Gas—liquid chromatography of amino acids in biological susbtances

Abstract

The development of a gas—liquid chromatographic (GLC) method for the quantitative analysis of amino acids in complex biological substances, specifically blood plasma and urine, has been achieved. The amino acids present in these physiological fluids were quantitatively isolated by ion-exchange methods and retained on the ion-exchange resin while the substances which interfere with the GLC analysis passed through the column and were discarded. The amino acids were then eluted from the column, derivatized to their N-trifluoroacetyl (N-TFA) n-butyl esters and analyzed by GLC. Quantitative recovery of the amino acids from the cation and anion-exchange clean-up columns, and amino acids in blood and urine were successfully carried out. Also, techniques for the analysis of amino acids over a wide range of concentrations were developed. Analyses were made on aliquots of human blood plasma containing only 200 μg of total amino acids, and the results obtained at this level of concentration were both accurate and precise. Further, quantitative data were obtained with samples containing only 20 μg of total amino acids, and semiquantitative analyses were performed on samples containing 2 μg of total amino acids. The data obtained by the GLC method were in excellent agreement with results by classical ion exchange. Investigations on acylation of the amino acid n-butyl esters have shown that the optimum molar ratio of TFAA/amino acids is 50:1 with regard to reproducibility of acylation, stability of the derivative, and maintenance of a small sample volume (> 75 μl).