High Performance Liquid Chromatographic Determination of Amino Acids in Biological Samples by Precolumn Derivatization with O-Phthaldialdehyde

Abstract

A suitable gradient system has been developed for rapid analysis of amino acids in biological samples using O-phthaldialdehyde as a precolumn derivatizing agent and fluorescence detection. Resolution of 21 amino acids has been accomplished with 3 μm Ultrasphere ODS column by using a multi-step gradient system of two solvents (0.1M sodium acetate, pH 7.2/methanol:tetrahydrofuran) in less than 1 hour. Within-assay and between-assay coefficients of variation of retention times and fluorescence yield show good reproducibility. The fluorometric detection response is linear from 25 to 500 pmoles with a minimum detection limit of less than 1 pmol. High resolution, rapid analysis and high sensitivity of this method facilitates amino acid analysis in samples of less than 1 mg of tissue.