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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
Author(s) -
SotoOtero R.,
MéndezAlvarez E.,
GalánValiente J.,
AguilarVeiga E.,
SierraMarcuño G.
Publication year - 1994
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130080304
Subject(s) - chemistry , chromatography , derivatization , amino acid , fluorescence , taurine , glycine , o phthalaldehyde , glutamine , cysteine , cysteic acid , high performance liquid chromatography , quantitative analysis (chemistry) , elution , labelling , biochemistry , cystine , enzyme , quantum mechanics , physics
A high‐performance liquid chromatographic method for the determination of aspartic acid, glutamic acid, glutamine, glycine, taurine, and γ‐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.