Gas-liquid chromatographic analysis of fluoropyrimidine nucleosides and fluorouracil in plasma and urine

A gas-liquid chromatographic method employing on-column alkylation and a nitrogen-sensitive detector was developed for the analysis of 5-fluoro-2′-deoxyuridine, 5-fluorouridine, and 5-fluorouracil in plasma and urine. Samples (0.72 ml) containing the fluoropyrimidine and internal standard (5-chloro-2′-deoxyuridine for nucleoside analyses and 6-methyluracil for 5-fluorouracil analyses) were prepared for gas-liquid chromatography by sequential cation-exchange and anion-exchange column chromatography. Recoveries of fluoropyrimidines were 71–95% over the concentration ranges studied. The dried eluate from the anion-exchange column was dissolved in p-tolyltrimethylammonium hydroxide in methanol before gas-liquid chromatographic analysis. Columns packed with either 3% SP-2100 on Supelcoport or 3% OV1 on Gas-Chrom Q were suitable for nucleoside analyses; a column packed with 0.75% Carbowax 20M-5% KOH on Chromsorb G was used for 5-fluorouracil analyses. The fluoropyrimidine nucleosides were well separated from each other and from the potentially interfering endogenous compounds 2′-deoxyuridine and uridine; 5-fluorouracil was well separated from uracil. Linear standard curves (peak area ratio method) were obtained for plasma containing 0.025 to 20 μg FdUrd (0.1 to 81 μ m) or 0.05 to 1.0 μg FUrd (0.2 to 3.8 μ m), and for urine containing 0.2 to 1.0 μg (0.8 to about 4 μ m) of the nucleosides. Standard curves for 5-fluorouracil (1.5 to 7.9 μ m) and 2′-deoxyuridine (0.9 to 4.4 μ m) were also linear. A measurable amount of 5-fluorouracil, equivalent to 4 to 7% of the 5-fluoro-2′-deoxyuridine injected, was formed from the nucleoside on the gas-liquid chromatographic column, requiring correction of 5-fluorouracil concentrations measured in the presence of 5-fluoro-2′-deoxyuridine. Applicability of the analytical method to determination of the pharmacokinetics of intravenous 5-fluoro-2′-deoxyuridine in the dog was demonstrated. Total, metabolic, and renal clearances of the drug were 42.8, 38.6, and 4.2 ml/kg/min, respectively.