Simultaneous HPLC determination of 5-fluorouracil and its metabolites in plasma of cancer patients

5-Fluorouracil (5-Fu) is a commonly used anticancer agent for treatment of solid tumours. Certain studies have reported conflicting results between individual plasma concentration levels and toxicity or therapeutic effects. For this reasons some authors proposed to evaluate the plasma levels of 5-Fu metabolites 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-5,6-dihydro-uracil. The aim of the present work is to develop and validate a new HPLC method simultaneously determining 5-fluorouracil and its three metabolites, to be used to study the plasma levels, therapeutic effects and toxicity in cancer patients. The analytes were separated on a 4.6 x 250 mm ODS1 (5 micro m) not end-capped column, operating at room temperature. Elution was performed under isocratic conditions, employing a 1.5 mM K(3)PO(4) mobile phase (pH 5). 5-Bromo-5,6-dihydro-uracil was used as internal standard. The limits of quantitation were 0.5 micro g/mL for 5-fluorouracil, 1 micro g/mL for 5-fluoro-5,6-dihydro-uracil, 3 micro g/mL for 5-fluoro-2'-deoxyuridine and 5-fluorouridine; the stability, recovery, linearity, accuracy and specificity of the compounds were evaluated according to the criteria widely accepted. Using this method we measured plasma samples of 18 cancer patients treated with folinic acid (100 mg/m(2)) by intravenous administration, followed by an i.v. bolus of 5-Fu (400 mg/m(2)). The concentration levels of 5-fluorouracil and for 5-fluoro-5,6-dihydro-uracil were detectable in all the subjects while 5-fluorouridine and 5-fluoro-2'-deoxyuridine were present only in eight patients.