Development and evaluation of a simultaneous strategy for pyrimidine metabolome quantification in multiple biological samples

Pyrimidines are critical nutrients and key biomolecules in nucleic acid biosynthesis and carbohydrate and lipid metabolism. Here, we proposed the concept of the pyrimidine metabolome, which covers 14 analytes in pyrimidine de novo and salvage synthetic pathways, and established a novel analytical strategy with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to efficiently illustrate pyrimidine transient distribution and dynamic balance. The lower limits of quantification (LLOQs) of all analytes were less than 10 ng/mL. Acceptable inter- and intra-day relative deviation (<15%) was detected, and good stability was obtained under different storage conditions. Metabolomics analysis revealed pyrimidine metabolic diversity in the plasma and brain among species, and a visualization strategy exhibited that pyrimidine biosynthetic metabolism is quite active in brain. Distinct metabolic features were also observed in cells with pyrimidine metabolomic disorders during proliferation and apoptosis. Absolute concentrations of pyrimidine metabolites in different bio-samples offered reference data for future pyrimidine studies.