Quantitative analysis of neuroactive amino acids in brain tissue by liquid chromatography using fluorescent pre-column labelling with o-phthalaldehyde and N-acetyl-L-cysteine.

Abstract

A high-performance liquid chromatographic method for the determination of aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid in brain tissue is described. Amino acids were derivatized with o-phthalaldehyde/N-acetyl-L-cysteine, a fluorescent labelling mixture, in the presence of 0.1 M borate buffer pH 9.5. The derivatization reaction was sensitive to the pH and concentration of borate buffer. A drift in the fluorescent response less than 4% was obtained with the reported conditions after 4 h of reaction. The resolution of the amino acid derivatives was accomplished in a reversed-phase column with a methanol gradient in 50 mM acetate buffer pH 5.5. These conditions also allowed the separation of the major tissue free physiological amino acids. L-Norvaline was used as an internal standard for both peak identification and quantification. Within-day and between-day precision 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 brain were examined.