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

Abstract

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.

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection

“…of NM, DA, Tyr, 5-HT, TA, and Trp that we measured using the proposed method were 1.32 ± 0.40, 1.14 ± 0.24, 150 ± 38, 0.48 ± 0.11, 0.51 ± 0.28, and 106 ± 47 nmol/mL urine from healthy subjects (n = 7). These values agree well with reported data [14,[16][17][18][19][20][21].…”

Rapid Isocratic Liquid Chromatographic Separation and Quantification of Tryptophan and Six kynurenine Metabolites in Biological Samples with Ultraviolet and Fluorimetric Detection

“…Most current analytical procedures involve relatively long runs of between 15 and 70 min for Trp and a small number of metabolites (up to 4),2–7 or 17–40 min for a larger number (6–9) 8–11. In more recent studies using LC-MS, faster separations were achieved.…”

“…For example, current HPLC methods can determine between 1 and 4 Trp metabolites in serum or urine in 15–70 min2–7 and, although faster methods have been developed for analysis of up to 6–9 kynurenine metabolites, a run still takes 17–40 min and most require additional pre-column sample deproteinisation and clean-up, and the use of gradient elution 8–11. More recently, HPLC-MS has been used for more rapid separation of kynurenines in two studies.…”

Chromatographic analysis of tryptophan metabolites

“…LC has been often used for this purpose. There are methods employing various detection modalities: UV absorbance , fluorescence , electrochemical methods as well as MS . The LC separation is mostly achieved on octadecyl silica (ODS) columns.…”