Improvement of high-performance liquid chromatography with fluorescence detection to measure amino acid neurotransmitters in the rat brain

Objective To improve the method of reversed phase high-performance liquid chromatography with fluorescence detection(HPLC-FL) to measure amino acid neurotransmitters in rat brain tissues.Methods Sodium phosphate-methanol-acetonitrile was used as the mobile phase.The reversed phase HPLC system with pre-column derivatization by o-phthalaldehyde and with fluorescence detection was used to measure the amino acid neurotransmitters including aspartate(Asp),glutamate(Glu),glutamine(Gln),glycine(Gly),γ-aminobutyric acid(GABA) and taurine(Tau) in the cerebral cortex,hippocampus,striatum,mesencephalon,cerebellum and hypothalamus of rats.Results The six kinds of amino acids were well separated using elution within 20 minutes.Good linear response was obtained for the 6 standards within the range of 6.25 to 400 μmol/L with correlation coefficients not less than 0.99.For these amino acids,good precisions was obtained by 1.38% to 7.59% for the intra-day precision(RSD,%) and 2.7% to 8.68% for the inter-day precision.The recovery of added amino acids was not less than 80%.The feasibility and advantages of this improved method were validated by determination of the 6 kinds of amino acid in 6 regions of brain samples from 5 rats.Conclusions The improved method of reversed phase HPLC with fluorescence detection is more sensitive,has better reproducibility,and can effectively separate and measure the amino acid neurotransmitters in rat brain.