Two-dimensional high-performance liquid chromatographic system for the determination of enantiomeric excess in complex amino acid mixtures: Single amino acid analysis

Abstract

An HPLC system that allows the determination of the enantiomeric composition of complex mixtures of amino acids such as those occurring in biological fluids ( e.g., serum, cerebrospinal fluid) and foods is described. d- and l-amino acids (including proline) can be determined. First, amino acid separation is achieved by means of an ion-exchange column by elution with a lithium chloride—lithium citrate buffer. Each peak corresponding to an individual amino acid can be switched to a reversed-phase column (C 18) and eluted with an aqueous solution containing chiral copper(II) complexes which perform chiral discrimination by a ligand-exchange mechanism. The method is very flexible as several chiral selectors and different types of detection ( e.g., UV, fluorescence) can be used. Moreover, it avoids unnecessary overrunning of the chiral system with the whole mixture, by switching only the peaks under investigation. It is possible to evaluate d- amino acids up to a 0.1 % d to d + l ratio in the nanomolar range. Postcolumn derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and fluorimetric detection were utilized for proline and hydroxyproline and with o-phthaldehyde for the other amino acids.