Prediction of clozapine metabolism by on-line electrochemistry/liquid chromatography/mass spectrometry

Combining electrochemical conversion, liquid chromatography and electrospray ionization mass spectrometry (EC/LC/ESI-MS) on-line allows the rapid identification of possible oxidation products of clozapine (CLZ) in the absence and in the presence of glutathione. CLZ is, depending on the applied potential, oxidized to various products in an electrochemical flow-through cell using a porous glassy carbon working electrode. Several hydroxylated and demethylated species are detected on-line using LC/MS. While hydroxy-CLZ is most abundant at a potential of 400 mV, demethylation occurs more readily at higher potentials (at around 700 mV versus Pd/H₂reference). In the presence of glutathione (GSH), various isomeric glutathione adducts and respective products of further oxidation can be identified. The thioadducts are characterized by tandem MS. Mono-GSH and bis-GSH derivatives can be seen in the chromatograms. The results correlate well with the cyclic voltammetric profile of CLZ. The data are relevant from a pharmacological point of view, since similar metabolites (phases I and II) have been reported in the literature. The EC/LC/MS and EC/MS methods should be valuable tools that can be used to anticipate and understand the metabolization patterns of molecules of pharmacological interest and to point out reactive intermediates.