Resumo

Background: The amino acids glutamate and glutamine play important physiological roles in cerebral neurotransmission, and disruption of these systems has implications in the pathophysiology of neuropsychiatric disorders. Objective: To validate a high pressure liquid chromatography (HPLC) method to simultaneously determine cerebral concentrations of glutamate and glutamine. Methods: We utilized derivatization of compounds of 1ml of sample with o-phthalaldehyde in the presence of 2-mercaptoethanol followed by extraction with iodoacetic acid (40% v/v) and buffer sodium borate pH11.5 following a chromatography on an C18 column. Compounds were excited at 260nm and emited at 455nm with fluorescence detection. Homoserine was used as the internal standard (IS). The mobile phase consisted of sodium phosphate buffer 0.04M; EDTA 0.2M and methanol (64.9:0.1:35 v/v). Results: The method was linear over a working range of 0.2-15ug/mL for the compounds analyzed. The quantification and detection limits were 0.5ug/mL e 0.2ug/mL, respectively. Precision for both intra-day and inter-day were 13.97% to glutamine and 12.33% to glutamate; and 12.42% to glutamine and 14.75% to glutamate respectively. The accuracy values ranged from 99,9 to 103.97% for glutamine and from 89.9 to 101.25% for glutamate The retention time (in minutes) was 7.7 for glutamine, 4.2 for glutamate and 9.4 for homoserine. Conclusion: We developed a selective, sensitive, and time-effective reversed-phase HPLC method for simultaneous analysis of glutamine and glutamate in mouse brain.