A simple method for the elimination of amine contaminants in buffers for single-column amino acid analysis in physiological samples at picomole levels

Abstract

A major problem in the use of single-column chromatography for the analysis of amino acids with fluorescence detection has been buffer contamination with amines. These elute in the basic amino acid region, causing major disturbances in the baseline (1,2). Thus, although the detection of very low levels of amino acids (10 −12 mol) is possible with the use of reagents such as fluorescamine (Fluram, Roche) and o-phthalaldehyde (Fluropa, Durrum), the irregular baseline especially in the basis region frequently allows one to reliably measure quantities of only 5 × 10 −10 mol. We present here a simple, reproducible method for the elimination of the amine contaminants of buffers. This procedure takes advantage of the fact that the product of the reaction of fluorescamine with an amine is fluorescent at basic pH (3), but rearranges to a nonfluorescent tetramic acid at acidic pH (4).