HPLC analysis of 5-FU and FdUMP in tissue and serum

Tissue and serum concentrations of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine 5' monophosphate (FdUMP) can be determined using a simple and sensitive reverse phase HPLC technique. Samples are extracted with ethyl acetate, evaporated to dryness, resuspended in the mobile phase (water/methanol [85:15]), and injected into a C18 muBondapack column (250 mm x 30 mm) with a flow rate of 1 ml/min. Both fluorescence (266 lambda Excitation and 350 lambda Emission) and UV (250 nm) are used for detection. Standard solutions of 5-FU and FdUMP produce a linear relationship (r2 = 0.985) with a detection sensitivity of 50 pg. Recovery following this extraction procedure is 78%. This analytical method is simple and convenient to use for the simultaneous detection of 5-FU and FdUMP.