Confirming testosterone administration by isotope ratio mass spectrometric analysis of urinary androstanediols / Cedric H. Shackleton, Andy Phillips, Tony Chang, Ye Li. - (Steroids 62 (1997) 4 (April); p. 379-387)

• PMID: 9090799
• DOI: 10.1016/s0039-128x(96)00253-x

Abstract

A gas chromatographic combustion isotope ratio mass spectrometric (GC/C/IRMS) method was used for studying the incorporation of exogenous testosterone enanthate into excreted urinary 5 alpha- and 5 beta-androstane-3 alpha, 17 beta-diols. A multistep but straightforward work-up procedure produced a simple GC chromatogram of urinary steroid acetates composed principally of two androstanediols and pregnanediol. It is anticipated that such a method may form the basis of a doping control test for testosterone that could be used as a primary method during major sporting events or alternatively as a verification technique. Urine samples from five individuals were collected before and after administration of testosterone enanthate (250 mg). The delta 13C0/1000 value of andro-stanediols was around -26 to -28 during the baseline period and decreased to about -29 to -30 in the days following synthetic testosterone administration. One of the other major steroids in the chromatogram, pregnanediol, was utilized as the "internal standard," because its delta 13C0/1000 values did not markedly change following testosterone administration, remaining at -25 to -27. In all subjects studied, the delta 13C0/1000 values for androstanediols were reduced sufficiently over 8 days to confirm administration of synthetic testosterone. Although steroids isolated from urine of normal individuals from 12 different countries gave values between -24 and -28, this seemed not to be related to nationality or region. The most likely variable is the proportion of plants with low and high carbon 13 content in the diet. This variable is likely to be more affected by individual food preferences than broad ethnic food divisions. In this paper, we propose a ratio of delta 13C0/1000 for androstanediols to pregnanediol as a useful discriminant of testosterone misuse, a value above 1.1:1.0 being indicative of such misuse. The work-up procedure was designed for batch analysis and to use only simple techniques, rather than employ further instrumentation, such as high-performance liquid chromatography (HPLC), in purifying steroids for GC/C/IRMS.

Carbon isotope ratio (delta13C) values of urinary steroids for doping control in sport / Adam T. Cawley  1 , Graham J. Trout, Rymantas Kazlauskas, Christopher J. Howe, Adrian V. George . - (Steroids 74 (2008) 3 (March); p. 379-392)

• PMID: 19056414
• DOI: 10.1016/j.steroids.2008.11.004

Abstract

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (delta(13)C) profiling study (n=1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This delta(13)C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to delta(13)C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n=100; consisting of three countries) is also presented with comparison to delta(13)C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.

CAS 2011/A/2499 Albert Subirats v. Fédération Internationale de Natation (FINA), award of 24 August 2011

Related case:
FINA 2011 FINA vs Albert Subirats
May 7, 2011

• Aquatics
• Swimming
• Doping (Whereabouts filing failure)
• Responsibility of the athlete for making accurate and complete whereabouts filings
• Responsibility of the anti-doping organization in the notification of the filing failures

1. It is the responsibility of each swimmer registered in the FINA Registered Testing Pool to report the required whereabouts information to the FINA office. Even when the athlete chooses to delegate whereabouts filings to a third party such as a national federation, he or she remains ultimately responsible at all times for making accurate and complete whereabouts filings. In particular, the athlete must make sure that such third party effectively forwards the whereabouts information to the anti-doping organization on time.

2. The anti-doping organization is responsible for making an accurate notification to the athlete. If it decides to notify the filing failure communication to the athlete’s national federation instead of directly to the athlete, it has to make sure that the athlete receives such communication from the national federation. If the athlete does not receive the filing failure communication from the national federation, he or she may not be declared to have committed any filing failure.

On 21 June 2011 the International Swimming Federation (FINA) Doping Panel decided to impose a 1 year period of ineligibility on the Venezuelan swimmer Albert Subirats for 3 Whereabouts Filing Failures in 2010 and 2011.

Hereafter in July 2011 the Athlete appealed the FINA decision with the Court of Arbitration for Sport (CAS).

Uncontested in this case is the fact that the Athlete always sent the whereabouts information timely to the Venezuelan Swimming Federation (VSF). However the VSF failed to forward such information to FINA, neither for the first quarter of 2010, nor for the fourth quarter of 2010, nor for the first quarter of 2011.

In addition, uncontested and supported by the documents in the file, is the fact that the FINA notified all three filing failures by letters of 25 February 2010, 11 November 2010 and 2 February 2011 addressed only to the VSF.
No failure notices were ever sent by FINA to the Athlete directly. Also uncontested is the fact that VSF forwarded these communications to the Athlete for the first time on 2 February 2011, i.e. after the third violation had already occurred.

Since it is undisputed that the Athlete did not receive any filing failure notice before the third whereabouts filing failure, the Panel concludes that the existence of a second and a third violation cannot be reproached to the Athlete.

Therefore the Court of Arbitration for Sport decides on 24 August 2011:

1.) The Appeal filed by Mr Albert Subirats is upheld.

2.) The decision rendered on 21 June 2011 by the FINA Doping Panel is overturned.

3.) The second and the third filing failure for the fourth quarter of 2010 and the first quarter of 2011 are cancelled.

4.) Mr Albert Subirats’ results are fully reinstated.

5.) (…).

6.) (…).

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

The application of carbon isotope ratio mass spectrometry to doping control / Adam T. Cawley, Ulrich Flenker. - (Journal of Mass Spectrometry 43 (2008) 7 (July); p. 854-864)

• PMID: 18523972
• DOI: 10.1002/jms.1437

Abstract

The administration of synthetic steroid copies is one of the most important issues facing sports. Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require methods of analysis that allow endogenous steroids to be distinguished from their synthetic analogs in urine. The ability to measure isotope distribution at natural abundance with high accuracy and precision has increased the application of Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) to doping control in recent years. GC-C-IRMS is capable of measuring the carbon isotope ratio (delta(13)C) of urinary steroids and confirm their synthetic origin based on the abnormal (13)C content. This tutorial describes some of the complexities encountered by obtaining valid delta(13)C measurements from GC-C-IRMS and the need for careful interpretation of all relevant information concerning an individual's metabolism in order to make an informed decision with respect to a doping violation.

CAS 2009/A/1870 World Anti-Doping Agency (WADA) v. Jessica Hardy & United States Anti-Doping Agency (USADA)

Related cases:

• AAA No. 77 190 00288 08 JENF USADA vs Jessica Hardy - Interim Award
  August 1, 2008
• AAA No. 77 190 00288 08 USADA vs Jessica Hardy
  May 30, 2009
• CAS 2011_O_2422 USOC vs IOC
  October 10, 2011
  
  

• Swimming
• Doping (Clenbuterol)
• Applicable law to the merits according to the principles of tempus regit actum and lex mitior
• Conditions for third parties to participate to a CAS procedure
• Provision granting a right to a third party to participate in the CAS proceedings
• Contaminated nutritional supplements and significant fault or negligence
• CAS review of a sanction imposed by a disciplinary body
• Negligence of an athlete with respect of the nutritional supplements
• Conditions for a Claimant to obtain a declaratory judgment

1. According to the principle “tempus regit actum”, new regulations do not apply retroactively to facts that occurred prior to their entry into force, but only for the future. However, according to the “lex mitior” principle, CAS Panels have the possibility to apply those rules subsequently entered into force which are more favourable to the athlete

2. In the CAS system a third entity can participate as a party to the arbitration proceedings already pending among other subjects in two situations, joinder or intervention, but subject to a common condition: that it agrees in writing to such participation or that it is bound by the same arbitration agreement binding the original parties to the dispute. The common condition is not satisfied if the third party has not agreed to participate in the arbitration, notwithstanding the opportunity to intervene granted by the Panel and if the same party is not bound by the same arbitration agreement binding the original parties to the dispute.

3. Under the FINA Rules, the IOC is given the right to appeal to CAS any decision rendered under the FINA Rules “where the decision may have an effect in relation to the Olympic Games, including decisions affecting eligibility for the Olympic Games”. Such provision grants a right of appeal to a subject that was not a party to the proceedings leading to the decision appealed against. Such provision, however, even if granting a right, does not create an obligation for the IOC to participate in the CAS proceedings: this obligation can rest only upon the IOC’s consent or a rule binding it – and the FINA Rules do not bind the IOC.

4. The fact that an adverse analytical finding is the result of the use of a contaminated nutritional supplement does not imply per se that the athlete’s negligence was “significant”. An athlete can avoid the risks associated with nutritional supplements by simply not taking them; but the use of a nutritional supplement “purchased from a source with no connection to prohibited substances, where the athlete exercised care in not taking other nutritional supplements” and the circumstances are “truly exceptional”, can give rise to “ordinary” fault or negligence and do not raise to the level of “significant” fault or negligence.

5. According to the CAS case law, the measure of the sanction imposed by a disciplinary body in the exercise of the discretion allowed by the relevant rules can be reviewed only when the sanction is evidently and grossly disproportionate to the offence.

6. An athlete’s behaviour is negligent if the Adverse Analytical Finding occurs years after that the risks connected to the use of nutritional supplements had first become known to athletes, since much information has been given and stringent warnings have been issued in this respect.

7. Swiss law subjects to stringent conditions the possibility for a Claimant to obtain from a Swiss court a declaratory judgment. Such conditions are also relevant in these arbitration proceedings, since Swiss law applies subsidiarily and Swiss law also defines the powers of adjudication of arbitration bodies having their seat in Switzerland. The prerequisites for a declaratory judgment are – in principle – threefold. The party seeking declaratory relief must show a legal interest to do so. The latter presupposes that the declaratory judgment is necessary to resolve a legal uncertainty that threatens the Claimant. A legal interest is missing if a declaratory judgment is insufficient or falls short of protecting the Claimant’s interests. Furthermore, the legal uncertainty must relate to the existence or non existence of a claim or a defined legal relationship between the parties to the dispute. Finally, there must be a certain urgency to resolve the uncertainty in order to protect the respective party’s right, i.e. there must be an immediate interest for solving the uncertainty now.

In July 2008 the United States Anti-Doping Agency (USADA) has reported an anti-doping rule violation against the Athlete Jessica Hardy after her A and B samples tested positive for the prohibitied substance Clenbuterol.

Following an Interim Award of 1 August 2008 the North American Court of Arbitration for Sport Panel (AAA) considered the Athlete's negligence with the use of contaminated supplements and decided in a Final Award on 30 May 2009 to impose a 1 year period of ineligibility on the Athlete.

Hereafter in June 2009 WADA appealed this AAA Panel Final Award of 30 May 2009 with the Court of Arbitration for Sport (CAS). In this matter both WADA and FINA withdrew in June 2009 their other filed appeals (CAS 2009/A/1852 & CAS 2009/A/1853) against the previous issued AAA Interim Award of 1 August 2008.

The CAS Panel notes that the AAA Final Award is challenged in this arbitration under several perspectives: both parties, in fact, dispute the measure of the sanction imposed on Hardy. On one side, the WADA submited that the AAA Panel wrongly held Hardy to be entitled to a reduction in the sanction under FINA DC 10.5.2 and therefore requested that a second year of suspension be imposed on Hardy.

On the other side, the Athlete argued that the sanction imposed on her, taking into account the IOC Rules, is excessive, and therefore requested that the Panel either declares that the IOC Rule is inapplicable or reduces the sanction to six months.

The Panel finds that the measure of the sanction cannot be determined taking into account the IOC Rules. In particular, the Panel finds that its approach does not prevent the Athlete Hardy from getting judicial relief and does, therefore, not amount to a denial of access to justice.

The sanction of the ineligibility for one year is proportionate to the kind of misconduct and level of negligence found with the Athlete. The prior body, i.e. the AAA Panel, therefore, was correct in imposing it on Hardy.

Therefore on 21 May 2010 the Court of Arbitration for Sport decides:

1.) The appeal filed by the World Anti-Doping Agency against the AAA Final Award issued on 30 May 2009 is dismissed.

2.) (…)

3.) (…)

4.) All other prayers for relief are dismissed.

Detecting growth hormone abuse in athletes / Richard I.G. Holt. - (Analytical and Bioanalytical Chemistry 401 (2011) 2 (August); p. 449-462).

• PMID: 21590497.
• DOI: 10.1007/s00216-011-5068-2

Abstract

It is believed that athletes have been abusing growth hormone (GH) for its anabolic and lipolytic effects since the early 1980s, at least a decade before endocrinologists began to treat adults with GH deficiency. There is an on-going debate about whether GH is performance enhancing. Although many of the early studies were negative, more recent studies suggest that GH improves strength and sprint capacity, particularly when it is combined with anabolic steroids. Although use of GH is banned by the World Anti-Doping Agency (WADA), its detection remains challenging. Two approaches have been developed to detect GH abuse. The first is based on measurement of pituitary GH isoforms; after injection of recombinant human GH, which comprises solely the 22-kDa isoform, endogenous production is down-regulated leading to an increase in the 22-kDa isoform relative to other isoforms. The second is based on measurement of markers of GH action. Insulin-like growth factor-I (IGF-I) and N-terminal pro-peptide of type III collagen (P-III-NP) increase in response to GH administration in a dose-dependent manner. When combined with discriminant function analysis, use of these markers differentiates between individuals taking GH and placebo. Subsequent studies have shown that the test is applicable across different ethnicities and is unaffected by injury. WADA regulations state that when analytes are measured by immunoassay, two assays are needed. Final validation of the marker test is currently being undertaken with modern commercially available immunoassays to finalise the threshold values to be used to determine whether a doping offence has been committed.

TAS 2000/A/270 David Meca-Medina & Igor Majcen v/FINA

Related cases:

• CAS 1999/A/234 David Meca-Medina vs FINA
• TAS 99/A/234 David Meca-Medina vs FINA
• CAS 1999/A/235 Igor Majcen vs FINA
• TAS 99/A/235 Igor Majcen vs FINA

David Meca-Medina (hereafter Appellant 1) is affiliated with the Spanish Swimming Federation, member of FINA, the International Federation governing swimming which is domiciled in Switzerland. Igor Majcen (hereafter Appellant 2) is affiliated with the Slovenian Swimming Federation, also a member of FINA.

The Appellants were each suspended for four years ("the decision") by the FINA doping panel on 8th August 1999, because they had tested positive for metabolites of Nandrolone, specifically norandrosterone ("NA"), as a result of doping control in a competition test conducted on January 31, 1999, after both Appellants took part in a long distance World Cup race in Salvador di Bahia in Brazil finishing first and second respectively.

On August 8, 1999, the FINA Doping Panel issued two separate decisions (“the decisions”) concerning Appellant 1, on the one hand and Appellant 2, on the other hand. In both cases, the Appellants were sentenced to a four-year ban.
By determinations [TAS 99/A/234 & TAS 99/A/235] dated 29 February 2000, the Court of Arbitration for Sport rejected the original appeal by both Appellants against the decisions (“the original award”).

In this Appeal the Court of Arbitration for Sport decides on 1 May 2001:

1.) The appeal of each Appellant is allowed to the extent of substituting a sentence of two years suspension for one of four years (such period to take account of any period already served, but to ignore the period from 20 April 2000 to date in which the Appellant's, pursuant to the arbitration agreement, have been free to compete).

2.) The present award is rendered without costs.

3.) Each party shall bear its own costs

High-sensitivity chemiluminescence immunoassays for detection of growth hormone doping in sports / Martin Bidlingmaier, Jennifer Suhr, Andrea Ernst, Zida Wu, Alexandra Keller, Christian J. Strasburger, Andreas Bergmann. - (Clinical Chemistry 55 (2009) 3 (March); p. 445-453)

• PMID: 19168559.
• DOI: 10.1373/clinchem.2008.112458

Abstract

Background:
Recombinant human growth hormone (rhGH) is abused in sports, but adequate routine doping tests are lacking. Analysis of serum hGH isoform composition has been shown to be effective in detecting rhGH doping. We developed and validated selective immunoassays for isoform analysis with potential utility for screening and confirmation in doping tests.

Methods:
Monoclonal antibodies with preference for pituitary hGH (phGH) or rhGH were used to establish 2 pairs of sandwich-type chemiluminescence assays with differential recognition of rhGH (recA and recB) and phGH (pitA and pitB). We analyzed specimens from volunteers before and after administration of rhGH and calculated ratios between the respective rec- and pit-assay results.

Results:
Functional sensitivities were <0.05 microg/L, with intra- and interassay imprecision < or =8.4% and < or =13.7%, respectively. In 2 independent cohorts of healthy subjects, rec/pit ratios (median range) were 0.84 (0.09-1.32)/0.81 (0.27-1.21) (recA/pitA) and 0.68 (0.08-1.20)/0.80 (0.25-1.36) (recB/pitB), with no sex difference. In 20 recreational athletes, ratios (median SD) increased after a single injection of rhGH, reaching 350% (73%) (recA/pitA) and 400% (93%) (recB/pitB) of baseline ratios. At a moderate dose (0.033 mg/kg), mean recA/pitA and recB/pitB ratios remained significantly increased for 18 h (men) and 26 h (women). After high-dose rhGH (0.083 mg/kg), mean rec/pit ratios remained increased for 32 h (recA/pitA) and 34 h (recB/pitB) in men and were still increased after 36 h in women.

Conclusions:
Using sensitive chemiluminescence assays with preferential recognition of phGH or rhGH, detection of a single injection of rhGH was possible for up to 36 h.

• CAS 1999/A/234 David Meca-Medina vs FINA
• CAS 1999/A/235 Igor Majcen vs FINA
• TAS 1999/A/234 David Meca-Medina v/ FINA
• TAS 1990/A/235 Igor Majcen v/ FINA

Related case:
CAS 2000/A/270 David Meca-Medina & Igor Majcen vs FINA
May 1, 2001

On 8 August 1999 the FINA Doping Panel decided to impose a 4 year period of ineligibility on the Spanish swimmer David Meca-Medina and the Slovenian swimmer Igor Majcen after their A and B samples tested positive for the prohibited substance 19-norandrosterone (Nandrolone).

Hereafter in August 1999 both Athletes appealed the FINA decision with the Court of Arbitration for Sport (CAS).

The Athletes argued that there were departures of the standards for testing and laboratories; the substance in question was not prohibited; the positive tests were caused by the ingestion of pork offal; and the imposed sanction was too severe.

The Panel rejects the arguments that it was not their urine which was analysed and finds that the duration of the transport to the laborary had no effect on the validity of the test. The Panel holds that the chain of custody including the documentation was complete and satisfactory.

Further the Panel finds that the Athletes failed to establish that any alleged departure from the procedure could lead to genuine doubt on the reliability of any finding. They also failed to demonstrate that the positive tests was the result of the ingestion of meat injected with Nandrolone.

The Panel establishes that the substances in question were prohibited as part of the “related substances” before they were expressly included in the newer lists of anabolic androgenic steroids.

The Court of Arbitration for Sport decides on 29 February 2000 with respect to David Meca-Medina:

1.) The appeal is rejected as far as it is filed on behalf of David Meca-Medina.
2.) The suspension of David Meca-Medina is confirmed for a duration of 4 years from August 20, 1999 under deduction of 77 days of provisional suspension (May 14, 1999 to July 30, 1999).
3.) All results achieved by David Meca-Medina between January 31, 1999 and August 19, 1999 shall be cancelled.
4.) The award is pronounced without costs, except for the Court Office fee of CHF 500,- which shall be kept by the CAS (art. R65.2 of the Code)
5.) David Meca-Medina is ordered to pay an amount of CHF 4’000,- (together with interest at 5% from the date of the decision) to Respondent FINA as a contribution towards its legal fees and expenses (art. R65.3 of the Code).

The Court of Arbitration for Sport decides on 29 February 2000 with respect to Igor Majcen:

1.) The appeal is rejected as far as it is filed on behalf of Igor Majcen.
2.) The suspension of Igor Majcen is confirmed for a duration of 4 years from August 20, 1999 under deduction of 77 days of provisional suspension (May 14, 1999 to July 30, 1999).
3.) All results achieved by David Meca-Medina between January 31, 1999 and August 19, 1999 shall be cancelled.
4.) The award is pronounced without costs, except for the Court Office fee of CHF 500,- which shall be kept by the CAS (art. R65.2 of the Code)
5.) Igor Majcen is ordered to pay an amount of CHF 4’000,- (together with interest at 5% from the date of the decision) to Respondent FINA as a contribution towards its legal fees and expenses (art. R65.3 of the Code).

A robust test for growth hormone doping : present status and future prospects / Anne E Nelson. Ken K Ho. - (Asian journal of andrology 10 (2008) 3 (May); p. 416-425).

• PMID: 18385903.
• DOI: 10.1111/j.1745-7262.2008.00395.x

Abstract

Although doping with growth hormone (GH) is banned, there is anecdotal evidence that it is widely abused. GH is reportedly used often in combination with anabolic steroids at high doses for several months. Development of a robust test for GH has been challenging because recombinant human 22 kDa (22K) GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22K and other GH isoforms. Administration of 22K GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method has been implemented; however, its utility is limited because of the short window of opportunity of detection. The second approach, which will extend the window of opportunity of detection, is based on the detection of increased levels of circulating GH-responsive proteins, such as insulin-like growth factor (IGF) axis and collagen peptides. Age and gender are the major determinants of variability for IGF-I and the collagen markers; therefore, a test based on these markers must take age into account for men and women. Extensive data is now available that validates the GH-responsive marker approach and implementation is now largely dependent on establishing an assured supply of standardized assays. Future directions will include more widespread implementation of both approaches by the World Anti-Doping Agency, possible use of other platforms for measurement and an athlete's passport to establish individual reference levels for biological parameters such as GH-responsive markers. Novel approaches include gene expression and proteomic profiling.