Do dried blood spots have the potential to support result management processes in routine sports drug testing?-Part 2: Proactive sampling for follow-up investigations concerning atypical or adverse analytical findings / Mario Thevis, Tiia Kuuranne, Andreas Thomas, Hans Geyer. - ( Drug Testing and Analysis 13 (2021) 3 (March); p. 505-509)

• PMID: 33538088
• DOI: 10.1002/dta.3011

Abstract

Capillary blood sampled as dried blood spot (DBS) has shown substantial potential as test matrix in sports drug testing in various different settings, enabling the analysis of numerous different drugs and/or their respective metabolites. In addition to established beneficial aspects of DBS specimens in general (such as the minimally invasive and non-intrusive nature, and simplified sample transport), a yet unexplored advantage of DBS in the anti-doping context could be the opportunity of preserving a source of information complementary to routine doping controls performed in urine or venous blood. Whenever follow-up investigations are warranted or required, frequently collected and stored (but yet not analyzed) DBS samples could be target-tested for the compound(s) in question, in order to contribute to results management and decision-making processes.

Keywords: adverse analytical finding; atypical finding; doping; dried blood spot; sport.

C19-steroids as androgen receptor modulators : design, discovery, and structure-activity relationship of new steroidal androgen receptor antagonists / Padma Marwah, Ashok Marwah, Henry A. Lardy, Hiroshi Miyamoto, Chawnshang Chang. - (Bioorganic & Medicinal Chemistry 14 (2006) 17 (1 September); p. 5933-5947)

• PMID: 16759873
• DOI: 10.1016/j.bmc.2006.05.022

Abstract

Dehydroepiandrosterone (DHEA), the most abundant steroid in human circulating blood, is metabolized to sex hormones and other C19-steroids. Our previous collaborative study demonstrated that androst-5-ene-3beta,17beta-diol (Adiol) and androst-4-ene-3,17-dione (Adione), metabolites of DHEA, can activate androgen receptor (AR) target genes. Adiol is maintained at a high concentration in prostate cancer tissue; even after androgen deprivation therapy and its androgen activity is not inhibited by the antiandrogens currently used to treat prostate cancer patients. We have synthesized possible metabolites of DHEA and several synthetic analogues and evaluated their role in androgen receptor transactivation to identify AR modulators. Steroids with low androgenic potential in PC-3 cell lines were evaluated for anti-dihydrotestosterone (DHT) and anti-Adiol activity. We discovered three potent antiandrogens: 3beta-acetoxyandrosta-1,5-diene-17-one 17-ethylene ketal (ADEK), androsta-1,4-diene-3,17-dione 17-ethylene ketal (OAK), and 3beta-hydroxyandrosta-5,16-diene (HAD) that antagonized the effects of DHT as well as of Adiol on the growth of LNCaP cells and on the expression of prostate-specific antigen (PSA). In vivo tests of these compounds will reveal their potential as potent antiandrogens for the treatment of prostate cancer.

Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanised liver, human urine samples and estimation of prevalence of its use in anti-doping samples / S. Kraiem, M.Y. Al-Jaber, H. Al-Mohammed, A.S. Al-Menhali, N.J. AlThani, M. Helaleh, W. Samsam, S. Touil, A. Beotra, C. Georgakopoulas, S. Bouabdallah, V. Mohamed-Ali, M. Almaadheed, - (Drug Testing and Analysis (2021) 23 March)

• PMID: 33759363
• DOI: 10.1002/dta.3032

Abstract

Ecdysteroids are of interest as potential sport performance enhancers, due to their anabolic effects. The current study aimed to analyse levels of the most abundant ecdysteroid, ecdysterone (20-hydroxyecdysone, 20-OHE) in easily available dietary supplements, and, outline an analytical strategy for its detection, and that, of its metabolites, 1) following administration of pure 20-OHE to uPA(+/+)-SCID mice with humanised liver, 2) in a human volunteer after ingestion of two supplements, one with a relatively low, and the other a high, concentration of 20-OHE, and, 3) to estimate the prevalence of use of 20-OHE in elite athletes (n=1000). Of the 16 supplements tested, only five showed detectable levels of 20-OHE, with concentrations ranging from undetectable up to 2.3 mg per capsule. Urine of uPA(+/+)-SCID urine showed the presence of 20-OHE and its metabolite, 14 deoxy ecdysterone, within 24 hours (h) of ingestion. In humans, both the parent and the metabolite were detectable within 2 to 5 h of ingestion, with the metabolite being detectable for longer than the parent. After ingestion of a low dose supplement, the parent and metabolite were detectable for 70 h and 48 h, while following the higher dose it was 96 h and 48 h, respectively. Analysis of urines from athletes (n=1000) confirmed four positives for 20-OHE, suggesting a prevalence of use of 0.4%. Prevalence of its use by elite athletes was relatively low, however, this needs to be confirmed in other populations, and with other related ecdysteroids.

iNADO Update (2021) 05 (3 May)
Institute of National Anti-Doping Organisations (iNADO)

Contents:

iNADO Community

• French Sport Organizations hold the 20th National Conference for Doping Free Sport
• A small NADO's Perspective - from the Importance of Education to Law Enforcement
• UNIL opens Facebook Group to share Anti-Doping Expertise

Bulletin Board

• iNADO Members elect new Board Members at Annual General Assembly
• Governance in Anti-Doping: How to meet the Challenges
• Capability Register of Member NADOs and RADOs is available now
• Teleconference with WADA for iNADO Members

Athlete's Voice

• Bridging the Gap: Researching Para-athlete's Experiences of Anti-Doping

People

• New Director General at NADA India
• AFLD announces new Secretary General

Science

• Doping Authority Netherlands publishes a Paper on the Prevalence of Doping Substances in Sport Nutrition Supplements
• New Detection Method developed for Prohibited Substances in Sample Analysis

Practical Development in Anti-Doping

• Restoring Trust in Sport: Corruption Cases and Solutions – newly published Book calls on WADA to clarify the Discretion available to NADOs in their Response to Sub-Elite and Recreational Level Athletes
• New EU Medical Device Regulation for Blood Sample Accessories
• ITA will oversee Anti-Doping Programmes at European Games

Feature of the Month

• Play True Day

iNADO Partners & Sponsors

• New at the Anti-Doping Knowledge Center

Related case:

ISR 2018 KNKF Decision Disciplinary Committee 2018001 T
March 26, 2018

On 26 March 2018 the ISR-KNKF Disciplinary Committee decided to impose a 4 year period of ineligibility on the Person after he tested positive for the prohibited substances Clenbuterol, Testosterone and 17α-methyl-5β-androstane-3α,17β-diol.

Hereafter in February 2021 the Dopingautoriteit has reported  that the Person had breached the imposed period of ineligibility through his participation in Germany into 2 powerlifting competitions.

After notification by the Doping Sanction Compliance Commission (CND) the Person failed to respond nor filed a statement in his defence. The CND renders a decision without a hearing based on the Parties' written submissions.

In support of the charges the Dopingautoriteit filed 2 online results lists as evidence showing that the Person had participated into the 2 powerlifting competitions in Germany in 2018 and in 2019.

Considering the evidence in this case the CND establishes that the Person through his participation into the 2 competitions in question indeed had breached the imposed period of ineligibility. Consequently a new period of ineligibility will be imposed on the Person without grounds for a reduced sanction.

Therefore the Doping Sanction Compliance Commission decides on 27 April 2021 to impose a new 4 year period of ineligibility on the Person starting on the date the current period of ineligibility shall end, i.e. on 4 January 2022.

The Person has not to pay the fees and expenses for this commission.

CAS 2020/ADD/12 Badminton World Federation v. Clement Krobakpo

In July 2020 the Disciplinary Committee of the Organizing Committee of the African Games decided to disqualify the the Nigerian badminton player Clement Krobakpo after his sample tested positive for the prohibited substances Clenbuterol, Heptaminol and Octodrine. The case was referred to the Badminton World Federation (BWF) and hereafter in August 2020 the BWF reported an anti-doping rule violation against the Athlete.

In September 2020 the BWF filed a request for Arbitration with the Anti-Doping Division of the Court of Arbitration for Sport (CAS ADD) as first-instance authority. The Sole Arbitrator renders a decision without a hearing based on the Parties' written submissions. 

The Athlete accepted the test result, admitted het violation and denied the intentional use of the substances. He could not explain the presence of the low concentration of Clenbuterol in his sample and suggested that imported contaminated meat in Nigeria had caused the positive test. Further he asserted that the energy drink Freedom Juice he had used was the source of Heptaminol and Octodrine. He had mentioned this product on the Doping Control Form while he was unaware that it contained the ingredient 2-aminoisoheptane (Octodrine). 

The BWF accepted that the energy drink Freed Juice was the source of the Heptaminol and Octodrine and that this violation was unintentional. Yet the BWF contended that the Athlete failed to establish the how the prohibited substance Clenbuterol had entered his system. 

The Sole Arbitrator concludes that the Athlete committed an anti-doping rule violation and that it is undisputed that the energy drink he had used was the source of the positive test. Further the Arbitrator holds that the Athlete only had offered speculation to explain the meat contamination and consequently that this defence must be rejected. Finally the Sole Arbitrator considers that there were substantial delays in the proceedings not attributed to the Athlete. 

Therefore the Court of Arbitration for Sport decides on 22 January 2021 that:

1. The request for arbitration filed by the Badminton World Federation on 25 September 2020 against Mr. Clement Krobakpo is upheld.
2. Mr. Clement Krobakpo committed an anti-doping rule violation in accordance with the Badminton World Federation’s Anti-Doping Rules applicable to the 2019 African Games in Rabat, Morocco.
3. Mr. Clement Krobakpo is sanctioned with a 4-year period of ineligibility starting from 16 October 2020.
4. All competitive results obtained by Mr. Clement Krobakpo from 25 August 2019 through the date of his provisional suspension are disqualified, with all resulting consequences, including forfeiture of medals, points and prizes.
5. (…).
6. (…).
7. All other motions or prayers for relief are dismissed.

CAS 2020/ADD/11 International Olympic Committee v. Vladimir Nikolov

Mr Vladimir Nikolo is a Bulgarina volleyball player at the London 2012 Olympic Games. 

In 2018, the International Olympic Committee (IOC) decided to perform further analyses on certain samples collected during the 2012 Olympic Games. These additional analyses were performed with analytical methods which were not available in 2012. 

In January 2020 the International Testing Agency (ITA), on behalf of the IOC, reported an anti-doping rule violation against the Athlete after his 2012 A and B samples tested positive for the prohibited substance Methyltestosterone. 

Hereafter in August 2020 the International Olympic Committee (IOC) filed a request for Arbitration with the Anti-Doping Division of the Court of Arbitration for Sport (CAS ADD) as first-instance authority. The Sole Arbitrator renders a decision without a hearing based on the Parties' written submissions. 

The Athlete accepted the test result, admitted the violation and denied intentional use. He was tested before without issues and could not explain how the substance had entered his system. He assumed that a contaminated supplement might have been the source of the positive test since only a very low concentration of the substance was found in his samples. 

The IOC contended that the presence of the prohibited substance had been established in the Athlete's sample and accordingly that he had committed an anti-doping rule violation. 

The Sole Arbitrator concludes that there is sufficient proof that the Athlete committed an anti-doping rule violation. Any mitigating circumstanced invoked by the Athlete in his defence, even if proven, are not relevant to establish whether an anti-doping rule violation was committed. The Sole Arbitrator deems that the Athlete may have an opportunity to explain the circumstances at a later stage of the prosecution of the anti-doping rule violation. 

Therefore the Court of Arbitration for Sport decides on 15 October 2020 that:

1. The request for arbitration filed by the International Olympic Committee on 28 August 2020 against Mr. Vladimir Nikolov is upheld.
2. Mr. Vladimir Nikolov committed an anti-doping rule violation in accordance with the International Olympic Committee’s Anti-Doping Rules applicable to the XXX Olympiad, London 2012.
3. Mr. Vladimir Nikolov shall return the Olympic diploma and pin he received on the occasion of the XXX Olympiad, London 2012.
4. (…).
5. (…).
6. All other motions or prayers for relief are dismissed.

CAS 2020/ADD/8 International Olympic Committee v. Martina Ratej

Ms Martina Ratej is a Slovenian Athlete competing in the Women’s Javelin throw event at the London 2012 Olympic Games. 

In 2018, the International Olympic Committee (IOC) decided to perform further analyses on certain samples collected during the 2012 Olympic Games. These additional analyses were performed with analytical methods which were not available in 2012. 

In January 2020 the International Testing Agency (ITA), on behalf of the IOC, reported an anti-doping rule violation against the Athlete after her 2012 sample tested positive for the prohibited substance Clostebol. 

Hereafter in May 2020 the International Olympic Committee (IOC) filed a request for Arbitration with the Anti-Doping Division of the Court of Arbitration for Sport (CAS ADD) as first-instance authority. The Sole Arbitrator renders a decision without a hearing based on the Parties' written submissions. 

The Athlete accepted the test result and denied the intentional use of the substance. She explained that she underwent treatment for a medical urgency and that the prescribed medication Trofodermin Creme, containing Clostebol, was the source of the positive test.

She asserted that she was tested before without issues and after medical treatment the Slovenian Anti-Doping Organization deemed that she did not need a TUE.  

The IOC contended that the presence of the prohibited substance had been established in the Athlete's sample and accordingly that she had committed an anti-doping rule violation. 

The Sole Arbitrator concludes that there is sufficient proof that the Athlete committed an anti-doping rule violation. Any mitigating circumstanced invoked by the Athlete in his defence, even if proven, are not relevant to establish whether an anti-doping rule violation was committed. The Sole Arbitrator deems that the Athlete may have an opportunity to explain the circumstances at a later stage of the prosecution of the anti-doping rule violation. 

Therefore the Court of Arbitration for Sport decides on 16 July 2020 that:

1. The request for arbitration filed by the International Olympic Committee on 6 May 2020 against Ms. Martina Ratej is upheld.
2. Ms. Martina Ratej committed an anti-doping rule violation in accordance with the International Olympic Committee’s Anti-Doping Rules applicable to the XXX Olympiad, London 2012.
3. The results obtained by Ms. Martina Ratej at the XXX Olympiad, London 2012 are disqualified with all resulting consequences including, if applicable, forfeiture of any medal, points and prizes.
4. (…).
5. (…).
6. All other motions or prayers for relief are dismissed.

2020/ADD/7 International Ski Federation v. Andrus Veerpalu
2020/ADD/13 International Ski Federation v. Andrus Veerpalu

During the FIS 2019 Nordic World Ski Championships in Austria from 19 February to 3 March 2019, the Austrian police raided the belongings of several athletes and athlete support personnel from Austria, Estonia and Kazakhstan on suspicion of violating Austrian anti-doping laws. Simultaneously, German police officers searched the medical practice of Dr Mark Schmidt in Erfurt, Germany. This joint police operation became publicly known as the Operation Aderlass. 

The police searched the hotel room of the Athlete Andrus Veerpalu in Seefeld during the raid and found a heavy box containing medical equipment, supplements and prohibited substances. The same hotel room was also used by the Athlete Alexey Poltoranin for prohibited blood doping treatments. 

Following the police raids in Seefeld and Erfurt, law enforcement authorities of Germany, Austria and Estonia initiated criminal investigations and proceedings against a number of athletes and support personnel.

The Austrian and German Police provided the International Ski Federation (FIS) with all the evidence and intelligence they had gathered in the context of the Operation Aderlass such as minutes of wire-taped conversations and in-person interrogations, mobile phone messages, surveillance photos and indices of confiscated items. 

As a result in September 2019 FIS openend proceedings against the Athlete Andrus Veerpalu and reported an anti-doping rule violation for Complicity and his failure to cooperate in full with the FIS investigations. After notification a provisional suspension was ordered and FIS filed a request for arbitration with the Anti-Doping Division of the Court of Arbitration for Sport (CAS ADD). 

FIS contended that the final report of the Austrian Police constitutes clear evidence that the Athlete committed anti-doping rule violations. The evidence shows that the Athlete granted access to his hotel room to Dr Mark Schmidt to perform blood transfusion on the Athlete Alexey Poltoranin thereby being an accomplice to Alexey Poltoranin’s admitted blood doping during the World Championships in Seefeld.

In addition FIS contended that there is evidence that that the Athlete purchased the prohibited product IGF-1 from one of Dr Mark Schmidt’s providers of prohibited substances. 

The Athlete disputed the jurisdiction of the CAS ADD in this case and denied that he had knowledge at all of the doping scheme admittedly organized and orchestrated by the manager of the Estonian team. He stated that he had no links to the Estonia team and worked only as a service staff member for the team of Kazakhstan.

The Athlete only acknowledged that in December 2016 he had picked-up a parcel. He was told that it contained medication for the Athlete Alexey Poltoranin and in return he gave an envelop with money to a man. 

The CAS ADD Sole Arbitrator establishes that the alteration in the disciplinary body from FIS Doping Panel to the CAS ADD in the 2019 FIS ADR being procedural, applied to the Athlete and confirms the jurisdiction of the CAS ADD to decide this matter. 

Considering the evidence in this case the Sole Arbitrator is comfortably convinced that the Athlete not only knew about but also was actively involved in allowing Dr Mark Schmidt to perform blood doping on Alexey Poltoranin by storing necessary equipment and grating access to his room. By doing so he committed an anti-doping rule violation by being intentionally complicit in the blood doping performed on Alexey Poltoranin. 

Further the Sole Arbitrator is convinced that by purchasing IGF-1 fromon of  Dr Schmidt’s providers on 4 December 2016 he intentionally aided and assisted the doping practice of an Estonian Athlete and other members of the Estonian Team. 

Therefore the Court of Arbitration for Sport decides on 17 March 2021 that:

1. The Anti-Doping Division of the Court of Arbitration for Sport has jurisdiction to decide on the subject matter of this dispute.
2. The request for arbitration filed by the International Ski Federation is admissible.
3. Andrus Veerpalu is found guilty of an anti-doping rule violation in accordance with Article 2.9 FIS ADR 2016.
4. Andrus Veerpalu is sanctioned with a 2-year period of ineligibility starting from the date of the final CAS ADD Award.
5. (…).
6. (…).
7. All other motions or prayers for relief are dismissed.

Arimistane : Degradation product or metabolite of 7-oxo-DHEA? / Dayamín Martínez Brito, Patrizia Leogrande, Cristiana Colamonici, Davide Curcio, Francesco Botre, Xavier de la Torre. - (Drug Testing and Analysis (2021) 30 March);

• PMID: 33783974
• DOI: 10.1002/dta.3036

Abstract

Rationale: The instability of androst-5-ene-3,7-dione structures under acidic conditions is known. The formation of arimistane from 7-oxo-DHEA, influenced by the conditions of sample extraction, and mainly derivatization reaction and gas chromatography (GC) injector temperature, was described earlier, potentially leading to misinterpretation of results. By using a liquid chromatography (LC)-mass spectrometry (MS) (LC-MS) we investigated the stability of the 7-oxo-DHEA in two different solvents (methanol and dimethyl sulfoxide [DMSO]), and the arimistane formation after the application common analytical procedures. Additionally, in vitro and in vivo studies of 7-oxo-DHEA were performed.

Methods: The stability of 7-oxo-DHEA was studied in solutions after 60 days storage at -20°C. In vitro studies were performed by incubating 7-oxo-DHEA with human liver microsomes (HLMs). Healthy volunteers collected urine samples before and after the administration of a single dose of 7-oxo-DHEA. Analyses were performed using high-performance LC (HPLC) coupled to a triple quadrupole mass spectrometer (MS/MS) and GC combustion isotope ratio mass spectrometry (GC-C-IRMS) following HPLC purification.

Results: 7-oxo-DHEA was stable after 60 days in DMSO while a protic solvent as methanol promotes the degradation of 7-oxo-DHEA to arimistane. HLM incubations showed no formation of arimistane and the sample preparation only influenced the degradation of 7-oxo-DHEA when solvolysis was applied. After the administration study the presence of arimistane also after the hydrolysis with β-glucuronidase (Escherichia coli) was observed while using β-glucuronidase/arylsulfatase (Helix pomatia) showed the presence of arimistane already in blank samples collected before administration.

Conclusions: Our results confirm arimistane as a valuable diagnostic marker of 7-oxo-DHEA administration, but also indicate that its formation is due to degradation processes rather than to metabolic biotransformation reactions.

Keywords: 7-oxo-DHEA; arimistane; degradation product; liquid chromatography-mass spectrometry; metabolism.