Sensitive analysis of asparagine and glutamine in physiological fluids and cells by precolumn derivatization with phenylisothiocyanate and reversed-phase high-performance liquid chromatography.

The analytical methodologies for the determination of free amino acids in plasma, serum, erythrocytes and leukemic cells are described. Deproteinization of the sample by methanol or organic acids is followed by derivatization with phenylisothiocyanate to form stable phenylthiocarbamylamino acid derivatives. The derivatives are separated by reversed-phase high-performance liquid chromatography in 80 min using a 5-microns C18 column (250 X 4 mm I.D.) and monitored by ultraviolet detection at 254 nm. Twenty physiological amino acids are resolved and quantified in plasma and erythrocyte samples. The resolution and sensitivity of the analytical method permitted unequivocal quantification of very low asparagine and glutamine levels in leukemic cells and growth media following treatment with asparaginase and glutaminase enzymes despite the presence of high aspartic and glutamic acid levels.