Abstract
A micellar electrokinetic chromatographic (MEKC) separation of glutamate (Glu) enantiomers fluorescently tagged with naphthalene-2,3-dicarboxaldehyde (NDA) is described. Chiral selectors tested included alpha-, beta-, and gamma-cyclodextrins, modified cyclodextrins, D-glucosamine, sucrose, taurocholate, and their binary mixtures. NDA-labeled Glu enantiomers were best resolved with beta-cyclodextrin in the presence of methanol as an organic modifier. Under the separation conditions, no other amino acids coelute with Glu enantiomers. Using NDA as the reagent, the labeling reaction proceeded readily in aqueous solution, and the spectroscopic properties of NDA fluorophore were optimum for the sensitive laser-induced fluorescence (LIF) detection. Glu enantiomers present in mass limited samples such as single neurons could be adequately quantified by coupling this separation with LIF detection. A detection limit of 0.57 microM Glu was obtained. Using the present method, D-Glu was detected in rat brain, and, for the first time, in ganglia and single neurons of Aplysia californica, an extensively studied neuronal model. Interestingly, the level of D-Glu was found to be higher than that of L-Glu in many Aplysia neurons.
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