A simple chromatographic method for the analysis of pyrimidines and their dihydrogenated metabolites

Fluorouracil (5-FU) is a pyrimidine analog widely used in the treatment of numerous malignancies. One major metabolic pathway is the reduction of its double bond by dihydropyrimidine dehydrogenase (DYPD), whose activity is a strong determinant of 5-FU plasma levels. Recent findings show that deficiencies in DYPD are less rare than generally assumed and play a major role in 5-FU toxic effects. We developped a simple, sensitive and accurate liquid chromatographic method that allows the simultaneous determination of uracil, 5-fluorouracil and their dihydrogenated metabolites in plasma. This method offers a useful tool for the detection of defects in pyrimidine degradation. HPLC was carried out by using Spherisorb ODS1 (10 cm) and ODS2 (25 cm) columns serially mounted, and 10 mM phosphate buffer, pH 3.0, as the mobile phase with UV detection at 205 nm. Many parameters, such as mobile phase pH, ionic strength, column temperature, were found to have a marked influence on the results. We calculated the ratio dihydrouracil/uracil and could describe a Gaussian distribution of this ratio in a population of 78 healthy volunteers.