Determination of D- and L-aspartate in cell culturing medium, within cellsof MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method

HPLC fluorometric methods have been used to analyze trace amounts Of D-amin o acids in biological samples. In this study, we established an expedient c olumn-switching fluorometric HPLC system that would improve the analysis Of D-amino, acids, in particular D-aspartate (Asp). Our system consists of th e fluorogenic derivatization of amino acids with NBD-F and two chromatograp hic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-ph ase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is si mplified and improved, where trichloroacetic acid is used for deproteinizat ion, and borate buffer, pH 9.5 is employed for the fluorescent derivatizati on. The detection limit for D-Asp in culturing medium is 5 nM. The resultin g peak heights correlated well with concentrations that ranged from 12.5 to Z50 nM for both D- and L-Asp. The present method was applied to determine D- and L-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels Of D- and L-Asp agree with those detect ed by our previous method. In addition, this method was used to measure D- and L-Asp levels in rat blood samples, and the results are consistent with the reported values. (C) 2001 Elsevier Science B.V. All rights reserved.