Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254

Accepted Manuscript Title: Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254 Author: Josef Dib Andreas Thomas Philippe Delahaut Eric Fichant Wilhelm Sch¨anzer Mario Thevis PII: DOI: Reference:

S0731-7085(16)30145-5 http://dx.doi.org/doi:10.1016/j.jpba.2016.03.027 PBA 10572

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

15-1-2016 9-3-2016 10-3-2016

Please cite this article as: Josef Dib, Andreas Thomas, Philippe Delahaut, Eric Fichant, Wilhelm Sch¨anzer, Mario Thevis, Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2016.03.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254 Josef Dib1, Andreas Thomas1, Philippe Delahaut2, Eric Fichant2, Wilhelm Schänzer1, and Mario Thevis1,3*

1

Center for Preventive Doping Research - Institute of Biochemistry, German Sport University

Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany 2

CER Groupe, Marloie, Belgium

3

European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn,

Germany

*corresponding author:

Mario Thevis, PhD Institute of Biochemistry - Center for Preventive Doping Research German Sport University Cologne Am Sportpark Müngersdorf 6 50933 Cologne, Germany Tel: +49 221 4982 7070 Fax: +49 221 4982 7071 Email: [email protected]

Keywords: sport, doping, mass spectrometry, exercise mimetic, metabolism

Graphical Abstract

Highlights     

In vitro- and in vivo-metabolism study of adipoR agonists AdipoRon and 112254. Identification of metabolites relevant for routine doping control assays. Characterization via high resolution/ high accuracy – tandem mass spectrometry. Overlapping patterns of phase-I metabolites were observed in vitro and in vivo. AdipoRon yielded phase-II metabolites in vivo (rat).

Abstract Peroxisome proliferator-activated receptors (PPARs), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), sirtuin 1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK) are regulators of transcriptional processes and effects of exercise and pseudoexercise situations. Compounds occasionally referred to as endurance exercise mimetics such as AdipoRon and 112254, both adiponectin receptor agonists, can be used to simulate the physiology of endurance exercise via pathways including these transcriptional regulators. Adiponectin supports fatty acid utilization and triglyceride-content reduction in cells and increases both the mitochondrial biogenesis and the oxidative metabolism in muscle cells. In routine doping control analysis, knowledge about phase-I and -II metabolic products of target analytes is essential. Hence, in vitro- and in vivo-metabolism experiments are frequently employed tools in preventive doping research to determine potential urinary metabolites for sports drug testing purposes, especially concerning new, (yet) unapproved compounds. In the present study, in vitro assays were conducted using human liver microsomal and S9 fractions, and rat in vivo experiments were performed using both AdipoRon and 112254. For AdipoRon, obtained samples were analyzed using liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry with both electrospray ionization or atmospheric-pressure chemical ionization techniques. Overall, more than five phase I-metabolites were found in vitro and in vivo, including particularly monohydroxylated and hydrogenated species. No phase IImetabolites were found in vitro; conversely, signals suggesting the presence of glucuronic acid or other conjugates in samples collected from in vivo experiment were observed, the structures of which were however not conclusively identified. Also for 112254, several phase-I metabolites were found in vitro, e.g. monohydroxylated and demethylated species. Here, no phase IImetabolites were observed neither using in vitro nor in vivo samples. Based on the generated

data, the implementation of metabolites and unmodified drug candidates into routine doping control protocols is deemed warranted for comprehensive sports drug testing programs until human elimination study data are available.

1. Introduction Substances aiming at simulating the effects of endurance exercise on skeletal muscle by triggering the same adaptations of the cardiopulmonary, endocrine, vascular, and neuromuscular system [1] are frequently referred to as exercise mimetics [2]. Endurance exercise further induces effects initiating the transdifferentiation of fast-twitch (type II) to slow-twitch (type I) myofibers, resulting in an increased aerobic capacity in skeletal muscle cells and, consequently, increased athlete performance [3]. Numerous transcriptional regulators are involved in these processes and therefore represent viable targets for exercise mimetic substances. Among these targets, the peroxisome proliferator-activated receptors (PPARs), which are nuclear receptors [4], the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) [5], sirtuin 1 (SIRT1) , a NAD-dependent deacetylase [6], and adenosine monophosphate-activated protein kinase (AMPK), an enzyme playing a critical role in the physiological energy balance [7], are of particular relevance [8]. While the development of such exercise mimetics aims at identifying and producing potential therapeutics ameliorating obesity, diabetes mellitus type II, and other health-related issues [2, 9], the associated increased mitochondrial biogenesis and aerobic capacity are factors that necessitate considerations also in the context of anti-doping research. Adiponectin is an endogenous protein secreted from adipocytes [10-13], supporting fatty acid utilization and triglyceride-content reduction in cells that leads to decreased insulin resistance [14]. It is further reported to exhibit an anti-atherosclerosis effect [15] and increases both the mitochondrial biogenesis and the oxidative metabolism in muscle cells [16]. To date two adiponectin receptors termed adiponectin receptor 1 and 2 (adipoR1 and adipoR2) are known. AdipoR1 is mainly found on skeletal muscle cells while adipoR2 predominates on liver cells [17,

18]. The aforementioned transcriptional regulators PPARs, PGC-1α/γ, SIRT1, and AMPK and consecutive pathways are activated by adiponectin via these receptors [14, 16], and two synthetic adipoR-agonists named AdipoRon and 112254 (Figure 1,compounds 1 and 2, respectively) were recently introduced [19, 20]. Due to an inherent potential for misuse in sports, proactive studies to enable the detection of these analytes in routine doping control specimens were initiated. Here, knowledge regarding phase-I and –II metabolic products of the respective compounds is required, and commonly employed strategies based on consecutive in vitro and in vivo studies were used [21-25] to provide the information required to ensure comprehensive doping controls concerning these new, emerging compounds. AdipoRon (1) and 112254 (2), which were synthesized and characterized as described elsewhere [26], were metabolized in vitro by human liver microsomes and S9 fraction, and administered to rats in an in vivo study. Metabolic products found were analyzed and characterized by liquid chromatography-high-resolution/high-accuracy tandem mass spectrometry (LC-HRMS(/MS)) and LC-MS³-experiments.

2. Materials and methods 2.1. Chemicals and reagents AdipoRon, 112254 and isotopically labeled analogs (Figure 1) were synthesized in-house and fully characterized as reported elsewhere [26]. Human liver microsomes were purchased from Life Technologies GmbH (Darmstadt, Germany) and S9 fraction from BD Gentest (Woburn, USA). 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) was obtained from Merck KGaA (Darmstadt, Germany). Both D-saccharic acid 1,4-lactone monohydrate (SL) and uridine 5'diphospho-glucuronosyltransferase (UDPGA) were purchased from Sigma-Aldrich (Steinheim, Germany) and nicotinamide adenine dinucleotide phosphate in the reduced form (NADPH) from

Roche Diagnostic GmbH (Mannheim, Germany). Deionized water used for all experiments was obtained from a Sartorius Stedim arium® pro UV (Guxhagen, Germany) apparatus. All organic solvents (analytical grade) were obtained from Honeywell (Seelze, Germany) and used without further purification.

2.2. In vitro metabolic assay An in vitro assay based on human liver enzyme fractions (microsomal and S9 fraction) was established for the investigation of metabolic reactions of AdipoRon (1) and 112254 (2) and both isotopically labeled compounds 3 and 4. For phase I metabolites, 10 µL of a stock solution of AdipoRon and 112254 (100 µM) was used and 70 µL of 50 mM phosphate buffer (pH 7.4) containing 5 mM of MgCl2 was added. A volume of 10 µL each of NADPH (5 mM) as co-factor and human liver microsomes (final protein concentration 2 mg/mL) was added, respectively, to reach a final volume of 100 µL. Both substances were incubated at 37 °C, with AdipoRon (1 and 3) was incubated for 2 h whereas 112254 (2 and 4) was incubated for 6 h. For the investigation on phase II metabolites, 10 µL of S9 fraction enzymes (final protein concentration 2 mg/mL) and either 10 µL of co-factor UDPGA (20 mM final concentration) for glucuronidation or PAPS (80 µM) for sulfonation were added after completion of the phase-I metabolism incubation time. In the samples subjected to glucuronidation, 10 µL of 1,4-saccharic acid lactone (5 mM) was added as glucuronidase inhibitor. Accordingly, adjusted volumes of the phosphate buffer were added while preparing the preceding phase I-metabolism reactions to allow for a final sample volume of 100 µL. The reaction was terminated by the addition of 300 µL ice-cold acetonitrile. The precipitated proteins were removed by centrifugation at 17,000 g and 4 °C for 5 min. The supernatant was transferred into a fresh tube and the acetonitrile was removed under reduced

pressure in a centrifuge. The residue was dissolved in 100 µL of a mixture of water and acetonitrile (90:10 v/v) and an aliquot of 3 µL was injected into the LC/ESI-HRMSsystem. All experiments were conducted with a minimum of three replicates. Furthermore, for every substance, samples were prepared to test for exclusivity of metabolic versus non-metabolic reactions. This comprised an additional incubation of a substrate blank sample, a co-factor blank sample, and an enzyme blank sample for each compound.

2.3. In vivo rat administration study A total of 6 Sprague Dawley rats each (weighing 200 – 300 g before administration study) were orally administered with a single dose of 1 mg (dissolved in 200 µL of dimethylsulfoxide) of AdipoRon (1) or 112254 (2), respectively. Over a period of five days, pooled urine samples were collected at 8 a.m. and 4 p.m. using a metabolic cage, with a negative control sample taken at 8 a.m. at day one before administration of the substances. Samples were stored at -18 °C until preparation and injected into the LC/ESI-HRMS-system. The animal studies were conducted with approval of the respective ethical committee and the laboratory followed the directive 2010/63/EU of the European parliament ant the council of September 22nd, 2010 on the protection of animals used for scientific purposes (approval #: LA1800104).

2.4. Urine sample preparation For the preparation of the urine samples of the rat administration study, 90 µL of urine were spiked with 10 µL of ISTD 4 (100 ng/mL in water) and 3 µL of the mixture were injected into the LC/ESI-HRMS-system.

2.5. Liquid chromatography – high resolution/high accuracy (tandem) mass spectrometry All LC/ESI-HRMS(/MS) measurements were performed using a Thermo Dionex Ultimate 3000 liquid chromatograph linked to a Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany). The column was a Thermo Scientific Hypersil Gold C8 (100 x 2.1 mm, 3 μm particle size) equipped with a Thermo Scientific Hypersil Gold pre-column (10 x 2.1 mm 3 μm particle size). The eluents were 0.2% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Gradient elution was started at 2% B increasing to 100% B within 6 min and maintained for 1 min followed by re-equilibration at 2% B for 3 min. The mass spectrometer was operated using a heated electrospray ionization source (HESI-II) in positive mode at a temperature of 300 °C, the temperature of the transfer capillary was set to 320 °C, and the spray voltage was 3.75 kV. Sheath and auxiliary gas flows were set to 30 and 10, respectively. The mass spectral resolution (full width at half maximum, FWHM) was set to 15,000. Nitrogen was used as sheath, auxiliary and collision gas delivered from a CMC nitrogen generator (CMC Instruments, Eschborn, Germany). All LC/APCI-HRMS(/MS) measurements were conducted using similar LC and MS parameters but employing the instrument’s APCI ion source with a vaporizer temperature set to 400 °C and a corona current of 5 µA. In order to separate two potentially regioisomeric species, the LC gradient was modified to start with 2% B increasing to 50% B in 7 min.

2.6. Liquid chromatography – tandem mass spectrometry (MS³)

All MS³-experiments were performed with an Agilent (Walbronn, Germany) 1290 Infinity liquid chromatograph linked to an ABSciex (Darmstadt, Germany) QTrap 6500 mass spectrometer. The column used was a Thermo Scientific Hypersil Gold C8 (100 x 2.1 mm, 3 μm particle size) with a Thermo Scientific Hypersil Gold pre-column (10 x 2.1 mm 3 μm particle size). The eluents were 5 mM ammonium acetate buffer with 0.1% acetic acid in water (mobile phase A) and acetonitrile (mobile phase B). Gradient elution was started at 2% B increasing to 100% B within 6 min, maintained for 1 min at 100 % of B followed by re-equilibration at 2% B for 3 min. The measurements were conducted in positive mode with an ionspay voltage set to 5.5 kV and the source temperature to 450 °C. The declustering potential was set to 100 V. For the MS³ experiments, the excitation energy was set to 0.2 V. A CMC nitrogen generator delivered the nitrogen used as sheath and collision gas.

3. Results and discussion 3.1. In vitro metabolism study As shown in Figures 2 and 3, four (M1.1-M1.4) and five (M2.1-M2.5) metabolites were observed after incubation under phase-I metabolic reaction conditions for AdipoRon (1) and 112254 (2), respectively. The accurate masses and elemental compositions together with the MSn-experiments of all product ions derived from the metabolic products are summarized in Table S1 (supplementary material). All metabolites and their product ions after collision-induced dissociation-experiments (CID) are in accordance with in vitro-generated metabolites of the isotopically labeled analogs (3 and 4, Figure 1). The ratios of the relative abundance of the in vitro-generated metabolites as the result of three technical replicates each demonstrate that for both substances (1 and 2) monohydroxylated species (M1.3 and M2.1) were the main phase-I

metabolites formed. Further investigations into the composition of the peak representing M1.3 revealed that the signal consisted of two species, which were eventually separated using a modified LC gradient elution program resulting in retention times of 4.72 and 5.19 min for the two analytes. Both compounds yielded identical product ions with slightly differing relative abundances (data not shown), suggesting the presence of two hydroxylated regioisomers of 1. For AdipoRon (1), the reduction of a carbonyl group and a dihydroxylation reaction were observed, while additional demethylated and differently monohydroxylated species were observed for 112254 (2). Corresponding product ion mass spectra and dissociation pathways for these minor metabolites (M1.1-M1.2, M1.4, M2.2-M2.5) are presented in the supplemental material (Figures S1-S7). Putative dissociation routes for the most abundant metabolic products derived from 1 and 2 are exemplified by means of M1.3 and M2.1 in the following.

3.1.1. Suggested dissociation pathway of metabolite M1.3 The protonated molecule of M1.3 was found at m/z 445. The product ion mass spectrum and proposed dissociation pathway are shown in Figures 4 (top) and 5 (bottom), respectively. The base peak at m/z 339 is suggested to result from the loss of a methylene-cyclohexadienone moiety (106 Da) from [M+H]+ as supported by accurate mass measurements (Table S1). Subsequently, ammonia (17 Da) is eliminated forming the product ion at m/z 322 before the mass of 76 Da, corresponding to cyclopentene, is lost yielding the product ion at m/z 256 as demonstrated in MSn experiments. The product ion at m/z 190 is proposed to be generated by the dissociation of 2-(4benzoylphenoxy)acetamide (255 Da) from the precursor ion at m/z 445 before it further dissociates into two species attributed to the hydroxytropylium cation (m/z 107) and protonated

tetrahydropyridine (m/z 84). The product ions found were corresponding with product ions formed after CID-experiments of the 13C2-labled analog (data not shown).

3.1.2. Suggested dissociation pathway of metabolite M2.1 Figures 4 (bottom) and 6 (bottom) illustrate the product ion mass spectrum and proposed dissociation pathway of the metabolite M2.1 that produced a protonated molecule at m/z 454. As before, all tentatively assigned ion structures and dissociation routes were corroborated by accurate mass, in vitro-experiments of the 2H8-labled analog (4, data not shown) and/or MSn measurements as summarized in Table S1. The base peak of the product ion mass spectrum recorded from [M+H]+ at m/z 454 with a collision energy of 30 eV was found at m/z 121, which is suggested to be composed of a methoxytropylium cation formed after the elimination of piperazine (86 Da) from the product ion at m/z 207. Further, in agreement with earlier studies [26], an intramolecular rearrangement enabling the elimination of the piperazine moiety (86 Da) was suggested, leading to the product ion at m/z 368. Finally, the loss of the methoxybenzyl group (122 Da) was proposed to yield the product ion at m/z 332, which subsequently forms the product ion at m/z 248 after elimination of the tetrahydropyrazine motif (84 Da).

3.1.3. Further discussions In previous studies, both AdipoRon (1) and 112254 (2) were found to allow for intramolecular rearrangement reactions leading to the elimination of piperidine and piperazine residues in CIDexperiments, respectively [26]. The herein presented in vitro-derived metabolites M2.1, M2.2

and M2.4 of 112254 (2) exhibited the same phenomenon accordingly (Figures 6, S4, and S6). Noteworthy, these molecules bear an intact methoxy group at the benzyl residue. Demethylation at that position results in the absence of the intramolecular elimination reaction in CIDexperiments (Figures S5 and S7), a possible explanation for which is the formation of a considerably more polar para-positioned hydroxyl group inhibiting the reported elimination reaction. The location (para) of the hydroxyl group is apparently essential for the inhibition of this dissociation pathway as M2.2 (Figure S4) is suggested to be metabolically hydroxylated at the benzyl residue (in ortho or meta positions) but the elimination of the piperazine moiety is still observed. Considering AdipoRon (1), no intramolecular elimination reaction was found for the in vitro-derived metabolites. Comparing this with the observations of 112254 (2), a possible reason is that both M1.3 (at least one species) and M1.4 (Figures 5 and S3, respectively) are suggested to be para-hydroxylated (C-28, Figure 1) at the benzyl-group and, concordantly, no elimination reaction occurred. For M1.1 and M1.2 (Figures S1 and S2, respectively), the elimination of water after hydrogenation of a carbonyl group affected the dissociation pattern fundamentally and a corresponding elimination reaction was not identified. Several additional signals with possibly relevant mass spectra were detected in both enzyme- and NADPH-blank, suggesting the formation of metabolites independent of the entire metabolic environment. For these signals, sum formulae were determined from accurate mass measurements of the intact molecules, and retention times suggested the formation of amine oxide species. In order to corroborate the formation of amine oxide species, the samples were analyzed using an APCI ion source as N-oxides have been reported to undergo decomposition reactions under APCI conditions by eliminating the nitrogen-bound oxygen atom (16 Da) [27]. In Figure S8, all amine oxide species suggested to be generated from AdipoRon (1) and 112254 (2) are depicted. The oxygenation site was unambiguous for AdipoRon (1) due to the observed mass

spectrometric dissociation pathway; conversely, equivocal data remained for 112254 (2) as two amenable nitrogen atoms exist in the piperazine residue. In Figures 5 and 6 (top), extracted ion chromatograms of ESI- and APCI-experiments are shown for the assigned N-oxides of AdipoRon (1) and 112254 (2). The abundant peak of the N-oxide with the retention time of 5.76 as observed in the ESI-based analysis (Figure 5c) was not detected with APCI ionization (Figure 5d). As a consequence however, a new peak at m/z 429 with a pursuant retention time of 5.78 min was observed (Figure 5b). An analogous behavior was found for 112254 (2), where the assumed Noxide with the retention time at 6.01 in the ESI-experiment (Figure 6c) is indicated in the peak profile of the APCI-experiment at m/z 438 (Figure 6b).

3.2. In vivo-metabolism study Urine samples collected post-administration of 1 mg of AdipoRon (1) and 112254 (2) to 6 test animals each were analyzed using the same analytical conditions as reported for the in vitro experiments. Extracted ion chromatograms of in vivo-generated metabolites are shown in Figures 2 and 3 (top right panels) for AdipoRon (1) and 112254 (2), respectively. Both chromatograms result from urine samples collected 8 hours after drug administration.

3.2.1. In vivo-derived metabolites of AdipoRon (1) AdipoRon (1) was found to form both phase-I and phase-II metabolites in vivo. While the intact and unmodified drug was observed in the urine samples collected 8 hours post-administration in one rat only, M1.1 and M1.2 were identified in all animals. These unconjugated metabolites were however not detected anymore in the pooled urine sample obtained at 24 h. Extracting accurate

masses of respective phase-II metabolites (i.e. glucuronides, sulfate and glutathione conjugates) from full scan data did also not allow for identifying conjugated species over the course of the excretion study; however, extracted ion chromatograms concerning the ion transitions 429/91 and 445/107 revealed signals at retention times preceding the intact adipoRon, indicating the presence of adipoRon conjugates decomposing in-source. These phase-II metabolites, detected in the urine sample collected 24 hours post-administration of AdipoRon (1), were subjected to additional experiments including the analysis by means of an alternative mass spectrometer and different sample preparation protocols. The use of a Waters (Eschborn, Germany) SYNAPT G2-S TOFMS also caused the in-source dissociation of the conjugate and thus did not contribute to further characterizing the nature of the phase-II metabolites (data not shown). Incubating an aliquot of the post-administration urine samples with β-glucuronidase (from E. Coli K12) for 1 h however resulted in a considerable decrease of the signal intensity of the first-eluting peak regarding the ion transition 429/91, concomitant with an increase of the peak of AdipoRon (1, Figure S9). Furthermore, in the extracted ion chromatogram of the ion transition of 445/107 the signal at 3.40 min disappeared under enzymatic hydrolysis conditions yielding the peak corresponding to M1.3 only (Figure S9, right). This supported the assumption that conjugation reactions forming glucuronides in the rat in vivo exist. Also the use of hydrochloric acid to hydrolyse potential conjugates resulted in significant alterations in the metabolite profiles as illustrated in Figure S9. Incubating a urine sample for 2 h at 70 °C with 1 M aqueous HCl reduced the number of measured analytes to adipoRon and M1.3 Since adipoRon (1) bears no hydroxyl group, conjugation reactions are likely yielding an N-glucuronide or a metabolite of adipoRon (1) is transformed into a glucuronide or sulfate conjugate in vivo and decomposes during the ionization process yielding a protonated species [M+H]+ at m/z 429. Here, further investigations concerning the determination of the structures of these phase-II-metabolites are warranted, particularly since

routine doping control analysis benefits from target analytes being traceable for as long as possible in sports drug testing specimens.

3.2.2. In vivo-derived metabolites of 112254 (2) Only two phase I-metabolites, namely M2.1 and M2.3 (Figure 3) were detected after oral administration of 112254 (2) in rat excretion study urine specimens. No phase II-metabolites (glucuronides, sulfate and glutathione conjugations) were observed and the intact drug candidate was excreted into urine also only at very low concentrations within the first 8-24 h. M2.1, the main metabolite found in in vitro experiments, was detected up to 24 h, and M2.3 up to 32 h in all animals. According to this study, M2.3 appears to be the most appropriate target analyte for routine doping control analysis for 112254 (2).

4. Conclusions New drug candidates designed to affect biochemical pathways that are assumed to be potentially performance-enhancing are always at risk to be abused by athletes for doping purposes. Consequently, preventive doping research aims at establishing assays for the detection of such compounds proactively and at early stages of drug development. PGC-1α, SIRT1, AMPK and the PPARs are examples for transcriptional regulators, the manipulation of which has been identified as relevant for doping controls in the past. AdipoRon and 112254 are both adiponectin receptor agonists, activating pathways affecting these transcriptional regulators. For an efficient inclusion into routine doping control assays, the knowledge about metabolites of the respective substances

are essential and both in vitro and animal in vivo studies were conducted to support the identification of suitable target analytes in the absence of data resulting from controlled human elimination studies. The obtained data support future anti-doping efforts and suggest further investigations particularly into the structures of phase-II metabolites for best-possible retrospectivity in sports drug testing.

Acknowledgements The study was supported by Antidoping Switzerland (Berne, Switzerland) and the Federal Ministry of the Interior of the Federal Republic of Germany (Bonn, Germany).

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Figures

Figure 1: Chemical structure of AdipoRon (1), 112254 (2), (4).

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C2-AdipoRon (3) and D8-112254

Figure 2: Top: Extracted product ion chromatograms of in vitro-generated metabolites (left) (M1.1-M1.4) of AdipoRon and the metabolites found in vivo 8 hours after administration of AdipoRon (right); Bottom: Proposed chemical structures of M1.1-M1.4.

Figure 3: Top: Extracted product ion chromatograms of in vitro-generated metabolites (left) (M2.1-M2.5) of 112254 and the metabolites found in vivo 8 hours after administration of 112254 (right); Bottom: Proposed chemical structures of M2.1-M2.5.

Figure 4: Top: Proposed dissociation pathway for the protonated precursor ion [M+H]+ of metabolite M1.3; Bottom: Proposed dissociation pathway for the protonated precursor ion [M+H]+ of metabolite M2.1.

Figure 5: Top: Extracted ion chromatograms of a) m/z 429 (ESI), b) m/z 429 (APCI), c) m/z 445 (ESI), d) m/z 445 (APCI); Bottom: Product ion mass spectrum of the protonated precursor ion [M+H]+ of metabolite M1.3 (m/z 445, retention time 5.19 min).

Figure 6: Top: Extracted ion chromatograms of a) m/z 438 (ESI), b) m/z 438 (APCI), c) m/z 454 (ESI), d) m/z 454 (APCI); Bottom: Product ion mass spectrum of the protonated precursor ion [M+H]+ of metabolite M2.1 (m/z 454, retention time 4.52 min).