QUANTITATIVE-ANALYSIS OF NEUROACTIVE AMINO-ACIDS IN BRAIN-TISSUE BY LIQUID-CHROMATOGRAPHY USING FLUORESCENT PRECOLUMN LABELING WITH O-PHTHALALDEHYDE AND N-ACETYL-L-CYSTEINE

A high-performance liquid chromatographic method for the determination of aspartic acid, glutamic acid, glutamine, glycine, taurine, and gam ma-aminobutyric acid in brain tissue is described. Amino acids were de rivatized with o-phthalaldehyde/N-acetyl-L-Cysteine, a fluorescent lab elling mixture, in the presence of 0.1 M borate buffer pH 9.5. The der ivatization reaction was sensitive to the pH and concentration of bora te buffer. A drift in the fluorescent response less than 4% was obtain ed with the reported conditions after 4 h of reaction. The resolution of the amino acid derivatives was accomplished in a reversed-phase col umn with a methanol gradient in 50 mM acetate buffer pH 5.5. These con ditions also allowed the separation of the major tissue free physiolog ical amino acids. L-Norvaline was used as an internal standard for bot h peak identification and quantification. Within-day and between-day p recision were less than 6.2%, and the accuracy ranged from 99.1 to 104 %. The applicability of the method was demonstrated in a study in rats in which the levels of the assayed amino acids in discrete areas of b rain were examined.