Fluorometric determination of L-tryptophan with methoxyacetaldehyde

It was observed in our laboratory that methoxyacetaldehyde induced strong fluorescence with L-tryptophan in the presence of NaNO2 at pH 2.75. The reaction mixture was heated at 80 degrees C for 15 min and the fluorescence was measured at the excitation wavelength, 253 nm and the emission, 450 nm. Amino acids other than L-tryptophan did not produce fluorescence under the conditions employed. A good linear working curve was observed between 25 pmol/50 microliters and 500 pmol/50 microliters of L-tryptophan. The limit of determination for L-tryptophan was 10 pmol/50 microliters. The coefficient of variation of the measurements (n = 5) was 1.5% for 250 pmol/50 microliters of L-tryptophan. The proposed method was successfully applied to the analyses of tryptophan in a pooled human serum. Total tryptophan was determined after deproteinization with trichloroacetic acid, and free tryptophan was determined using the ultrafiltrate of a pooled human serum. Analytical recoveries of total and free tryptophan were 98.8% and 99.1%, respectively. The fluorescent products of L-tryptophan and methoxyacetaldehyde were characterized as beta-carboline and 1-methoxymethyl-beta-carboline by the use of liquid chromatography-mass spectrometry (LC-MS) and HPLC.