Determination of urinary tryptophan and its metabolites along the nicotinic acid pathway by high performance liquid chromatography with ultraviolet detection

A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites on the nicotinic acid pathway. Kynurenine, tryptophan, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid and xanthurenic acid were isocratically eluted and completely resolved with a mobile phase of acetonitrile + sodium acetate buffer, pH 4.76 (4:96, v/v). The flow rate was 0.8 mL/min at the beginning and was then linearly increased to 1.2 after 6 min; after 14 min the flow was augmented from 1.2 to 2 mL/min. The effluent was monitored with a variable UV detector set at 254 nm for the first five peaks and at 280 and 325 nm for the penultimate peak and final peak. Analytical recoveries of the compounds after deproteinization varied between 64% and 98%. The reported method should enable one to examine easily, extensively, quantitatively and routinely urinary tryptophan and the most important metabolites of the nicotinic acid pathway.