Stereoisomers in sports drug testing: Analytical strategies and applications.

Analytics employed in modern doping controls are designed to cover an extensive range of rather diverse classes of substances, all of which are banned in sport according to the list of prohibited substances and methods of doping, resulting from their potential to be performance-enhancing and/or harmful to health. Many of these bioactive substances or their metabolites are chiral, which are comprehensively characterized and, if appropriate analytical approaches are applied, can be clearly identified. In sports drug testing, the enantiomeric composition of relevant compounds is not considered in all instances, although differences of isomers concerning their biological activity have been established. To date, the separation of stereoisomers in doping controls is only applied for selected target compounds, but with the development of efficient chiral chromatographic stationary phases, the added value of information on e.g. racemic shifts during the metabolic biotransformation reactions of drugs has been recognized. The immense variability of the substance classes represents however a major challenge, especially because both 'classic' doping agents belonging to the category of lower molecular mass molecules (e.g. stimulants, ß2-agonists, betablockers, corticoids, etc.) as well as larger molecules from the category of peptides and proteins necessitate consideration. In the present (mini)review, the current status of analytical techniques in the field of doping control analysis of stereoisomers is highlighted and critically reviewed.

In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples.

SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-9009 in an associated plasma and urine doping sample. Liquid chromatography-high-resolution mass spectrometry was used to verify the presence of the N-dealkylated metabolite (M1) in both matrices, with a low concentration of the parent compound and additional N-desalkyl metabolites (M2 and M3) detected in the plasma sample as supporting evidence of administration. To the best of the authors' knowledge, this is the first report of an adverse analytical finding in an equine sample for SR-9009 or its metabolites in equine doping control.

Annual banned-substance review: Analytical approaches in human sports drug testing 2020/2021.

Most core areas of anti-doping research exploit and rely on analytical chemistry, applied to studies aiming at further improving the test methods' analytical sensitivity, the assays' comprehensiveness, the interpretation of metabolic profiles and patterns, but also at facilitating the differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds and comprehending the athlete's exposome. Further, a continuously growing number of advantages of complementary matrices such as dried blood spots have been identified and transferred from research to sports drug testing routine applications, with an overall gain of valuable additions to the anti-doping field. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2020 and September 2021 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified in the World Anti-Doping Agency's 2021 Prohibited List.

Nontargeted detection of designer androgens: Underestimated role of in vitro bioassays.

Androgens, both steroidal and nonsteroidal in nature, are among the most commonly misused substances in competitive sports. Their recognized anabolic and performance enhancing effects through short- and long-term physiological adaptations make them popular. Androgens exist as natural steroids, or are chemically synthesized as anabolic androgenic steroids (AAS) or selective androgen receptor modulators (SARMs). In order to effectively detect misuse of androgens, targeted strategies are used. These targeted strategies rely heavily on mass spectrometry, and detection requires prior knowledge of the targeted structure and its metabolites. Although exquisitely sensitive, such approaches may fail to detect novel structures that are developed and marketed. A nontargeted approach to androgen detection involves the use of cell-based in vitro bioassays. Both yeast and mammalian cell androgen bioassays demonstrate a clear ability to detect AAS and SARMS, and if paired with high resolution mass spectrometry can putatively identify novel structures. In vitro cell bioassays are successfully used to characterize designer molecules and to detect exogenous androgens in biological samples. It is important to continue to develop new and effective detection approaches to prevent misuse of designer androgens, and in vitro bioassays represent a potential solution to nontargeted detection strategies.

A cell-free bioassay for the detection of androgens.

Androgens remain abused performance-enhancing drugs in sports. Technologies based on mass spectrometry can detect all forms of androgens but fail if the androgen represents a novel structure. A bioassay detects androgens based on function rather than structure. To date, there has been limited adoption of cell-based in vitro bioassays as a screening tool for nontargeted androgen detection because they require expert personnel and specialized equipment to perform. We now describe the development of a cell-free version of an androgen in vitro bioassay. Stage 1 involved in vitro transcription/translation reactions (IVTT) using a DNA template encoding an enhancer/androgen response element (ARE) regulatory region upstream of a minimal promoter that drives expression of a reporter protein. The assay detected testosterone across the concentration range of 106.7 to 0.0144 ng/ml (3.7 × 10-7 to 5 × 10-11 M), with an EC50 of 6.63 ng/ml (23 nM). To reduce complexity, Stages 2-4 of development included just in vitro transcription (IVT) reactions, whereby the output was an RNA molecule. Stage 2 involved directly labelling the RNA molecule with fluorophore-labelled nucleotide triphosphates, Stage 3 involved reverse transcription-polymerase chain reaction (PCR) of the RNA molecule, and Stage 4 utilized an RNA aptamer, Mango II, as its RNA output. The Stage 4 product detected testosterone across the range of 106.7-0.0001 ng/ml (3.7 × 10-7 to 5 × 10-13 M), with an EC50 of 0.04 ng/ml (0.155 nM). Further to this, we show that the Stage 4 product can detect other androgenic molecules. Relative to cell-based bioassays, the Stage 4 product is easy to perform and could be developed into a routine, high-throughput, nontargeted androgen screen.

Detection and longitudinal distribution of GW1516 and its metabolites in equine hair for doping control using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: GW1516 is a peroxisome proliferator-activated receptor-δ (PPAR-δ) agonist that is banned in horseracing and equestrian sports. Long-term detection and longitudinal distribution of GW1516 in the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control. METHODS: Mane hairs were divided into three segments (0-7, 7-15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using a Q-Exactive instrument. This method was successfully validated and applied to post-administration samples to confirm the presence of GW1516 and its metabolites and estimate the uptake amounts of GW1516. RESULTS: After administration of 150 mg of GW1516 to a thoroughbred mare, GW1516 was detected in one of two segments of all mane hairs, and four metabolites, namely GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), and 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), were also identified. The longitudinal distribution analysis results showed that the maximum uptake of GW1516 into hair (approximately 0.05 pg/mg) was observed at around 13 weeks post-administration and GW1516 could be detected and confirmed up to 6 months post-administration. CONCLUSIONS: The parent drug GW1516 was identified as the most appropriate monitoring target in equine hair for controlling its misuse in horses. The use of hair analysis could extend the detection time of GW1516 to at least 6 months after the administration of 150 mg of GW1516 to a thoroughbred mare.

Metabolic studies of hypoxia-inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control.

The transcriptional activator hypoxia-inducible factor (HIF) is a vital arbitrator in the performance of cellular responses lacking oxygen supply in aerobic organisms. Because these compounds are capable of enhancing the organism's capacity for molecular oxygen transport, they possess great potential for abuse as a performance-enhancing agent in sports. A comprehensive study of the metabolic conversion of the most popular HIF stabilisers such as IOX2, IOX3 and IOX4 using equine liver microsomes (in vitro) is reported. The parents and their metabolites were identified and characterised by liquid chromatography-mass spectrometry in negative ionisation mode using a QExactive high-resolution mass spectrometer. Under the current experimental condition, a total of 10 metabolites for IOX2 (three phase I and seven phase II), nine metabolites for IOX3 (four phase I and five phase II) and five metabolites for IOX4 (three phase I and two phase II) were detected. The outcome of the present study is as follows: (1) all the three IOX candidates are prone to oxidation, results in subsequent monohydroxylated, and some dihydroxylated metabolites. (2) Besides oxidation, there is a possibility of hydrolysis and de-alkylation, which results in corresponding carboxylic acid and amide, respectively. (3) The glucuronide and sulphate conjugate of the parent drugs as well as the monohydroxylated analogues were observed in this study. The characterised in vitro metabolites can potentially serve as target analytes for doping control analysis.

Doping detection in animals: A review of analytical methodologies published from 1990 to 2019.

Despite the impressive innate physical abilities of horses, camels, greyhounds, or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti-doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices published from 1990 to 2019 were considered. A total of 317 studies were included, of which 298 were related to horses, demonstrating significant advances toward the development of doping detection methods for equine sports. However, analytical methods for the detection of doping agents in sports involving other species are lacking. Due to enhanced accuracy and specificity, chromatographic analysis coupled to mass spectrometry detection is preferred over immunoassays. Regarding biological matrices, blood and urine remain the first choice, although alternative biological matrices, such as hair and feces, have been considered. With the increasing number and type of drugs used as doping agents, the analytes addressed in the published papers are diverse. It is very important to continue to detect and quantify these drugs, recognizing those that are most frequently used, in order to punish the abusers, protect animals' health, and ensure a healthier and genuine competition.

An overview on performance and image enhancing drugs (PIEDs) confiscated in Italy in the period 2017-2019.

CONTEXT: The illegal market of counterfeit and falsified medicines and supplements containing unlabeled pharmaceuticals is expanding worldwide. They are usually referred to by the term "performance and image enhancing drugs" (PIEDs) and are mainly steroids, stimulants, hormones, and drugs for erectile dysfunction. PIEDs are easily accessible through the online or black markets. We analyzed over 400 such medicines confiscated in Italy in the period 2017-2019, to determine their composition. METHODS: Confiscated products were analyzed by gas chromatography/mass spectrometry and liquid chromatography/high-resolution mass spectrometry, in order to ascertain their composition and to evaluate the correspondence between what was declared on the label and the actual content, or to identify unknown products. RESULTS: The most commonly found substance was anabolic steroids, found in 64% of products, with 11% containing hormone modulators, 6% stimulants, 6% sexual enhancers (mainly sildenafil) and other drugs, including thyroid hormones, melanin stimulators, and vitamins. These substances were often in mixtures. The products were often mislabeled, containing contaminants in addition to the drug declared, or consisted of a drug completely different from the one reported on the label. Fifteen percent of products had a qualitative composition completely different from that declared, while 10% of products showed cross-contamination with other drugs, mainly testosterone esters, probably due to the presence of residues of other drugs in the production line. In addition, 11% of products were not labeled, so their purported composition was unknown. DISCUSSION: PIEDs pose a threat to public health. The main risks are related to the intrinsic toxicity of the substances found, especially when taken without a therapeutic indication. Another issue is related to the mislabeling of the fake medicines, and the poor-quality standard of counterfeit product preparation, with additional risks of the presence of other toxic ingredients or microbial contamination. CONCLUSIONS: The use of counterfeit products is a public health concern, as it constitutes a high risk for consumer health. It is mainly caused by the uncontrolled use of steroids, stimulants, sexual enhancers, and other medicaments, without medical indication or supervision, with variable and unknown compositions and doses, as well as other contaminants as a result of the absence of good manufacturing practices.

Annual banned-substance review: Analytical approaches in human sports drug testing 2019/2020.

Analytical chemistry-based research in sports drug testing has been a dynamic endeavor for several decades, with technology-driven innovations continuously contributing to significant improvements in various regards including analytical sensitivity, comprehensiveness of target analytes, differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds, assessment of alternative matrices for doping control purposes, and so forth. The resulting breadth of tools being investigated and developed by anti-doping researchers has allowed to substantially improve anti-doping programs and data interpretation in general. Additionally, these outcomes have been an extremely valuable pledge for routine doping controls during the unprecedented global health crisis that severely affected established sports drug testing strategies. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2019 and September 2020 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified the World Anti-Doping Agency's 2020 Prohibited List.

An in vitro assay approach to investigate the potential impact of different doping agents on the steroid profile.

The steroid profile, that is, the urinary concentrations and concentration ratios of selected steroids, is used in sports drug testing to detect the misuse of endogenous steroids such as testosterone. Since several years, not only population-based thresholds are applied but also the steroid profile is monitored via the Athlete Biological Passport whereby the individual reference ranges derived from multiple test results of the same athlete are compared to population-based thresholds. In order to maintain a high probative force of the passport, samples collected or analyzed under suboptimal conditions should not be included in the longitudinal review. This applies to biologically affected or degraded samples and to samples excluded owing to the presence of other substances potentially (or evidently) altering the steroid profile. Nineteen different doping agents comprising anabolic steroids, selective androgen receptor modulators, selective estrogen receptor modulators, ibutamoren, and tibolone were investigated for their effect on the steroid profile using an androgen receptor activation test, an androgen receptor binding assay, an aromatase assay, and a steroidogenesis assay. The in vitro tests were coupled with well-established liquid chromatography/mass spectrometry-based analytical approaches and for a subset of steroidal analytes by gas chromatography/mass spectrometry. The variety of tests employed should produce a comprehensive data set to better understand how a compound under investigation may impact the steroid profile. Although our data set may allow an estimate of whether or not a substance will have an impact on the overall steroid metabolism, predicting which parameter in particular may be influenced remains difficult.

LC-HRMS characterization of the skin pigmentation and sexual enhancers melanotan II and bremelanotide sold on the black market of performance and image enhancing drugs.

The spread of performance and image enhancing drugs (PIEDs) often requires forensic toxicology laboratories to identify unknown compounds without reference standards. We characterized the PIEDs melanotan II and bremelanotide, not legally marketed, in eight unknown samples confiscated by police together with anabolic steroids, hormone modulators, sexual enhancers and stimulants, intended for the black market of bodybuilders, using liquid chromatography-high resolution/high accuracy Orbitrap mass spectrometry (LC-HRMS). The characterization was carried out by the accurate mass measurements of MH+ ionic species, the study of their isotopic patterns and the associated relative isotopic abundance (RIA) values, as well as the accurate mass measurements of collision-induced product ions obtained in fragmentation experiments. LC-HRMS confirmed itself as a powerful analytical tool to elucidate the elemental composition and structural characteristics of unknown compounds.

Linking long noncoding RNAs (lncRNAs) and doping detection.

In the fight against doping, efficient methods for detecting substances or biomarkers are still being improved. Indirect methods are an interesting alternative for the detection of substances misuse longitudinally. Here we shed lights the long non-coding RNAs (lncRNAs) as a possible biomarkers due to their characteristics such as tissue-specific expression and strict regulation.

Caffeine and Doping-What Have We Learned since 2004.

Caffeine is a naturally occurring plant alkaloid and is found in plant constituents such as coffee and cocoa beans, tea leaves, guarana berries and the kola nut [...].

Analysis of doping control test results in individual and team sports from 2003 to 2015.

Background: Determining the prevalence of doping in sport might be useful for anti-doping authorities to gauge the effectiveness of anti-doping policies implemented to prevent positive attitudes toward doping. Using questionnaires and personal interviews, previous investigations have found that the prevalence of doping might be different among different sports disciplines; however, there is no sport-specific information about the proportion of adverse and atypical findings (AAF) in samples used for doping control. The aim of the present investigation was to assess the differences in the frequency of adverse analytical and atypical findings among sports using the data made available by the World Anti-Doping Agency. Methods: The data included in this investigation were gathered from the Testing Figures Reports made available annually from 2003 to 2015 by the World Anti-Doping Agency. These Testing Figures Reports include information about the number of samples analyzed, the number of AAFs reported, and the most commonly found drugs in the urine and blood samples analyzed. A total of 1,347,213 samples were analyzed from the individual sports selected for this investigation, and 698,371 samples were analyzed for disciplines catalogued as team sports. Results: In individual sports, the highest proportions of AAF were 3.3% ± 1.0% in cycling, 3.0% ± 0.6% in weightlifting, and 2.9% ± 0.6% in boxing. In team sports, the highest proportions of AAF were 2.2% ± 0.5% in ice hockey, 2.0% ± 0.5% in rugby, and 2.0% ± 0.5% in basketball. Gymnastics and skating had the lowest proportions at (≤1.0%) for individual sports, and field hockey, volleyball and football had the lowest proportions for team sports (≤1.4%). Conclusion: As suggested by the analysis, the incidence of AAF was not uniform across all sports disciplines, with the different proportions pointing to an uneven use of banned substances depending on the sport. This information might be useful for increasing the strength and efficacy of anti-doping policies in those sports with the highest prevalence in the use of banned substances.

Potential Risk of Higenamine Misuse in Sports: Evaluation of Lotus Plumule Extract Products and a Human Study.

Since 2017, higenamine has been added to the World Anti-Doping Agency (WADA) prohibited list as a ß2-agonist prohibited at all times for sportspersons. According to WADA's report, positive cases of higenamine misuse have been increasing yearly. However, higenamine occurs naturally in the Chinese herb lotus plumule-the green embryo of lotus (Nelumbo nucifera Gaertn) seeds-commercially available as concentrated powder on the Asian market. This study evaluated the major phytochemical components of lotus plumule products using an appropriate extraction method, followed by a human study in which the products were orally administered in multiple doses to investigate the risk of doping violations. Comparing various extraction methods revealed that optimized microwave-assisted extraction exhibited the highest extraction efficiency (extraction time, 26 min; power, 1046 W; and temperature, 120 °C). Subsequently, the alkaloids in lotus plumule products were quantitatively confirmed and compared. Human study participants (n = 6) consumed 0.8 g of lotus plumule (equivalent to 679.6 µg of higenamine) three times daily for three consecutive days. All participants' urinary higenamine concentrations exceeded the WADA reporting cut-off of 10.0 ng/mL. Accordingly, lotus plumule consumption may engender adverse analytical findings regarding higenamine. Athletes should avoid consuming lotus plumule-containing products during in- and out-of-competition periods.

Annual banned-substance review - Analytical approaches in human sports drug testing.

Within the complex construct of today's antidoping work, continuously updated routine doping controls, as well as advancements in sampling and analysis have been of particular relevance and importance. New analytes of existing classes of prohibited substances are frequently included into sports drug testing assays, analytical approaches are optimized to allow for better sensitivities, selectivity, and/or faster turnaround times, and research dedicated to addressing analytical issues concerning scenarios of both (potentially) inadvertent doping and new emerging doping agents is constantly conducted. By way of reviewing and summarizing, this annual banned-substance review evaluates the literature published between October 2018 and September 2019 offering an in-depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2019 Prohibited List.