Development, validation and application of a novel liquid chromatography tandem mass spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-fluorouracil, α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine in human plasma

•The UH2/U is a plausible index of 5-FU response and toxicity.•There is a case for therapeutic drug monitoring of 5-FU, also metabolites FUH2, FUPA and FβAL.•An LC–MS/MS assay was developed for U, UH2, 5-FU, FUH2, FUPA and FβAL in plasma.•The assay was shown to be sensitive, accurate, precise and robust.•It was further validated in plasma from 10 cancer patients receiving 5-FU.