Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system
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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.
KeywordsChiral capillary electrophoresis Neurotransmitter amino acids d-Aspartate NBD-F Dual cyclodextrin system
This work has been supported by the Hungarian National Scientific Research Fund (OTKA 63415 and 73219) and TÁMOP-4.2.1/B-09/1/KMR-2010-0001.
Conflict of interest
The authors have declared no conflict of interest.
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