Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system

Abstract

Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of D-Asp 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.