Simultaneous HPLC analysis of hydrophobic prodrugs and their hydrophilic metabolites in biological media without sample preparation. Application to the biotransformation studies of pronucleotides derived from 5-fluorouracil

Improvements of an "on-line cleaning" HPLC method for analysis of biological samples are presented: (i) the use of cleaning precolumns filled with hydrophobic stationary phases instead of the hydrophilic ones previously used to eliminate the biological matrix: (ii) the combination in the mobile phase of anionic and cationic pairing reagents in order to retain on the precolumn all the metabolites, whatever their hydrophilicity and ionicity are. Such modifications allowed to study the biotransformation of prodrugs of 5-Fluorouracil, designed to act as antitumoral pronucleotides.