A high performance liquid chromatography method with electrochemical detection of gamma-aminobutyric acid, glutamate and glutamine in rat brain homogenates

Determination of γ-aminobutyric acid (GABA), glutamate (Glu) and glutamine (Gln) in animal models has been important to understand the normal function and clinical aspects of some neurological diseases. Quantification of these amino acid transmitters has conventionally been performed by using a high performance liquid chromatography (HPLC) system. This paper describes an improved HPLC method with electrochemical detection for glutamate, glutamine and GABA determination in brain homogenates. The protocol is based on a precolumn derivatization of amino acids with o-phthalaldehyde and sodium sulfite, a separation through a C18, 5μm particle size column and an isocratic elution. Several modifications of previous works on methanol percentage, pH, temperature, flow rate and derivatization solution concentration were done to obtain a suitable protocol for amino acid quantification in brain homogenate samples. Total elution time is 35min approximately. Technical requirements and laboratory expenses of this new protocol are minimal. This technique showed high linearity, repeatability and accuracy.