2-DIMENSIONAL HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SYSTEM FOR THE DETERMINATION OF ENANTIOMERIC EXCESS IN COMPLEX AMINO-ACID MIXTURES - SINGLE AMINO-ACID-ANALYSIS

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.