Ines Möller

In vitro phase I metabolism of the synthetic cannabimimetic JWH-018

A potent synthetic cannabinoid receptor agonist, JHW-018, was recently detected as one of the most prominent active agents in abusively used incenses such as Spice and other herbal blends. The high pharmacological and addictive potency of JWH-018 highlights the importance of elucidating the metabolism of JWH-018, without which a meaningful insight into its pharmacokinetics and its toxicity would not be possible. In the present study, the cytochrome P450 phase I metabolites of JWH-018 were investigated, after in vitro incubation of the drug with human liver microsomes, followed by liquid chromatography–tandem mass spectrometry analysis. This revealed monohydroxylation of the naphthalene ring system, the indole moiety, and the alkyl side chain. In addition, observations were made of dihydroxylation of the naphthalene ring system, and the indole moiety, or as result of a combination of monohydroxylations of both the naphthalene ring system and the indole moiety or the alkyl side chain, or a combination of monohydroxylations of both the indole ring system and the alkyl side chain. There is also evidence of trihydroxylation at different locations of the hydroxyl groups in the molecule. Furthermore, dehydration of the alkyl side chain, in combination with both monohydroxylation and dihydroxylation as well as arene oxidation of the naphthalene ring system, combined with both monohydroxylation and dihydroxylation at different sites of oxidation were found. N-dealkylation also in combination with both monohydroxylation and dihydrodiol formation of the N-dealkylated metabolite was detected. Finally, a metabolite was found carboxylated at the alkyl side chain.

Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO‐mimetic peptide for sports drug testing purposes

Erythropoietin (EPO) and other erythropoiesis-stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO-mimetic peptides is under development from which peginesatide (also referred to as Hematide™), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase-III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO-mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL).

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.

Mass spectrometric detection of peginesatide in human urine in doping control analysis.

Erythropoiesis-stimulating agents (ESAs) have frequently been confessed to be illicitly used in elite sports due to their endurance enhancing effects. Recently, peginesatide, the first representative of a new generation of ESAs, referred to as Erythropoietin (EPO)-mimetic peptides, obtained approval in the USA under the trade name Omontys(®) for the treatment of anaemic patients. Lacking sequence homology with EPO, it consists of a pegylated homodimeric peptide of approximately 45 kDa, and thus, specific approaches for the determination of peginesatide in blood were developed as conventional detection assays for EPO do not allow for the analysis of the EPO-mimetic peptides. However, as urine specimens are the most frequently provided doping control samples and pharmacokinetic studies conducted in rats and monkeys revealed the excretion of the pegylated peptide into urine, a detection method for peginesatide in urine would be desirable. A mass spectrometric assay in human urine was developed consisting of protein precipitation with acetonitrile followed by proteolytic digestion after the removal of the acetonitrile fraction under reduced pressure. Purification and concentration of the resulting proteotypic target peptide was accomplished by means of solid-phase extraction on strong cation-exchange resin prior to liquid chromatographic-tandem mass spectrometric analysis. Method validation was performed for qualitative purposes and demonstrated specificity, precision, linearity as well as sufficient sensitivity (limit of detection: 0.5 ng/ml) while proof-of-concept for the applicability of the assay for the determination of peginesatide in authentic urine samples was obtained by analyzing animal in vivo specimens collected after a single i.v. administration of peginesatide over a period of 4 days.

Iodine speciation using thin-layer chromatography coupled to inductively coupled plasma-mass spectrometry by means of an extraction device

Thin-layer chromatography (TLC) has been coupled to inductively coupled plasma-mass spectrometry (ICP-MS) using an extraction device, which allows a nearly complete recovery of compounds from thin-layer chromatograms within short extraction times, small volumes and without contamination. Iodinated X-ray contrast agents were separated and analyzed with the newly developed setup. Iodine speciation analysis was carried out with deviations between the expected and the measured concentrations in spiked urine samples from 3.2 to 16.6%.

Mass spectrometry-based characterization of new drugs and methods of performance manipulation in doping control analysis

Efficient and comprehensive sports drug testing necessitates frequent updating and proactive, preventive anti-doping research and the early implementation of new, emerging drugs into routine doping controls is an essential aspect. Several new drugs and drug candidates with potential for abuse, including so-called Rycals (ryanodine receptor calstabin complex stabilizers, for example, S-107), hypoxia-inducible factor (HIF) stabilizers and peroxisome-proliferator-activated receptor (PPAR) δ agonists (for example, GW1516), were studied using different mass spectrometry- and ion mobility-based approaches and their gas-phase dissociation behaviors were elucidated. The detailed knowledge of fragmentation routes allows a more rapid identification of metabolites and structurally related, presumably “tailor-made”, analogs potentially designed for doping purposes. The utility of product ion characterization is demonstrated, in particular, with GW1516, for which oxidation products were readily identified in urine samples by means of diagnostic fragment ions, as measured using high-resolution/high-accuracy mass spectrometry and higher energy collision-induced dissociation (HCD).

Synthesis, Mass Spectrometric Characterization, and Analysis of the PPARδ Agonist GW1516 and Its Major Human Metabolites: Targets in Sports Drug Testing

The elucidation of metabolic pathways and the detection of emerging therapeutics potentially enhancing athletic performance are of paramount importance to doping control authorities to protect the integrity of elite sports. A new drug candidate belonging to the family of the peroxisome proliferator-activated receptor-delta agonists termed GW1516 (also referred to as GW501516) has been prohibited by the World Anti-Doping Agency in 2009 due to its potential to artificially increase endurance. Consequently, sports drug testing laboratories need to establish detection methods enabling the identification of the intact substance and/or its metabolite(s) that unambiguously prove the presence or absence of the target substances in doping control specimens. Simulating human metabolic reactions using liver microsomal preparations, minute amounts of possible urinary metabolites were obtained that were characterized by mass spectrometry-based methods. Subsequently, the most abundant metabolic products were chemically synthesized and as well characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Finally, GW1516 and two oxidized metabolites were implemented in a routine doping control analytical assay based on liquid chromatography-(tandem) mass spectrometry (LC-MS/MS), which was tested for its -fitness-for-purpose using spiked urine samples.

Detection of peginesatide in equine serum using liquid chromatography-tandem mass spectrometry for doping control purposes

Erythropoietin (EPO) and its recombinant analogues are suspected to be illicitly administered to horses for performance enhancing purposes and, consequently, prohibited in equine sports. Recently, a new erythropoiesis-stimulating agent, peginesatide (Omontys, formerly referred to as Hematide), belonging to the upcoming class of EPO-mimetic peptides, received approval for the treatment of anaemia in humans with chronic kidney disease on dialysis. As the pegylated dimeric peptide of approximately 45 kDa without sequence homology to EPO is not detectable by conventional EPO detection assays, specific methods are bound to be established for horse sports drug testing. Thus, by fortifying equine serum with peginesatide, an approach consisting of a proteolytic digestion with subtilisin after protein precipitation was developed, eventually targeting a proteotypic and xenobiotic pentapeptide which is easily accessible to liquid chromatography-tandem mass spectrometry analysis. The method was validated for qualitative purposes and demonstrated to be specific, precise (relative standard deviations below 14%), sensitive (limit of detection 10 ng mL−1) and linear. Being simple, cost-effective and readily transferable to other doping control laboratories, a mass spectrometric assay for the detection of therapeutic concentrations of peginesatide in equine serum is, in terms of preventive doping research, applicable to routine analysis shortly after approval of the drug.

Using electrochemistry for metabolite simulation and synthesis in preventive doping research: application to the Rycal S107 and the PPARδ-agonist GW1516

In view of the numerous newly emerging drug candidates, it is a constant challenge for doping control laboratories to keep sports drug testing procedures up-to-date, thus, allowing for comprehensive analyses. Therefore, it is of major importance to elucidate the biotransformation pathway of potentially performance-enhancing therapeutics as early as possible in order to determine analytical targets that can reliably identify drug abuse. Usually, in vivo and/or in vitro assays with rodents and/or liver cell preparations, respectively, serve this purpose. However, these models for metabolism simulation are time-consuming and uneconomical for the generation and isolation of larger amounts of metabolite reference material for thorough method development. In this work, electrochemistry (EC) in hyphenation with liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS) was used to study the metabolic fate of the ryanodine receptor-calstabin-complex stabilizer (“Rycal”) S107 and the peroxisome proliferator-activated receptor (PPAR) δ agonist GW1516. High resolution/high accuracy MS and tandem MS were utilized and supported by experiments with stable isotope-labeling for S107, or human liver microsomes (HLM) and S9 fraction for GW1516. The results were compared with recently published in vitro data. For both potential doping agents, the purely instrumental approach succeeded in generating all of the known in vitro phase I metabolites. Mono-oxygenated products like N- and/or S-oxides were generated, next to demethylated and dehydrogenated species (S107) or bis-oxygenated products (e.g. the GW1516 sulfone). This demonstrates the value of EC as a fast and easy method for metabolism simulation while facilitating the generation of pure reference material for preventive doping research.