Simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry

In forensic analysis, the identification of urine or human urine among unknown liquids plays an important role. Urea, uric acid, and creatinine are major organic compounds found in human urine. Previous studies have reported that the concentration quotients of these three compounds can be used as an index for the identification of human urine. Here we describe a method for the simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry (LC/MS), with the aim of forensic identification of human urine. Separation of the three analytes was achieved by hydrophilic interaction chromatography, using a TSK gel Amide-80 column with a mobile phase composed of acetonitrile and ammonium acetate aqueous solution, coupled with detection using a mass spectrometer. For quantification, melamine and violuric acid were used as internal standards. Human urine samples were pretreated for LC/MS analysis by dilution with LC mobile phase, followed by centrifugation and filtration. The analytes and internal standards were separated within 9 min. The linear ranges were 2.0-40.0, 0.10-1.60, and 0.13-2.00 mg/mL for urea, uric acid, and creatinine, respectively, with correlation coefficients > 0.99. The intra- and inter-day accuracies of the analytes were - 10.6% to 7.4%, and the precision was within 7.6%. For all analytes, no significant matrix effects were observed and recoveries ranged from 95.4% to 104.6%. Quantitative results of 3 analytes were obtained within their linear range from 10 human urine samples and the quotients, UA/UN × 20 and UA/Cre, were calculated based on previous reports.