Precolumn phenylisothiocyanate derivatization and liquid chromatography of free amino acids in biological samples

Abstract

A method for the analysis of free amino acids using precolumn phenylisothiocyanate derivatization and liquid chromatography was tested on a range of sample matrices: bovine plasma, dry cat food, dry infant milk formula and potatoes. The chromatographic resolution of the phenylthiocarbamyl derivatives of free amino acids was satisfactory, with few unidentified compounds present. Standard deviations for triplicate analyses were generally less than 10% of their means. 5-Sulfosalicylic acid (SSA) and a small pore membrane ultrafilter were tested for the deproteinization of bovine plasma. Recoveries of standards were highest when 1% and 4% SSA were used for deproteinization (98% average recovery), and lower (91%) when the ultrafilter was used. The 4% SSA treatment gave higher average free amino acid contents and protein removal than the other deproteinization techniques. These higher values were not due to protein hydrolysis by SSA. Interaction of free amino acids with plasma protein remaining after deproteinization may account for the lower free amino acid contents measured after 1% SSA and ultrafilter treatment. Deproteinization of plasma with 4% (w/v) (final concentration after mixing with the sample) SSA is recommended.