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

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 5.27×10-4μg·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.