Generation and identification of reactive metabolites by electrochemistry and immobilized enzymes coupled on-line to liquid chromatography/mass spectrometry.

The detection of reactive metabolites using conventional in vivo and in vitro techniques is hampered because the intermediately formed reactive species are prone to covalent binding to cellular macromolecules. Therefore, the application of improved methods is required. The on-line coupling of an electrochemical reactor and horseradish peroxidase immobilized on magnetic microparticles with liquid chromatography/mass spectrometry (EC/LC/MS or HRP/LC/MS) allows the direct detection of reactive metabolites of the model compounds amodiaquine, amsacrine, and mitoxantrone, which are all known for readily binding to cellular macromolecules after metabolization by cytochrome P450. EC/LC/MS and HRP/LC/MS experiments were compared to rat liver microsome incubations and proved to be valuable complementary methods since reactive quinone, quinone imine, and quinone diimine species could be detected directly and not only after trapping with glutathione. Furthermore, N-dealkylation and N-oxidation of amodiaquine were successfully simulated by electrochemical oxidation reactions, as well as the formation of an aldehyde. Therefore, EC/LC/MS and HRP/LC/MS are promising tools for the identification of both reactive and stable metabolites in drug development.