Urinary nitrite/nitrate ratio measured by isotope-dilution LC–MS/MS as a tool to screen for urinary tract infections

Urinary tract infections (UTIs) are the most common type of nosocomial infection. Traditionally, the presence of white blood cells and microorganisms in the urine provides objective evidence for UTI diagnosis. Here, we describe the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the nitrite and nitrate levels in urine and investigate the potential of this method for UTI diagnosis. LC–MS/MS analysis was performed in positive electrospray ionization mode. After adding 15N-labeled internal standards and derivatizing with 2,3-diaminonaphthalene (DAN), the urinary nitrite content was directly analyzed by LC–MS/MS, whereas the urinary nitrate was first reduced to nitrite before derivatization and LC–MS/MS analysis. The derivatization of nitrite and enzymatic reduction of nitrate were optimized. This method was then applied to 241 healthy subjects and 73 UTI patients. Optimization tests revealed that 1mL of crude urine required at least 6.25μmol of DAN to completely derivatize nitrite and 2.5U of nitrate reductase to completely reduce nitrate to nitrite. Urinary analysis showed that the urinary concentration of nitrite and the nitrite/nitrate ratio were higher in UTI patients than in healthy subjects. Compared with the dipstick-based urinary nitrite test and using LC–MS/MS to determine the nitrite concentration (sensitivity: 23–25%), the nitrite/nitrate ratio was significantly more sensitive (95%) and exhibited a satisfactory specificity (91%) in the screening of UTIs. Taken together, the nitrite/nitrate ratio, which reflects the reducing ability of pathogenic bacteria, could be a better method for the diagnosis of UTIs that is not subject to variations in urine specimen quality. [Display omitted] •We developed a LC-MS/MS method to measure nitrite and nitrate in human urine.•Derivatization of nitrite and enzymatic reduction of nitrate were optimized.•UTI patients had significantly higher urinary ratio of nitrite to nitrate.•The nitrite/nitrate ratio had significantly decreased after antibiotic treatment.•The nitrite/nitrate ratio is a better method for UTIs diagnosis than nitrite alone.