Liquid chromatography-tandem mass spectrometric assay for the analysis of uracil, 5,6-dihydrouracil and beta-ureidopropionic acid in urine for the measurement of the activities of the pyrimidine catabolic enzymes

A liquid chromatography-tandem mass spectrometric assay for the determination of uracil, 5,6-dihydrouracil and beta-ureidopropionic acid in urine was developed to measure the activities of enzymes involved in pyrimidine breakdown. The assay was required to investigate the relation between the uracil-dihydrouracil ratio and toxicities observed after treatment with fluoropyrimidines drugs. After addition of stable isotopically labelled internal standards, the analytes were isolated from a 100-microl urine sample using liquid-liquid extraction with ethyl acetate-2-propanol. Compounds were separated on an Atlantis dC18 column, using ammonium acetate-formic acid in water as the eluent. The eluate was totally led into an electrospray interface with positive ionisation and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the range 1.6-1600 microM, using both, artificial urine and pooled urine as matrices. Intra-day precisions were < or = 8% and inter-day precisions were < or = 10%. Accuracies between 91 and 108% were found. The analytes were chemically stable under all relevant conditions and the assay was successfully applied in two clinical studies of cancer patients treated with 5-fluorouracil or capecitabine.