ABSTRACT
Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time.
Subject(s)
Doping in Sports , Sports , Doping in Sports/methods , Substance Abuse Detection/methods , DNA/genetics , TransgenesABSTRACT
2-(Dimethylamino)ethan-1-ol (Deanol) is a widely produced chemical used by both industry and consumers in a variety of applications. Meclofenoxate, a stimulant classified on the World Anti-Doping Agency Prohibited List, metabolizes into deanol and, presumably, its main metabolite deanol-N-oxide. Hence, using liquid chromatography-tandem mass spectrometry, a quantitative detection method for deanol-N-oxide in urine was developed. Subsequently, the urinary excretion of deanol-N-oxide after oral application of 130 mg of deanol was determined in six volunteers, and urine samples of a cohort of 180 male and female athletes from different sports were analyzed. In addition, urinary deanol-N-oxide was determined in an exploratory study with one volunteer ingesting 250 mg of meclofenoxate. The developed test method allowed for limits of detection and quantification for deanol-N-oxide at 0.05 and 0.15 µg/mL, respectively. Urinary deanol-N-oxide cmax levels were found between 100 and 250 µg/mL 2-5 h post-administration of 130 mg of deanol. Similarly, urine samples collected after the administration of 250 mg of meclofenoxate exhibited cmax levels of 115 µg/mL. In contrast, deanol-N-oxide urine concentrations of pre-administration specimens and 180 routine doping control urine sample were between 0.3 and 1.3 µg/mL and below limit of quantification and 1.8 µg/mL, respectively. The study suggests that the use of deanol and meclofenoxate results in significantly elevated urinary deanol-N-oxide levels. Whether or not monitoring deanol-N-oxide in doping controls can support decision-making processes concerning the detection of meclofenoxate use necessitates further investigations taking into consideration the elimination kinetics of 4-chlorophenoxyacetic acid, the main metabolite of meclofenoxate, and deanol-N-oxide.
Subject(s)
Deanol , Doping in Sports , Humans , Male , Female , Meclofenoxate , Mass Spectrometry , Eating , Substance Abuse Detection/methodsABSTRACT
Higenamine is prohibited in sports as a ß2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.
Subject(s)
Annona , Fruit , Substance Abuse Detection , BiomarkersABSTRACT
Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance-enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2-Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts.
Subject(s)
Anabolic Agents , Receptors, Androgen , Humans , Receptors, Androgen/metabolism , Androgens/metabolism , Mass Spectrometry/methods , Chromatography, Liquid/methods , Androgen Antagonists , Microsomes, Liver/metabolism , Substance Abuse Detection/methods , Anabolic Agents/analysisABSTRACT
RATIONALE: Chlorphenesin is an approved biocide frequently used in cosmetics, and its carbamate ester is an approved skeletal muscle relaxant in certain countries for the treatment of discomfort related to skeletal muscle trauma and inflammation. A major urinary metabolite is 4-chlorophenoxy acetic acid (4-CPA), also known as para-chlorophenoxyacetate, which is also employed as a target analyte in sports drug testing to detect the use of the prohibited nootropic stimulant meclofenoxate. To distinguish between 4-CPA resulting from chlorphenesin, chlorphenesin carbamate, and meclofenoxate, urinary metabolite profiles of chlorphenesin after legitimate use were investigated. METHODS: Human administration studies with commercially available sunscreen containing 0.25% by weight of chlorphenesin were conducted. Six study participants dermally applied 8 g of sunscreen and collected urine samples before and up to 7 days after application. Another set of six study participants applied 8 g of sunscreen on three consecutive days, and urine samples were also taken for up to 5 days after the last dosing. Urine specimens were analyzed using liquid chromatography-high resolution (tandem) mass spectrometry, and urinary metabolites were identified in accordance with literature data by accurate mass analysis of respective precursor and characteristic product ions. RESULTS: In accordance with literature data, chlorphenesin yielded the characteristic urinary metabolites, chlorphenesin glucuronide, chlorphenesin sulfate, and 3-(4-chlorophenoxy)-2-hydroxypropanoic acid (4-CPP), as well as the common metabolite 4-CPA. 4-CPA and 4-CPP were observed at similar abundances, with urinary concentrations of 4-CPA reaching up to ~1500 and 2300 ng/mL after single and multiple sunscreen applications, respectively. CONCLUSION: 4-CPA is a common metabolite of meclofenoxate, chlorphenesin, and chlorphenesin carbamate. Monitoring the diagnostic urinary metabolites of chlorphenesin provides conclusive supporting evidence of whether chlorphenesin or the prohibited nootropic meclofenoxate was administered.
Subject(s)
Chlorphenesin , Chromatography, High Pressure Liquid/methods , Sunscreening Agents , Tandem Mass Spectrometry/methods , Chlorphenesin/chemistry , Chlorphenesin/metabolism , Chlorphenesin/urine , Female , Humans , Limit of Detection , Male , Reproducibility of Results , Sunscreening Agents/analysis , Sunscreening Agents/chemistry , Sunscreening Agents/metabolismABSTRACT
RATIONALE: Exhaled breath (EB) has been demonstrated to be a promising alternative matrix in sports drug testing due to its non-invasive and non-intrusive nature compared with urine and blood collection protocols. In this study, a pilot-test system was employed to create drug-containing aerosols simulating EB in support of the analytical characterization of EB sampling procedures, and the used analytical method was extended to include a broad spectrum of prohibited substances. METHODS: Artificial and authentic EB samples were collected using sampling devices containing an electret filter, and doping agents were detected by means of liquid chromatography and tandem mass spectrometry with unispray ionization. The analytical approach was characterized with regard to specificity, limits of detection, carry-over, recovery and matrix effects, and the potential applicability to routine doping controls was shown using authentic EB samples collected after single oral dose applications of glucocorticoids and stimulants. RESULTS: The analytical method was found to be specific for a total of 49 model substances relevant in sports drug testing, with detection limits ranging from 1 to 500 pg per cartridge. Both ion suppression (-62%) and ion enhancement (+301%) effects were observed, and all model compounds applied to EB sampling devices were still detected after 28 days of storage at room temperature. Authentic EB samples collected after the oral administration of 10 mg of prednisolone resulted in prednisolone findings in specimens obtained from 3 out of 6 participants up to 2 h. In octodrine, dimethylamylamine (DMAA) and isopropylnorsynephrine post-administration EB samples, the drugs were detected over a period of 50, 48, and 8 h, respectively. CONCLUSIONS: With the analytical approach developed within this study, the identification of a broad spectrum of prohibited doping agents in EB samples was accomplished. Application studies and stability tests provided information to characterize EB as a potential matrix in sports drug testing.
Subject(s)
Breath Tests/methods , Chromatography, Liquid/methods , Doping in Sports , Tandem Mass Spectrometry/methods , Adult , Female , Humans , Illicit Drugs/analysis , Limit of Detection , Linear Models , Male , Middle Aged , Reproducibility of Results , Young AdultABSTRACT
Isopropylnorsynephrine (isopropyloctopamine, deterenol, 4-(1-hydroxy-2-(isopropylamino)ethyl)phenol), a beta-selective and direct-acting adrenergic agonist, has been reported in the past as declared as well as non-declared ingredient of dietary supplements. The proven biological activity and the structural similarity of isopropylnorsynephrine to substances classified as prohibited compounds according to the World Anti-Doping Agency's (WADA's) regulations could necessitate the inclusion of this sympathomimetic amine into routine doping control analytical assays. Therefore, information on urinary metabolites is desirable in order to allow for an efficient implementation of target compounds into existing multi-analyte testing procedures, enabling the unequivocal identification of the administration of isopropylnorsynephrine by an athlete. In a pilot study setting, urine samples were collected prior to and after the oral application of ca. 8.7 mg of isopropylnorsynephrine, which were subjected to liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. The intact drug, hydroxylated and/or glucurono- or sulfo-conjugated isopropylnorsynephrine were detected up to 48 h post-administration, with isopropylnorsynephrine sulfate representing the most abundant urinary target analyte. No relevant amounts of the dealkylation product (octopamine) were observed, indicating that merely moderate adaptations of existing test methods (or data evaluation strategies) are required to include isporpoylnorsynephrine in antidoping analytics, if required.
ABSTRACT
The possibility of nutritional supplement contamination with minute amounts of the selective androgen receptor modulator (SARM) ostarine has become a major concern for athletes and result managing authorities. In case of an adverse analytical finding (AAF), affected athletes need to provide conclusive information, demonstrating that the test result originates from a contamination scenario rather than doping. The aim of this research project was to study the elimination profiles of microdosed ostarine and characterize the time-dependent urinary excretion of the drug and selected metabolites. Single- and multi-dose administration studies with 1, 10, and 50 µg of ostarine were conducted, and collected urine samples were analyzed by LC-MS/MS following solid-phase extraction or enzymatic hydrolysis combined with liquid-liquid extraction. In the post-administration samples, both the maximum urine concentrations/abundance ratios and detection times of ostarine and its phase-I and phase-II metabolites were found to correlate with the administered drug dose. With regard to the observed maximum levels of ostarine, the time points of peak urinary concentrations/abundance ratios, and detection windows, a high inter-individual variation was observed. However, the study demonstrated that a single oral dose of as little as 1 µg can be detected for up to 9 (5) days by monitoring ostarine (glucuronide), and hydroxylated metabolites (especially M1a) appear to offer a considerably shorter detection window. The obtained data on ostarine (metabolite) detection times and urinary concentrations following different administration schemes support the interpretation of AAFs, in particular when scenarios of proven supplement contamination are discussed and supplement administration protocols exist.
Subject(s)
Anilides/administration & dosage , Anilides/urine , Dietary Supplements/analysis , Eating/physiology , Food Contamination/analysis , Substance Abuse Detection/methods , Administration, Oral , Anabolic Agents/administration & dosage , Anabolic Agents/urine , Doping in Sports/prevention & control , Dose-Response Relationship, Drug , Eating/drug effects , Humans , Liquid-Liquid Extraction/methods , Liquid-Liquid Extraction/standards , Male , Receptors, Androgen/metabolism , Solid Phase Extraction/methods , Solid Phase Extraction/standards , Substance Abuse Detection/standards , Yogurt/analysisABSTRACT
To date, blood (and serum) as well as urine samples are the most commonly collected specimens for routine doping controls, which allow for the analytical coverage of an extensive set of target analytes relevant to sports drug testing programs. In the course of studies to identify potential alternative matrices to complement current testing approaches, exhaled breath (EB) has been found to offer advantageous properties especially with regard to the sample collection procedure, which is less invasive, less intrusive, and less time-consuming when compared to conventional blood and urine testing. A yet unaddressed question has been the potential contribution of oral fluid (OF) to EB samples. The current investigation focused on characterizing an electret membrane-based EB collection device concerning a potential introduction of OF during the sampling procedure. For that purpose, EB and OF samples collected under varying conditions from a total of 14 healthy volunteers were tested for the presence of abundant salivary proteins using bottom-up proteomics approaches such as SDS-PAGE followed by tryptic digestion and chromatographic-mass spectrometric analysis. The trapping baffles integrated into the mouthpiece of the EB collection device were found to effectively retain OF introduced into the unit during sample collection as no saliva breakthrough was detectable using the established analytical approach targeting predominantly the highly abundant salivary α-amylase. Since α-amylase was found unaffected by storage, smoking, food intake, and exercise, it appears to be a useful marker to reveal possible OF contaminations of EB collection devices.
Subject(s)
Breath Tests/methods , Membranes, Artificial , Proteins/analysis , Saliva/chemistry , Adult , Breath Tests/instrumentation , Equipment Design , Female , Humans , Male , Middle Aged , Proteomics/methods , Specimen Handling/instrumentation , Substance Abuse Detection/instrumentation , Substance Abuse Detection/methods , Young AdultABSTRACT
Cobaltous ions (Co2+ ) stabilize HIFα, increase endogenous erythropoietin (EPO) production, and may, therefore, be used as a performance-enhancing substance. To date, the dosage necessary to stimulate erythropoiesis is unknown. The aim of this study was, therefore, to determine the minimum dosage necessary to increase erythropoietic processes. In a first double-blind placebo-controlled study (n = 5), single oral Co2+ dosages of 5 mg (n = 6) and 10 mg (n = 7) were administered to healthy young men. Cubital venous blood and urine samples were collected before and up to 24 hours after Co2+ administration. In a second study, the same daily Co2+ dosages were administered for five days (placebo: n = 5, 5 mg: n = 9, 10 mg: n = 7). Blood and urine samples were taken the day before administration and at day 3 and day 5. Plasma [EPO] was elevated by 20.5 ± 16.9% at 5 hours after the single 5-mg administration (p < 0.05) and by 52.8 ± 23.5% up to 7 hours following the 10-mg Co2+ administration (p < 0.001). Urine [Co2+ ] transiently increased, with maximum values 3-5 hours after Co2+ ingestion (5 mg: from 0.8 ± 1.1 to 153.6 ± 109.4 ng/mL, 10 mg: from 1.3 ± 1.7 to 338.0 ± 231,5 ng/mL). During the five days of Co2+ application, 5 mg showed a strong tendency to increase [EPO], while the 10-mg application significantly increased [EPO] at day 5 by 27.2 ± 26.4% (p < 0.05) and the immature reticulocyte fraction by 49.9 ± 21.7% (p < 0.01). [Ferritin] was decreased by 12.4 ± 10.4 ng/mL (p < 0.05). An oral Co2+ dosage of 10 mg/day exerts clear erythropoietic effects, and 5 mg/day tended to increase plasma EPO concentration.
Subject(s)
Cobalt/administration & dosage , Cobalt/pharmacology , Erythropoiesis/drug effects , Adult , Blood Cell Count/statistics & numerical data , Cobalt/pharmacokinetics , Cobalt/urine , Double-Blind Method , Erythropoietin/blood , Ferritins/blood , Humans , Male , Trace Elements/pharmacokinetics , Trace Elements/pharmacology , Trace Elements/urineABSTRACT
Introduction: Cobalt ions (Co2+) stabilize HIFα and increase endogenous erythropoietin (EPO) production creating the possibility that Co2+ supplements (CoSupp) may be used as performance enhancing substances. The aim of this study was to determine the effects of a small oral dosage of CoSupp on hemoglobin mass (Hbmass) and performance with the objective of providing the basis for establishing upper threshold limits of urine [Co2+] to detect CoSupp misuse in sport. Methods: Twenty-four male subjects participated in a double-blind placebo-controlled study. Sixteen received an oral dose of 5 mg of ionized Co2+ per day for 3 weeks, and eight served as controls. Blood and urine samples were taken before the study, during the study and up to 3 weeks after CoSupp. Hbmass was determined by the CO-rebreathing method at regular time intervals, and VO2max was determined before and after the CoSupp administration period. Results: In the Co2+ group, Hbmass increased by 2.0 ± 2.1% (p < 0.001) while all the other analyzed hematological parameters did not show significant interactions of time and treatment. Hemoglobin concentration ([Hb]) and hematocrit (Hct) tended to increase (p = 0.16, p = 0.1) and also [EPO] showed a similar trend (baseline: 9.5 ± 3.0, after 2 weeks: 12.4 ± 5.2 mU/ml). While mean VO2max did not change, there was a trend for a positive relationship between changes in Hbmass and changes in VO2max immediately after CoSupp (r = 0.40, p = 0.11). Urine [Co2+] increased from 0.4 ± 0.3 to 471.4 ± 384.1 ng/ml (p < 0.01) and remained significantly elevated until 2 weeks after cessation. Conclusion: An oral Co2+ dosage of 5 mg/day for 3 weeks effectively increases Hbmass with a tendency to increase hemoglobin concentration ([Hb]) and hematocrit (Hct). Because urine Co2+ concentration remains increased for 2 weeks after cessation, upper limit threshold values for monitoring CoSupp can be established.
ABSTRACT
Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22â¯kDa, the GHRPs are short (3-6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22â¯kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124â¯Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195â¯Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration.
Subject(s)
Doping in Sports , Human Growth Hormone/analysis , Oligopeptides/analysis , Substance Abuse Detection/methods , Chromatography, Liquid , Humans , Tandem Mass SpectrometryABSTRACT
Analytical chemistry represents a central aspect of doping controls. Routine sports drug testing approaches are primarily designed to address the question whether a prohibited substance is present in a doping control sample and whether prohibited methods (for example, blood transfusion or sample manipulation) have been conducted by an athlete. As some athletes have availed themselves of the substantial breadth of research and development in the pharmaceutical arena, proactive and preventive measures are required such as the early implementation of new drug candidates and corresponding metabolites into routine doping control assays, even though these drug candidates are to date not approved for human use. Beyond this, analytical data are also cornerstones of investigations into atypical or adverse analytical findings, where the overall picture provides ample reason for follow-up studies. Such studies have been of most diverse nature, and tailored approaches have been required to probe hypotheses and scenarios reported by the involved parties concerning the plausibility and consistency of statements and (analytical) facts. In order to outline the variety of challenges that doping control laboratories are facing besides providing optimal detection capabilities and analytical comprehensiveness, selected case vignettes involving the follow-up of unconventional adverse analytical findings, urine sample manipulation, drug/food contamination issues, and unexpected biotransformation reactions are thematized.
Subject(s)
Doping in Sports , Mass Spectrometry/methods , Substance Abuse Detection/methods , Athletes , Designer Drugs/analysis , Doping in Sports/methods , Food Contamination/analysis , Humans , Performance-Enhancing Substances/urine , Urine Specimen Collection/methodsABSTRACT
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.
Subject(s)
Breath Tests/methods , Chromatography, Liquid/methods , Doping in Sports , Substance Abuse Detection/methods , Tandem Mass Spectrometry/methods , Adult , Anabolic Agents/analysis , Androgens/analysis , Central Nervous System Stimulants/analysis , Female , Humans , Limit of Detection , Male , Middle Aged , Models, Chemical , Reproducibility of Results , Young AdultABSTRACT
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.
Subject(s)
Doping in Sports , Glucuronides/chemistry , Glucuronides/metabolism , Glycine/analogs & derivatives , Isoquinolines/chemistry , Prolyl-Hydroxylase Inhibitors/chemistry , Athletes , Chromatography, Liquid , Glycine/chemistry , Humans , Limit of Detection , Substance Abuse Detection , Tandem Mass Spectrometry , UrinalysisABSTRACT
BACKGROUND: Substances developed for therapeutic use are also known to be misused by athletes as doping agents and, outside of regulated sport, for image-enhancement. This has generated a market for counterfeit doping substances. Counterfeit doping agents may be of poor pharmaceutical quality and therefore constitute health risks to consumers. OBJECTIVES: This study aims to investigate the pharmaceutical quality of 1,190 doping products seized at the Swiss border. METHODS: Swiss customs authorities seize incoming shipments potentially containing doping agents. Qualitative and semiquantitative analyses were performed in order to test for prohibited doping substances. The main analytical methods utilized for characterizing confiscated compounds were liquid chromatography-high resolution mass spectrometry, polyacrylamide gel electrophoresis with subsequent in-gel tryptic digestion and identification of peptidic compounds using nanoliquid chromatography-tandem mass spectrometry, and electrochemiluminescence immuno assay. RESULTS: For 889 (75%) of the analyzed products, the label suggested the content of anabolic agents, for 146 samples (12%) peptide hormones or growth factors, and for 113 items (9%) antiestrogens, aromatase inhibitors or other metabolic modulators. For the majority of the investigated products, the pharmaceutical quality was an unsatisfactory standard: nonapproved substances were detected and less than 20% of the products contained the claimed substance in the respective amount. CONCLUSION: A comprehensive sample of confiscated doping products was analyzed, allowing for monitoring of developments regarding the use of doping substances in Switzerland and for anticipating future trends and challenges in sports drug testing. An alarming number of tested products was of substandard pharmaceutical quality.
Subject(s)
Performance-Enhancing Substances , Anabolic Agents/analysis , Doping in Sports , Growth Hormone/analysis , Humans , Performance-Enhancing Substances/analysis , SwitzerlandABSTRACT
Research into developing anabolic agents for various therapeutic purposes has been pursued for decades. As the clinical utility of anabolic-androgenic steroids has been found to be limited because of their lack of tissue selectivity and associated off-target effects, alternative drug entities have been designed and are commonly referred to as selective androgen receptor modulators (SARMs). While most of these SARMs are of nonsteroidal structure, the drug candidate MK-0773 comprises a 4-aza-steroidal nucleus. Besides the intended therapeutic use, SARMs have been found to be illicitly distributed and misused as doping agents in sport, necessitating frequently updated doping control analytical assays. As steroidal compounds reportedly undergo considerable metabolic transformations, the phase-I metabolism of MK-0773 was simulated using human liver microsomal (HLM) preparations and electrochemical conversion. Subsequently, major metabolic products were identified and characterized employing liquid chromatography-high-resolution/high- accuracy tandem mass spectrometry with electrospray (ESI) and atmospheric pressure chemical ionization (APCI) as well as nuclear magnetic resonance (NMR) spectroscopy. MK-0773 produced numerous phase-I metabolites under the chosen in vitro incubation reactions, mostly resulting from mono- and bisoxygenation of the steroid. HLM yielded at least 10 monooxygenated species, while electrochemistry-based experiments resulted predominantly in three monohydroxylated metabolites. Elemental composition data and product ion mass spectra were generated for these analytes, ESI/APCI measurements corroborated the formation of at least two N-oxygenated metabolites, and NMR data obtained from electrochemistry-derived products supported structures suggested for three monohydroxylated compounds. Hereby, the hydroxylation of the A-ring located N- bound methyl group was found to be of particular intensity. In the absence of controlled elimination studies, the produced information enables the implementation of new target analytes into routine doping controls and expands the focus of anti-doping efforts concerning this new anabolic agent.
Subject(s)
Doping in Sports , Receptors, Androgen , Substance Abuse Detection , Anabolic Agents , Androgens , Humans , Hydroxylation , Spectrometry, Mass, Electrospray IonizationABSTRACT
This retrospective study evaluates the content, the destination and the source of 960 postal items seized by the Swiss customs authorities at the Swiss border between 2013 and 2014. The packages were seized because they contained at least one prohibited doping product as identified by the Swiss law on encouraging sports and physical activity. A total number of 1825 different doping products were confiscated from these parcels, accounting for an average of 1.9 doping products per seized item. In 74% of the cases, where seizures were made, anabolic androgenic steroids, mostly testosterone esters, were discovered. An obvious trading channel for doping products was identified in this study. The seized compounds were predominately manufactured in Asian countries, but sent to Switzerland mostly via South Eastern Europe countries. Due to the unique collaboration between the Swiss customs authorities and the national anti-doping agency, this study uncovered an alarming trend of illegal doping product trafficked to Switzerland.
ABSTRACT
LG121071 is a member of the tetrahydroquinolinone-based class of selective androgen receptor modulator (SARM) drug candidates. These nonsteroidal compounds are supposed to act as full anabolic agents with reduced androgenic properties. As SARMs provide an alternative to anabolic androgenic steroids, they represent an emerging class of potential doping substances abused by athletes for illicit performance enhancement. According to the World Anti-Doping Agency's regulations, SARMs are banned substances and part of the Prohibited List since 2008. In consideration of the increasing number of adverse analytical findings in doping controls caused by SARMs abuse, potential drug candidates such as LG121071 have been proactively investigated to enable a timely integration into routine testing procedures even though clinical trials are not yet complete. In the present approach, the collision-induced dissociation (CID) of LG121071 was characterized by means of electrospray ionization-high resolution/high accuracy mass spectrometry, MS(n), and isotope labeling experiments. Interestingly, the even-electron precursor ion [Mâ¯+â¯H](+) at m/z 297 was found to produce a radical cation at m/z 268 under CID conditions, violating the even-electron rule that commonly applies. For doping control purposes, metabolites were generated in vitro and a detection method for urine samples based on liquid chromatography-tandem mass spectrometry was established. The overall metabolic conversion of LG121071 was modest, yielding primarily mono-, bis- and trishydroxylated species. Notable, however, was the identification of a glucuronic acid conjugate of the intact drug, attributed to an N-glucuronide structure. The sample preparation procedure included the enzymatic hydrolysis of glucuronides prior to liquid-liquid extraction, allowing intact LG121071 to be measured, as well as the corresponding phase-I metabolites. The method was characterized concerning inter alia lower limit of detection (0.5 ng mL(-1) in urine), recovery (40%), and intra-/interday precision (2.3% to 11.7%) to assess its fitness for purpose. Prospectively, the assay can serve as detection method for LG121071 in drug testing and/or doping controls.