o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide...

A rapid and ultrasensitive fluorescence amino acid analysis system has been developed which utilizes omicron-phthaldialdehyde as a precolumn derivatizing agent. omicron-Phthaldialdehyde in the presence of mercaptan reacts rapidly with primary amino acids to form intensely fluorescent derivatives. These derivatives are analyzed with good selectivity by high-performance liquid chromatography employing 3-microns particle size reversed-phase columns. Resolution of the amino acid derivatives is accomplished with a methanol gradient in 0.1 M aqueous sodium acetate, pH 7.2. The quantitation of the individual amino acid derivatives is reproducible within an average relative deviation of +/- 1.5% and has a detection limit of less than 100 fmoles. Amino acid mixtures obtained by either enzymatic or acid hydrolysis of polypeptides are efficiently resolved with an analysis time of less than 18 min. Methods for the amino acid analysis of physiological fluids such as serum, urine and cerebrospinal fluid were also developed which employ the above separation procedure for the identification and quantitation of amino acids and other biological amines. Mixtures which contained as many as 48 components were resolved with an analysis time of less than 50 min.