Glucuronidation of Prodrug Reactive Site: Isolation and Characterization of Oxymethylglucuronide Metabolite of Fosphenytoin

Abstract

Abstract Background: This investigation was undertaken to identify the structure of a novel immunoreactive metabolite derived from fosphenytoin that has been hypothesized previously as present in sera from renally impaired patients receiving this prodrug. Methods: The metabolite was isolated from uremic sera using solid-phase extraction and HPLC. Structural analysis was performed using HPLC–tandem mass spectrometry, nuclear magnetic resonance (NMR), deuterium exchange, and chemical derivatization. Immunoreactivity was evaluated using a fluorescence polarization immunoassay. Results: The metabolite had a parent ion at m/z 457 in the negative-ion mode and fragmented to yield the m/z 251 of phenytoin, as well as other mass fragments of phenytoin. Mass fragments associated with glucuronic acid were also present. The chromatographic peak corresponding to this metabolite demonstrated immunoreactivity sufficient to lead to falsely increased reported values for phenytoin immunoassays. The observed immunoreactivity was also proportional to the relative concentration of the metabolite in collected fractions. Analysis by NMR indicated the presence of phenyl groups with chemical shifts identical to those of phenytoin, as well as the presence of a methylene bridge, which was consistent with the same methylene bridge present on the phosphate ester of fosphenytoin. Comparative analysis of serum samples from renally impaired patients receiving phenytoin vs fosphenytoin using multiple reaction monitoring quantification demonstrated that this metabolite was associated with fosphenytoin administration. Conclusions: A unique immunoreactive oxymethylglucuronide metabolite derived from fosphenytoin has been isolated from sera from uremic patients receiving this prodrug.