A novel UHPLC-MS/MS approach for simultaneous quantification of pyrimidine metabolites in human biofluids

Pyrimidine intermediates, essential components of DNA/RNA, serve as energy transducers and signaling mediators in cellular communication pathways. However, research on pyrimidine metabolism in acute kidney injury (AKI) is hindered by the limited variety of metabolites and poorly understood underlying mechanisms. To date, no liquid chromatography coupled to mass spectrometry (LC-MS) method has achieved comprehensive coverage of pyrimidine metabolism. In this study, we developed an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous detection of 10 pyrimidine metabolites. The method demonstrated a linear range of 1.2–2400 ng/mL, with reproducible recovery rates and consistent matrix effect. Both intra-day and inter-day accuracy and precision were within acceptable limits. Most metabolites exhibited stability under various conditions, including room temperature, autosampler, −40°C preservation, and after three freeze-thaw cycles. The method enabled extensive detection of pyrimidine metabolites, ensuring effective separation of all target compounds and achieving satisfactory peak shapes, particularly for the challenging monophosphate nucleotides. The method was successfully applied to matched plasma-urine biofluids from critically ill patients. In AKI inpatients, plasma levels of uracil, thymidine, and 2’-deoxyuridine were significantly reduced, while cytosine and cytidine levels were elevated. Additionally, a decrease in urinary cytidine concentration was observed in AKI patients. These findings demonstrate disturbances in the pyrimidine metabolic pathway in AKI patients, suggesting that the onset of AKI in critically ill patients may be closely associated with the accumulation of uremic solutes. The reliable quantification provided novel insights into the disease pathogenesis of AKI.