Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of DAsp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4- fluoro-7-nitro-2,1,3- benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3 mM 6-monodeoxy-6-mono(3-hydroxy)- propylamino-β-cyclodextrin (HPA-β-CD), further improvement of the chemical selectivity was required because of the high excess of L-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8 mM heptakis(2,6-di-O- methyl)-β-cyclodextrin and 5 mM HPA-β- CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for D-Asp and D-Glu were 17 and 9 nM, respectively, while the limit of quantification for both analytes was 50 nM. This is the lowest quantification limit reported so far for NBD-tagged D-Asp and D-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the D-enantiomer contributed 1-2 % of the total aspartate content, corresponding to 17-45 nmol/g wet tissue.
ASJC Scopus subject areas
- Analytical Chemistry