Confirming chiral high-performance liquid chromatographic separations with stereospecific enzymes

Abstract

Identification of optical isomers of amino acids, separated by chiral high-performance liquid chromatography, has proved to be difficult. Despite highly selective separation techniques, identification of peaks based on retention times alone is usually uncertain, particularly in assays of complex biological samples in which interfering compounds are eluted with the peak of interest. We describe here an approach for increasing the certainty of identification of peaks of d- and l-amino acids by the use of stereospecific amino acid oxidase and racemase enzymes. A portion of the sample is first incubated with the enzymes. The amino acids in the treated and untreated portions are then chromatographed and the chromatograms of the samples with and without enzyme incubation are compared. The differences are used to help identify the amino acids.