Simultaneous determination of N-acetylaspartylglutamate and N-acetylaspartate in rat brain homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

Abstract

Simultaneous determination method of N-acetyl- l-aspartyl- l-glutamate (NAAG), an endogenous agonist at type3 metabotropic glutamate receptor, and its degradation product, N-acetyl- l-aspartate (NAA) was developed by using reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4- N, N-dimethylaminosulfonyl-7- N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. The detection limits of NAAG and NAA were approximately 12 and 34 fmol on the column, respectively (signal to noise ratio 3). The proposed HPLC method was applied to determine NAAG and NAA simultaneously in the rat brain homogenate. Both concentrations of NAAG and NAA in the male rat cerebrum (13 weeks old) were 5.7 ± 0.30 and 2.1 × 10 2 ± 9.2 nmol/mg protein, respectively ( n = 6), while those in the hippocampus were 6.8 ± 0.48 and 1.9 × 10 2 ± 8.5 nmol/mg protein, respectively ( n = 5). Hippocampal NAA concentration was significantly increased in the ketamine-treated rats as compared to the control rats ( p < 0.01).