Background: Plasma concentrations of fluoropyrimidine exhibit a wide interindividual variability that depends mainly on the activity of dihydropyrimidine dehydrogenase, its major catabolic enzyme. Patients with low dihydropyrimidine dehydrogenase activity are at an 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 Liquid chromatography coupled to tandem mass spectrometry (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 the reaction monitoring mode after 2 transitions per analyte and 1 per internal standard. The data obtained with this technique were retrospectively gathered for uracil metabolism phenotyping before 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 mcg/L, with extreme values of 3.9 and 81.6 mcg/L; 78 patients (15%) had uracil concentration ≥16 mcg/L, that is, above the threshold of decreased enzyme activity and initial dose reduction.