WADA ... Olympics ...

[Michael Scally MD]

Cawley AT, Blakey K, Waller CC, McLeod MD, Boyd S, et al. Detection and metabolic investigations of a novel designer steroid: 3-chloro-17α-methyl-5α-androstan-17β-ol. Drug Testing and Analysis. http://onlinelibrary.wiley.com/doi/10.1002/dta.1832/abstract

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid-related compounds.

Subsequent gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α- and 3β- isomers of the novel compound; 3-chloro-17α-methyl-5α-androstan-17β-ol.

Synthesis of authentic reference materials followed by comparison of NMR, GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) data confirmed the finding of a new ‘designer’ steroid.

Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid.

Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α-chloro-17α-methyl-5α-androstan-17β-ol using equine and human S9 liver fractions were performed.

For equine, GC-MS/MS analysis identified the diagnostic 3α-chloro-17α-methyl-5α-androstane-16α,17β-diol metabolite. For human, the 17α-methyl-5α-androstane-3α,17β-diol metabolite was found.

Results from these studies were used to verify the ability of GC-MS/MS precursor-ion scanning techniques to support untargeted detection strategies for designer steroids in anti-doping analyses.

 

[Michael Scally MD]

de Hon O, Kuipers H, van Bottenburg M. Prevalence of doping use in elite sports: a review of numbers and methods. Sports Med. 2015;45(1):57-69. http://link.springer.com/article/10.1007/s40279-014-0247-x

The prevalence of doping in elite sports is relevant for all those involved in sports, particularly for evaluating anti-doping policy measures. Remarkably, few scientific articles have addressed this subject so far, and the last review dates back to 1997. As a consequence, the true prevalence of doping in elite sports is unknown.

Even though it is virtually impossible to uncover the exact prevalence of a prohibited activity such as doping, various methods are available to uncover parts of this particular problem, which enables the circumvention (to a certain degree) of the issues of truthfulness, definition problems and the limits of pharmacological evidence.

This review outlines the various methods that exist and presents the scarce data available in this area. It is concluded that a combination of questionnaires using the Randomised Response Technique and models of biological parameters is able to provide the statistical possibilities to reveal accurate estimates of this often undisclosed practice.

Data gathered in this way yield an estimation of 14-39% of current adult elite athletes who intentionally used doping. These period prevalences have been found in specific sub-groups of elite athletes, and the available data suggest that the prevalence of doping is considerably different between sub-groups with varying types of sport, levels and nationalities.

The above-mentioned figure of 14-39% is likely to be a more accurate reflection of the prevalence of intentional doping in elite sports than that provided by doping control test results (estimate of doping: 1-2% annually) or questionnaire-based research (estimations between 1 and 70% depending on sport, level and exact definitions of intent and doping).

In the future, analytical science may play a more important role in this topic if it may become feasible to detect very low concentrations of prohibited substances in sewage systems downstream of major sporting events. However, it is clear that current doping control test results show a distinct underestimation of true doping prevalence.

It does not seem feasible to distil better estimates of the prevalence of doping based on performance indicators or ego documents because of the various existing effects that influence athletic performance.

Such information can only be used as extra information to augment the accuracy of prevalence rates that have been found by using other techniques. True doping prevalence studies have been scarce in elite sports so far.

With the correct application of the available scientific methods, preferably using harmonised definitions of the terms 'doping' and 'elite sports', more information on this topic may be gathered in a relatively short time.

This would assist anti-doping professionals in the future in order to evaluate the effects of possible anti-doping measures, and better anti-doping policies would serve athletes who compete without doping.

The existing anti-doping measures seriously impact the lives of elite athletes and their immediate entourage, which imposes a moral burden to evaluate these measures in the best possible way.

 

[Michael Scally MD]

Strahm E, Mullen JE, Garevik N, Ericsson M, Schulze JJ, et al. Dose-dependent testosterone sensitivity of the steroidal passport and GC-C-IRMS analysis in relation to the UGT2B17 deletion polymorphism. Drug Test Anal. http://onlinelibrary.wiley.com/doi/10.1002/dta.1841/abstract

The newly implemented Steroid Module of the Athlete Biological Passport has improved doping tests for steroids. A biomarker included in this passport is the urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratio, a ratio greatly affected by a deletion polymorphism in UGT2B17. Suspect urine doping tests are further analyzed with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the origin of the androgen.

In this study, we investigated the sensitivity of the steroidal module and the IRMS analysis, in subjects administered with three doses of testosterone enanthate (500, 250, and 125 mg), in relation to the UGT2B17 polymorphism.

All subjects carrying the UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4.

On the other hand, using the athlete biological passport and IRMS analysis, all three doses could be detected to a high degree of sensitivity. The concentrations of all steroids included in the steroidal module were dose dependently increased, except for epitestosterone which decreased independent of dose. The decrease in epitestosterone was significantly associated with circulatory levels of testosterone post dose (rs =0.60 and p=0.007).

In conclusion, these results demonstrate that administration of a single dose of 125-500 mg testosterone enanthate could be detected using the athlete biological passport, together with IRMS. Since IRMS is sensitive to testosterone doping independent of UGT2B17 genotype, also very small changes in the steroidal passport should be investigated with IRMS.

 

[Michael Scally MD]

Leaked IAAF doping files reveal 'extraordinary extent of cheating'
http://www.bbc.com/sport/0/athletics/33749208

The Sunday Times and German broadcaster ARD/WRD have obtained access to the results of 12,000 blood tests from 5,000 athletes.

According to the newspaper, the evidence - which has been seen by the BBC - reveals the "extraordinary extent of cheating" by athletes at the world's biggest events.

The investigation used two of the world's "foremost anti-doping experts", scientists Robin Parisotto and Michael Ashenden, to review the data. The files belong to world governing body the International Association of Athletics Federations (IAAF), but have been leaked by a whistleblower.

According to the experts, the database reveals:

· A third of medals (146, including 55 golds) in endurance events at the Olympics and World Championships between 2001 and 2012 were won by athletes who have recorded suspicious tests. It is claimed none of these athletes have been stripped of their medals.

· More than 800 athletes - one in seven of those named in the files - have recorded blood tests described by one of the experts as "highly suggestive of doping or at the very least abnormal".

· A top UK athlete is among seven Britons with suspicious blood scores.

· British athletes - including Olympic champion heptathlete Jessica Ennis-Hill - have lost out in major events to competitors who were under suspicion.

· Ten medals at London 2012 were won by athletes who have dubious test results.

· In some finals, every athlete in the three medal positions had recorded a suspicious blood test.

· Russia emerges as "the blood testing epicentre of the world" with more than 80% of the country's medals won by suspicious athletes, while Kenya had 18 medals won by suspicious athletes.

· Stars such as Britain's Mo Farah and Jamaican sprinter Usain Bolt recorded no abnormal results.

· Athletes are increasingly using blood transfusions and EPO micro-doses to boost the red cell count.

 

[Michael Scally MD]

Speed Bumps: Why It’s So Hard to Catch Cheaters in Track and Field
http://www.propublica.org/article/speed-bumps-why-its-so-hard-to-catch-cheaters-in-track-and-field

A cache of leaked blood tests showed hundreds of track athletes have recorded results “suggestive of doping.”

With the 2015 world track championships about to start, a look at why anti-doping tests are so ineffective. Here’s a look at why drug tests will never snare every cheater.

 

[Michael Scally MD]

Usain Bolt vs. Justin Gatlin: 100 meters for the soul of track?
http://www.si.com/more-sports/2015/08/20/2015-iaaf-world-championships-usain-bolt-justin-gatlin#

On Sunday evening in Beijing (Sunday morning in the U.S.), Usain Bolt and Justin Gatlin will contest the 100 meters at the world championships (assuming each man survives the preliminary heats without injury or uncharacteristically slow running).

But Sunday’s race (which you can watch, delayed, on NBC later that afternoon, or dig up on a live morning feed somewhere in the corners of the Internet) is fundamentally different from almost any showdown in the history of the sport.

It is, on a basic level, the fastest man in history against the fastest man of the year.

But it’s much more: In a sport plagued more than ever by revelations of widespread PED use, it is being cast as a race for the future of track and field.

If Bolt wins, it is a triumph of the clean athlete and a repudiation of the doping culture, proof that greatness is possible without pharmaceutical assistance.

If Gatlin wins, it’s cheating rewarded with money and medals, a trying period in the sport’s history made ever more trying.

Joy vs. sadness.

Good vs. evil.

Light vs. darkness.

 

[ergomaniac]

Leaked IAAF doping files reveal 'extraordinary extent of cheating'
http://www.bbc.com/sport/0/athletics/33749208

The Sunday Times and German broadcaster ARD/WRD have obtained access to the results of 12,000 blood tests from 5,000 athletes.

According to the newspaper, the evidence - which has been seen by the BBC - reveals the "extraordinary extent of cheating" by athletes at the world's biggest events.

The investigation used two of the world's "foremost anti-doping experts", scientists Robin Parisotto and Michael Ashenden, to review the data. The files belong to world governing body the International Association of Athletics Federations (IAAF), but have been leaked by a whistleblower.

According to the experts, the database reveals:

· A third of medals (146, including 55 golds) in endurance events at the Olympics and World Championships between 2001 and 2012 were won by athletes who have recorded suspicious tests. It is claimed none of these athletes have been stripped of their medals.

· More than 800 athletes - one in seven of those named in the files - have recorded blood tests described by one of the experts as "highly suggestive of doping or at the very least abnormal".

· A top UK athlete is among seven Britons with suspicious blood scores.

· British athletes - including Olympic champion heptathlete Jessica Ennis-Hill - have lost out in major events to competitors who were under suspicion.

· Ten medals at London 2012 were won by athletes who have dubious test results.

· In some finals, every athlete in the three medal positions had recorded a suspicious blood test.

· Russia emerges as "the blood testing epicentre of the world" with more than 80% of the country's medals won by suspicious athletes, while Kenya had 18 medals won by suspicious athletes.

· Stars such as Britain's Mo Farah and Jamaican sprinter Usain Bolt recorded no abnormal results.

· Athletes are increasingly using blood transfusions and EPO micro-doses to boost the red cell count.

wada considering banning countries for doping
http://reuters.com/article/2015/08/18/us-athletics-doping-wada-idUSKCN0QN28Z20150818

the world anti doping association (wada) is considering a blanket ban on countries whose athletes regularly dope in the wake of a series of damaging blows for the sport in recent days, according to its president craig reedie.
he told cnn in an interview that such a deterrent could be "a pretty blunt instrument" in the war on drugs but he was waiting on a report from his independent commission before deciding whether to push forward with the strategy.
the world athletics' governing body (iaaf) has come under fire for not following up suspicious test results from more than 800 athletes between 2001 and 2012, based on an investigation by britains sunday times and german broadcaster ard/wrd.

 

[ergomaniac]

Cawley AT, Blakey K, Waller CC, McLeod MD, Boyd S, et al. Detection and metabolic investigations of a novel designer steroid: 3-chloro-17α-methyl-5α-androstan-17β-ol. Drug Testing and Analysis. http://onlinelibrary.wiley.com/doi/10.1002/dta.1832/abstract

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid-related compounds.

Subsequent gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α- and 3β- isomers of the novel compound; 3-chloro-17α-methyl-5α-androstan-17β-ol.

Synthesis of authentic reference materials followed by comparison of NMR, GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) data confirmed the finding of a new ‘designer’ steroid.

Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid.

Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α-chloro-17α-methyl-5α-androstan-17β-ol using equine and human S9 liver fractions were performed.

For equine, GC-MS/MS analysis identified the diagnostic 3α-chloro-17α-methyl-5α-androstane-16α,17β-diol metabolite. For human, the 17α-methyl-5α-androstane-3α,17β-diol metabolite was found.

Results from these studies were used to verify the ability of GC-MS/MS precursor-ion scanning techniques to support untargeted detection strategies for designer steroids in anti-doping analyses.

ergo-log.com/doping-hunters-stumble-across-a-completely-new-anabolic-steroid-3-chloro-17-alpha-methyl-5-alpha-androstan-17-beta-ol.html

"we haven't been able to find any information on 3-chloro-17-alpha-methyl-5-alpha-androstan-17-beta-ol in any scientific journals, textbooks, or patents. we would seem therefore to be looking at a completely new anabolic steroids. which means we also have no idea about the safety of this component.
we haven't been able to trace any sales points for the new steroid on the web. that means that 3-chloro-17-alpha-methyl-5-alpha-androstan-17-beta-ol isn't being marketed as a designer supplement and is therefore within reach of everyone who can afford to pay twenty bucks or so. that would suggest that 3-chloro-17-alpha-methyl-5-alpha-androstan-17-beta-ol is pretty exclusive, and that its perhaps being produced for athletes who have to take doping tests.
current doping tests don't pick up the new steroid. the Australians, research will make it possible to alter the tests so they can do so."

 

[Michael Scally MD]

Russian Athletes Part of State-Sponsored Doping Program, Report Finds
http://www.nytimes.com/2015/11/10/s...te-sponsored-doping-program-report-finds.html

GENEVA — Top Russian athletes, including Olympians and winners of prestigious events like the Chicago marathon, have for years participated in a systematic doping program that involved some of Russia’s sports officials, the World Anti-Doping Agency said on Monday.

The agency released a lengthy report here that described a pervasive doping culture among Russia’s sports programs, evoking notorious drug regimes like the state-run doping system of East Germany. The report recommended that Russia be suspended from competition.
https://wada-main-prod.s3.amazonaws.com/resources/files/wada_independent_commission_report_1_en.pdf

The report implicated athletes, coaches, trainers, doctors and various Russian institutions, including the country’s anti-doping agency and an accredited laboratory in Moscow that handled testing for the 2014 Winter Olympics in Sochi.

...

 

[Michael Scally MD]

Drugs Pervade Sport in Russia, World Anti-Doping Agency Report Finds
http://www.nytimes.com/2015/11/10/s...te-sponsored-doping-program-report-finds.html

 

[Michael Scally MD]

The Latest: Mutko says head of Moscow lab resigns
http://bigstory.ap.org/article/7fe77c20178e4a7d9a1d91ab292a3193/latest-putin-spokesman-doping-charges-appear-unfounded

 

[Michael Scally MD]

Kremlin denies damning claims of widespread doping scandal in Russia
http://mashable.com/2015/11/10/russia-doping-scandal-denial/#7CIUfMJ2pqqz

 

[tenpoundsleft]

Side comment - weird that something as innocuous as DHEA is banned by so many sports organizations. Don't many question whether that does anything? I'm taking 25mg daily, but that's just because my doc says so. Organized sports, the more you learn about them, the dumber they appear.

 

[Michael Scally MD]

I.O.C. Wants New Global Antidoping Body by 2018
http://www.nytimes.com/2015/12/11/sports/olympics/ioc-proposes-new-global-antidoping-body.html

The International Olympic Committee’s executive board recommended Thursday that a restructured antidoping system with independent oversight be created in time for the Winter Olympics in 2018.

Such a system would take the responsibility for testing and results management out of the hands of sports organizations, including international federations, and place it firmly under the leadership of the World Anti-Doping Agency, the I.O.C. said.

In a statement released after meetings in Lausanne, Switzerland, the I.O.C.’s executive board called on sports organizations to transfer their doping control operations to the new umbrella organization and to give it financing that, at least initially, would equal what they were spending on existing antidoping efforts.

Olympic leaders recommended an independent drug-testing entity at a meeting in October. WADA is forming a working group to examine the proposal and is to report to the WADA board in May.

...

 

[Michael Scally MD]

Teenage British Junior Champion Admits EPO Doping
http://www.nytimes.com/reuters/2015/12/11/sports/cycling/11reuters-cycling-england-evans.html

LONDON — Fears that the use of performance-enhancing drugs in amateur cycling are on the rise increased with a confession by a British junior national champion to having used the banned blood booster EPO.

Eighteen year-old Gabriel Evans said in a statement on a time-trialling website and an interview with Cycling Weekly magazine that he had been tempted to dope after losing the national 25-mile time trial title in August and having watched a BBC documentary on doping.

"A lot of it was curiosity. On 3 August 2015 I bought EPO for the first time," he wrote.

"On 11 August 2015 I traveled to France for a week's training camp with the family of a then-team mate.

"With me I brought one vial of EPO. This was found by the teammate’s father who presented evidence to UK Anti-Doping.

"UKAD contacted me shortly after to arrange a deposition, in which I promptly admitted to all wrongdoing."

Evans won the national junior 10-mile time trial in September but has subsequently been stripped of the title and dismissed by two amateur clubs, London Dynamo and Catford CC Equipe/Banks.

…

 

[Michael Scally MD]

Impact of UGT2B17 Gene Deletion on the Steroid Profile

Testosterone is mainly excreted as a glucuronide conjugate after its metabolism by uridine diphosphate (UDP) and glucuronosyl transferase (UGT). UGT2B7, UGT2B15, and UGT2B17 are known to be the main glucuronidation catalysts of androgens and their metabolites in humans.

Testosterone is mainly conjugated by UGT2B17 and to a lesser extent by UGT2B15. The main androgen substrate of UGT2B15 is androstane-3α 17β-diol. The actions of UGT2B17 are 96% in common with those of UGT2B15.

The enzyme UGT2B7 also has the capacity to conjugate epitestosterone, while testosterone is a poor substrate for this enzyme.

It has been established that a deletion polymorphism in the gene that codes for UGT2B17 correlates highly with testosterone levels in urine.

Thus, subjects lacking this gene have been found to show a T/E ratio lower than 0.4

Martin-Escudero P, Munoz-Guerra J, Del Prado N, et al. Impact of UGT2B17 gene deletion on the steroid profile of an athlete. Physiol Rep 2015;3(12). Impact of UGT2B17 gene deletion on the steroid profile of an athlete - Martín-Escudero - 2015 - Physiological Reports - Wiley Online Library

The measurement of the testosterone to epitestosterone ratio (T/E ratio) in urine is often used as a marker for testosterone administration in the doping control field. This study examines the frequencies of the different expression forms of the UGT2B17 gene, and assesses their effects on this marker in volunteer subjects.

The sample for this descriptive study was composed of male and female athletes aged between 16 and 55 years old who practiced different sports disciplines. All participants underwent a sports-medical physical examination, and subsequently provided 10 urine samples consecutively over a period of 48 h. The dependent variable examined was T/E and the main independent variable was the UGT2B17 gene polymorphism.

During 1 year, 1410 urine samples were obtained from 141 athletes. The frequencies of the three genotypes were as follows: wt homozygotes (ins/ins) 48.2% (n = 68), mutant homozygotes (del/del) 12.1% (n = 17), and heterozygotes (ins/del) 39.7% (n = 56). Genotype distributions varied significantly (P < 0.001) according to ethnicity, 80% of Asian subjects being homozygous for the gene deletion (del/del) compared to 6.9% of Caucasian subjects.

A multivariate analysis adjusted for genotype, age, sex, and sports discipline revealed that athletes with the del/del polymorphism showed a significantly lower mean T/E than heterozygotes (ins/del). In contrast, homozygous athletes for the gene insertion (ins/ins) showed higher mean T/E ratios than heterozygotes (ins/del). UGT2B17 gene deletion has a strong influence on the T/E ratio in urine, which is the most efficient indicator of testosterone prohormone misuse.

Others factors studied seem not to have such an impact. The genotyping of UGT2B17 is an important source of information for understanding steroid profiling in the doping control field; therefore it is suggested that it be included in the Athletes Biological Passport.

 

[Michael Scally MD]

Bejder J, Aachmann-Andersen NJ, Bonne TC, Olsen NV, Nordsborg NB. Detection of erythropoietin misuse by the Athlete Biological Passport combined with reticulocyte percentage. Drug Testing and Analysis.
Detection of erythropoietin misuse by the Athlete Biological Passport combined with reticulocyte percentage - Bejder - 2015 - Drug Testing and Analysis - Wiley Online Library

The sensitivity of the adaptive model of the Athlete Biological Passport (ABP) and reticulocyte percentage (ret%) in detection of recombinant human erythropoietin (rHuEPO) misuse was evaluated using both a long-term normal dose and a brief high dose treatment regime.

Sixteen subjects received either 65 IU rHuEPO × kg-1 every second day for two weeks (normal-dose), 390 IU rHuEPO × kg-1 on three consecutive days (high-dose), or frequent placebo treatment for 13 days in a randomized, placebo-controlled, double-blind crossover design.

Blood variables were measured 4, 11, and 25 days following treatment initiation. The ABP based on haemoglobin concentration ([Hb]) and OFF-hr score ([Hb] – 60 × √ret%) yielded atypical profiles following both normal-dose and high-dose treatment (0 %, 31 %, 13 % vs. 21 %, 33 %, 20 % at days 4, 11, and 25 after normal and high dose, respectively).

Including ret% as a stand-alone marker for atypical blood profiles increased (P < 0.05) the sensitivity of the adaptive model at day 11 to 63 % and 67 % for normal-dose and high-dose rHuEPO administration, respectively.

In conclusion, ~30 % of subjects injecting a normal-dose rHuEPO for two weeks or a high-dose rHuEPO for three days will present an atypical ABP profile. Including ret% as a stand-alone parameter improves the sensitivity two-fold.

 

[Michael Scally MD]

Forsdahl G, Erceg D, Geisendorfer T, et al. Detection of testosterone esters in blood. Drug Test Anal 2015;7(11-12):983-9. Detection of testosterone esters in blood - Forsdahl - 2015 - Drug Testing and Analysis - Wiley Online Library

Injections of synthetic esters of testosterone are among the most common forms of testosterone application.

In doping control, the detection of an intact ester of testosterone in blood gives unequivocal proof of the administration of exogenous testosterone.

The aim of the current project was to investigate the detection window for injected testosterone esters as a mixed substance preparation and as a single substance preparation in serum and plasma.

Furthermore, the suitability of different types of blood collection devices was evaluated.

Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes, were tested.

A clinical study with six participants was carried out, comprising a single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido((R)) ) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanon((R)) ).

Blood was collected throughout a testing period of 60 days.

The applied analytical method for blood analysis included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection.

All investigated testosterone esters could be detected in post-administration blood samples.

The detection time depended on the type of ester administered.

Furthermore, results from the study show that measured blood concentrations of especially short-chained testosterone esters are influenced by the type of blood collection device applied.

The testosterone ester detection window, however, was comparable.

 

[Michael Scally MD]

Revisiting the Metabolism of 19-Nortestosterone Using Isotope Ratio and High Resolution/High Accuracy Mass Spectrometry

Highlights
· The metabolism of nortestosterone was studied using the deuterated substance.
· Metabolites were identified from human urine by means of hydrogen isotope ratio mass spectrometry and, where possible, characterized by GC–MS, GC-HRMS, and LC-HRMS(/MS).
· Artefact formation during commonly employed sample preparation steps was identified.
· Elimination of deuterium atoms by metabolic reactions were observed.
· Established metabolites such as norandrosterone glucuronide and sulfate were confirmed as best-possible options for doping control purposes.

Piper T, Schanzer W, Thevis M. Revisiting the metabolism of 19-nortestosterone using isotope ratio and high resolution/high accuracy mass spectrometry. J Steroid Biochem Mol Biol. Revisiting the metabolism of 19-nortestosterone using isotope ratio and high resolution/high accuracy mass spectrometry

The synthetic anabolic androgenic steroid 19-nortestosterone is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) due to its performance-enhancing effects. Today, doping controls focus predominantly on one main urinary metabolite, 19-norandrosterone glucuronide, which offers the required detection windows for an appropriate retrospectivity of sports drug testing programs.

As 19-norandrosterone can also be found in urine at low concentrations originating from in situ demethylation of other abundant steroids or from endogenous production, the exogenous source of 19-norandrosterone needs to be verified, which is commonly accomplished by carbon isotope ratio analyses.

The aim of this study was to re-investigate the metabolism of 19-nortestosterone in order to probe for additional diagnostic long-term metabolites, which might support the unambiguous attribution of an endo- or exogenous source of detected 19-nortestosterone metabolites.

Employing a recently introduced strategy for metabolite identification, threefold deuterated 19-nortestosterone (16,16,17-2H3-NT) was administered to one healthy male volunteer and urine samples were collected for 20 days. Samples were prepared with established methods separating unconjugated, glucuronidated and sulfated steroids, and analytes were further purified by means of high-performance liquid chromatography before trimethylsilylation.

Deuterated metabolites were identified using gas chromatograph/thermal conversion/isotope ratio mass spectrometer comprising an additional single quadrupole mass spectrometer. Additional structural information was obtained by gas chromatography/time-of-flight mass spectrometry and liquid chromatography/high resolution mass spectrometry.

In general, sulfo-conjugated metabolites were excreted for a longer time period than the corresponding glucuronides. Several unexpected losses of the arguably stable isotope labels were observed and characterized, attributed to metabolic reactions and sample preparation procedures.

The detection window of one of the newly detected metabolites was higher than currently used metabolites. The suitability of this metabolite to differentiate between endo- or exogenous sources could however not be verified conclusively.

 

[Michael Scally MD]

Longterm Detection of Anabolic Steroid Metabolites in Urine
https://www.agilent.com/cs/library/applications/5990-5748EN.pdf

A method for the longterm detection of anabolic steroid metabolites in urine was developed using both the Agilent 6890N network GC system with an Agilent 5975B Series GC/MSD and the Agilent 7000 Series Triple Quadrupole GC/MS. Monitoring the sulfate conjugates of these metabolites, instead of the routine glucuronide metabolites allowed detection of methenolone for a post-administration period that was almost twice as long. This was achieved using GC/MS in SIM mode. Performing the analysis on the Triple Quadrupole GC/MS in SRM mode extends the detection period even further, to almost three times as long as the conventional glucuronide approach.


Schänzer W, Delahaut P, Geyer H, Machnik M, Horning S. Long-Term Detection and Identification of Metandienone and Stanozolol Abuse in Athletes by Gas Chromatography-High-Resolution Mass Spectrometry. J Chromatogr B Biomed Appl. 1996;687:93-108. Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry

The misuse of anabolic androgenic steroids (AAS) in human sports is controlled by gas chromatography-mass spectrometric analysis of urine specimens obtained from athletes. The analysis is improved with modern high-resolution mass spectrometry (HRMS). The detection and identification of metabolites of stanozolol (I) [3′-hydroxystanozolol (II) and 4β-hydroxystanozolol (III)] and metandienone (IV) [17β-methyl-5β-androst-1-ene-3α,17α-diol (V) and 18-nor-17,17-dimethyl-5β-androsta-1, 13-dien-3α-ol (VI)] with GC-HRMS at 3000 resolution yielded a large increase in the number of positive specimens. A total of 116 anabolic steroid positives were found in this laboratory in 1995 via GC-MS and GC-HRMS screening of 6700 human urine specimens collected at national and international sporting events and at out-of-competition testing. Of the 116 positive cases, 41 were detected using conventional (quadrupole) GC-MS screening. The other 75 positives were identified via GC-HRMS screening. To confirm the HRMS screening result, the urine sample was reanalyzed using a specific sample workup procedure to selectively isolate the metabolites of the identified substance. II and III were selectively isolated via immunoaffinity chromatography (IAC) using an antibody which was prepared for methyltestosterone and shows high cross reactivity to II and III. V and VI were isolated using high-performance liquid chromatography (HPLC) fractionation.


Fast and Comprehensive Doping Agent Screening in Urine by Triple Quadrupole GC/MS
http://www.analytical.com/fullaccess/novedad31file1.pdf

A rapid method was developed on the Agilent 7000 Series Triple Quadrupole GC/MS system to screen for more than 150 doping agents in seven classes of substances, at or below WADA MRPLs [1]. A short capillary column, rapid scan speed and hydrogen as carrier gas enable a run time of less than 8 minutes.


Parr MK, Schanzer W. Detection of the misuse of steroids in doping control. The Journal of Steroid Biochemistry and Molecular Biology 2010;121(3-5):528-37. Detection of the misuse of steroids in doping control

The list of prohibited substances of the World Anti-Doping Agency (WADA) classifies the administration of several steroids in sports as doping. Their analysis is generally performed using urine specimen as matrix. Lots of the steroids are extensively metabolised in the human body. Thus, knowledge of urinary excretion is extremely important for the sensitive detection of steroid misuse in doping control. The methods routinely used in steroid screening mainly focus on substances, that are excreted unconjugated or as glucuronides. Common procedures include deconjugation using a β-glucuronidase enzyme. Following extraction and concentration the analytes are submitted to LC-MS(/MS) analysis and/or GC-MS(/MS) analyses. Besides the classical steroids, more and more products appear on the market for “dietary supplements” containing steroids that have never been marketed as approved drugs, mostly without proper labelling of the contents. To cover the whole range of potential products comprehensive screening tools have to be utilised in addition to the classical methods.

Endogenous steroids, e.g. testosterone, represent a special group of compounds. As classical chemical methodology is incapable of discriminating synthetic hormones from the biosynthesised congeners, the method of steroid profiling is used for screening purpose. Additionally, based on isotope signatures a discrimination of synthetic and natural hormones can be achieved.