A relatively simple and sensitive high-performance liquid chromatographic (HPLC) method is described for measuring galocitabine (Ro 09-1390) and its meatbolites, i.e. 5'-deoxy-fluorocytidine (5'-DFCR), 5'-deoxy-fluorouridine (5'-DFUR) and 5-fluorouracil (5-FU), in blood for the purpose of studying pharmacokinetics and toxicokinetics in small animals. The procedure for blood includes deproteinization with acetonitrile. Blood components were separated on a reversed-phase C18 column with a linear gradient of acetonitrile and water and detected at a wavelength of 270 nm. The between-day relative standard deviation (RSD) was less than 10% for all compounds at concentrations of 10-100 micrograms ml-1. The calibration curves obtained from the analysis of blood samples were linear and the correlation coefficients ranged from 0.997 to 0.999. The calculated determination limits were 6.9 micrograms ml-1 for galocitabine, 3.0 micrograms ml-1 for 5'-DFCR, 4.0 micrograms ml-1 for 5'-DFUR and 3.7 micrograms ml-1 for 5-FU.
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