iNADO Update (2022) 11 (7 November)
Institute of National Anti-Doping Organisations (iNADO)

Contents:

iNADO Community

• iNADO Presentation Report - NADOs acting as Sport Integrity Agencies
•  Insights Report: Practices around calling Athletes within the final five minutes of the testing time slot
• Testing Process for Athletes with Disability

Bulletin Board

• iNADO Stakeholder Survey designed by OAKS Consultancy
• Working Together Towards Excellence: iNADO Board hosted Webinars to Present the 5-year Strategic Plan
• iNADO Webinar: New Way of Working
• iNADO Webinar Summary: WADA NADO EAG Elections Candidates Introductory Webinars

Practical Development in Anti-Doping

• iNADO ADAMS Working Group
• iNADO Members: Data Protection and Privacy Policy

Athlete's Voice

• Léa Krüger - Advocating for Athletes and Clean Sport
• Sharing Global Stories: Inclusion of Athletes' Voice in the Eduation Sessions of NADA Germany

People

• Tony Josiah appointed as Director of Education, Insight and Global Engagement at UKAD
• Kim Højgaard Ravn, new CEO of Anti-Doping Denmark

iNADO Partners & Sponsors

• New at the Anti-Doping Knowledge Center

Annual banned-substance review: analytical approaches in human sports drug testing / Mario Thevis, Tiia Kuuranne, Hans Geyer

• Drug Testing and Analysis 14 (2022) 11 November
• PMID: 36369629
• DOI: 10.1002/dta.3408

Contents:

• Introduction
• Anabolic Agent
  • Anabolic-androgenic steroids (AASs)
  • Initial testing procedures (ITP): Comprehensive screening, metabolism studies
  • Other anabolic agents
  • Steroid profiling in urine and blood
  • Confirmatory testing procedures – IRMS
• Peptide Hormones, Growth Factors, Related Substances, and Mimetics
  • Erythropoietin-receptor agonists
  • Growth hormone (GH), its fragments and releasing factors
• β2‐Agonists, Hormone and Metablolic Modulators, and diuretics
• Stimulants, Glucocorticoids, and Cannabinoids
• Manipulation of blood and blood components
• Chemical and Physical Manipulation and Gene Doping
• Conclusion

Abstract

Also in 2021/2022, considerable efforts were invested into advancing human sports drug testing programs, recognizing and taking into account existing as well as emerging challenges in anti-doping, especially with regard to substances and methods of doping specified in the World Anti-Doping Agency's 2022 Prohibited List. In this edition of the annual banned-substance review, literature on recent developments published between October 2021 and September 2022 is summarized and discussed. Focus is put particularly on enhanced analytical approaches and complementary testing options in human doping controls, appreciating the exigence and mission in anti-doping and, equally, the contemporary “new normal” considering, for example, the athlete's exposome versus analytical sensitivity and applicable anti-doping regulations for result interpretation and management.

Identification of equine in vitro metabolites of seven non-steroidal selective androgen receptor modulators for doping control purposes / Charlotte Cutler, Marjaana Viljanto, Polly Taylor, Pamela Hincks, Simon Biddle, Peter Van Eenoo

• Drug Testing and Analysis 14 (2022) 2 (February), p. 349-370
• PMID: 34714606
• DOI: 10.1002/dta.3189

Abstract

Selective androgen receptor modulators, SARMs, are a large class of compounds developed to provide therapeutic anabolic effects with minimal androgenic side effects. A wide range of these compounds are available to purchase online and thus provide the potential for abuse in sports. Knowledge of the metabolism of these compounds is essential to aid their detection in doping control samples. In vitro models allow a quick, cost-effective response where administration studies are yet to be carried out. In this study, the equine phase I metabolism of the non-steroidal SARMs GSK2881078, LGD-2226, LGD-3303, PF-06260414, ACP-105, RAD-140 and S-23 was investigated using equine liver microsomes. Liquid chromatography coupled to a QExactive Orbitrap mass spectrometer allowed identification of metabolites with high resolution and mass accuracy. Three metabolites were identified for both GSK2881078 and LGD-2226, four for LGD-3303 and RAD-140, five for PF-06260414, twelve for ACP-105 and ten for S-23. The equine metabolism of GSK-2881078, LGD-2226, LGD-3303 and PF-06260414 is reported for the first time. Although the equine metabolism of ACP-105, RAD-140 and S-23 has previously been reported, the results obtained in this study have been compared with published data.

Human In Vivo Metabolism and Elimination Behavior of Micro-Dosed Selective Androgen Receptor Modulator RAD140 for Doping Control Purposes / Felicitas Wagener, Luisa Euler, Christian Görgens, Sven Guddat, Mario Thevis

• Metabolites 12 (2022) 7, p. 1-13
• PMID: 35888790
• PMCID: PMC9325264
• DOI: 10.3390/metabo12070666

Abstract

RAD140 is a selective androgen receptor modulator which has been abused in sporting competitions. Its use is prohibited by the World Anti-Doping Agency (WADA) for athletes at all times. In addition to its illicit use, adverse analytical findings of RAD140 in doping control samples might result from other scenarios, e.g., the ingestion of contaminated dietary supplements. The differentiation between samples resulting from such contamination scenarios and intentional doping presents a considerable challenge, as little is known about the metabolism and elimination behavior of RAD140 in humans. In this study, six micro-dose excretion studies with five adult male volunteers each were conducted, and urine samples were analyzed by means of LC-HRMS/MS. Multiple metabolites, firstly detected in human urine, are described in this study. The sample preparation included an enzymatic hydrolysis step, which facilitated the estimation of RAD140 concentrations in urine. The elimination profiles and detection times for six metabolites as well as the intact drug are presented. The method was extensively characterized and deemed fit-for-purpose. The metabolite ratios were investigated for their predictive power in estimating the dose of RAD140 intake. The presented data will aid in better case result management in future doping cases involving RAD140.

Determination of growth hormone releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography mass spectrometry / Andreas Thomas, Sebastian Höppner, Hans Geyer, Wilhelm Schänzer, Michael Petrou, Dorota Kwiatkowska, Andrzej Pokrywka, Mario Thevis

• Analytical and Bioanalytical Chemistry 401 (2011) 2 (August), p. 507-516
• PMID: 21298258
• DOI: 10.1007/s00216-011-4702-3

Abstract

A family of small peptides has reached the focus of doping controls representing a comparably new strategy for cheating sportsmen. These growth hormone releasing peptides (GHRP) are orally active and induce an increased production of endogenous growth hormone (GH). While the established test for exogenous GH fails, the misuse of these prohibited substances remains unrecognized. The present study provides data for the efficient extraction of a variety of known drug candidates (GHRP-1, GHRP-2, GHRP-4, GHRP-5, GHRP-6, alexamorelin, ipamorelin, and hexarelin) from human urine with subsequent mass spectrometric detection after liquid chromatographic separation. The used method potentially enables the retrospective evaluation of the acquired data for unknown metabolites by means of a non-targeted approach with high-resolution/high-accuracy full-scan mass spectrometry with additional higher collision energy dissociation experiments. This is of great importance due to the currently unknown metabolism of most of the targets and, thus, the method is focused on the intact peptidic drugs. Only the already characterised major metabolite of GHRP-2 (D-Ala-D-2-naphthylAla-L-Ala, as well as its stable isotope-labelled analogue) was synthesised and implemented in the detection assay. Method validation for qualitative purpose was performed with respect to specificity, precision (<20%), intermediate precision (<20%), recovery (47-95%), limit of detection (0.2-1 ng/mL), linearity, ion suppression and stability. Two stable isotope-labelled internal standards were used (deuterium-labelled GHRP-4 and GHRP-2 metabolite). The proof-of-principle was obtained by the analysis of excretion study urine samples obtained from a single oral administration of 10 mg of GHRP-2. Here, the known metabolite was detectable over 20 h after administration while the intact drug was not observed.

Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin / Ekaterina Semenistaya, Irina Zvereva, Andreas Thomas, Mario Thevis, Grigory Krotov, Grigory Rodchenkov

• Drug Testing and Analysis 7 (2015) 10 (October), p. 919-925
• PMID: 25869809
• DOI: 10.1002/dta.1787

Abstract

Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti-Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti-doping control we have investigated metabolites of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid-phase extraction on weak cation exchange cartridges and analyzed by means of nano-liquid chromatography - high resolution mass spectrometry. Six metabolites of GHRP-1 were identified. GHRP-1 in the parent form was not detected. GHRP-1 (2-4) free acid was detected in urine up to 27 h. GHRP-2, GHRP-2 free acid and GHRP-2 (1-3) free acid were detected in urine up to 47 h after administration. GHRP-6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra-pressure liquid chromatography-tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration.

Detection by LC-MS/MS of HIF stabilizer FG-4592 used as a new doping agent : Investigation on a positive case / C. Buisson, A. Marchand, I. Bailloux, A. Lahaussois, L. Martin, A. Molina

• Journal of Pharmaceutical and Biomedical Analysis 121 (20 March 2016), p. 181-187
• PMID: 26808067
• DOI: 10.1016/j.jpba.2016.01.029

Abstract

Stabilizing the labile factor HIF (hypoxia inducible factor) for therapeutic use has led to the development of various molecules by pharmaceutical companies. These HIF stabilizers show promising erythropoiesis stimulating capacities and are of great interest for patients with chronical kidney disease and anemia. Amongst them FG-4592 from FibroGen is now under phase 3 of clinical studies. While this drug is still under investigation, a parallel market already allows to buy this product, which could be tempting for some athletes willing to increase their performances. To avoid such a use for doping purpose, WADA has listed HIF stabilizers and FG-4592 in particular as prohibited substances since 2011 and some anti-doping laboratories have developed a technique of identification of FG-4592 in urine. Here, we described the first case ever identified by an anti-doping laboratory of an athlete using FG-4592. Detection and confirmation in urinary samples was performed by LC-MS/MS. In addition, potential indirect markers erythropoietin (EPO) and hematological parameters followed in the Athlete Biological Passport (ABP) were analyzed during and after the period of use but showed no profound alterations. Only ABPS (abnormal blood profile score) reached (but did not exceed) the upper limit proposed by the ABP adaptive model just after the period of use of FG-4592. Altogether this case sends a warning for anti-doping laboratories which now must strengthen surveillance on HIF stabilizers and develop sensitive methods of detection for this new class of drugs.

lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG-4592) and its main metabolites into routine doping controls /  Daniel Eichner, Ryan M. Van Wagoner, Mitch Brenner, James Chou, Scott Leigh, Lee R. Wright, Lee A. Flippin, Michael Martinelli, Oliver Krug, Wilhelm Schänzer, Mario Thevis

• Drug Testing and Analysis 9 (2017) 11-12 (November-December), p. 1768-1778)
• Special Issue: 35th Cologne workshop: Advances in sports drug testing
• PMID: 28378453
• DOI: 10.1002/dta.2202

Abstract

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

CAS 2017/A/5357 World Anti-Doping Agency (WADA) & Fédération Internationale de Basketball (FIBA) v. Hellenic National Council for Combating Doping (ESKAN) & Olga Chatzinikolaou

• Basketball
• Doping (cocaine metabolites)
• Standard of proof to rebut the intentional committing of an anti-doping rule violation
• Balance of probability and source of prohibited substance

1. The standard of proof by which an athlete can rebut the presumption of having intentionally committed an anti-doping rule violation or establish specific facts or circumstances is on the balance of probability.

2. The standard of proof of “balance of probability” has to be understood in the way that in case a CAS panel is offered several alternative explanations for the ingestion of the prohibited substance but it is satisfied that one of them is more likely than not to have occurred, the athlete has met the required standard of proof regarding the means of ingestion of the prohibited substance. It remains irrelevant that there may also be other possibilities of ingestion, as long as they are considered by the panel to be less likely to have occurred. In other words, for the panel to be satisfied that a means of ingestion is demonstrated on a balance of probability simply means, in percentage terms, that it is satisfied that there is a 51% chance of it having occurred. The athlete thus only needs to show that one specific way of ingestion is marginally more likely than not to have occurred.

In April 2017 the Hellenic National Council for Combating Doping (ESKAN) has reported an anti-doping rule violation against the basketball player Olga Chatzinikolaou after her A and B samples tested positive for the prohibited substance Cocaine.

Nevertheless the ESKAN Disciplinary Committee accepted the Athlete's explanation that there had been passive inhalation of Cocaine out-of-competition at an afternoon dinner more than 12 hours prior to the game and the Doping Control. Accordinghly the Disciplinary Committee decided on 17 July 2017 not to impose a sanction on the Athlete.

Hereafter in October 2017 the World Anti-Doping Agency (WADA) and the International Basketball Federation (FIBA) appealed the ESKAN Decision with the Court of Arbitration for Sport (CAS).

Furthermore the Athlete's samples were transferred from the Athens Lab to the Lausanne Lab. Reanalysis of these samples revealed that the found concentration of Cocaine was not consistent with her alleged passive inhalation of Cocaine more than 12 hours prior to the game.

WADA and FIBA requested the Panel to set aside the Appealed Decision and to impose a sanction of 4 years. They contended that the Athlete had the burden to demonstrate with corroboration evidence that the violation was not intentional and to prove how the Cocaine had entered her system.

The Athlete denied the intentional use of the substance and requested for a reduced sanction. She asserted that the violation occurred out-of-competition and unrelated to sport performance.

The Sole Arbitrator determines that it was undisputed that the presence of of prohibited substance had been established in the Athlete's samples and accordingly that she committed an anti-doping rule violation.

Following assessment of the evidence the Sole Arbitrator deems finds that the Athlete failed to establish, on a balance of probabilities, her lack of intent nor the source of the Cocaine and the metabolites found in her system.

Therefore the Court of Arbitration for Sport decides on 31 May 2018 that:

1.) The appeal filed on 3 October 2017 by the World Anti-Doping Agency with the Court of Arbitration for Sport against the decision of the Hellenic National Council for Combatting Doping (ESKAN) dated 17 July 2017 is admissible.

2.) The decision of the Hellenic National Council for Combatting Doping (ESKAN) dated 17 July 2017 is set aside.

3.) Ms Olga Chatzinikolaou committed an anti-doping rule violation according to Article 3.1 of the Greek law n° 4373.

4.) Ms Olga Chatzinikolaou is sanctioned with a four (4) year period of ineligibility, starting on 31 May 2018. The period of provisional suspension served by Ms Olga Chatzinikolaou between 13 April 2017 and 17 July 2017, shall be credited against the four-year period of ineligibility to be served.

(…)

7.) All other motions or requests for relief are dismissed.

CAS 2016/A/4596 Fédération Internationale de Football Association (FIFA) v. Saudi Arabian Olympic Committee (SAOC) & Saudi Arabian Anti-Doping Committee (SAADC) & Mohammed Mohammed Noor Adam Hawsawi

• Football
• Doping (Amfetamine)
• Admissibility of new arguments presented after the hearing
• Obligation to report the concentration of a non-threshold substance
• Differing concentrations of the prohibited substance in the A and B samples
• Intent
• Out-of-competition ingestion
• Context unrelated to sport performance
• Effect of a partly respected provisional suspension on the period of ineligibility

1. Arguments that were not contained in the answer and do not appear to have been advanced in any of the proceedings before the first instance body but were submitted for the first time after the conclusion of the CAS hearing in a document which the CAS panel in charge directed should be strictly limited to responding to the arguments and expert evidence adduced at that hearing are late and inadmissible.

2. For a non-threshold substance, it is the mere presence of the substance in an athlete’s body rather than the amount or concentration of the substance, that constitutes an anti-doping violation. There is nothing in the WADA International Standard for Laboratories (ISL) or the WADA Code that requires a laboratory to specify the concentration of a non-threshold substance in an A-sample or B-sample as a precondition to recording an adverse analytical finding. Article 6 (2) and (3) of the FIFA Anti-Doping Regulations also makes it clear that, in the case of a non-specified substance such as Amfetamine, it is the mere presence of the substance in a player’s bodily sample that establishes an anti-doping violation.

3. The analysis of a B-sample is intended to confirm the presence of a prohibited substance. However this does not mean that it is either intended or expected to yield identical values as the A-sample. The WADA ISL makes clear that, in the case of a non-threshold substance, the laboratory method for analysing the B-sample is not aimed at having identical results or at gaining information on the background or quantification, but only at confirming the presence of the prohibited substance. In other terms, the ISL only requires the identification in the B-sample of the same prohibited substance that was found in the A-sample, and it does not require the chromatograms or the quantities or the ‘background noises’ to be exactly the same.

4. An athlete who is aware that the substance s/he is consuming has powerful pharmacological properties but takes no steps to verify the origin or content of the substance, purchases it from unknown vendors in unlabelled packages, with no indication of the ingredients or origin must know or have known that there is a significant risk that his/her consumption of the substance involves a significant risk of anti-doping violation.

5. It is extremely difficult, if not impossible, to infer the date of ingestion from the level of concentration in a sample without also knowing the size of the dose and further information about the individual’s metabolic rate. Therefore, the mere fact that the concentration of a substance in a sample is relatively low does not establish that it was ingested out of competition.

6. The ingestion of a prohibited substance such as Amfetamine with a view to alleviate chronic joint pain is not “unrelated to sport performance”. Professional footballers are regularly required to engage in high impact, high intensity cardiovascular exercise which places considerable physical demands upon the individual’s body and joints. The effect of a chronic joint condition is likely to be at its most acute – and to have the greatest inhibiting impact – during such periods of intense physical activity. Against this backdrop, the deliberate use of a powerful artificial stimulant to reduce or remove chronic joint pain is likely to have a significant and non-incidental effect on sport performance. Accordingly, the consumption of the substance cannot be characterised as being “unrelated to sport performance”.

7. If an athlete only respected some but not all of a provisional suspension, s/he is not entitled to receive credit for any period of the provisional suspension. An athlete’s obligation to respect a provisional suspension in order to receive credit for that period of ineligibility applies to the provisional suspension as a whole and not merely to a portion of it. It is incumbent upon the athlete, as the subject of a provisional suspension, to abide by the terms of the suspension and to exercise due care and responsibility in ascertaining what sporting activities and events fall within its scope.

In November 2015 the Saudi Arabian Anti Doping Committee (SAADC) has reported an anti-doping rule violation against the football player Mohammed Mohammed Noor Adam Hawsawi after his A and B samples tested posititive for the prohibited substance Amfetamine.

Consequently on 28 February 2016 the SAOC Hearing Panel decided to impose a 4 year period of ineligibility on the Athlete, starting on the date of the decision. Following the Athlete's appeal on 17 April 2016 the SAOC Appeal Panel decided to annul the sanction of 4 years. Instead the Appeal Panel imposed only a period of ineligibility for the time already served by the Athlete until the date of the Appeal Decision.

Hereafter the International Football Federation (FIFA) appealed the SAOC Appeal Panel Decision with the Court of Arbitration for Sport (CAS). FIFA requested the Panel to set aside the SAOC Appeal Panel Decision and to impose a 4 year period of ineligibility on the Athlete.

The Athlete denied that he had committed an anti-doping rule violation nor that he had acted intentionally. He requested the Panel to impose no period of ineligiblity or alternatively only a reduced sanction with credit for the time already served.

Also the Athlete disputed the validity of the test results and argued that the Lausanne Lab failed to specify the concentration of Amfetamine in his B-sample. He asserted that the Lab misidentified the presence of Amfetamine due to incorrect parameters were used during chemical analysis of a urine sample. Instead the Lab didn't identifiy the presence of the non-prohibited substance β-Methylphenethylamine in his samples.

Following assessment of the evidence the Panel concludes that:

• the Laboratory carried out the tests of the Athlete’s A-sample and B-sample in accordance with the WADA ISL and did not mistakenly identify a non-prohibited substance as Amfetamine; and
• the Laboratory was not required to specify the concentration of Amfetamine in the Player’s B-sample, which was irrelevant to the question whether he had committed an anti-doping violation.

In view of the Athlete's conduct the Panel concludes that the Athlete's anti-doping rule violation was intentional whereas there are a number of facts that substantially undermined the credibility of the Athlete's version of events.

The Panel deems that the Athlete failed to demonstrate that the substance was used out-of-competition and in a context unrelated to sport performance. Furthermore the Panel considers that the Athlete had participated in two matches during the period of his provisional suspension.

Therefore the Court of Arbitration for Sport decides on 1 March 2017 that:

1. The appeal filed by the Fédération Internationale de Football Association on 9 May 2016 is upheld.
2. The decision rendered by the Appeal Panel of the Saudi Arabian Olympic Association dated 17 April 2016 is set aside.
3. Mohammed Mohammed Noor Adam Hawsawi is suspended for a period of four (4) years as from 15 December 2016, with credit given for any period of suspension already served.
4. (…).
5. (…).
6. All other motions or prayers for relief are dismissed.