Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts

Methodology that permits complete analysis of the intracellular metabolites of 5-fluorouracil (FUra) has been developed. A high-pressure liquid chromatography system that is capable of separating all metabolites of FUra found in acid-soluble cell extracts is described. In addition to the expected FUra metabolites, FUDP-hexoses were found to be present in large amounts in L121O cells treated with FUra. Improved procedures that permit quantitation of the FdUMP which is covalently bound to dTMP synthetase, as well as the total intracellular FdUMP levels are described; the latter is accomplished by dissociation of the FdUMP-dTMP synthetase complex in sonicated cell extracts followed by phosphatase treatment and subsequent high-pressure liquid chromatography analysis of FdUrd. An example of the integrated methodology in which all metabolites of FUra metabolism are analyzed over a 6-h exposure period of L1210 cells to [6-³H]FUra is provided.