Adverse Analytical Findings Research Articles

Unravelling the threat of contamination in elite sports: Exploring diverse sources impacting adverse analytical findings and the risk of inadvertent exposure to prohibited substances

In recent years, increasing concerns have emerged regarding athletes being exposed to various sources of contamination that could result in an adverse analytical finding (AAF), which is considered a positive doping test and may lead to the athlete's sanction. This review aims to examine the potential sources of contamination. Firstly, exogenous sources such as food, water, supplements, and medications will be described, along with endogenous sources, primarily arising from the athlete’s physiological condition via the biotransformation of Medications. Finally, other hypothetical contaminations arising from sample collection procedures, poor transport or storage, and laboratory conditions will be discussed. Despite some legislative efforts to regulate the production of food and supplements, contamination remains a significant concern in the context of anti-doping, necessitating athletes to stay vigilant against the risks of inadvertent uptake of illicit products. Increased knowledge of the potential sources of contamination is essential for all parties involved in the fight against doping, including athletes, support personnel, legitimate supplement product manufacturers, and the anti-doping and scientific community. Such insights can contribute to developing the most effective strategy for preventing contamination and, most importantly, reducing the risk of inadvertent AAFs.

Heptaminol hair testing to highlight octodrine contamination in supplements, responsible for a doping adverse analytical finding

Octodrine, also known as dimethylhexylamine (DMHA), is a central nervous stimulant. When ingested by humans, octodrine is rapidly metabolized to heptaminol, the main compound present in the blood. Due to their stimulant activities, octodrine and its metabolite are specified substances in section S6b “specified stimulants” of the World Anti-Doping Agency (WADA) Prohibited List, prohibited in competition. But in some cases, they can be responsible for an adverse analytical finding (AAF) following contamination, with a low concentration in the urine. In such cases, a hair test can distinguish doping from contamination according to the concentration measured, or the absence of the target drug. However, no data are available in the literature. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of octodrine and heptaminol in hair, and to apply it to an athlete with an AAF with heptaminol (141 and 136 ng/mL in urine samples A and B after correction for specific gravity) and octodrine (16 ng/mL, not given by the WADA laboratory in sample A as being below the minimum request level of 50 ng/mL).All the supplements taken by the athlete were analysed, and pubic hair (black, 1.5–2 cm long, no head hair available) and toenail clippings (0.1 cm) were sampled two months after AAF. A supplement was identified as the source of contamination, containing octodrine at a concentration range 16–34 µg/g. The athlete had taken 13 g daily (0.2–0.4 mg octodrine) for 20 days prior to the AAF (total dose 4–8 mg). His pubic hair and toenail clippings contained only heptaminol (30 pg/mg and 3 pg/mg, respectively). A controlled study was carried out on a volunteer who took a single 13 g dose of the contaminated supplement (single dose of octodrine 0.2–0.4 mg). His urine was positive for both compounds, with concentrations very close to those of the athlete on the day of the AAF after the same delay between intake and sampling (around 4 h). One month later, his head hair and pubic hair showed only heptaminol at 2 and 3 pg/mg, respectively, and toenail clippings were totally negative for both compounds. The contamination was accepted by the WADA commission, but the athlete was still banned for 9 months for having consumed supplements purchased on the internet.

Long-Term Excretion of Roxadustat in Urine.

Roxadustat (FG-4592), an orally active hypoxia-inducible factor prolyl hydroxylase stabilizer, has been shown to enhance erythropoiesis by increasing endogenous erythropoietin. It is indicated for the treatment of anemia and chronic kidney disease and is approved for clinical use in several countries, including the European Union, Japan and others. Due to its reasonably anticipated performance-enhancing effect in athletes, roxadustat is prohibited for use in sports at all times. A few cases of adverse analytical findings in routine doping controls have been reported worldwide, some of which were claimed to be the result of contaminated dietary supplements. The present study offers new data demonstrating the long-term excretion pattern of roxadustat. Even after a single-dose administration, roxadustat can remain detectable in urine for 8 months, albeit at very low concentrations (<10 pg/mL). Following three times a week treatment with 70 to 100 mg of roxadustat, the drug was still detectable in the urine of anemic patients for between 9 and 18 months after treatment was discontinued. Lastly, an athlete who admitted use of roxadustat for almost a year (50 mg 3 to 5 times a week) has now tested positive multiple times over the course of 15 months (the first test being 12 months after the drug was discontinued), with an estimated concentration of roxadustat between 3 and 8 pg/mL. Altogether, these findings indicate the unusually prolonged terminal excretion kinetics of roxadustat, a property that testing authorities should consider in their results management process.

Investigations into the Concentrations and Metabolite Profiles of Doping Agents and Antidepressants in Human Seminal Fluid Using Liquid Chromatography-Mass Spectrometry.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called adverse analytical finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offenses and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from nonathletes were analyzed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e., samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the nonapproved proliferator-activated receptor delta agonist GW1516) in a total of 81 samples, with 91% of these suspected cases being verified by the confirmation method. In addition to the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. SIGNIFICANCE STATEMENT: This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

Investigation of the Physiological and Post‐training Effects of Ecdysteroid Supplementation by Multivariate Analysis of the Human Serum Metabolome

ABSTRACTThis work aims to characterize the serum profile of athletes after the administration of ecdysteroids, natural steroid hormones recently reported to enhance athletic performance. The combination of mass spectrometry and chemometric tools may allow to differentiate physiological effects from post‐training and intake‐driven effects. Serum samples were collected from 46 healthy male volunteers and divided into four groups: control (two capsules/day of Peak Ecdysone without training), placebo (two capsules without ecdysteroids with training), Ec1 (two capsules/day Peak Ecdysone with training), and Ec2 (eight capsules/day Peak Ecdysone with training). Metabolic profiling was measured using a SCIEX Triple Quadrupole LC‐MS/MS system coupled with the Biocrates AbsoluteIDQ p180 kit, which allows quantitation of a large panel of metabolites that were subjected to multivariate analysis. Unsupervised analysis of the data found no significant differences between the placebo and the ecdysteroid supplementation groups. By merging Ec1 and Ec2 into a single group, coded as treated, a clear discrimination between the control and placebo groups was observed. Phosphatidylcholines were among the most significant features of ecdysteroids administration, showing a dose‐dependent effect in Ec1 and Ec2 groups. As specific metabolic phenotypes can result from years of training, the discrimination of physiological effects from those caused by the administration of banned substances can be a valuable analytical strategy for the interpretation of adverse analytical findings in the anti‐doping field.

Interest of hair tests to discriminate a tail end of a doping regimen from a possible contamination in case of challenging an anti-doping rule violation. V. Case reports involving trimetazidine, a drug where the concentration after a single 20 mg dose has been established.

The identification of trimetazidine, a medicine used for treating stable angina pectoris and for preventing angina attacks, has been recently observed in doping cases involving high profile athletes from various countries over the world. In all the files where the authors have been involved, the urine concentration of trimetazidine was low (<2 ng/mL), and the athletes argued that contamination was the source of their adverse analytical finding. It is possible to challenge imposed sanctions in relation to an adverse analytical finding, but it is the responsibility of the athlete to demonstrate he/she is innocent and can qualify for no fault or negligence. When the delay between the urine collection and the notification of the violation was not too long (less than 6months), these athletes requested a head hair test. Trimetazidine was analyzed by an original LC-MS/MS method involving pH 9.5 borate buffer overnight incubation of 20 mg and subsequent solvents extraction in presence of trimetazidine-D8 used as internal standard. Linearity was verified from 1 to 200 pg/mg (R2 = 0.9987). Limit of detection of the method was 0.1 pg/mg. The hair specimen of a male subject, collected 4 weeks after single oral ingestion of 20 mg trimetazidine, tested positive at 146 pg/mg in the corresponding segment. Concentrations of trimetazidine measured in several hair specimens (n = 5) collected from athletes challenging their anti-doping rule violation were below 1 pg/mg, which is consistent with incidental exposure due to contamination. This is the first evidence that trimetazidine is incorporated in human hair after a single therapeutic dose administration.

Complementary information concerning the suspected interindividual transmission of GW1516, a substance prohibited in sport, through intimate contact: a case report

PurposeInadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed.MethodsTo probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography–tandem mass spectrometry. Further, drug-residue testing using patients’ ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry.ResultsIn one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete’s doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid.ConclusionsThe combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments.

Chiral analysis of ostarine in commercially available products and doping control urine samples

Various substances classified as prohibited in sport by the World Anti-Doping Agency (WADA) comprise one or more chiral centers. One such substance is enobosarm ((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2‑hydroxy-2-methylpropanamide, S-ostarine, S-22, MK-2866, GTx-024, VERU-024). Although ostarine is not an approved drug, it has been readily available via Internet-based suppliers, and to date it is unknown to what extent R-ostarine, S-ostarine, or a mixture of both is used in these products. Likewise, such information is not available with regards to urine samples that returned an adverse analytical finding in the context of sports drug testing programs and, hence, this pilot-study aimed at establishing a test method to provide first insights into the presence of R- and S-ostarine in commercially available products and in authentic doping control urine samples.Here, a chiral liquid chromatographic - mass spectrometric method, employing a teicoplanin-containing (chiral) stationary phase that allows the differentiation of R- and S-enantiomers of ostarine, is presented. Ostarine products were extracted and diluted prior to analysis, while urine samples were prepared for analysis by enzymatic hydrolysis followed by liquid-liquid extraction.Seven products declaring to contain ostarine were investigated, five of which were ordered via the Internet and two were confiscated and made available by a customs authority. Only 2 of the 5 purchased products contained ostarine. In these two products and the confiscated products, only the ostarine S-enantiomer was found. Further, five authentic doping control urine samples containing the target analyte were subjected to chiral analysis, also confirming the sole presence of the S-ostarine enantiomer.The developed approach proved suitable for the separation of ostarine enantiomers, which adds further information e.g. in the context of illicit drug production, trafficking, and doping controls, especially when additional details are desirable that might assist in determining the origin of a drug or drug residue.

Evidence of ostarine excretion in oral fluid after a single controlled oral administration

The presence of ostarine, a selective androgen receptor modulator (SARM) in an athlete’s urine specimen constitutes one of the most frequent anti-doping rules violation as the drug is listed as a member of the S1.2 class “other anabolic agents” of the World Anti-doping Agency Prohibited List, forbidden in- and out-competition. It is possible to challenge this violation but it is at the charge of the athlete to prove innocence. The conditions to evidence no fault or negligence are mostly based on 2 points: 1. the athlete must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete that the violation was not intentional.Some months before the Olympic games, a female athlete was suspended by a national anti-doping agency because of an adverse analytical finding for ostarine. She claimed that her violation was due to drug transfer when kissing her boyfriend, who did not inform her about his ostarine daily intake. To document this claim (excretion of ostarine in oral fluid in sufficient amounts), a male volunteer ingested 17.3 mg of ostarine (dose verified by 1H NMR). Oral fluid was collected over 8 h using the NeoSal™ collection device and was tested by liquid chromatography coupled to tandem mass spectrometry. Maximal ostarine concentration was 468 ng/mL at T + 15 min, which can also be partially attributed to mouth contamination. Ostarine was detectable during the whole period of test, with concentrations at 1–2 ng/mL after T + 4 h. These results support drug transfer during kissing and subsequent possible contamination of the partner.

Body fluid contamination in the context of an adverse analytical finding in doping: About a case involving ostarine

Ostarine, also known as MK-2866 or enobosarm, is a selective androgen receptor modulator (SARM). It has anabolic properties and as such is widely used in doping, accounting in 2021 for 25 % of the adverse analytical findings (AAF) among the class S1.2 “Other anabolic agents” of products banned by the World Anti-Doping Agency, to which it belongs. But in some cases, it can be responsible for an AAF following contamination. We report the case of an athlete who contaminated herself by exchanging body fluids while kissing her boyfriend, who took 25 mg per day of MK-2866 for 9 days prior to the athlete's AAF (urinary concentration evaluated at 13 ng/mL) without her knowledge. Both subjects came to our lab for hair testing. The athlete's hair was black and slightly frizzy. Six segments of 2 cm then 7 × 3 cm (33 cm) were analysed and showed increasing concentrations, from 2 pg/mg on the first segment to 17.8 pg/mg on the last segment. The boyfriend's hair, light-brown, analyzed on 4 × 2 cm, also showed increasing values, from 65 to 143 pg/mg. These gradients of concentration in the hair’s athlete and in her boyfriend were compatible with external contamination of the hair, confirmed by analysis of washing baths, pillowcases (150 pg on each), and the athlete's hairbrush (250 pg).Fingernails were also contaminated, with 21 pg/mg in the athlete and 1041 pg/mg in the boyfriend, with highly contaminated washing baths, and toenails were less contaminated, with 2 pg/mg in the athlete and 17.3 pg/mg in the boyfriend. Urine samples taken 35 days after the start of MK-2866 treatment showed a value of 3690 ng/mL in the boyfriend and 5.7 ng/mL in the athlete. After 6 days off, these concentrations were 3.3 ng/mL and 0.1 ng/mL, respectively. A controlled transfer study was carried out 12 days after discontinuation (urine concentrations returned to negative level). After administration of 17 mg (the 25 mg/mL vial having been controlled at 17 mg/mL), urine samples were taken from the boyfriend and the athlete (n = 10 for each) for more than 25 h after they had been living normally with each other (regular kissing in particular). The boyfriend's urine concentrations ranged from 681 ng/mL to 12822 ng/mL (Tmax = 8:30 hrs), and the athlete's from 0.3 ng/mL to 13 ng/mL with Tmax = 8:30 hrs, i.e. at 22:30 hrs, which corresponded exactly to the time of collection of the urine that showed AAF, with a similar concentration. The dose ingested by the athlete was estimated at 15 µg. These results demonstrate the transfer of ostarine via body fluids between two subjects, with a high risk of AAF in one athlete, as observed in our case.

Determination of higenamine in common natural spices by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Higenamine (HG) is a β2 receptor agonist and was explicitly added to the Prohibited List of the World Anti-Doping Agency in 2017. This compound is prohibited in both in- and out-of-competition athletes and falls under the category of nonthreshold substances. Because of HG presence in numerous plants, as evidenced by a growing body of research data, an exception was made for HG in the TD2017MRPL document, in which adverse analytical findings (AAFs) were not reported if the urinary HG concentration was less than 10ng/mL. In this study, a comprehensive and systematic analysis of the HG content in five batches of samples from each of the 48 natural spices selected for this investigation was conducted using UPLC-MS/MS technology. Method validation was carried out in accordance with the ICH Analytical Procedures and Methods Validation for Drugs and Biologics Guidance, and the experimental results demonstrated that the method provided appropriate sensitivity, precision, stability, linearity, and accuracy. HG was detected for the first time in Houttuynia cordata, Zingiber officinale, Cinnamomum cassia, Stevia rebaudiana, Piper nigrum, Siraitia grosuenorii, Platycodon grandiflorus, and Myristica fragrans. Furthermore, the content of HG was found to vary significantly among the different plant parts of Nelumbo nucifera, such as rhizomes, leaves, seeds, and plumules. This paper provides systematic and comprehensive data to support the safe use of spices in athletes' diets, thereby reducing the risk of food-sourced doping violations.

Elimination profile of low-dose chlortalidone and its detection in hair for doping analysis-Implication for unintentional non-therapeutic exposure.

Chlortalidone (CLT) is a thiazide-type diuretic with high affinity for the erythrocyte carbonic anhydrase. Therapeutically, it is mostly used to treat edema and hypertension due to liver cirrhosis, heart insufficiency, or renal dysfunction. Although diuretics and masking agents are prohibited by the World Anti-Doping Agency (WADA) at all times in sports, substances belonging to this category are constantly detected in athlete samples, according to WADA's annual testing figures. Within this group of structurally diverse compounds, a threshold of 20 ng/mL has been introduced for six substances solely due to their presence as contaminants in other permitted drugs because of pharmaceutical production processes. In a recent presumptive doping case with a low urinary CLT concentration, the question of unintentional doping, for example, by contaminated non-steroidal anti-inflammatory drug intake, arose. To examine this potential scenario, a co-elimination of low-dose CLT and hydrochlorothiazide (HCTA; 20 × 50 μg, 0.2 mg/day each) was conducted on five consecutive days in two volunteers. Urine samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Moreover, we examined the incorporation of CLT in scalp hair. HCTA is rapidly excreted renally in comparatively high concentrations. In contrast, the elimination of CLT is considerably slower (terminal elimination half-life extended by a factor of 12) and, consequently, much less concentrated in corresponding urine samples (45 and 53 ng/mL, respectively). Conversely, a higher hair incorporation of chlorthalidone was observed with simultaneous dosing of both. The results suggest that an unintentional intake of sub-therapeutic CLT doses due to contamination might result in an adverse analytical finding.

Prevalence of nandrolone preparations with endogenous carbon isotope ratios in Australia

AbstractNandrolone and its prohormones, including 19‐norandrost‐4‐ene‐3,17‐dione and 19‐norandrost‐4‐ene‐3β,17β‐diol, are anabolic steroids forbidden at all times in sports according to the World Anti‐Doping Code Prohibited List and its metabolite 19‐norandrosterone (19NA) is the preferred urinary target compound to identify their abuse. In recent years, an increasing number of 19NA isotope ratio mass spectrometry (IRMS) cases have arisen that, based on the initial testing procedure, were likely to result in an adverse analytical finding but were concluded negative after IRMS analysis. The current study was therefore set up to gain a better insight on the prevalence of nandrolone preparations with endogenous carbon isotope ratio values in Australia. Suitable workplace (non‐athlete) urine samples that had previously been reported positive for 19NA were identified and analysed on IRMS. A total of 82% of the samples that were analysed were reported with enriched carbon isotope ratios of 19NA (i.e., 19NA greater than −26‰). This indicates that there is a high prevalence of nandrolone‐containing anabolic androgenic steroid preparations in Australia that have ‘endogenous’ carbon isotope ratios which reduces the ability to identify exogenous nandrolone.

DOPING CONTROL IN MALE SOCCER PLAYERS IN BRAZIL: 10 YEARS OF FOLLOW-UP.

To understand the Adverse Analytical Finding (AAF) that have occurred in Brazilian soccer in a recent 10-year period, comparing them to international data, to know the Brazilian profile. A review of the AAR in the Doping Control Commission database of the Brazilian Football Association from 2008 to 2017. The AAR in professional male soccer players between 2008 and 2017 were considered. The sample selected in this research was composed of 40,092 doping tests, with 113 AAR, identified in 18 different competitions (0.28%) in the professional category, in Brazilian national and state competitions between 2008 and 2017, flagged in doping control exams through urine samples. Stimulants were detected most frequently (31.0%), followed by glucocorticoids (21.2%), diuretics, and masking agents (19.5%). The Brazilian Championship series did not show a relationship with any of the World Anti-Doping Agency (WADA) groups of substances. Series A showed 0.07% of AAR, Series B 0.21%, Series C 0.75% and Series D 1.49. The rate of AAR in Brazilian soccer was 0.28%, lower than the average for all soccer worldwide, and shows similar percentages among field positions. Stimulants were the most prevalent drugs. The national elite soccer competitions showed significantly fewer cases than the lower divisions. Level of Evidence II; Retrospective Study.

Hair and dietary supplements testing to identify contamination with roxadustat in an adverse analytical finding

Roxadustat is an oral inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylase, which increases endogenous erythropoiesis. WADA has included roxadustat and other HIF stabilizers on its list of prohibited substances. We describe here the case of an elite athlete (female, 31 years old, 168 cm and 53 kg) with an adverse analytical finding (AAF) with concentration of roxadustat in her urine at 0.289 ng/mL in the A sample and 0.529 ng/mL in the B sample (83% higher than A). A stability study was carried out, showing total stability of roxadustat at this concentration in urine exposed to light for 50 h, so photoisomerization degradation cannot explain the difference in concentration. Her urine had been completely negative in a control test carried out three days previously, while roxadustat had been shown to be present in urine for at least 20 days after administration of pharmacologically effective doses to an athlete. Hair concentration was 0.39 and 0.35 pg/mg in the segments corresponding to the presumed period of intake, with few adjacent segments also positive (0.29–0.33 pg/mg), likely explained by cosmetic treatments. Concentrations found in a patient treated with a pharmacologically active dose (between 100 and 120 mg 3 days a week) were more than 100 times higher (between 41 and 57 pg/mg). Numerous supplements and pharmaceuticals taken by the athlete were analyzed. Only collagen powder showed the presence of roxadustat, at a very low but highly variable concentration (100 pg/g-1000 pg/g). A female volunteer (58 years old, 169 cm and 65 kg), taking this powder at the same doses as the athlete (10 g of powder 5 times for 6 days) presented 7 roxadustat-positive urine samples (although lower than those observed in the athlete) out of 34 sampled over 7 days, the difference in powder sampling location, age, weight, height, pharmacokinetic parameters variability and level of sporting activity between the athlete and the volunteer probably explaining the difference in concentrations observed. All these results could be consistent with an AAF due to contamination by dietary supplements, which are becoming increasingly common due to the current exposome of athletes in our society.

SFC-MS/MS for orthogonal separation of hydroxylated 17α-methyltestosterone isomers.

Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS)methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometrymethod was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.

Annual banned-substance review 16th edition-Analytical approaches in human sports drug testing 2022/2023.

In this 16th edition of the annual banned-substance review on analytical approaches in human sports drug testing, literature on recent developments in this particular section of global anti-doping efforts that was published between October 2022 and September 2023 is summarized and discussed. Most recent additions to the continuously growing portfolio of doping control analytical approaches and investigations into analytical challenges in the context of adverse analytical findings are presented, taking into account existing as well as emerging challenges in anti-doping, with specific focus on substances and methods of doping recognized in the World Anti-Doping Agency's 2023 Prohibited List. As in previous years, focus is put particularly on new or enhanced analytical options in human doping controls, appreciating the exigence and core mission of anti-doping and, equally, the conflict arising from the opposingly trending extent of the athlete's exposome and the sensitivity of instruments nowadays commonly available in anti-doping laboratories.

Prevalence of adulteration in dietary supplements and recommendations for safe supplement practices in sport.

The prevalence of dietary supplement use among athletes continues to rise with 60-80% of athletes often reporting current or previous use of dietary supplements. While select dietary ingredients have been shown to improve acute performance and enhance training adaptations over time, it is important to still consider the risk vs. reward for athletes before opting to consume a dietary supplement. Previous work has indicated that certain dietary supplements may pose risks for inadvertent doping, may be susceptible to mislabelling, could be banned by certain governing bodies of sport, or pose health risks for certain populations. The purpose of the current narrative review is to summarize the prevalence of adulteration in dietary sport supplement products, outline the risks of inadvertent doping for athletes, and highlight best practices regarding safe supplementation strategies. Analytical studies have found anywhere from 14 to 50% of samples analyzed from dietary supplement products have tested positive for anabolic agents or other prohibited substances. It is important for the consumer to adhere to safe supplementation strategies, which include following serving size recommendations, cross-referencing ingredient profiles with the list of prohibited substances, choosing quality products that have been verified by a third-party certification program, and being cognizant of consuming multiple dietary supplement products with overlapping ingredient profiles. Once these practices have been considered, it is reasonable for an athlete to utilize dietary supplements as a strategy to optimize performance and health, with a low risk of failing a drug test (adverse analytical finding) and experiencing adverse events.

Investigation of in vitro generated metabolites of LGD-4033, a selective androgen receptor modulator, in homogenized camel liver for anti-doping applications.

The use of selective androgen receptor modulators (SARM) in sports is prohibited by the World Anti-Doping Agency (WADA) due to their potential as performance-enhancing drugs, offering an unfair advantage. LGD-4033 is a SARM known for its similarities to anabolic steroids and can be easily purchased online, leading to increased availability and misuse. Adverse analytical findings have revealed the presence of SARMs in dietary supplements. Although LGD-4033 misuse has been reported in human sports over the years, concerns also arise regarding its illicit use in animal sports, including camel racing. Although various studies have investigated the metabolism of LGD-4033 in humans, horse, and other species, there is limited research specifically dedicated to racing camels. This study focuses on the in vitro metabolism of LGD-4033 in homogenized camel liver using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to identify and characterize the metabolites. The findings indicated the presence of 12 phase I metabolites and 1 phase II metabolite. Hydroxylation was responsible for the formation of the main phase I metabolites that were identified. A glucuronic acid conjugate of the parent drug was observed in this study, but no sulfonic acid conjugate was found. The possible chemical structures of these metabolites, along with their fragmentation patterns, were identified using MS. These findings provide valuable insights into the metabolism of LGD-4033 in camels and aid in the development of effective doping control methods for the detection of SARMs in camel racing.

Detection of capromorelin in urine following oral and dermal routes of administration.

Capromorelin is a growth hormone secretagogue. Despite promising results to alleviate muscle-wasting in the elderly, it has not advanced further in human development. Subsequent studies demonstrated capromorelin's ability to increase food intake in animals, leading to approval in the United States and Europe as an appetite stimulant for cats (Elura) and dogs (Entyce). Capromorelin is prohibited in sports due to its ability to stimulate growth hormone production and enhance performance. However, given that its veterinary preparation is formulated as a highly concentrated solution (20 or 30 mg/mL) delivered orally, incidental ingestion or dermal absorption may result in an adverse analytical finding (AAF) by way of direct exposure during oral administration to a pet. An administration study was conducted by either oral or transdermal application of capromorelin solution to mimic the scenario of inadvertent exposure to the drug. Ingestion of 30 μg of capromorelin orally (equivalent to 1 μL of Entyce) resulted in detectable amounts of capromorelin in urine for up to 48 h after administration with a maximum urinary concentration of 7ng/mL. Importantly, when applied directly to the skin on the hands in larger quantities mimicking a pet administration exposure scenario (30 mg or 1mL of Entyce), capromorelin was also detected reaching a maximum urinary concentration of 0.7ng/mL. Athletes and testing authorities should be aware of the risk of an AAF arising due to incidental exposure to veterinary preparations of capromorelin. To our knowledge, before 2022, no positive test for capromorelin had ever been reported.