World Anti-Doping Agency Research Articles

Whispers in the Blood: Leveraging MicroRNAs for Unveiling Autologous Blood Doping in Athletes.

The prevalence of autologous blood transfusions (ABTs) presents a formidable challenge in maintaining fair competition in sports, as it significantly enhances hemoglobin mass and oxygen capacity. In recognizing ABT as a prohibited form of doping, the World Anti-Doping Agency (WADA) mandates stringent detection methodologies. While current methods effectively identify homologous erythrocyte transfusions, a critical gap persists in detecting autologous transfusions. The gold standard practice of longitudinally monitoring hematological markers exhibits promise but is encumbered by limitations. Despite its potential, instances of blood doping often go undetected due to the absence of definitive verification processes. Moreover, some cases remain unpenalized due to conservative athlete-sanctioning approaches. This gap underscores the imperative need for a more reliable and comprehensive detection method capable of unequivocally differentiating autologous transfusions, addressing the challenges faced in accurately identifying such prohibited practices. The development of an advanced detection methodology is crucial to uphold the integrity of anti-doping measures, effectively identifying and penalizing instances of autologous blood transfusion. This, in turn, safeguards the fairness and equality essential to competitive sports. Our review tackles this critical gap by harnessing the potential of microRNAs in ABT doping detection. We aim to summarize alterations in the total microRNA profiles of erythrocyte concentrates during storage and explore the viability of observing these changes post-transfusion. This innovative approach opens avenues for anti-doping technologies and commercialization, positioning it as a cornerstone in the ongoing fight against doping in sports and beyond. The significance of developing a robust detection method cannot be overstated, as it ensures the credibility of anti-doping efforts and promotes a level playing field for all athletes.

Competence in Sports Pharmacy among Pharmacy Students in Norway.

Pharmacists are competent to promote the proper use of medicines. According to the International Pharmaceutical Federation, pharmacists must develop competence in sports pharmacy and the contents of the World Anti-Doping Agency code. This explorative study aimed to identify the status of sports pharmacy in pharmacy education in Norway and competence in sports pharmacy among Norwegian pharmacy students. The study curricula of pharmacy education were examined for the content of sports pharmacy. An online questionnaire was also developed and distributed among pharmacy students. The anonymous survey collected demographic information and data on competence in sports pharmacy. Data from 122 participants were analyzed. Only 22.5% of pharmacy students had acquired a form of training in sports pharmacy and 91.7% wished to gain higher competence. In total, 40.2% of respondents were uncomfortable in advising athletes and trainers on medication use in sports. Study year was found to correlate with competence level with a significant difference between the 3rd year (bachelor) and 5th year (master) students. Age, institution, and number of years engaged in sports were not associated with competence level. The inclusion of sports pharmacy in pharmacy programs holds practical relevance for enhancing competency levels. This implementation can be realized through the integration of sports pharmacy modules and/or the incorporation of research-based activities.

Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay.

Hypoxia-inducible factor (HIF) stabilizers are listed in the World Anti-Doping Agency's prohibited list as they can increase aerobic exercise capacity. The rapid pace of emergence of highly structurally diverse HIF stabilizers could pose a risk to conventional structure-based methods in doping control to detect new investigational drugs. Therefore, we developed a strategy that is capable of detecting the presence of any HIF stabilizer, irrespective of its structure, by detecting biological activity. Previously developed cell-based HIF1/2 assays were optimized to a stable format and evaluated for their screening potential toward HIF stabilizers. Improved pharmacological characterization was established by the stable cell-based formats, and broad specificity was demonstrated by pharmacologically characterizing a diverse set of HIF stabilizers (including enarodustat, IOX2, IOX4, MK-8617, JNJ-42041935). The methodological (in solvent) limit of detection of the optimal HIF1 stable bioassay toward detecting the reference compound roxadustat was 100 nM, increasing to 50-100 ng/mL (corresponding to 617-1233 nM in-well) in matching urine samples, owing to strong matrix effects. In a practical context, a urinary limit of detection of 1.15 μg/mL (95% detection rate) was determined, confirming the matrix-dependent detectability of roxadustat in urine. Pending optimization of a universal sample preparation strategy and/or a methodology to correct for the matrix effects, this untargeted approach may serve as a complementing method in antidoping control, as theoretically, it would be capable of detecting any unknown substance with HIF stabilizing activity.

Carbon isotope ratios of phenethylamine and its urinary metabolite phenylacetylglutamine.

Phenethylamine (PEA) is a naturally occurring trace amine that acts as a modulator in the central nervous system. It is widely sold as a dietary supplement and advertised for its mood enhancing effects and should support weight loss. It is prohibited in sports and itemized as a stimulant on the Prohibited List issued by the World Anti-Doping Agency (WADA). After oral administration of PEA, its urinary concentration is found only slightly elevated while metabolites of PEA show a significant increase. Besides 2-(2-hydroxyphenyl)acetamide sulfate, especially phenylacetylglutamine (PAG) was found at significantly elevated urinary concentrations after the administration. Due to large inter- and intra-individual variations in urinary concentrations of all metabolites, establishing a concentration or concentration ratio-based threshold remained complicated to unambiguously identify post-administration samples. In accordance with the approach employed in detecting testosterone misuse, the applicability of isotope ratio mass spectrometry to differentiate between endogenously elevated concentrations and PEA administrations was investigated. A method encompassing solid-phase extraction combined with acetylation and high-performance liquid chromatography (HPLC)-based clean-up was developed and validated for PEA. The more abundant metabolite PAG was purified by a direct injection approach on the HPLC and could be analyzed without the need for derivatization. Both methods were validated considering applicable WADA regulations. A reference population encompassing n = 57 samples was investigated to establish population-based thresholds considering the carbon isotope ratios (CIRs) found at natural abundance for PAG. The derived threshold was tested for its applicability by re-analysis of numerous post-administration samples encompassing single- and multi-dose trials.

QuEChERS as alternative extraction procedure in doping analyses.

QuEChERS is a dispersive solid phase extraction commonly applied in food analysis for residues, such as pesticides or mycotoxins for more than 20 years. Due to the quick and easy sample preparation procedure, a QuEChERS method based on ammonium acetate combined with formic acid in acetonitrile was tested for the preparation of urine samples for doping control purposes. Testing urine samples with different pH and specific gravity, using the combination of 10M ammonium acetate with 3% formic acid in acetonitrile, 312 out of 342 tested compounds could be extracted at their respective minimum required performance levels according to the World Anti-Doping Agency (WADA) technical documents. For nine selected analytes representing six categories of WADA's Prohibited List, we validated the QuEChERS extraction method fulfilling WADA's requirements for a confirmation procedure of the nonthreshold substances investigated. Especially for the intact stanozolol-glucuronides analyzed by high-resolution mass spectrometry, the described extraction method might be an alternative for confirmation procedures as it is time- and cost-saving compared with the commonly applied solid phase extraction.

Critical Legal Reading of World Anti-Doping Agency’s Gene Doping Guidance

The genetic barrier negatively affected competitive athletic performance until the advent of gene therapy and genetic manipulation, which cast doubt on and impacted the legitimacy of sporting events. At that a critical point, the World Anti-Doping Agency (WADA) interfered in such experimentation and application with a serious attempt to curb the problem and set things back on track with the healthiest standards in light of bioethics. However, the major problem that the agency has encountered and which this legal study wants to raise, and address is the legal consequences that result from the lack of a reliable method that provides sufficient evidence and definitive answers to confirm whether cellular and gene doping are occurring or not. This is study aims to demonstrate that the procedures for accusing players of using genetic modification and genetic change techniques are incorrect and insufficient and may be harmful. The suspicion must be interpreted in favour of the accused (i.e., the athlete) in accordance with the general principles of penal codification. However, some of the Agency's procedures are not compatible with the provisions of international treaties, not to mention they conflict with the Punitive legislation of numerous countries.

The Uses of Anabolic Androgenic Steroids Among Athletes; Its Positive and Negative Aspects- A Literature Review.

The use of anabolic androgenic steroids (AAS) for strength training and muscle building is a widespread practice among athletes and young individuals. Athletes and bodybuilders are using these substances for various purposes, such as enhancing muscle mass, strengthening their bodies, and enhancing their performances. AAS exert a wide range of physiological effects that result in the activation of central signaling, resulting in adverse effects. Moreover, excessive use of AAS which can be categorized as AAS abuse; is linked to biological and psychological pathologies, which can lead to mortality. Complications arising from steroid abuse involve both cellular and physiological complications. Cellular complications arise when activation of signaling proteins like mTOR, Akt, etc. leads to alteration in protein synthesis pathways, cell cycle, oxidative stress, and apoptosis, contributing to damage at the cellular level. Physiological complications are evident with cardiovascular pathologies, including an altered lipid profile, cardiac hypertrophy, hypogonadism after discontinuation of AAS, and modulation of GABA receptors in the brain, all contributed by the androgen receptor signaling. Clinical complications budding from these altered physiological processes lead to clinical effects like testicular dysfunction, acne, gynecomastia, and neuropsychiatric disorders. Despite potential therapeutic benefits, AAS use is prohibited by the World Anti-Doping Agency (WADA) due to concerns over adverse health effects. This review highlights the molecular mechanisms, physiological processes, and clinical complications arising from the excessive use of AAS among athletes.

Age-Markers on the Red Blood Cell Surface and Erythrocyte Microparticles may Constitute a Multi-parametric Strategy for Detection of Autologous Blood Transfusion

BackgroundAutologous blood transfusion is one of the illicit strategies, banned by the World Anti-Doping Agency, to increase the levels of hemoglobin, with a consequent improvement in the delivery of oxygen to tissues. At present, this practice is detectable exclusively by the individual, longitudinal monitoring of hematological biomarkers, as in the hematological module of the Athlete Biological Passport; but this indirect approach may suffer from different confounding factors. We are presenting a multi-parametric, analytical strategy to detect autologous blood transfusions by targeting the modification of the red blood cells during storage. We focused on the assessment of “storage lesions”, targeting (i) membrane proteins: Glycophorin-A and Band 3 complex, (ii) biomarkers of oxidative stress: Peroxiredoxin-2, (iii) biomarkers of senescence: CD47 and Phosphatidylserine, (iv) erythrocytes microparticles.ResultsAll of the above markers were monitored, by immunological and flow cytofluorimetric methods, on samples of stored whole blood collected at different time intervals, and on fresh blood samples, collected for official doping control tests, mixed “ex vivo” to simulate an autotransfusion. Although anonymized before the delivery to the laboratory, it was possible to mix samples belonging to the same subject based on the “athlete biological passport” code. Our results showed that the irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the “aging” of the erythrocytes during storage are closely related to: (i) the reduced concentration, on the erythrocyte membrane, of Band 3 protein (decrease of 19% and of 39% after 20 and 40 days of storage respectively) and of glycophorin A (− 47% and − 63% respectively); (ii) the externalization of phosphatidyl serine (with a five-fold increase after 20 days and a further 2× increase after 40 days); (iii) the reduced concentration of CD47; and (iv) increased levels of erythrocyte microparticles.ConclusionsThe most promising method to detect the presence of transfused blood in whole blood samples can be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena.

Testing for clomiphene in keratinous matrices using LC–MS/MS in doping purpose: Is a single intake of clomiphene detectable in hair and nail clippings?

Clomiphene is a selective estrogen receptor modulator. It is indicated for the treatment of female infertility issues but in sport, it can be misused to stimulate endogenous testosterone secretion in men. Therefore, it has been prohibited at all times by the World Anti-doping Agency. The aim of this study was to get data to be able to interpret concentrations in athletes. A healthy volunteer (male, 62 years-old) ingested a single therapeutic dose of clomiphene (Clomid™, 50 mg). Strands of hair (blond, 4 cm) were collected one month after the ingestion. Body hair (beard, axillary, pubic and chest hair), and finger and toenails were collected over 4–5 months.A previous method was modified to identify and quantify clomiphene in keratinous matrices. 30 mg of specimen were sonicated and incubated in 1 mL of methanol, in presence of 200 pg of clomiphene-D5 (internal standard). After centrifugation and evaporation of the organic phase, the samples were analyzed using LC-MS/MS. Linearity was verified in hair and nail clippings between 1 and 500 pg/mg. The limits of detection and quantification were determined at 0.3 and 1 pg/mg respectively.The study demonstrated that clomiphene tested positive in all the analyzed specimens at 9 pg/mg in head hair, from 28 to 486 pg/mg (body hair) and from 4 to 57 pg/mg (nails).Clomiphene was identified for the first time in multiple keratinous matrices. This study demonstrated that a single oral therapeutic dose is detectable in keratinous matrices over a long period of time.

Anti-Doping Policy, Health, and Harm

ABSTRACT The anti-doping policies of the World Anti-Doping Agency (WADA) aim to promote a level playing field and protect the health of the athlete. Anti-doping policy discourages research using performance enhancing drugs (PEDs) or methods and prohibits athlete support personnel, including healthcare providers, from providing advice, assistance, or aid to an athlete or others seeking to use, or using PEDs until harm has occurred. Athletes are individually responsible for the presence of a prohibited substance in their bodies and face sanction regardless of knowledge or intent. I will argue (i) that WADA policies fail to meet the epistemic and control conditions needed to adequately assign individual responsibility and (ii) that transferring responsibility for use of PEDs solely to the athlete results in harm. WADA is drifting perilously close to the morally impermissible ‘doing of harm’ to athletes by initiating or sustaining a causal sequence of events that can result in harm.

Detection of anabolic agents including selective androgen receptor modulators in samples outside of sport.

Selective androgen receptor modulators (SARMs) are prohibited by the World Anti-Doping Agency (WADA) since 2008. Similarly, to anabolic androgenic steroids (AAS), SARMs are detrimental to health not only in athletes but also in the general population. However, studies of the occurrence of SARMs outside of sport are scarce. Swedish healthcare samples from the Drugs of Abuse Laboratory at Karolinska were analyzed using WADA-accredited screening methods at the Doping Control Laboratory in Stockholm to estimate the frequency of SARM use outside of the WADA laboratories. Twenty (4%) of the male urine samples (n = 542) were positive for SARMs, whereas none of the analyzed female samples (n = 100) contained any SARMs. The top three SARMs found were LGD-4033 followed by RAD140 and ostarine. Two or more SARMs were found in >50% of the SARM-positive samples. AASs were identified in 40% of samples containing SARMs. A difference between genders was observed where 34% male and 7% female samples contained AAS. Many samples displayed testosterone/epitestosterone values indicative of testosterone intake, without presence of other AAS, and hence, there is a risk that these samples are being falsely reported as negative. Our results indicate that SARM use might be a concern outside of sport. Subsequently, in addition to AAS, the healthcare system should also be informed about SARM abuse and the associated adverse side effects.

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.

Some historical aspects for introduction of the doping statistics

The relevance of the investigation consists in the interest in the doping development during holding competitions in different historical times. Efforts, aspiration and intention to win somebody in something (mostly in speed, strength and agility) led to searching, production, usage and spread of stimulant drugs for their using in competitive matches to raise the image and status in the social environment. Aim of the research consists in the introduction and implementation of statistics of usage by sportsmen of stimulant drugs in the history of competitive matches of different ranks. Results of the research. Doping history developed alongside with the history of competitive matches. The athletes' attempts to outdo their rivals caused the search of external means and their utilisation for the sportsmen' own bodily use to get the possibility making superhuman efforts for achieving the set goal. Over time pharmacology interfered to the world gaming culture. Production and spreading of stimulant drugs of physical capacity for their own financial enrichment were the main aim of some pharmaceutical companies. In the modern sports - and game history more than 200 sportsmen passed away because of doping, more sportsmen were discqualified from participation in contests for two years, and after repeated use they got lifelong disqualification. For fight against stimulant drugs five groups of forbidden drugs and also forbidden methods were distinguished by the World anti-doping agency. Conclusions. Big game cannot do without stimulants. Many specialists say, that in sports training still there are unused reserves which enable sportsmen show record results. But functional abilities of the organism have a certain limit, which is difficult to jump over without doping.

The association between subjective anti-doping knowledge and objective knowledge among Japanese university athletes: a cross-sectional study.

This study aimed to assess the association between subjective anti-doping knowledge (subjective ADK) and objective anti-doping knowledge (objective ADK) among Japanese university athletes, framed within the context of the Theory of Planned Behavior (TPB). Eligible participants were 486 university athletes [320 men (65.8%), 166 women; mean age of 18.9 ± 1.0 years]. The participants categorized themselves in terms of the quality of their anti-doping knowledge. This assessment resulted in an independent variable coded as "(1) substantial lack of adequate knowledge," "(2) some lack of adequate knowledge," "(3) fair amount of knowledge" or "(4) good amount of knowledge." Objective ADK was assessed using the Athlete Learning Program about Health and Anti-Doping (ALPHA) test, a set of questions derived from the ALPHA-a former World Anti-Doping Agency e-learning program. The test comprises 12 questions (four choices each; passing index: ≧10 points or 80% correct answer rate). ANCOVA was conducted using subjective ADK as an independent variable and ALPHA scores as a dependent variable, adjusting for confounding factors (anti-doping experience). The ALPHA corrected answer rate across subjective ADK levels for the group were 73.10% for "(1) substantial lack of adequate knowledge," 71.97% for "(2) some lack of adequate knowledge," 75.18% for "(3) fair amount of knowledge" and 72.86% for "(4) good amount of knowledge." Comparison between different levels of subjective ADK revealed no significant differences in ALPHA score considering the main effects or any of their interactions. The present results revealed that Japanese university athletes' subjective ADK did not match their objective ADK. In the context of the TPB, there may be limitations in the perceived behavioral control in anti-doping knowledge. Even if athletes view doping as a wrongful act and have formed attitudes and subjective norms to comply with the rules, the results suggest that errors may occur in the composition of behavioral intentions due to a lack of knowledge. This could lead to the possibility of facing the risk of unintentional anti-doping rule violations. It highlights the need for targeted educational interventions to align subjective ADK of athletes with their objective ADK.

Computational intelligence spectrophotometric scenarios for screening and quantification of single-dose triple therapy banned by the World Anti-Doping Agency in some sports

Efficient, accurate, and cost-effective spectrophotometric scenarios via innovative methods have been established, validated, and confirmed for the synchronous determination andanalyzingof the single-dose triple anti-hypertensive therapy of Atenolol (ATN), Amiloride hydrochloride (AMI) and Hydrochlorothiazide (HCT). This therapy is explicitly prohibited in various sports by the World Anti-Doping Agency (WADA) due to unfair advantage for dishonest athletes during athletic competition, it has performance-enhancing effect as well as it can lessen the detection of some doping drugs. Novel intelligence spectrophotometric solutions based on coupling of data analysis and spectral manipulation including three methods, namely, ratio conversion (RC), derivative ratio transformation (DDT) and advanced derivative ratio transformation (ADDT). Linearity ranges were 25.0–255.0 µg/mL, 2.0–23.0 µg/mL and 2.0–24.0 µg/mL for ATN, AMI and HCT, respectively. All suggested approaches were validated and confirmed in compliance with ICH guidelines, and the results were satisfactory and within approved bounds. The findings obtained using the suggested procedures and those obtained using the officially and reported methods were statistically compared, and there was no discernible difference. Evaluation of the greenness of the indicated methods was performed and compared with the reported HPLC method via utilizing the Analytical greenness metric tool [AGREE] whereas it confirmed that the applied methods are environmentally beneficial. The accomplished results together with the easiness, accessibility, low-cost and affordability of the indicated methods supported their suitability for routine quality control investigation of authentic materials, pharmaceutical preparations in addition their efficacy in cited drugs’ screening and quantification in spiked human urine.

Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing.

The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti-Doping Agency anti-doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)-based sample authentication with a multi-target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA-profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE-product detection by MALDI-TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8ml) and DBS (20 μl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high-throughput format with an overall assay turnaround time of approximately 11 h. SNP-based DNA typing via MALDI-TOF MS thus represents a high throughput-capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings.

Investigation of the content of anabolic steroids in food supplements used in sports practice

The goal of this study is to establish the presence or absence of anabolic androgenic steroids in food supplements implemented in sports practice, to assess the health risk from intake of food supplements with quality and content discrepancies in conflict with the recommendations. The authors analysed 23 samples of food supplements by using liquid chromatographic methods. The presence/absence of substances with steroid structure was determined by considering the resulting chromatograms and using reference substances. The analysis of the resulting data showed that 11 out of 23 samples of food supplements did not contain anabolic steroid substances, while different amounts of androgenic steroid substances were identified in the other 12 samples. The results from the analyses confirmed the hypothesis formulated by the authors for possible presence of undeclared steroid ingredients in food supplements, and enabled the following conclusion: more than half of the tested food supplements contained undeclared anabolic steroids, all banned by the World Anti-Doping Agency, and their number in one of the samples could be more than one. The authors also revealed that the information on the product label and package concealed the undeclared ingredients of the food supplement and claimed for high efficacy and safety. Highlights The findings of the analyses with chromatographic methods confirmed the hypothesis set by the authors for the possible presence of undeclared steroid ingredients in food supplements (FS) and point to the following highlights: More than half of the tested FS (52.2%) contained undeclared anabolic steroids and their number in one of the samples could be more than one (sample 16 contained 9 steroid substances). The label and product package information conceal the undeclared ingredients of the food supplement and claim high efficacy and safety. All anabolic steroids detected in the composition of the tested FS have been banned by the World Anti-Doping Agency (WADA).

Effectiveness of blood steroidal passport markers for detecting testosterone abuse in Asians.

The World Anti-Doping Agency (WADA) has introduced an Athlete Biological Passport (ABP) with a steroidal module, which is intended for the monitoring of longitudinal profiles of an athlete's steroid variables in urine to identify endogenous anabolic androgenic steroids that are administered exogenously. It has been in use since 2014. The prevalence of UGT2B17 gene deletion with relatively low levels of testosterone (T) glucuronide in urine is high in the Asian region. There are cases in which urinary T is below the detection limit in specific urine samples, for example, diluted urine, urine collected from females, or urine collected from UGT2B17 deletion individuals. Additional steroid markers T, 4-androstenedione (A4), and the T/A4 ratio in serum were newly added to the ABP steroidal module by WADA in 2023 to compensate for the urine steroid profile. In this study, populations of blood steroid markers in Asians (n = 510) were investigated and classified according to UGT2B17 polymorphism to confirm the effectiveness of blood steroid markers in monitoring Asian athletes. No significant difference in the T/A4 ratio was observed between the genotypes. Furthermore, an administration study of T enanthate in females (n = 10) who were classified according to UGT2B17 genotypes was performed. The concentration of T and the T/A4 ratio were found to be significantly increased in all post-administration samples until 15 days after administration (p < 0.01). The overall results supported the high effectiveness of subject-based monitoring for serum T and T/A4 ratio for recently identified shortcomings in the detection of T abuse in Asians.

In silico and in vitro human metabolism of IOX2, a performance-enhancing doping agent

IOX2 is a potent inhibitor of prolyl hydroxylase 2, a key enzyme in the regulation of hypoxia-inducible factor (HIF) and oxygen homeostasis. As such, it can be used to enhance athletic performance and is currently banned by the World Anti-Doping Agency (WADA). Detection of metabolites is critical to demonstrate drug use in doping. However, there is currently little data on IOX2 human metabolism. Our aim was to identify relevant biomarkers of IOX2 use in humans. For this purpose, IOX2 was incubated with 10-donor-pooled human hepatocytes for 3 h, incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC–HRMS/MS), and LC–HRMS/MS data were screened with Compound Discoverer (Thermo Scientific) for a comprehensive identification of IOX2 metabolites. Additionally, IOX2 human metabolites were predicted with GLORYx open-access software (University of Hamburg, Germany) to assist in the LC–HRMS/MS analysis and data mining. Thirteen metabolites were identified, oxidation at the quinolinyl group, O-glucuronidation, and combinations being predominant biotransformations. The results were consistent with previous animal studies and a single case of oral microdose administration. We suggest hydroxyquinolinyl-IOX2 as major biomarker of IOX2 use in biological samples, glucuronide hydrolysis being critical to increase IOX2 and hydroxyquinolinyl-IOX2 detectability in urine.

Cationic exchange SPE combined with triple quadrupole UHPLC-MS/MS for detection of GHRHs in urine samples

The use of growth hormone-releasing hormones (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Considering the complexity of urine samples and the low concentrations at which these analytes should be detected, analyzing GHRHs is a challenging task. In most of the studies, GHRHs are analyzed using UHPLC-HRMS with an orbitrap. The present developed and validated method for some GHRHs (tesamorelin, CJC-1295, sermorelin (GRF 1–29), sermorelin (3–29)-NH2, somatorelin) is based on the triple quadrupole UHPLC/MS-MS method with solid phase extraction (SPE) with weak cation exchange and is able to detect concentrations as low as 0.2 ng/mL (LOD), a limit of quantification (LOQ) at 0.6 ng/mL, and linearity across the range of 0.1 ng/mL to 1.2 ng/mL. The present method developed by our doping control laboratory was validated according to WADA technical documents for selectivity, limit of detection (LOD), carryover, reliability of detection, stability and recovery. The results show that the method has adequate recoveries and sensitivity, hence, it can be employed for routine screening in anti-doping laboratories.