Quantitative Analysis of Uric Acid Metabolites in Urine by High Performance Liquid Chromatography - Mass Spectrometry Using Silica Hydride Columns

Background: Monitoring of uric acid levels is of significant importance for the prevention of conditions such as gout and kidney disease. Likewise, the metabolites produced from reaction of uric acid with reactive oxygen species are indicative of the level of oxidative stress. Quantitative methods are therefore needed for measuring these compounds in biological sample matrices. Objective: To investigate the use of silica hydride-based HPLC columns operating in aqueous normal phase mode to retain and separate uric acid from its metabolites in both reference standard solutions and urine samples. Method: LC-MS instrumentation operating in positive ion mode was used for the detection of the analyte peaks. An internal standard of tyrosine was chosen in order to normalize the detector response. Two stationary phases based on silica hydride were investigated. Results: The diol-based silica hydride column showed superior chromatographic resolution of the four analyte peaks. As polar molecules, all four were readily retained in the aqueous normal phase mode. Another metabolite, triuret, was also possibly observed, although lack of an available standard precluded definitive confirmation. Quantitative data demonstrated uric acid linearity in the range 0.2–1.6 mg/dL. Conclusion: The diol functional groups appeared to impart beneficial secondary selectivity to the separation, which was not observed using the other silica hydride-based column. The preliminary quantitative data showed that the developed method approach could be suitable for the analyses of uric acid and its metabolites in biological samples.