HPTLC method for effective separation of L-glutamic acid and GABA present in brain tissue extracts

Abstract

L-glutamic acid and ϒ-aminobutyric acid (GABA) show similar physicochemical properties, so separating them by thin-layer chromatography (TLC) is difficult. TLC is currently a widely used method because of its simplicity, flexibility, and low cost compared to liquid or gas chromatography. This paper shows the TLC separation of [3H]glutamic acid from [3H]GABA formed by incubating the former with the membrane or the cytosolic fraction obtained from the locus coeruleous on the brain. Such separation was achieved with a better resolution than the literature reported. Radiolabeled compounds were extracted from the tissue and seeded on an HPTLC plate. In addition, placed the L-glutamic acid and GABA standards in different lanes. The plates were developed in a solvent system of butan-1-ol, acetic acid, and water = 5:3:2 (v:v), containing 0.4% ninhydrin as a derivatizing agent. HPTLC was used to obtain better resolving power, faster development times, less sample diffusion, and lower solvent consumption than TLC. Results showed that the L-glutamic acid and GABA standards were adequately separated, with a resolution of 2.2. Moreover, the studied tissue transformed 9% of the radiolabeled glutamate into GABA. The L-glutamic acid was converted into a fluorescent tautomer from Schiff's base, detectable on a λ=302 nm. However, when the HPTLC dried, the tautomer was identified as GABA for its Rf=0.32, and the luminescence disappeared. The resolution between both components was excellent (Rs=2.2). In conclusion, it was possible to develop a reproducible method from the data of this study, which could attract the interest of researchers.