Metabolism of 4-hydroxyandrostenedione and 4-hydroxytestosterone: Mass spectrometric identification of urinary metabolites

5 Jan 2007

Metabolism of 4-hydroxyandrostenedione and 4-hydroxytestosterone : Mass spectrometric identification of urinary metabolites / Maxie Kohler, Maria K. Parr, Georg Opfermann, Mario Thevis, Nils Schlörer, Franz-Josef Marner, Wilhelm Schänze. - (Steroids 72 (2007) 3 (March); p. 278-286)

  • PMID: 17207827
  • DOI: 10.1016/j.steroids.2006.11.018


Abstract

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography-mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3beta-hydroxy-5alpha-androstane-4,17-dione (2) and 3alpha-hydroxy-5beta-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3xi,4xi-dihydroxy-5xi-androstan-17-one (4, 6-11) except 3alpha,4alpha-dihydroxy-5beta-androstan-17-one (5). Out of the 17beta-hydroxylated analogs 4-hydroxytestosterone (18), 3beta,17beta-dihydroxy-5alpha-androstan-4-one (19), 3alpha,17beta-dihydroxy-5beta-androstan-4-one (20), 5alpha-androstane-3beta,4beta,17beta-triol (21), 5alpha-androstane-3alpha,4beta,17beta-triol (26) and 5alpha-androstane-3alpha,4alpha,17beta-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation.

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

3 Jul 2007

Preventive doping control analysis : liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids / Costas G. Georgakopoulos, Ariadni Vonaparti, Marianna Stamou, Polyxeni Kiousi, Emmanouil Lyris, Yiannis S. Angelis, George Tsoupras, Bernhard Wuest, Michel W.F. Nielen, Irene Panderi, Michalis Koupparis. - (Rapid Communicaton in Mass Spectrometry 21 (2007) 15 (15 August); p. 2439-2446)

  • PMID: 17610244
  • DOI: 10.1002/rcm.3103


Abstract

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.

Screening for benfluorex and its major urinary metabolites in routine doping controls

30 Nov 2010

Screening for benfluorex and its major urinary metabolites in routine doping controls / Mario Thevis, Gerd Sigmund, Vassilios Gougoulidis, Simon Beuck, Nils Schlörer, Andreas Thomas, Dorota Kwiatkowska, Andrzej Pokrywka, Wilhelm Schänzer. - (Analytical and Bioanalytical Chemistry 401 (2011) 2 (August); p. 543–551)

  • PMID: 21116611
  • DOI: 10.1007/s00216-010-4455-4


Abstract

Benfluorex [1-(m-trifluoromethylphenyl)-2-(β-benzoyloxyethyl)aminopropane] has been widely used for the treatment of atherogenic metabolic disorders and impaired carbohydrate metabolism (particularly in obese type-II diabetic patients) as well as an anorectic drug. Due to its potentially performance-enhancing properties, benfluorex has been added to the list of prohibited compounds and methods of doping by the World Anti-Doping Agency (WADA) in 2010, necessitating the implementation of the drug as well as its major metabolites into routine doping control procedures. In the present study, human urinary metabolites of benfluorex were characterized by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) as well as liquid chromatography-electrospray ionization-high resolution/high accuracy tandem mass spectrometry (LC-ESI-MS/MS). Commonly employed sports drug testing approaches consisting of liquid-liquid extraction followed by GC-MS or urine dilution and immediate LC-MS/MS analysis were expanded and validated with regard to specificity, recovery (48-54%, GC-MS only), intra- and interday precision (<25%), limits of detection (5-8 ng/mL for LC-MS/MS and 80 ng/mL for GC-MS), and ion suppression (for LC-ESI-MS/MS only) to allow the detection of benfluorex metabolites 1-(m-trifluoromethylphenyl)-2-(2-hydroxyethyl)aminopropane (M1), 1-(m-trifluoromethylphenyl)-2-(2-carboxymethyl)aminopropane (M2), and 1-(m-trifluoromethylphenyl)-2-aminopropane (M3) as well as the glucuronic acid conjugate of M1.

Comprehensive plasma-screening for known and unknown substances in doping controls

19 Mar 2010

Comprehensive plasma-screening for known and unknown substances in doping controls / Andreas Thomas, Sven Guddat, Maxie Kohler, Oliver Krug, Wilhelm Schänzer, Michael Petrou, Mario Thevis. - (Rapid Communicaton in Mass Spectrometry 24 (2010) 8 (30 April); p. 1124-1132)

  • PMID: 20301105
  • DOI: 10.1002/rcm.4492


Abstract

Occasionally, doping analysis has been recognized as a competitive challenge between cheating sportsmen and the analytical capabilities of testing laboratories. Both have made immense progress during the last decades, but obviously the athletes have the questionable benefit of frequently being able to switch to new, unknown and untested compounds to enhance their performance. Thus, as analytical counteraction and for effective drug testing, a complementary approach to classical targeted methods is required in order to implement a comprehensive screening procedure for known and unknown xenobiotics. The present study provides a new analytical strategy to circumvent the targeted character of classical doping controls without losing the required sensitivity and specificity. Using 50 microL of plasma only, the method potentially identifies illicit drugs in low ng/mL concentrations. Plasma provides the biological fluid with the circulating, unmodified xenobiotics; thus the identification of unknown compounds is facilitated. After a simple protein precipitation, liquid chromatographic separation and subsequent detection by means of high resolution/high accuracy orbitrap mass spectrometry, the procedure enables the determination of numerous compounds from different classes prohibited by the World Anti-Doping Agency (WADA). A new hyphenated mass spectrometry technology was employed without precursor ion selection for higher collision energy dissociation (HCD) fragmentation experiments. Thus the mass spectra contained all the desired information to identify unknown substances retrospectively. The method was validated for 32 selected model compounds for qualitative purposes considering the parameters specificity, selectivity, limit of detection (<0.1-10 ng/mL), precision (9-28%), robustness, linearity, ion suppression and recovery (80-112%). In addition to the identification of unknown compounds, the plasma samples were simultaneously screened for known prohibited targets.

Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination - insights from metandienone metabolism

31 Jul 2012

Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination - insights from metandienone metabolism / Maria Kristina Parr, Andy Zöllner, Gregor Fußhöller, Georg Opfermann, Nils Schlörer, Mirela Zorio, Matthias Bureik, Wilhelm Schänzer. - (Toxicology Letters 213 (2012) 3 (18 September); p. 381-391)

  • PMID: 22885098
  • DOI: 10.1016/j.toxlet.2012.07.020


Abstract

The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner-Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC-MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize metandienone leading to the formation of 11β-hydroxymetandienone and 18-hydroxymetandienone. Following Wagner-Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of metandienone was confirmed by analysis of post-administration samples of metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes.

A steroidomic approach for biomarkers discovery in doping control

9 Aug 2011

A steroidomic approach for biomarkers discovery in doping control / Julien Boccard, Flavia Badoud, Elia Grata, Samia Ouertani, Mohamed Hanafi, Gérard Mazerolles, Pierre Lantéri, Jean-Luc Veuthey, Martial Saugy, Serge Rudaz. - (Forensic Science International 213 (2011) 1-3 (10 December); p. 85-94)

  • PMID: 21831550
  • DOI: 10.1016/j.forsciint.2011.07.023


Abstract

Anti-doping authorities have high expectations of the athlete steroidal passport (ASP) for anabolic-androgenic steroids misuse detection. However, it is still limited to the monitoring of known well-established compounds and might greatly benefit from the discovery of new relevant biomarkers candidates. In this context, steroidomics opens the way to the untargeted simultaneous evaluation of a high number of compounds. Analytical platforms associating the performance of ultra-high pressure liquid chromatography (UHPLC) and the high mass-resolving power of quadrupole time-of-flight (QTOF) mass spectrometers are particularly adapted for such purpose. An untargeted steroidomic approach was proposed to analyse urine samples from a clinical trial for the discovery of relevant biomarkers of testosterone undecanoate oral intake. Automatic peak detection was performed and a filter of reference steroid metabolites mass-to-charge ratio (m/z) values was applied to the raw data to ensure the selection of a subset of steroid-related features. Chemometric tools were applied for the filtering and the analysis of UHPLC-QTOF-MS(E) data. Time kinetics could be assessed with N-way projections to latent structures discriminant analysis (N-PLS-DA) and a detection window was confirmed. Orthogonal projections to latent structures discriminant analysis (O-PLS-DA) classification models were evaluated in a second step to assess the predictive power of both known metabolites and unknown compounds. A shared and unique structure plot (SUS-plot) analysis was performed to select the most promising unknown candidates and receiver operating characteristic (ROC) curves were computed to assess specificity criteria applied in routine doping control. This approach underlined the pertinence to monitor both glucuronide and sulphate steroid conjugates and include them in the athletes passport, while promising biomarkers were also highlighted.

World Athletics Russia Taskforce Report to the Council Meeting - 28 July 2021

28 Jul 2021

Russia Taskforce Report to Council Meeting of 28 July 2021 / Rune Andersen. - Monaco : World Athletics, 2021



In its report to the Council, the Russia Taskforce confirmed that the suspended Russian Federation (RusAF) was making satisfactory progress against the milestones and KPIs set out in the Reinstatement Plan and had paid the latest invoice of US$431,848 for the reinstatement costs incurred by World Athletics in the quarter ending 31 March 2021. 

The Taskforce is hopeful that, under the guidance of Acting President Irina Privalova, and with the assistance of the three international experts appointed to assist in the Reinstatement Plan, RusAF will continue to meet the remaining milestones and KPIs to satisfy all of the conditions set for its reinstatement to membership of World Athletics.

Consequently, the Council resolved that the Authorised Neutral Athlete (ANA) process would remain in effect and that the Council would consider in November whether the ANA quota of 10 athletes that applied to certain competitions in 2021 should be maintained or revised for competitions staged in 2022.

It further decided that the status of RusAF’s membership would be put on the agenda for the 53rd World Athletics Congress to be held in November, where the congress will be asked to vote on the following resolution:

Congress resolves, in exercise of its powers under Article 13.7 of the World Athletics Constitution, that the suspension of RusAF’s membership of World Athletics will continue until Council decides that all of the conditions set by Council from time to time for the revocation of RusAF’s suspension and the consequent reinstatement of RusAF’s membership have been met.

However the Taskforce warned that if RusAF stopped making satisfactory progress against the KPIs and milestones in the Reinstatement Plan, or failed to reimburse World Athletics' reinstatement costs on a timely basis in this period, it would recommend that the Council withdraw the proposed resolution to Congress, and propose instead that Congress resolves to expel RusAF from membership with immediate effect. The Taskforce trusts that this will not be necessary.

CAS OG_2020_06 World Athletics vs Alex Wilson & Antidoping Switzerland & Swiss Olympic

27 Jul 2021
  • CAS OG 20/06 World Athletics v. Alex Wilson, Swiss Anti-Doping & Swiss Olympic
  • CAS OG 20/08 WADA v. Alex Wilson, Swiss Anti-Doping & Swiss Olympic

In April 2021 Antidoping Switzerland reported an anti-doping rule violation against the Swiss Athlete Alex Wilson after his A and B samples tested positive for the prohibited substance Trenbolone.

After notification a provisional suspension was ordered which was callenged by the Athlete in May 2021. The Athlete requested the Disciplinary Chamber of Swiss Olympic to lift the provisional suspension in order to participate in the outdoor season and the Tokyo Olympic Games.

The Athlete argued that the source of the positive test was  contaminated meat he had ingested in a restaurant in Las Vegas, USA, in March 2021. On 2 July 2021 the Disciplinary Chamber of Swiss Olympic decided to lift the provisional suspension imposed on the Athlete.

Hereafter on 22 and 24 July 2021 both World Athletics and WADA appealed the decision of the Disciplinary Chamber with the CAS Ad Hoc Division. They requested the Panel to set aside the Appealed Decision and for the imposition of the provisional suspension with immediate effect.

The Athlete raised a number of challenges to jurisdiction of the CAS Ad Hoc Division. However the Panel deems that it has jurisdiction to hear the filed applications and that they are admissible.

Based on the evidence the Panel concludes that the Athlete has not satisfied it that, on the balance of probabilities, it is likely or probable that the Trenbolone came from contaminated meat in the circumstances presently described by the Athlete.

The Panel finds that the evidence available so far clearly shows that the provisional suspension imposed on the Athlete should not have been lifted by the Disciplinary Chamber. It follows that the decision of the Disciplinary Chamber of 2 July 2021 should not be reinstated and, thus, the mandatory provisional suspension shall be reinstated with immediate effect.

Therefore the CAS Ad Hoc Panel decides on 27 July 2021:

  1. The Ad Hoc Division of the Court of Arbitration for Sport has jurisdiction to entertain the Application filed by World Athletics on 22 July 2021 and by WADA on 24 July 2021.
  2. The applications filed by World Athletics and by WADA on 22 and, respectively, 24 July 2021 are upheld.
  3. The Decision rendered on 2 July 2021 by the Disciplinary Chamber of Swiss Olympic is set aside.
  4. The provisional suspension imposed on Mr Alex Wilson by Antidoping Switzerland on 28 April 2021 shall be reinstated with immediate effect.
  5. Each Party shall bear its own legal costs and other expenses incurred by this procedure.

CAS OG_2020_04 Maxim Agapitov vs IOC

24 Jul 2021

CAS OG 20/04 Maxim Agapitov v. International Olympic Committee

Mr. Maxim Agapitov is a retired weightlifter and currently the acting President of the European Weightlifting Federation and President of the Russian Weightlifting Federation. As an Athlete he was sanctioned in 1994 for committing an anti-doping rule violation, 27 years ago.

After recent revelations on widespread doping practices at the International Weightlifiting Federation (IWF) the IOC rendered in June 2021 conditions for the accreditation for IWF officials for the Tokyo 2020 Olympic Games.

In July 2021 the IOC decided to withdraw the accreditation for the Tokyo 2020 Olympic Games due to Mr. Agapitov did not meet the criteria for IWF officials as he had “a personal history linked to any anti-doping rule violation and/or sanction”.

Hereafter on 21 July 2021 Mr. Agapitov filed an application with the CAS Ad Hoc Division against the IOC with respect to the withdrawal of his accreditation for the Tokyo 2020 Olympic Games.

Mr. Agapitov requested the Panel to set aside the IOC Decision regarding the withdrawal of his accreditation, to approve his participation and to reinstate his accreditation for the Tokyo 2020 Olympic Games.

The IOC argued that there there were 3 grounds for the withdrawal of Mr. Agapitov's accreditation:

  • his 1994 anti-doping rule violation;
  • the presence of his name in the IWF independent investigation report drafted by Professor Richard McLaren;
  • the official functions of Mr. Agapitov within weightlifting federations at the Russian or European level.

The CAS ad Hoc Panel finds that the Mr. Agapitov's application is admissble, that it has jurisdiction of the present dispute whereas it shall review this matter de novo.

The Panel assessed the IOC criteria in question and concludes that the absence of any limitation in time with respect to the expression of “a personal history linked to any anti-doping rule violation and/or sanction”, 27 years is far beyond the time that could be reasonably considered as being part of a “personal history” likely to adversely affect the reputation of the sport of weightlifting at the Tokyo 2020 Olympic Games.

Furthermore, the Panel finds that the 1994 violation was committed by Mr. Agapitov during his athlete’s life while he is now exercising new functions as a sports official.

Finally, the Panel deems that Mr. Agapitov’s 1994 violation is not even relevant or related to the IWF’s governance problems and its officials’ reprehensible conducts towards doping, which have generated the issuance of the IOC’s criteria.

Quite to the contrary, Prof. McLaren, certainly the best positioned person to make any finding in this respect, has convincingly testified that Mr. Agapitov had an affirmative approach in fighting against doping and in restructuring the governance of the IWF.

As such, in the opinion of the Panel, the withdrawal of Mr. Agapitov's accreditation does not actually serve the purpose pursued by the IOC and could even be perceived as counterproductive in that respect.

Therefore the CAS Ad Hoc Panel decides on 24 July 2021:

  1. The application filed by Mr Maxim Agapitov 21 July 2021 is admissible and upheld.
  2. The decision of the IOC to withdraw the accreditation of Mr. Maxim Agapitov is set aside.
  3. The accreditation delivered to Mr. Maxim Agapitov in June 2021 for the Games of the XXXII Olympiad in Tokyo shall be reinstated in full.

iNADO Update #2021-08

2 Aug 2021

iNADO Update (2021) 08 (2 August)
Institute of National Anti-Doping Organisations (iNADO)



Contents:

iNADO Community

  • Anti-Doping Agency of Kenya launches own E-Learning Platform 
  • Few places still available for Masters in “Doping Studies and Analysis of Anti-Doping Policies”, by the UNESCO Chair
  • Annual Reports 2020

Bulletin Board

  • Luisa Parente (ABCD) becomes eighth Board Member of iNADO
  • Participate in our webinar and needs assessment: Clean Sport Education Repository
  • Save the Date – iNADO Workshop 2022
  • Best Practices in Research: Important Aspects during the Webinar

Athlete's Voice

  • A note to acknowledge your efforts and a call for Kaizen in Athlete Voice

People

  • Farewell to Nicole Sapstead, UK Anti-Doping
  • Farwell to Herman Ram, Dopingautoriteit
  • iNADO Live Chat  - Catherine Ordway

Science

  • FINCIS collaborates on a Study to identify the Anti-Doping Knowledge and educational Needs of Finnish Pharmacists
  • Report of the European Commission maps Member States´ Legislation to assist in the Fight against Doping in Sport

Practical Development in Anti-Doping

  • IPC publish Doping Control Guide for Testing Athletes in Para Sport
  • Japan Anti-Doping Agency Education Package

Feature of the Month

  • iNADO's #MembersWednesday on Twitter

iNADO Partners & Sponsors

  • New at the Anti-Doping Knowledge Center
Category
  • Legal Source
  • Education
  • Science
  • Statistics
  • History
Country & language
  • Country
  • Language
Other filters
  • ADRV
  • Legal Terms
  • Sport/IFs
  • Other organisations
  • Laboratories
  • Analytical aspects
  • Doping classes
  • Substances
  • Medical terms
  • Various
  • Version
  • Document category
  • Document type
Publication period
Origin