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Abstract: A rapid high-performance liquid chromatography method has been developed for simultaneous determination of capecitabine and its metabolites: 5′-deoxy-5-fluorocytidine (5′-DFCR), 5′-deoxy-5-fluorouridine (5′-DFUR) and 5-fluorouracil (5-FU). 5′-DFCR was synthesized by hydrolyzing capecitabine using commercially available carboxyl esterase (CES) and characterized by NMR, mass spectrometry and elemental analysis. Base-line separations between capecitabine, 5′-DFCR, 5′-DFUR and 5-FU were found with symmetrical peak shapes on a Discovery RP-amide C16 column using 10mM ammonium acetate at pH 4.0 and methanol as the mobile phase. The retention times of capecitabine, 5′-DFCR, 5′-DFUR and 5-FU were 8.9, 5.0, 5.3 and 3.0min, respectively. Linear calibration curves were obtained for each compound across a range from 1 to 500μgml−1. The intra- and inter-day relative standard deviations (%RSD) were <5%. A single-step protein precipitation method was employed for separation of the analytes from bio-matrices. Greater than 85% recoveries were obtained for capecitabine, 5′-DFCR, 5′-DFUR and 5-FU from bio-fluids including mouse plasma, mouse serum and rabbit bile. [Copyright &y& Elsevier]