Josef Dib

Mildronate (Meldonium) in professional sports – monitoring doping control urine samples using hydrophilic interaction liquid chromatography – high resolution/high accuracy mass spectrometry

To date, substances such as Mildronate (Meldonium) are not on the radar of anti‐doping laboratories as the compound is not explicitly classified as prohibited. However, the anti‐ischemic drug Mildronate demonstrates an increase in endurance performance of athletes, improved rehabilitation after exercise, protection against stress, and enhanced activations of central nervous system (CNS) functions. In the present study, the existing evidence of Mildronates usage in sport, which is arguably not (exclusively) based on medicinal reasons, is corroborated by unequivocal analytical data allowing the estimation of the prevalence and extent of misuse in professional sports. Such data are vital to support decision‐making processes, particularly regarding the ban on drugs in sport. Due to the growing body of evidence (black market products and athlete statements) concerning its misuse in sport, adequate test methods for the reliable identification of Mildronate are required, especially since the substance has been added to the 2015 World Anti‐Doping Agency (WADA) monitoring program. In the present study, two approaches were established using an in‐house synthesized labelled internal standard (Mildronate‐D3). One aimed at the implementation of the analyte into routine doping control screening methods to enable its monitoring at the lowest possible additional workload for the laboratory, and another that is appropriate for the peculiar specifics of the analyte, allowing the unequivocal confirmation of findings using hydrophilic interaction liquid chromatography‐high resolution/high accuracy mass spectrometry (HILIC‐HRMS). Here, according to applicable regulations in sports drug testing, a full qualitative validation was conducted. The assay demonstrated good specificity, robustness (rRT=0.3%), precision (intra‐day: 7.0–8.4%; inter‐day: 9.9–12.9%), excellent linearity (R>0.99) and an adequate lower limit of detection (<10 ng/mL).

Characterization of a non‐approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase‐I metabolites for human sports drug testing

RATIONALE Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). METHODS In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. RESULTS By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono- and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. CONCLUSIONS The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.

Analyses of Meldonium (Mildronate) from Blood, Dried Blood Spots (DBS), and Urine Suggest Drug Incorporation into Erythrocytes

Initially developed in the late 1970s for veterinary applications due to proposed growth-promoting effects in animals [5], meldonium has become an approved drug in selected Eastern European countries and is the subject of ongoing clinical trials focusing the compound’s anti-ischemic and cardioprotective properties [2, 3, 12, 15] as well as potential applications regarding diabetes, neurodegenerative disorders, and bronchopulmonary diseases. In the context of athletic performance, beneficial effects on the individuals’ physical working capacity, increased endurance performance, and accelerated recovery after physical activity were discussed [4, 10, 11], mentioning oral doses of meldonium of up to 2.0 g per day over 2–3 weeks in the course of pre-competition preparation phases [4]. In 2015, the World Anti-Doping Agency (WADA) initiated a one-year monitoring program [22] regarding the prevalence of meldonium (mildronate) in doping controls. Obtained data demonstrated a considerable extent of meldonium use by athletes [8, 16], which was further corroborated by a significant number of declarations of use and analytical findings at the Baku 2015 European Games [18]. Subsequently, the WADA Prohibited List that became effective in January 2016 [24] classified meldonium as banned under S4 (Hormone and Metabolic Modulators). Pharmacokinetic properties of meldonium were reported for singleand multiple-dose administration studies with healthy volunteers [25], where the drug’s elimination was monitored in plasma over 24 h post-administration and characterized by nonlinear pharmacokinetics. To date, doping controls are based on urine and blood as test matrices, and a variety of alternative matrices including amongst others dried blood spots (DBS) and dried plasma spots (DPS) have been considered lately [20]. Consequently, the knowledge about factors that potentially influence the elimination of target analytes is of particular importance to sports drug testing and, to the best of our knowledge, the role of erythrocytes and their ability to affect detection windows of meldonium in doping controls (e. g., by incorporation) has not been investigated. Therefore, in the context of a pilot study, DBS, whole blood (Na2EDTA), and urine samples were collected from 2 healthy male volunteers who orally administered meldonium either as single dose (500 mg) or as multi-dose (3 × 500 mg/day over a period of 6 consecutive days). The study protocol was approved by the local ethics committee of the National Institute for Sports Research (Bucharest, Romania, approval number #162/2016), written consent was obtained from the study participants, and the study was conducted in accordance with ethical standards in sports medicine and exercise science [9]. DBS samples were collected prior to and post-administration up to 16 days using standard DBS collection cards (Whatman DMPK-C, GE Heathcare Europe, Freiburg, Germany), dried at room temperature, and stored at + 4 °C in a plastic bag with desiccant until analysis. Na2-EDTA-stabilized whole blood specimens (3.5 mL) were sampled within the multi-dose study on day 4 and day 28 post-administration, and aliquots (4 × 20 μL) were immediately spotted on DBS cards. Further, following centrifugation of the blood samples at 1 000 × g for 15 min at 10 °C, the plasma was separated from the red blood cell (RBC) fraction, and 200 μL of the RBCs (retained for deposit onto DBS cards) was subsequently washed twice with 600 μL of phosphate-buffered saline (pH 7.4). The obtained plasma and washed erythrocytes were spotted onto DBS cards (four 20 μL aliquots each) and were also stored at + 4 °C in a plastic bag with desiccant until analysis. Online sample preparation of DBS was performed using a DBS card autosampler (DBSA) directly coupled to an automated solid-phase extraction (SPE) cartridge exchange module (SPExos) (Gerstel GmbH, Mulheim a.d.R., Germany). The sample preparation protocol was adapted from a previous application and was optimized to meet the current requirements [21]. In brief, the spots were extracted by means of flow-through desorption technology using 1 200 μL of acetonitrile/water (70:30, v/v), which included the online-addition of stable isotope-labeled meldonium (triply deuterated, TRC Toronto, Canada) as internal standard. Sample purification was performed by means of online-SPE using hydrophilic interaction liquid chromatography (HILIC) SPE cartridges. The target compounds were eluted onto the analytical column (Hypersil Gold C8, 2.1 mm × 30 mm, 1.9 μm particle size) via the LC mobile phase applying a gradient program with A: 5 mM ammonium acetate buffer (pH 3.5) and B: acetonitrile. LC-HR-MS/MS analysis was performed with a Thermo Dionex Ultimate 3000 liquid chromatograph interfaced to a Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany). Data were acquired in full scan mode with concomitant targeted higher energy collisional dissociation (HCD) experiments (precursor ion: m/z 147.1126, normalized collision energy: 40). The total sample-to-sample cycle time was 13 min. In addition to blood sampling, post-administration urine specimens were collected over a period of up to 49 days. These samples were subjected to analysis using a hydrophilic interaction liquid chromatography-high resolution high accuracy mass spectrometry approach (HILIC-HR-MS) published previously [8]. The analytical method for DBS measurements was validated for qualitative result evaluation purposes according to current guidelines of the International Standard for Laboratories (ISL) of the World Anti-Doping Code (WADC) [23]. Investigated parameters included specificity, carry-over, LOD (20 ng/mL), robustness, matrix interferences, and linearity (0–2 000 ng/mL), which allowed for estimating meldonium concentration levels in DBS by means of calibration curves prepared and analyzed with each batch of cards. Based on the method validation results ( ●▶ Table 1), the fitness-for-purpose of the assay was demonstrated. A total of 8 DBS samples collected prior to and up to 16 days post-administration of a single-dose (500 mg) of meldonium were analyzed using the automated isotope-dilution mass spectrometric approach. Maximum concentration levels were

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

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 pseudo-exercise 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 II-metabolites 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 II-metabolites 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.

Studies on the collision-induced dissociation of adipoR agonists after electrospray ionization and their implementation in sports drug testing.

AdipoR agonists are small, orally active molecules capable of mimicking the protein adiponectin, which represents an adipokine with antidiabetic and antiatherogenic effects. Two adiponectin receptors were reported in the literature referred to as adipoR1 and adipoR2. Activation of these receptors stimulates mitochondrial biogenesis and results in an improved oxidative metabolism (via adipoR1) and increased insulin sensitivity (via adipoR2). Hence, adipoR agonists are potentially performance enhancing substances and targets of proactive and preventive anti-doping measures. In this study, two adipoR agonists termed AdipoRon and 112254 as well as two isotopically labeled internal standards (ISTDs) were synthesized in three-step reactions. The products were fully characterized by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS) and density functional theory (DFT) computation. Collision-induced dissociation pathways following electrospray ionization were suggested based on the determined elemental compositions of product ions, comparison to product ions derived from labeled analogs (ISTDs), H/D-exchange experiments and the results of DFT calculations. The most abundant product ions were found at m/z 174, tentatively assigned to protonated 1-benzyl-1,2,3,4-tetrahydropyridine for AdipoRon, and m/z 207, suggested as protonated 1-(4-methoxybenzyl)piperazine, for 112254. Notably, the loss of the heterocyclic ring (i.e. piperazine and piperidine, respectively) in a supposedly intramolecular elimination reaction was observed in both cases. A qualitative determination of both AdipoR agonists in human plasma was established and fully validated for doping control purposes. Validation items such as recovery (86-89%), specificity, linearity, lower limit of detection (1 ng/ml), intraday (3-18%) and interday (5-16%) precision as well as ion suppression or enhancement were determined. Based on these findings adipoR agonists can be implemented in sports drug testing procedures.

Mass spectrometric characterization of the hypoxia-inducible factor (HIF) stabilizer drug candidate BAY 85-3934 (molidustat) and its glucuronidated metabolite BAY-348, and their implementation into routine doping controls

The development of new therapeutics potentially exhibiting performance-enhancing properties implicates the risk of their misuse by athletes in amateur and elite sports. Such drugs necessitate preventive anti-doping research for consideration in sports drug testing programmes. Hypoxia-inducible factor (HIF) stabilizers represent an emerging class of therapeutics that allows for increasing erythropoiesis in patients. BAY 85-3934 is a novel HIF stabilizer, which is currently undergoing phase-2 clinical trials. Consequently, the comprehensive characterization of BAY 85-3934 and human urinary metabolites as well as the implementation of these analytes into routine doping controls is of great importance. The mass spectrometric behaviour of the HIF stabilizer drug candidate BAY 85-3934 and a glucuronidated metabolite (BAY-348) were characterized by electrospray ionization-(tandem) mass spectrometry (ESI-MS(/MS)) and multiple-stage mass spectrometry (MSn ). Subsequently, two different laboratories established different analytical approaches (one each) enabling urine sample analyses by employing either direct urine injection or solid-phase extraction. The methods were cross-validated for the metabolite BAY-348 that is expected to represent an appropriate target analyte for human urine analysis. Two test methods allowing for the detection of BAY-348 in human urine were applied and cross-validated concerning the validation parameters specificity, linearity, lower limit of detection (LLOD; 1-5 ng/mL), ion suppression/enhancement (up to 78%), intra- and inter-day precision (3-21%), recovery (29-48%), and carryover. By means of ten spiked test urine samples sent blinded to one of the participating laboratories, the fitness-for-purpose of both assays was provided as all specimens were correctly identified applying both testing methods. As no post-administration study samples were available, analyses of authentic urine specimens remain desirable. Copyright

Monitoring 2‐phenylethanamine and 2‐(3‐hydroxyphenyl)acetamide sulfate in doping controls

2-Phenylethanamine (phenethylamine, PEA) represents the core structure of numerous drugs with stimulant-like properties and is explicitly featured as so-called specified substance on the World Anti-Doping Agency (WADA) Prohibited List. Due to its natural occurrence in humans as well as its presence in dietary products, studies concerning the ability of test methods to differentiate between an illicit intake and the renal elimination of endogenously produced PEA were indicated. Following the addition of PEA to the Prohibited List in January 2015, retrospective evaluation of routine doping control data of 10 190 urine samples generated by combined gas chromatography-mass spectrometry and nitrogen phosphorus-specific detection (GC-MS/NPD) was performed. Signals for PEA at approximate concentrations > 500 ng/mL were observed in 31 cases (0.3%), which were subjected to a validated isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) test method for accurate quantification of the target analyte. Further, using elimination study urine samples collected after a single oral administration of 250 mg of PEA hydrochloride to two healthy male volunteers, two tentatively identified metabolites of PEA were observed and evaluated concerning their utility as discriminative markers for PEA intake. The ID-LC-MS/MS approach was extended to allow for the simultaneous detection of PEA and 2-(3-hydroxyphenyl)acetamide sulfate (M1), and concentration ratios of M1 and PEA were calculated for elimination study urine samples and a total of 205 doping control urine samples that returned findings for PEA at estimated concentrations of 50-2500 ng/mL. Urine samples of the elimination study with PEA yielded concentration ratios of M1/PEA up to values of 9.4. Notably, the urinary concentration of PEA did increase with the intake of PEA only to a modest extent, suggesting a comprehensive metabolism of the orally administered substance. Conversely, doping control urine samples with elevated (>50 ng/mL) amounts of PEA returned quantifiable concentrations of M1 only in 3 cases, which yielded maximum ratios of M1/PEA of 0.9, indicating an origin of PEA other than an orally ingested drug formulation. Consequently, the consideration of analyte abundance ratios (e.g. M1/PEA) is suggested as a means to identify the use of PEA by athletes, but further studies to support potential decisive criteria are warranted.

Mass spectrometric characterization of the selective androgen receptor modulator (SARM) YK‐11 for doping control purposes

RATIONALE Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics targeting inter alia conditions referred to as cachexia and sarcopenia. Due to their anabolic properties, the use of SARMs is prohibited in sports as regulated by the World Anti-Doping Agency (WADA), and doping control laboratories test for these anabolic agents in blood and urine. In order to accomplish and maintain comprehensive test methods, the characterization of new drug candidates is critical for efficient sports drug testing. Hence, in the present study the mass spectrometric properties of the SARM YK-11 were investigated. METHODS YK-11 was synthesized according to literature data and three different stable-isotope-labeled analogs were prepared to support the mass spectrometric studies. Using high-resolution/high-accuracy mass spectrometry following electrospray ionization as well as electron ionization, the dissociation pathways of YK-11 were investigated, and characteristic features of its (product ion) mass spectra were elucidated. These studies were flanked by density functional theory (DFT) computation providing information on proton affinities of selected functional groups of the analyte. RESULTS AND CONCLUSIONS The steroidal SARM YK-11 was found to readily protonate under ESI conditions followed by substantial in-source dissociation processes eliminating methanol, acetic acid methyl ester, and/or ketene. DFT computation yielded energetically favored structures of the protonated species resulting from the aforementioned elimination processes particularly following protonation of the steroidal D-ring substituent. Underlying dissociation pathways were suggested, supported by stable-isotope labeling of the analyte, and diagnostic product ions for the steroidal nucleus and the D-ring substituent were identified. Further, trimethylsilylated YK-11 and its deuterated analogs were subjected to electron ionization high-resolution/high-accuracy mass spectrometry, complementing the dataset characterizing this new SARM. The obtained fragment ions resulted primarily from A/B- and C/D-ring structures of the steroidal nucleus, thus supporting future studies e.g. concerning metabolic pathways of the substance. Copyright