Novel Method for Spectrophotometric Determination of L-Tryptophan in the Enzymatic Resolution of DL-N-Acetyl-Tryptophan

Abstract.

A novel spectrophotometric method for the determination of tryptophan (Trp) has been proposed. The sensitive and simple method was based upon the finding that NaNO₂ and Trp formed a diazotized product in nitric acid medium, and that a bright magenta color developed for the product on coupling to N-(1-naphthyl) ethylenediamine dihydrochloride (NEDA) which was stable for at least 40 min. The absorption maximum was observed at 550 nm, and the apparent molar absorptivity was 8.48×10³ L·mol⁻¹·cm⁻¹. A linear dependence of absorbance on Trp concentration allowed sensitive quantification of Trp over the range of 1–20 µg mL⁻¹, and the detection limit was 0.5 µg mL⁻¹. The method has been successfully applied to the determination of L-Trp in the DL-N-acetyltryptophan enzymatic resolution system. The relative standard deviations (n=6) and recoveries were found to be 0.97–2.21% and 97.5–101.3%, respectively. The accuracy of the new method was confirmed by the results from capillary electrophoresis. Furthermore, the interference study showed that DL-N-acetyltryptophan (substrate), CoCl₂ (activator), and other common amino acids did not exhibit any detectable interference with Trp determination.