Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection

Abstract

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze–thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1 mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient ( r) > 0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0–106.4%; Kyn, 97.9–106.9%; Kyna, 98.5–105.6%; Trp, 96.7–105.2% and 5-HIAA, 96.1–99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.