A simple and sensitive fully validated HPLC-UV method for the determination of 5-fluorouracil and its metabolite 5,6-dihydrofluorouracil in plasma

The authors developed a simple and sensitive, fully validated HPLC-UV method for the determination of both 5-FU and its metabolite DHFU in small-volume plasma samples. The analytes were separated on a 4.6 x 250 mm ID Atlantis dC18 5-microm column with isocratic elution at room temperature. Chlorouracil was used as internal standard. The analytes were detected with an UV diode array detector. DHFU was detected at 205 nm, 5-FU at 266 nm, and chlorouracil at both wavelengths. The limits of quantification in plasma were 0.040 mug /mL for 5-FU and 0.075 microg/mL for DHFU. Linearity, accuracy, precision, recovery, dilution, freeze-thaw stability, and stability in the sample compartment were evaluated. The method appeared linear over a range from 0.04 to 15.90 microg/mL for 5-FU and from 0.075 to 3.84 microg/mL for DHFU. The method appeared very suitable for therapeutic drug monitoring and pharmacokinetic studies of 5-FU because of its simple extraction and small sample volume. Problems in earlier published methods with interfering peaks and variable retention times were overcome. The method appeared also to be suitable for detection of uracil and its metabolite dihydrouracil in plasma.