title: 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 1mL/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.
authors.affiliation: Shanghai Key Laboratory of Vascular Biology at Ruijin Hospital and Shanghai Institute of Hypertension, Shanghai Jiao Tong University School of Medicine, 197 Ruijin 2nd Road, Shanghai 200025, China. jzhao021@yahoo.com
authors.forename: Jianxing
authors.initials: J
authors.lastname: Zhao
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Peihua
authors.initials: P
authors.lastname: Ni
authors.forename: Bingxin
authors.initials: B
authors.lastname: Xu
authors.forename: Xuemei
authors.initials: X
authors.lastname: Luo
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhan
authors.forename: Pingjin
authors.initials: P
authors.lastname: Gao
authors.forename: Dingliang
authors.initials: D
authors.lastname: Zhu
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003404:Creatinine / Q000652:urine*; D004355:Drug Stability; D005260:Female; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000652:urine; D007736:Kynurenic Acid / Q000652:urine; D007737:Kynurenine / Q000652:urine*; D016014:Linear Models; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014364:Tryptophan / Q000652:urine*
chemical_list: D007737:Kynurenine; D006897:Hydroxyindoleacetic Acid; D014364:Tryptophan; D003404:Creatinine; D007736:Kynurenic Acid