Application of high-performance liquid chromatography to the determination of free amino acids in physiological fluids

Multiple-step gradient systems were used for the analysis of free amino acids in physiological fluids by high-performance liquid chromatography with fluorescence detection on two reversed-phase C18 columns. Standard amino acids, plasma or urine samples were subjected to derivatization with ophthalaldehyde in the presence of mercaptoethanol before the separation was performed. More than 22 amino acids were separated in less than 1 h on either 5-micron Ultrasphere-ODS columns or 5-micron Resolve C18 columns by using a two-solvent system. The correlation of the integrated peak areas with the concentration of all amino acids showed a linear relationship between 10 and 150 pmol per 20-microliter injection for all The method has a lower detection limit of less than 1 pmol per 20-microliter injection for all amino acids. Quantitative analysis of micro amounts of amino acids in plasma and urine by the internal standard method gave highly reproducible results with a mean coefficient of variation of less than 3% and r2 = 0.999. Because of the simplicity of the method its application provides a better means for closely monitoring the patients undergoing dialysis and treatment for renal disorders. These results are compared with those from classical ion-exchange chromatography.