Development and validation of a robust LC-MS/MS method for the simultaneous quantification of doxifluridine and its two metabolites in beagle dog plasma

A reverse-phase HPLC method with detection by mass spectrometry is described for the simultaneous determination of doxifluridine and its two active metabolites, 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd), in beagle dog plasma. The optimal chromatographic separation was achieved on a Waters Xterra®C18 column (4.6 × 250 mm i.d., 5 μm particle size) with a mobile phase of 0.1% formic acid in a mixture of 99% methanol and purifid water (99:1, v/v). The developed method was validated in beagle dog plasma with a lowest limit of quantification of 0.05 μg/mL for both doxifluridine and 5-FU, and 0.2 μg/mL for 5-FUrd. Doxifluridine and its two metabolites were stable under the analysis conditions, and intra- and inter-day accuracies exceeded 92.87%, with a precision variability ≤11.34% for each analyte. Additionally, the method for quantifying doxifluridine and its two metabolites, 5-FU and 5-FUrd, in beagle dog plasma was applied successfully to the analysis of pharmacokinetic samples.