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

Abstract

HPLC fluorometric methods have been used to analyze trace amounts of d-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of d-amino acids, in particular d-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for d-Asp in culturing medium is 5 n M. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 n M 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 detected 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.