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

An HPLC system that allows the determination of the enantiomeric composition of complex mixtures of aminoacids such as those occurring in biological fluids (e.g., serum, cerebrospinal fluid) and foods is described. d- and l-aminoacids (including proline) can be determined. First, aminoacid 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 aminoacid can be switched to a reversed-phase column (C18) 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- aminoacids 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 aminoacids.