Determination of free amino acids in biological samples: problems of quantitation.

An automatic on-line high-performance liquid chromatographic method was developed to study the effects of various precipitating agents and delayed deproteinization procedures on the estimation of plasma levels of amino acids. The optimized method for analysis is based on pre-column derivatization with o-phthalaldehyde in the presence of 2-mercaptoethanol. The separation of 25 amino acids is accomplished within 45 min on a 5-microns C18 column, using a multi-step gradient with two solvents. The method is sensitive and reproducible, and the relationship between the fluorescence intensity and concentration is linear for each amino acid over a wide concentration range.