A ferrocene-based reagent for the conjugation and quantification of reactive metabolites.

In the present study, a method for the analysis of reactive metabolites via liquid chromatography (LC) with inductively coupled plasma-mass spectrometry (MS) was developed. A ferrocenyl-modified glutathione (GSH) reagent, consisting of GSH and succinimidyl-3-ferrocenylpropionate, was synthesized. Derivatization of the tripeptide was performed at the N-terminus, leaving the nucleophilic thiol group vacant for the attack of electrophilic compounds. The potential of ferrocenylpropionate (FP)-GSH as a trapping agent for reactive metabolites was investigated using an electrochemical flow-through cell for metabolism simulation coupled online to a LC system with electrospray ionization mass spectrometric detection. The pharmaceuticals amodiaquine, an antimalarial agent, and clozapine, an antipsychotic compound, served as model substances. By proving the successful adduct formation between the reactive metabolite and ferrocene-labeled GSH, it could be shown that FP-GSH is an effective trapping agent which eases routine reversed-phase LC analyses. In contrast to GSH, which is usually used for the conjugation of reactive metabolites and where the resulting adducts often show no or only very little retention, FP-GSH facilitates the detection of the corresponding metabolite adducts due to higher retention times.

Metabolic studies of tetrazepam based on electrochemical simulation in comparison to in vivo and in vitro methods.

During the last 2 years, the knowledge on the metabolic pathway of tetrazepam, a muscle relaxant drug, was expanded by the fact that diazepam was identified as a degradation product of tetrazepam. The present study demonstrates that this metabolic conversion, recently discovered by in vivo studies, can also be predicted on the basis of a purely instrumental method, consisting of an electrochemical cell (EC) coupled to online liquid chromatography (LC) and mass spectrometry (MS). By implementing a new electrochemical cell type into the EC-LC-MS set-up and by an enhanced oxidation potential range up to 2V, one limitation of the electrochemical metabolism simulation, the hydroxylation of alkanes and alkenes, has been overcome. Instead of commonly used flow-through cell with a porous glassy carbon working electrode, a wall-jet cell with exchangeable electrode material was used for this study. Thereby, the entire metabolic pathway of tetrazepam, in particular including the hydroxylation of the tetrazepam cyclohexenyl moiety, was simulated. The electrochemical results were not only compared to microsomal incubations, but also to in vivo experiments, by analysing urine samples from a patient after tetrazepam delivery. For structure elucidation of the detected metabolites, MS/MS experiments were performed. The comparison of electrochemistry to in vitro as well as to in vivo experiments underlines the high potential of electrochemistry as a fast screening tool in the prediction of metabolic transformations in drug development.

Electrochemistry meets enzymes: instrumental on-line simulation of oxidative and conjugative metabolism reactions of toremifene.

The metabolism of the selective estrogen receptor modulator toremifene was simulated in an on-line electrochemistry/enzyme reactor/liquid chromatography/mass spectrometry system. To simulate the oxidative phase I metabolism, toremifene was oxidized in an electrochemical (EC) flow-through cell at 1,500 mV vs. Pd/H2 to its phase I metabolites, some of which are reactive quinoid species. In the presence of glutathione-S-transferase (GST), these quinoid compounds react with glutathione, which is also the common detoxification mechanism in the body. While reacting with glutathione, the chlorine atom is eliminated from the toremifene moiety. Due to higher conversion rates, GST supplied in continuous flow proved to be more efficient than using immobilized GST on magnetic microparticles. In the absence of GST, not all GSH adducts are formed, proving the necessity of a phase II enzyme to simulate the complete metabolic pathway of xenobiotics in an on-line EC/LC/MS system.

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

On-line electrochemistry/liquid chromatography/mass spectrometry (EC/LC/MS) was employed to mimic the oxidative metabolism of the fungicide boscalid. High-resolution mass spectrometry and MS/MS experiments were used to identify its electrochemical oxidation products. Furthermore, the introduction of a second electrochemical cell with reductive conditions provided important additional information on the oxidation products. With this equipment, hydroxylation, dehydrogenation, formation of a covalent ammonia adduct, and dimerization were detected after initial one-electron oxidation of boscalid to a radical cation. On-line reaction with glutathione yielded different isomeric covalent glutathione adducts. The results of the electrochemical oxidation are in good accordance with previously reported in vivo experiments, showing that EC/LC/MS is a useful tool for studying biotransformation reactions of various groups of xenobiotics.

Quantification of electrochemically generated iodine-containing metabolites using inductively coupled plasma mass spectrometry.

For the risk assessment of drug candidates, the identification and quantification of their metabolites is required. The majority of analytical techniques is based on calibration standards for quantification of the metabolites. As these often are not readily available, the use of inductively coupled plasma mass spectrometry (ICPMS) is an attractive alternative for drugs containing heteroatoms. In this work, the online coupling of electrochemistry (EC), liquid chromatography (LC), and ICPMS is presented. The antiarrhythmic agent amiodarone, which contains two iodine atoms, is oxidized in an electrochemical flow-through cell under N-dealkylation and deiodination. The metabolites that are generated at different EC potentials are identified by electrospray ionization (ESI) mass spectrometry, compared to those from rat liver microsomal incubations and quantified by ICPMS. Phase-optimized LC, a recent approach for high-performance isocratic separations, is used to avoid the ICPMS calibration problems known to occur with gradient separations. The potential of the complementary use of ESI-MS and ICPMS for the qualitative and quantitative analysis of drug metabolites becomes apparent in this work.

Covalent protein modification by reactive drug metabolites using online electrochemistry/liquid chromatography/mass spectrometry.

We present a rapid and convenient method to perform and evaluate the covalent protein binding of reactive phase I metabolites. The oxidative metabolism of the drugs paracetamol, amodiaquine, and clozapine is simulated in an electrochemical (EC) flow-through cell, which is coupled online to an LC/MS system. Adduct formation of the reactive metabolites with the proteins beta-lactoglobulin A and human serum albumin proceeds in a reaction coil between EC cell and injection system of the HPLC system. The formed drug-protein adducts are characterized with online time-of-flight mass spectrometry, and the modification site is localized using FTICR-mass spectrometry. Due to its simple setup, easy handling, and short analysis times, the method provides an interesting tool for the rapid risk assessment of covalent protein binding as well as for the synthesis of covalent drug-protein adducts in high purity and high yield.

Use of an electrochemically synthesised metabolite of a selective androgen receptor modulator for mass spectrometry-based sports drug testing.

The elucidation of the metabolism of new therapeutics is a major task for pharmaceutical companies and of great interest for drug testing laboratories. The latter in particular need to determine the presence or absence of drugs or their metabolic products in urine to test for a misuse of these compounds. Commonly, in vitro or animal models are used to mimic the human metabolism and produce potential targets in amounts allowing for method development. An alternative route based on electrochemical reactions of drugs was reported to allow for the generation of selected metabolites. The utility of this approach for doping control purposes was demonstrated with a novel class of anabolic agents termed selective androgen receptor modulators (SARMs). An arylpropionamide- derived drug candidate was subjected to electrochemical "metabolism" and a major phase-I- metabolite, resulting from the elimination of a substituted phenol residue as identified in in vitro experiments, was generated and characterised using liquid chromatography/nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. The metabolite was included in routine doping control procedures based on liquid chromatography/tandem mass spectrometry and has served as a reference compound for 5000 doping control specimens.

Biomimetic modeling of oxidative drug metabolism : Strategies, advantages and limitations.

The prediction of drug metabolism is an important task in drug development. Besides well-established in vitro and in vivo methods using biological matrices, several biomimetic models have been developed. This review summarizes three different nonenzymatic strategies, including metalloporphyrins as surrogates of the active centre of cytochrome P450, Fenton's reagent, and the electrochemical oxidation of drug compounds. Although none of the systems can simulate the whole range of cytochrome P450-catalyzed reactions adequately, the biomimetic models show some advantages over standard in vitro methods. For example, metalloporpyhrin catalysts allow the synthesis of certain metabolites in sufficient amounts and with sufficient purities to permit characterization and further pharmacological and toxicological tests. The electrochemical generation of metabolites coupled on-line to liquid chromatography/mass spectrometry is a promising tool for studying reactive metabolites and can be applied in automated high-throughput screening approaches. In this paper, detailed comparisons with cytochrome P450 catalysis are drawn, advantages and disadvantages of the respective methods are revealed, and possible applications are discussed.

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.

Simulation of the detoxification of paracetamol using on-line electrochemistry/liquid chromatography/mass spectrometry.

On-line electrochemistry/liquid chromatography/mass spectrometry was used to simulate the detoxification mechanism of paracetamol in the body. In an electrochemical flow-through cell, paracetamol was oxidized at a porous glassy carbon working electrode at a potential of 600 mV vs. Pd/H2 with formation of a quinoneimine intermediate. The quinoneimine further reacted with glutathione and/or N-acetylcysteine to form isomeric adducts via the thiol function. The adducts were characterized on-line by liquid chromatography/mass spectrometry. These reactions are similar to those occurring between paracetamol and glutathione under catalysis by cytochrome P450 enzymes in the body.