵敏,特异性强,重复性好,适用于海马中GLU和GABA的含量测定。

OBJECTIVE To establish a method for the content of glutamic acid and γ-aminobutyric acid in hippocampus of rat by O-phthalaldehyde（OPA） pre-column derivatization high performance liquid chromatograph-fluorescence （HPLC-FLU）. METHODS The separation and determination were carried out by linear gradient elution under following conditions： Alltima C18（250 mm×4.6 mm, 5 μm）column, 50 mmol·L^-1 acetic acid solution （pH 6.8）, methanol and trifluorofuran as mobile phase, flow-rate was 1.0 mL·min^-1. The fluorescence excitation and emission wavelengths were set at λEx 338 and λEm 425 nm, respectively. RESULTS The linear range of the method was 1.03-20.6 μg·mL^-1（r=0.999 2） for glutamic acid（Glu） and 1.13-22.6 μg·mL^-1（r=0.999 7） for r-aminobutyric acid（GABA）. The detection limits of GLU and GABA were 5.27×10^-4 μg·mL^-1,and 7.35×10^-4 μg·mL^-1, respectively, when the ratio of signal to noise was 3. The inter-assay precisions were 3.4% and 3.5%, respectively. The recoveries were in the range of 90%-96% and 85%-91%. CONCLUSION The method is simple, sensitive, reliable and has strong specificity and good repetition. It is suitable for the content determination of GLU, GABA in hippocampus of rat by HPLC.