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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 t