Gas chromatographic measurement of urinary 5-fluoro-2′-deoxyuridine levels after barium salt precipitation and sephadex LH-20 cleanup

Abstract

A method is presented for the quantitative determination of 5-fluoro-2′-deoxyuridine (FdUrd) in human urine. An internal standard (IS), 5-chloro-2′-deoxyuridine, is added to the sample (1.0 ml). The urinary phosphates and sulfates are removed by precipitation as their barium salts, FdUrd, and IS are subsequently isolated on a small column of strong anion-exchange resin. The crude extract obtained is further purified by chromatography on Sephadex LH-20; the resulting eluate is collected and evaporated to dryness. The residue is then permethylated using potassium- tert-butoxide in dimethyl sulfoxide and methyl iodide; the derivatized nucleosides are reextracted from the reaction mixture and analyzed on a glass capillary column coated with OV-17, coupled to a thermionic nitrogen-selective detector. Reproducible quantitative results can be obtained down to urinary concentrations of 50 ng/ml; recovery values average 70% in the range of 0–1 μg/ml. The method was applied to the monitoring of the urinary FdUrd excretion in two cancer patients.