Measurement of Free Amino Acids in Human Biological Fluids by High-performance Liquid Chromatography

Favourable analytical conditions allowing amino acid analysis in biological fluids, acquired from small human biopsy specimens, were achieved by considering various derivatization methods, the mode of detection and the column used. By using o-phthalaldehyde-3-mercaptopropionic acid as derivatization agent (high sensitivity and stability) and fluorescence detection (excitation at 330 nm, emission at 450 nm), excellent separation of 26 amino acids was obtained in the lower pmol range (1-10 pmol). The reproducibility of the retention times was better than 1.0% for the majority of amino acids and the results from high-performance liquid chromatography (HPLC), compared favourably with those of conventional amino acid analysis (r = 0.97). HPLC technology facilitates amino acid analysis in biopsy specimens of less than 1 mg of tissue.