Phenotyping of uracil and 5-fluorouracil metabolism using LC-MS/MS for prevention of toxicity and dose adjustment of fluoropyrimidines.

BACKGROUND: Plasma concentrations of fluoropyrimidine exhibit a wide inter-individual variability that depends mainly on the activity of dihydropyrimidine dehydrogenase (DPD), its major catabolic enzyme. Patients with low DPD activity are at increased risk of overexposure and often severe, sometimes lethal toxicity. This study aimed to develop a quick and easy bioanalytical method for the simultaneous determination of endogenous uracil (U), exogenous 5-fluorouracil (5-FU), and their respective 5,6-dihydro-metabolite in human plasma using LC-MS/MS. METHODS: After protein precipitation, the compounds were purified using liquid-liquid extraction. Chromatographic separation was conducted using a Cortecs T3 column and binary gradient elution. Detection and quantification were performed in the positive electrospray ionization and selected reaction monitoring mode following two transitions per analyte and one per internal standard. The data obtained with this technique were retrospectively gathered for uracil metabolism phenotyping prior to fluoropyrimidine treatment (as enforced by national regulations) in a large group of 526 patients. RESULTS: The analytical response was linear (r > 0.99 for all compounds), and it yielded a lower limit of quantification of 2 ng.mL for U and UH2 as well as 4 ng.mL for 5-FU and 5,6-dihydro-5-FUH2. The median uracil concentration in 526 patients was 10.6 µg/L, with extreme values of 3.9 and 81.6 µg/L; 78 patients (15%) had uracil concentration ≥16 µg.L, i.e. above the threshold of decreased enzyme activity and initial dose reduction.