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

Abstract

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/H(2) reference). 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.

Application of On-line Electrochemistry/Electrospray/Tandem Mass Spectrometry to a Quantification Method for the Antipsychotic Drug Zotepine in Human Serum

“…[7][8][9][10][11][12][13][14][15] It has also been reported that an electrochemical reaction coupled on-line to LC-MS is a promising approach in drug metabolism and protein research. [16][17][18][19][20][21][22][23] However, there are few reports on applications to methods for determining organic compounds in biological samples using this technique. 24 The combination of LC-MS/MS and the on-line EC reaction is potentially more selective as a drug quantification method, in biological samples, than ordinary LC-MS/MS.…”

On-line electrochemistry/liquid chromatography/mass spectrometry for the simulation of pesticide metabolism

“…Therefore, the on-line EC/MS system was expanded by introduction of an HPLC separation of the oxidation products before MS analysis (EC/LC/MS). Using this set-up, valuable information on the polarity of the oxidation products and on the formation of different isomers can be obtained, as was shown in our group for the antipsychotic agent clozapine [15] and for paracetamol [16]. Particularly for the investigation of reactive metabolites, on-line EC/LC/MS proved to offer some interesting aspects concerning the preparation [17] and the direct detection of short-lived species that often cannot be seen in biological systems [18].…”

Electrochemical Methods for the In Vitro Assessment of Drug Metabolism

“…To improve this system and obtain more information about metabolic reactions, different modifications have also been utilized. For example, the incorporation of trapping agents (i.e., glutathione (GSH) or other thiols) that can form stable adducts with the reactive intermediates generated in the electrochemical cell is commonly used to detect the interactions of metabolites (Lohmann & Karst, 2006;van Leeuwen et al, 2005;). Other electrochemical applications useful for metabolic studies include the development of enzyme-based biosensors, antibodies and DNA, among others (Joseph et al, 2003).…”

“…Furthermore, the simulation of phase II reactions, including conjugation with GSH or other thiols from electrochemically generated phase I metabolites, has also been achieved. For example, the metabolism of clozapine, including both phase I and phase II reactions, has successfully been mimicked (van Leeuwen et al, 2005), as well as the detoxification of acetaminophen (Lohmann & Karst, 2006) and the conjugation of diclofenac (Madsen et al, 2008a) by the electrochemical oxidation of the parent drugs.…”

“…The ternary combination of EC-LC-MS is able to detect reaction products with lifetimes on the order of several minutes; therefore, short-lived intermediates are not detected, as they react to more stable products during the chromatographic run. Direct EC-MS allows the detection of less stable compounds with lifetimes of a few seconds (van Leeuwen et al, 2005). In figure 2, a schematic representation of both systems is shown.…”