The achievement of mass balance by simultaneous quantification of floxuridine prodrug floxuridine 5-fluorouracil 5-dihydrouracil α-fluoro-β-ureidopropionate α-fluoro-β-alanine using LC-MS

摘要

5-fluoro-2′-deoxyuridine (floxuridine, 5-FdUrd) and 5-fluorouracil (5-FU) are widely used for the treatment of colorectal cancers. The mechanisms of action of 5-FdUrd and 5-FU, as well as the biochemical pathway responsible for their metabolism, are well understood. Identification of every metabolite and achieving mass balance by conventional UV absorption-based HPLC analysis are not feasible because the metabolites beyond 5-FU in the 5-FdUrd metabolic pathway are undetectable by UV light. We therefore established a mass spectrometry method, designed for fast and convenient analysis, for simultaneously measuring 5-FdUrd, 5-FU, and their metabolites. Linearity, precision and accuracy were validated in the concentration ranges studied for each compound. Hydrolysis studies of 5-FdUrd and amino acid mono ester prodrugs of 5-FdUrd in Capan-2 cell homogenates were carried out and the achievement of mass balance was established with this method (recovery of 5′-O-L-leucyl-FdUrd was 96.6 – 108.2% and that of 5-FdUrd was 79.4 – 117.4%). This simple LC-MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies.