Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry.

Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes' urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033-substances listed under anabolic substances (S1) on the World Anti-Doping Agency's Prohibited List-in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02-0.40 ng/mL) and in sf (0.01-0.25 ng/mL) as well as of LGD-4033 in bp (0.21-2.00 ng/mL) and in sf (0.03-0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine.

Investigations on the in vivo metabolism of 5α-androst-2-en-17-one.

RATIONALE: The anabolic steroid 5α-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as a steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2ß,3α-dihydroxy-5α-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance. METHODS: The metabolism of 2EN was reinvestigated after oral administration of twofold-deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high-accuracy/high-resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9 days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations. RESULTS: More than 15 different metabolites still containing the deuterium label were detected after administration. The presence of steroids exhibiting a 5ß-configuration was unexpected as the administered 2EN features a 5α-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5ß-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9 days after administration. CONCLUSIONS: The new metabolites identified in this study enable the detection of the misuse of 2EN for up to 9 days. The conversion of a 5α-steroid to urinary metabolites with 5ß-configuration has not been reported so far and should be further investigated.

Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis.

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1'N-glucuronide and 17-epistanozolol-2'N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards. Examination of excretion study samples shows large detection windows for the phase-II metabolites stanozolol-1'N-glucuronide and 17-epistanozolol-1'N-glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid-phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given.

Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis.

The anabolic-androgenic steroid methylstenbolone (MSTEN; 2α,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one) is available as a so-called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post-administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α-dimethyl-5α-androst-1-ene-3ß,17ß-diol and 2α,17α-dimethyl-5α-androst-1-ene-3α,17ß-diol) were synthesized and characterized by NMR.

SARCOSYL-PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins.

PEGylation of recombinant proteins and synthetic peptides aims to generate biopharmaceuticals with altered physical properties. The modification may lead to a prolonged serum half-life caused by decreased receptor-mediated endocytosis and/or delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEPO) used in the treatment of anemia due to chronic kidney disease, has also been abused by athletes as performance-enhancing drug. While it can be detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the sensitivity of the test is significantly lower compared to other epoetins. By replacing SDS with sarcosyl in the sample and running buffers, the interaction between SDS and the PEG group of the protein no longer reduces the affinity of the monoclonal anti-EPO antibody (clone AE7A5) to the protein chain. Contrary to SDS, sarcosyl only binds to the amino acid chain of the PEGylated protein and thus leads to a sharper electrophoretic band and enhanced antibody binding. While the method was originally developed for anti-doping purposes, it may also be useful for the electrophoretic separation and immunological detection of other PEGylated proteins. Protocols for urine and serum are presented. They are also applicable for the general detection of EPO-based erythropoiesis-stimulating agents (ESA) in these matrices.

Studies on the in vivo metabolism of the SARM YK11: Identification and characterization of metabolites potentially useful for doping controls.

A steroidal compound was recently detected in a seized black market product and was identified as (17α,20E)-17,20-[(1-methoxyethylidene) bis (oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11). This compound is described to possess selective androgen receptor modulator- and myostatin inhibitor-like properties. As YK11 is an experimental drug candidate and a non-approved substance for humans, scientific data on its metabolism is scarce. Due to its steroidal backbone and the arguably labile orthoester-derived moiety positioned at the D-ring, substantial metabolic conversion in vivo was anticipated. To unambiguously detect urinary metabolites of YK11, an elimination study with six-fold deuterated YK11 was conducted. Post-administration specimens were analyzed using hydrogen isotope ratio mass spectrometry coupled to single quadrupole mass spectrometry to identify metabolites alongside basic mass spectrometric data. Further characterization of those metabolites relevant to sports drug testing was accomplished using gas chromatography-high resolution-high accuracy mass spectrometry. Fourteen deuterated urinary metabolites were detected comprising unconjugated, glucuronidated, and sulfoconjugated metabolites. As expected, no intact YK11 was observed in the elimination study urine samples. While the unconjugated metabolites disappeared within 24 hours post-administration, both glucuronidated and sulfated metabolites were traceable for more than 48 hours. The chemical structures of the two most promising glucuronidated metabolites (5ß-19-nor-pregnane-3α,17ß,20-triol and 5ß-19-nor-pregnane-3α,17ß-diol-20-one) were verified by in-house synthesis of both metabolites and confirmed by nuclear magnetic resonance analysis. In order to elucidate their potential in sports drug testing, both were successfully implemented into the currently applied analytical method for the detection of anabolic agents.

Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays.

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10-8 M), 4-hydroxyandrost-4-ene-3,17-dione (10-8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

Nutrition Status of Young Elite Female German Football Players.

PURPOSE: To investigate energy intake, energy expenditure, and the nutritional status of young female elite football players using 7-day food and activity records and blood parameters. METHODS: A total of 56 female elite football players [14.8 (0.7) y] completed the requested food and activity protocols. Misreporting was assessed by the ratio of energy intake to energy expenditure. The food records were analyzed concerning energy and macronutrient and micronutrient intakes, and energy expenditure was calculated using predictive equations. Hematological data and 25-hydroxyvitamin D serum concentrations were determined. RESULTS: Mean energy intake was 2262 (368) kcal/d [40.5 (7.0) kcal/kg/d] and estimated EE averaged 2403 (195) kcal/d. Fifty-three percent of the players exhibited an energy availability <30 kcal/kg lean body mass; 31% of the athletes consumed <5 g/kg carbohydrates and 34% consumed <1.2 g/kg proteins. A large proportion of players (%) had intakes below the recommended daily allowance of folate (75%), vitamin D (100%), iron (69%), and calcium (59%). Ferritin and 25-hydroxyvitamin D serum levels were below the recommendations of 59% and 38%, respectively. CONCLUSIONS: A remarkable number of players failed to meet the energy balance and the recommended carbohydrate and protein intakes. Low iron and 25-hydroxyvitamin D serum levels were observed showing a suboptimal nutrition status of some young female football players. As a consequence, strategies have to be developed for a better information and application of sport nutrition practice among young female football players.

Detection of the Human Anti-ActRII Antibody Bimagrumab in Serum by Means of Affinity Purification, Tryptic Digestion, and LC-HRMS.

PURPOSE: Inhibitors of the ActRII signaling pathways represent promising therapeutics for the treatment of muscular diseases, but also pose risks as performance-enhancing agents in sports. Bimagrumab is a human anti-ActRII antibody which was found to increase muscle mass and function by blocking ActRII signaling. As it has considerable potential for being misused as doping agent in sports, the aim of this study was to develop a mass spectrometric detection assay for doping control serum samples. EXPERIMENTAL DESIGN: Within this study, a detection method for Bimagrumab in human serum was developed, which combines ammonium sulfate precipitation and affinity purification with proteolytic digestion and LC-HRMS. To facilitate the unambiguous identification of the diagnostic peptides, an orthogonal IM separation was additionally performed. RESULTS: The assay was successfully validated and the analysis of clinical samples demonstrated its fitness for purpose for an application in routine doping control analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Although no myostatin inhibitors have obtained clinical approval yet, the proactive development of detection methods for emerging doping agents represents a key aspect of preventive doping research. The presented approach will expand the range of available tests for novel protein therapeutics and can readily be modified to include further target analytes.

Detection of SARMs in doping control analysis.

The class of selective androgen receptor modulators (SARMs) has been the subject of intense and dedicated clinical research over the past two decades. Potential therapeutic applications of SARMs are manifold and focus particularly on the treatment of conditions manifesting in muscle loss such as general sarcopenia, cancer-associated cachexia, muscular dystrophy, etc. Consequently, based on the substantial muscle- and bone-anabolic properties of SARMs, these agents constitute substances with significant potential for misuse in sport and have therefore been added to the Word Anti-Doping Agency's (WADA's) Prohibited List in 2008. Since then, numerous adverse analytical findings have been reported for various different SARMs, which has underlined the importance of proactive and preventive anti-doping measures concerning emerging drugs such as these anabolic agents, which have evidently been misused in sport despite the fact that none of these SARMs has yet received full clinical approval. In this review, analytical data on SARMs generated in the context of research conducted for sports drug testing purposes are summarized and state-of-the-art test methods aiming at intact drugs as well as diagnostic urinary metabolites are discussed. Doping control analytical approaches predominantly rely on chromatography hyphenated to mass spectrometry, which have allowed for appropriately covering the considerable variety of pharmacophores present in SARMs such as the non-steroidal representatives ACP-105, BMS-564929, GLPG0492 (DT-200), LG-121071, LGD-2226, LGD-4033/VK 5211, ostarine/enobosarm, RAD-140, S-40503, etc. as well as steroidal compounds such as MK-0773 and YK-11.

Potential of GHB phase-II-metabolites to complement current approaches in GHB post administration detection.

Recently, phase-II-metabolites of γ-hydroxybutyric acid (GHB), namely GHB-ß-O-glucuronide and GHB-4-sulfate, were implemented in the scope of drug testing methods The clearance of GHB from the circulation is extremely fast due to its incorporation into the metabolic pathway of the citrate cycle. The elimination half-life of GHB from blood was reported to be dose dependent between 30 and 50min resulting in narrow detection windows of less than 12h after illicit administration or cases of drug facilitated sexual assault regardless of the biological matrix used. As sulfated metabolites tend to show prolonged half-lives and slower elimination kinetics compared to unmodified or glucuronidated drugs, the potential of GHB-4-sulfate in prolonging the detection of GHB administration was assessed. Its urinary concentrations were determined in n=100 samples from athletes and n=50 samples from sport students, and the resulting data were used to calculate a preliminary reference population-based threshold for urinary GHB-sulfate concentration. The threshold was then compared to concentrations found in post-administration urine samples collected from 3 volunteers who administered GHB within the setting of a clinical trial. Due to the large inter-individual variability of concentrations found in the reference population, GHB-4-sulfate itself was not suitable to prolong the detection times for GHB applications, even when specific gravity-corrected values were used. Therefore, a metabolomics-based approach was applied to the reference population samples and evaluated regarding other urinary metabolites that potentially correlate with the urinary excretion of GHB-4-sulfate and GHB-ß-O-glucuronide in order to find a suitable marker to normalize urinary concentrations. The most promising candidate was found at a molecular mass of 321.0696 and was preliminarily identified as ß-citryl-glutamic acid.

Characterization of in vitro generated metabolites of selected peptides <2 kDa prohibited in sports.

With an increasing number of prohibited substances in doping controls, knowledge about their metabolism is crucial for efficient analysis. While for low molecular mass molecules, standard protocols for in vitro metabolism experiments are well established, the situation with peptidic drugs has been shown to be substantially more heterogeneous and complex. Two principle strategies aiming at simulating the metabolism of lower molecular mass peptides in vitro are presented within this study. The prohibited peptides ARA-290, GHRP-3, and Peforelin, with a to-date unknown metabolism, were chosen as model compounds for these experiments and metabolism after incubation with different blood specimens (EDTA-, heparin-, citrate-plasma, and serum) and exposure to recombinant amidase were investigated. The characterization of in vitro generated drug-derived peptidic analytes was accomplished by means of liquid chromatography coupled to high resolution mass spectrometry. Identification of the generated metabolites was ensured by dedicated high resolution product ion experiments after liquid chromatographic separation. While extensive exopeptidase-driven metabolism was observed for ARA-290 (with one main metabolite PyrEQLERALN), GHRP-3 and Peforelin were found to exhibit a considerable metabolic stability with a low tendency for deamidation only. Copyright © 2017 John Wiley & Sons, Ltd.

Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry.

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted. Copyright © 2017 John Wiley & Sons, Ltd.

Immunoaffinity techniques coupled to mass spectrometry for the analysis of human peptide hormones: advances and applications.

INTRODUCTION: The accurate and comprehensive determination of peptide hormones from biological fluids has represented a considerable challenge to analytical chemists for decades. Besides long-established bioanalytical ligand binding assays (or ELISA, RIA, etc.), more and more mass spectrometry-based methods have been developed recently for purposes commonly referred to as targeted proteomics. Eventually the combination of both, analyte extraction by immunoaffinity and subsequent detection by mass spectrometry, has shown to synergistically enhance the test methods' performance characteristics. Areas covered: The review provides an overview about the actual state of existing methods and applications concerning the analysis of endogenous peptide hormones. Here, special focus is on recent developments considering the extraction procedures with immobilized antibodies, the subsequent separation of target analytes, and their detection by mass spectrometry. Expert commentary: Key aspects of procedures aiming at the detection and/or quantification of peptidic analytes in biological matrices have experienced considerable improvements in the last decade, particularly in terms of the assays' sensitivity, the option of multiplexing target compounds, automatization, and high throughput operation. Despite these advances and progress as expected to be seen in the near future, immunoaffinity purification coupled to mass spectrometry is not yet a standard procedure in routine analysis compared to ELISA/RIA.

Determination of LongR3-IGF-I, R3-IGF-I, Des1-3 IGF-I and their metabolites in human plasma samples by means of LC-MS.

According to the regulations of the World Anti-Doping Agency (WADA), growth promoting peptides such as the insulin-like growth factor-I (IGF-I) and its synthetic analogues belong to the class of prohibited compounds. While several assays to quantify endogenous IGF-I have been established, the potential misuse of synthetic analogues such as LongR3-IGF-I, R3-IGF-I and Des1-3-IGF-I remains a challenge and superior pharmacokinetic properties have been described for these analogues. Within the present study, it was demonstrated that the target peptides can be successfully detected in plasma samples by means of magnetic beads-based immunoaffinity purification and subsequent nanoscale liquid chromatographic separation with high resolution mass spectrometric detection. Noteworthy, the usage of a specific antibody for LongR3-IGF-I enables the determination in low ng/mL levels despite the presence of an enormous excess of endogenous human IGF-I. In addition, different metabolism studies (in-vitro and in-vivo) were performed using sophisticated strategies such as incubation with skin tissue microsomes, degradation in biological fluids (for all analogues), and administration to rats (for LongR3-IGF-I). Herewith, several C-and N-terminally truncated metabolites were identified and their relevancy was additionally confirmed by in-vivo experiments with rodents. Especially for LongR3-IGF-I, a metabolite ((Des1-11)-LongR3-IGF-I) was identified that prolonged the detectability in-vivo by a factor of approximately 2. The method was validated for qualitative interpretation considering the parameters specificity, identification capability, recovery (26-60%), limit of detection (0.5ng/mL), imprecision (<25%), linearity, stability, and matrix effects. A stable isotope labelled (15N)-IGF-I was used as internal standard to control all sample preparation steps.

Do we excrete what we eat? Analysis of stable nitrogen isotope ratios of human urinary urea.

RATIONALE: Natural stable nitrogen isotope ratios (δ15 N) are frequently used for the determination of provenance and dietary assessment of recent and ancient humans. Although individual δ15 N values typically correspond to the dietary δ15 N composition, they are also affected by metabolic conditions. Preferred matrices for the measurement of human δ15 N values have been hair, nail or blood. The goal of this study was to validate a novel approach for the assessment of the δ15 N values from urinary urea, the principal end-product of human N metabolism. METHODS: The method, which involves the precipitation of urea from urine using xanthydrol, was validated using fortified urea solutions. Intra- and inter-individual variance of the δ15 N values of urinary urea was determined from samples obtained from multiple human subjects. RESULTS: Precipitation with xanthydrol did not alter the δ15 N values of urea. The mean δ15 N value in urinary urea from human subjects from Germany was +4.4 ± 0.6 ‰, which corresponds to the estimated dietary composition. It falls below previously reported δ15 N values for human tissue and blood samples. Longitudinal analyses over 7 days illustrate short-time changes linked to varying protein intake. CONCLUSIONS: Our results indicate that δ15 N values can be measured reliably from human urine and that the method is suitable to monitor rapid dietary and metabolic changes of an individual. Our findings further confirm that urinary urea is depleted in 15 N compared with human tissue but within the range of the δ15 N composition of the diet. Copyright © 2017 John Wiley & Sons, Ltd.

Expanding analytical options in sports drug testing: Mass spectrometric detection of prohibited substances in exhaled breath.

RATIONALE: Continuously refining and advancing the strategies and methods employed in sports drug testing is critical for efficient doping controls. Besides improving and expanding the spectrum of target analytes, alternative test matrices have warranted in-depth evaluation as they commonly allow for minimal-/non-invasive and non-intrusive sample collection. In this study, the potential of exhaled breath (EB) as doping control specimen was assessed. METHODS: EB collection devices employing a non-woven electret-based air filter unit were used to generate test specimens, simulating a potential future application in doping controls. A multi-analyte sports drug testing approach configured for a subset of 12 model compounds that represent specific classes of substances prohibited in sports (anabolic agents, hormone and metabolic modulators, stimulants, and beta-blockers) was established using unispray liquid chromatography/tandem mass spectrometry (LC/MS/MS) and applied to spiked and elimination study EB samples. The test method was characterized concerning specificity, assay imprecision, and limits of detection. RESULTS: The EB collection device allowed for retaining and extracting all selected model compounds from the EB aerosol. Following elution and concentration, LC/MS/MS analysis enabled detection limits between 5 and 100 pg/filter and imprecisions ranging from 3% to 20% for the 12 selected model compounds. By means of EB samples from patients and participants of administration studies, the elimination of relevant compounds and, thus, their traceability in EB for doping control purposes, was investigated. Besides stimulants such as methylhexaneamine and pseudoephedrine, also the anabolic-androgenic steroid dehydrochloromethyltestosterone, the metabolic modulator meldonium, and the beta-blocker bisoprolol was detected in exhaled breath. CONCLUSIONS: The EB aerosol has provided a promising proof-of-concept suggesting the expansion of this testing strategy as a complement to currently utilized sports drug testing programs.

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 © 2017 John Wiley & Sons, Ltd.

lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG-4592) and its main metabolites into routine doping controls.

The utility of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors as a therapeutic means of treating patients suffering from anaemia has been demonstrated for various clinical settings. However, besides this intended use, HIF stabilizers can be the subject of misuse in amateur and elite sports due to their erythropoietic properties, as recently proven by several cases of adverse analytical findings in doping control testing. Consequently, to allow for adequate and comprehensive test methods, knowledge of the drug candidates' metabolism and analytical options enabling appropriate detection windows in sports drug testing samples (i.e., blood and urine) is essential to doping control laboratories. In the present study, a novel HIF prolyl hydroxylase inhibitor referred to as Roxadustat (FG-4592) and main plasma- and urine-derived metabolites were investigated in the context of routine doping control analytical approaches. Liquid chromatography-mass spectrometry-based test methods were used to study the target analytes' dissociation pathways following electrospray ionization and collision-induced dissociation. Diagnostic precursor-product ion pairs were selected to enable the implementation of the intact drug Roxadustat and selected metabolites into multi-analyte initial testing procedures for plasma and urine specimens. The assays were validated in accordance to guidelines of the World Anti-Doping Agency (WADA) and results demonstrated the suitability (fitness-for-purpose) of the employed analytical methods with detection limits ranging from 0.05 to 1 ng/mL and 1 to 5 ng/mL for urine and plasma, respectively. Subsequently, elimination study plasma and urine samples collected up to 167 h post-administration were analyzed using the validated methods, which suggested the use of different target analytes for blood and urine analyses with FG-4592 and its glucuronide, respectively, for optimal detection windows. Additionally, a light-induced rearrangement product (photoisomer) of Roxadustat resulted in the formation of an additional compound of identical mass. Copyright © 2017 John Wiley & Sons, Ltd.