Simon Beuck

Hypoxia‐inducible factor stabilizers and other small‐molecule erythropoiesis‐stimulating agents in current and preventive doping analysis

Increasing the bloods capacity for oxygen transport by erythropoiesis-stimulating agents (ESAs) constitutes a prohibited procedure of performance enhancement according to the World Anti-Doping Agency (WADA). The advent of orally bio-available small-molecule ESAs such as hypoxia-inducible factor (HIF) stabilizers in the development of novel anti-anaemia therapies expands the list of potential ESA doping techniques. Here, the erythropoiesis-stimulating properties and doping relevance of experimental HIF-stabilizers, such as cobaltous chloride, 3,4-dihydroxybenzoic acid or GSK360A, amongst others, are discussed. The stage of clinical trials is reviewed for the anti-anaemia drug candidates FG-2216, FG-4592, GSK1278863, AKB-6548, and BAY85-3934. Currently available methods and strategies for the determination of selected HIF stabilizers in sports drug testing are based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). For the support of further analytical assay development, patents claiming distinct compounds for the use in HIF-mediated therapies are evaluated and exemplary molecular structures of HIF stabilizers presented. Moreover, data concerning the erythropoiesis-enhancing effects of the GATA inhibitors K7174 and K11706 as well as the lipidic small-molecule ESA PBI-1402 are elucidated the context of doping analysis.

Investigation of the in vitro metabolism of the emerging drug candidate S107 for doping-preventive purposes

The metabolic fate of the emerging drug candidate S107, possessing the potential for misuse as performance-enhancing agent in sports, was investigated by in vitro phase I and II experiments with human microsomal and S9 liver enzymes. The metabolites were identified by liquid chromatography-mass spectrometry with electrospray ionisation in positive mode (LC-ESI-MS/MS). Their collision-induced dissociation behaviour was studied by high-resolution/high accuracy Orbitrap MS(n) analysis, supported by stable isotope labelling, H/D-exchange experiments and density functional theory calculations. Monooxygenation accounted for the main phase I metabolic transformation due to N- and S-oxidation of the 1,4-benzothiazepine core, as substantiated by chemical synthesis, selective reduction methods and characteristic APCI in source fragmentation behaviour of the metabolites. Another dominant metabolic pathway was demethylation, yielding the N- and O-demethylated metabolite, respectively. The latter was further conjugated by glucuronidation as well as sulfonation in subsequent phase II metabolic reactions, whereas the N-demethylated metabolite was not amenable to conjugation. The active drug molecule itself was converted to two glucuronic acid conjugates, which are proposed to consist of two quaternary S107-N(+)-glucuronide isomers. All glucuronides were susceptible to enzymatic hydrolysis with β-glucuronidase (Escherichia coli). A comprehensive LC-ESI-MS(/MS)-based detection method for urine was developed and its fitness for purpose was assessed. The assay can serve as a potential screening and/or confirmation method for S107 in clinical drug testing and doping control analysis in the future.

Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator andarine (S-4) for routine doping control purposes

Selective androgen receptor modulators (SARMs) are potent anabolic agents with tissue-selective properties. Due to their potential misuse in elite sport, the World Anti-Doping Agency (WADA) has prohibited SARMs since 2008, and although no representative drug candidate has yet received full clinical approval, recent findings of SARMs illegally sold via the internet have further supported the need to efficiently test for these compounds in doping controls. In the present communication, the mass spectrometric characterization of urinary metabolites of the SARM Andarine (also referred to as S-4) compared with earlier in vitro and animal studies is reported. Liquid chromatography interfaced to high-resolution/high-accuracy (tandem) mass spectrometry was used to identify phase I and II metabolites, confirming the predicted target analytes for sports drug testing purposes including the glucuronic acid conjugates of the active drug, its monohydroxylated and/or deacetylated product, the hydrolysis product resulting from the removal of the compounds B-ring, as well as the sulfate of the monohydroxylated and the deacetylated phase I metabolite. The obtained data will support future efforts to effectively screen for and confirm the misuse of the non-approved drug candidate Andarine.

Unusual mass spectrometric dissociation pathway of protonated isoquinoline-3-carboxamides due to multiple reversible water adduct formation in the gas phase

The study of the collision-induced dissociation behavior of various substituted isoquinoline-3-carboxamides, which are amongst a group of drug candidates for the treatment of anemic disorders (e.g., FG-2216), allowed for the formulation of the general mechanisms underlying the unusual fragmentation behavior of this class of compounds. Characterization was achieved with high-resolution/high accuracy LTQ-Orbitrap tandem mass spectrometry of the protonated precursor ions. Presented data were substantiated by the synthesis and analysis of proposed gas-phase intermediate structures and stable isotope labeled analogues, as well as by density functional theory calculations. Exemplary, CID of protonated N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-yl)carbonyl]glycine gives rise to the isoquinoline-3-carboxy-methyleneamide product ion which nominally eliminates a fragment of 11 u. This was attributed to the loss of methyleneamine (−29 u) and a concomitant spontaneous and reversible water addition (+18 u) to the resulting acylium ion to yield the protonated isoquinoline-3-carboxylic acid. The same water addition pattern is observed after loss of propylene (−42 u). A further nominal loss of 10 u is explained by the elimination of carbon monoxide (−28 u) followed by another water adduct formation (+18 u) to yield the protonated 1-chloro-3,4,7-trihydroxy-isoquinoline. The source of the multiple gas-phase water addition remained unclear. This atypical fragmentation pattern proved to be highly characteristic for all studied and differentially substituted isoquinoline-3-carboxamides, and offers powerful analytical tools for the establishment of a LC/MS(/MS) based screening procedure for model HIF-stabilizers and their potential metabolites in clinical, forensic and sports drug testing.

Does the analysis of the enantiomeric composition of clenbuterol in human urine enable the differentiation of illicit clenbuterol administration from food contamination in sports drug testing

RATIONALE Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzyl alcohol) is approved for human and veterinary use primarily for the treatment of pulmonary afflictions. Despite the authorized administration in cases of medical indications, the misuse of clenbuterol in animal husbandry as well as elite and amateur sport has frequently been reported, arguably due to growth-promoting properties. Due to various recent incidences of doping control specimens containing clenbuterol, strategies towards the discrimination of a surreptitious application from unintended intake via animal-derived edibles or dietary supplements were required. METHODS The enantiomeric compositions of clenbuterol in human urine samples derived from administration studies with therapeutic amounts of the β(2)-agonist and authentic doping control specimens were determined. Due to the facts that therapeutic clenbuterol consists of a racemic mixture of (+)- and (-)-stereoisomers and that the first mentioned (dextrorotatory) stereoisomer is retained to a greater extent in edible animal tissue, the differentiation of a recent administration of therapeutic (and thus racemic) clenbuterol from food contamination (stereoisomerically depleted clenbuterol) was considered. Employing deuterated clenbuterol as internal standard, the target analytes were extracted from human urine by means of concerted liquid-liquid and solid-phase extractions and subjected to chiral liquid chromatography hyphenated to high resolution/high accuracy mass spectrometry with electrospray ionization. RESULTS Both enantiomers of clenbuterol were baseline separated and relative abundances of corresponding labeled and unlabeled stereoisomers were determined, demonstrating that the therapeutic use of clenbuterol results in racemic mixtures in urine for at least 24 h while adverse analytical findings presumably originating from food contaminations can yield (-)-clenbuterol-depleted pairs of analytes. CONCLUSIONS The determination of relative abundances of clenbuterol enantiomers can indicate the ingestion of clenbuterol via contaminated food; however, depletion of (-)-clenbuterol in edible animal tissue is time-dependent and thus results can still be inconclusive as to the inadvertent ingestion of clenbuterol when clenbuterol administration to animals was conducted until slaughter.

Determination of salbutamol and salbutamol glucuronide in human urine by means of liquid chromatography-tandem mass spectrometry.

The determination of salbutamol and its glucuronide in human urine following the inhalative and oral administration of therapeutic doses of salbutamol preparations was performed by means of direct urine injection utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and employing d(3)-salbutamol and d(3)-salbutamol glucuronide as internal standards. Unconjugated salbutamol was detected in all administration study urine samples. Salbutamol concentrations following inhalation were commonly (99%) below 1000 ng/ml whereas values after oral administration frequently (48%) exceeded this threshold. While salbutamol glucuronide was not detected in urine samples collected after inhalation of the drug, 26 out of 82 specimens obtained after oral application contained salbutamol glucuronide with a peak value of 63 ng/ml. The percentage of salbutamol glucuronide compared to unconjugated salbutamol was less than 3%. Authentic doping control urine samples indicating screening results for salbutamol less than 1000 ng/ml, showed salbutamol glucuronide concentrations between 2 and 6 ng/ml, whereas adverse analytical findings resulting from salbutamol levels higher than 1000 ng/ml, had salbutamol glucuronide values between 8 and 15 ng/ml. The approach enabled the rapid determination of salbutamol and its glucuronic acid conjugate in human urine and represents an alternative to existing procedures since time-consuming hydrolysis or derivatization steps were omitted. Moreover, the excretion of salbutamol glucuronide in human urine following the administration of salbutamol was proven.

Characterization of in vitro generated metabolites of the selective androgen receptor modulators S‐22 and S‐23 and in vivo comparison to post‐administration canine urine specimens

Selective androgen receptor modulators (SARMs) have great therapeutic potential in various diseases including cancer cachexia, sarcopenia, and osteoporosis, and the number of drug candidates has been growing over the last decade. The SARM drug candidates S-22 and S-23 belong to one of the most advanced groups of androgen receptor modulators and are based on an arylpropionamide-derived core structure. Due to their anabolic effects, SARMs have been prohibited in elite sports and have been a subject of sports drug testing programmes since January 2008. Consequently, the structure of analytically useful urinary metabolites should be elucidated to provide targets for sensitive and retrospective analysis. In the present study, the phase-I and -II metabolism of S-22 and S-23 was simulated using hepatic human enzymes, and resulting metabolites were characterized by means of state-of-the-art mass spectrometric approaches employing high resolution/high accuracy Orbitrap mass spectrometry. Subsequently, the newly defined target compounds including the glucuronic acid conjugates of S-22 and S-23, their corresponding monohydroxylated and bishydroxylated analogs, as well as their B-ring depleted counterparts were implemented into an existing routine doping control procedure, which was examined for its specificity for the added substances. In order to obtain proof-of-concept data for authentic urine specimens, canine urine samples collected up to 72 h after oral administration of S-22 to dogs were analyzed using the established approach outlining the capability of the presented assay to detect the glucuronide of S-22 as well as the B-ring-depleted metabolite (M3) in all samples following therapeutic (31.4 µg/kg) dosing. Finally, M3 was chemically synthesized, characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry, and chosen as primary target for future doping control analyses.

Doping control analysis of emerging drugs in human plasma – identification of GW501516, S‐107, JTV‐519, and S‐40503

An important aspect of preventive doping research is the rapid implementation of tests for emerging drugs with potential for misuse into routine doping control assays. New therapeutics of different classes such as PPARdelta-agonists (e.g. GW501516), ryanodine-calstabin-complex stabilizers (e.g. S-107 and JTV-519), and selective androgen receptor modulators (SARMs, e.g. S-40503) are currently used for the treatment of particular medical conditions such as metabolic syndrome, cardiac arrhythmia, debilitating diseases and osteoporosis, respectively. Due to their being at an early stage of clinical trials and the limited availability of data on the metabolism and possible renal elimination of the active drugs, the development of protocols for doping control analyses of plasma specimens could be an option for the detection of the circulating agents. The mass spectrometric fragmentation of four emerging drug candidates (GW501516, S-107, JTV-519, and S-40503) was elucidated by positive electrospray ionization and collision-induced dissociation using a high resolution/high accuracy mass spectrometer. A screening and confirmation procedure was established based on liquid chromatography/tandem mass spectrometry requiring a volume of 100 microL of plasma. Proteins were precipitated using acetonitrile, the specimens were centrifuged and the supernatant analyzed using a triple-quadrupole mass spectrometer employing multiple reaction monitoring of diagnostic ion transitions. The method was validated with regard to specificity, limits of detection (0.4-8.3 ng/mL), recoveries (72-98%), intraday and interday precisions (12-21%), and ion suppression/enhancement effects.

Development of liquid chromatography‐tandem mass spectrometry‐based analytical assays for the determination of HIF stabilizers in preventive doping research

Hypoxia-inducible factor (HIF) stabilizers increase blood haemoglobin levels after oral administration and their use in sports was recently banned by the World Anti-Doping Agency. For the support of analytical assay development, the metabolic fate of two model HIF stabilizers, based on the isoquinoline-3-carboxamide scaffold of the lead drug candidate FG-2216, was assessed by in vitro methods. The analytes were identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative ionization mode using an API 4000 Qtrap as well as an exactive high resolution-high accuracy MS. The model HIF stabilizer N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-carbonyl)-amino]-acetic acid (1), was converted into 3 major phase I metabolites by hydroxylation, dealkylation, and dehydrogenation. The structures of the hydroxylated and the dealkylated metabolites were confirmed by LC-coupled nuclear magnetic resonance spectroscopy. Moreover, glucuronic acid conjugates of the active drug and one of the dealkylated phase I metabolite were identified. Hydroxylation of model compound 2 (N-[(1-chloro-4-hydroxy-isoquinolin-3-carbonyl)-amino]-acetic acid) yielded two metabolites, regioisomeric to the dealkylated product of 1. Mass spectral data of compounds 1 and 2, as well as a structure-related analogue were included into a multi-target analytical assay based on direct injection and LC-MS/MS analysis of human urine. The method was validated for quantitative purposes. In an approach of preventive doping research, more comprehensive screening methods applying precursor ion (m/z 166) and neutral loss (-10 Da) scans were developed, allowing for the detection of unknown metabolites and structurally analogous HIF stabilizers emerging from ongoing lead structure developments.

Screening for the calstabin-ryanodine receptor complex stabilizers JTV-519 and S-107 in doping control analysis

Recent studies outlined the influence of exercise on the stability of the skeletal muscle calstabin1-ryanodine receptor1-complex, which represents a major Ca(2+) release channel. The progressive modification of the type-1 skeletal muscle ryanodine receptor (RyR1) combined with reduced levels of calstabin1 and phosphodiesterase PDE4D3 resulted in a Ca(2+) leak that has been a suggested cause of muscle damage and impaired exercise capacity. The use of 1,4-benzothiazepine derivatives such as the drug candidates JTV-519 and S-107 enhanced rebinding of calstabin1 to RyR1 and resulted in significantly improved skeletal muscle function and exercise performance in rodents. Due to the fact that the mechanism of RyR1 remodelling under exercise conditions were proven to be similar in mice and humans, a comparable effect of JTV-519 and S-107 on trained athletes is expected, making the compounds relevant for doping controls. After synthesis of JTV-519, S-107, and a putative desmethylated metabolite of S-107, target compounds were characterized using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-high-resolution/high-accuracy Orbitrap mass spectrometry. Collision-induced dissociation pathways were suggested based on the determination of elemental compositions of product ions and H/D-exchange experiments. The most diagnostic product ion of JTV-519 was found at m/z 188 (representing the 4-benzyl-1-methyl piperidine residue), and S-107 as well as its desmethylated analog yielded characteristic fragments at m/z 153 and 138 (accounting for 1-methoxy-4-methylsulfanyl-benzene and 4-methoxy-benzenethiol residues, respectively). The analytes were implemented in existing doping control screening procedures based on liquid chromatography, multiple reaction monitoring and simultaneous precursor ion scanning modes using a triple quadrupole mass spectrometer. Validation items such as specificity, recovery (68-92%), lower limit of detection (0.1-0.2 ng/mL), intraday (5.2-18.5%) and interday (8.7-18.8%) precision as well as ion suppression/enhancement effects were determined.