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.
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.
Soto-Otero
R
R
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Santiago de Compostela, Spain.
Méndez-Alvarez
E
E
Galán-Valiente
J
J
Aguilar-Veiga
E
E
Sierra-Marcuño
G
G
D000111:Acetylcysteine; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001224:Aspartic Acid / Q000032:analysis; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D005973:Glutamine / Q000032:analysis; D005998:Glycine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000032:analysis; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D005680:gamma-Aminobutyric Acid / Q000032:analysis; D009764:o-Phthalaldehyde; D016875:tau Proteins / Q000032:analysis
D000596:Amino Acids; D005456:Fluorescent Dyes; D005971:Glutamates; D007211:Indoles; D016875:tau Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D009764:o-Phthalaldehyde; D005998:Glycine; D000111:Acetylcysteine