Soto-Otero R, Méndez-Alvarez E, Galán-Valiente J, Aguilar-Veiga E, Sierra-Marcuño G
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Santiago de Compostela, Spain.
Biomed Chromatogr. 1994 May-Jun;8(3):114-8. doi: 10.1002/bmc.1130080304.
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.
描述了一种用于测定脑组织中天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和γ-氨基丁酸的高效液相色谱法。氨基酸在0.1 M pH 9.5硼酸盐缓冲液存在下，用邻苯二甲醛/N-乙酰-L-半胱氨酸（一种荧光标记混合物）进行衍生化。衍生化反应对硼酸盐缓冲液的pH值和浓度敏感。在报道的条件下反应4小时后，荧光响应的漂移小于4%。氨基酸衍生物的分离在反相柱中进行，流动相为含甲醇梯度的50 mM pH 5.5醋酸盐缓冲液。这些条件也能分离主要的组织游离生理氨基酸。L-正缬氨酸用作峰识别和定量的内标。日内和日间精密度均小于6.2%，准确度范围为99.1%至104%。该方法的适用性在一项对大鼠的研究中得到了验证，该研究检测了脑不同区域中所测氨基酸的水平。
