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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2011-09, Vol.879 (26), p.2720-2725
• Main Authors: Zhao, Jianxing, Chen, Hong, Ni, Peihua, Xu, Bingxin, Luo, Xuemei, Zhan, Yiming, Gao, Pingjin, Zhu, Dingliang
• Format: Article
• Language: English
• Subjects: 5-Hydroxyindole-3-acetic acid; Adult; ambient temperature; Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; centrifugation; Chromatography, High Pressure Liquid - methods; Chromium; correlation; creatinine; Creatinine - urine; detection limit; Detectors; Drug Stability; Female; fluorescence; fluorometry; Fundamental and applied biological sciences. Psychology; General pharmacology; High performance liquid chromatography; Humans; Hydroxyindoleacetic Acid - urine; Kynurenic acid; Kynurenic Acid - urine; Kynurenine; Kynurenine - urine; Linear Models; Male; Medical sciences; Metabolites; Pharmacology. Drug treatments; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence - methods; Spectrophotometry, Ultraviolet - methods; Tryptophan; Tryptophan - urine; Ultraviolet; urinalysis; Urine; Wavelengths
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.07.035

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.