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[Michael Scally MD]

A New Sulphate Metabolite as a Long-Term Marker of Metandienone [Dianabol] Misuse

Highlights
· A new sulphate metabolite was detected as a long-term marker of metandienone use.
· It was characterized by GC–MS/MS and LC–MS/MS.
· It is 18-nor-17β-hydroxymethyl-17α-methyl-androst-1,4,13-triene-3-one sulphate.
· It improves the retrospectivity compared to previously reported metabolites.

Gomez C, Pozo OJ, Garrostas L, Segura J, Ventura R. A new sulphate metabolite as a long-term marker of metandienone misuse. Steroids 2014;78(12-13):1245-53. https://www.sciencedirect.com/science/article/pii/S0039128X13002018

Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing.

Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) after hydrolysis with beta-glucuronidase and liquid-liquid extraction.

It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied.

The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC-MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods.

One of the metabolites (M1) was identified and characterised by GC-MS/MS and LC-MS/MS as 18-nor-17beta-hydroxymethyl-17alpha-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17beta-hydroxymethyl-17alpha-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).

 

[Michael Scally MD]

Andersen DW, Linnet K. Screening for Anabolic Steroids in Urine of Forensic Cases Using Fully Automated Solid Phase Extraction and LC-MS-MS. J Anal Toxicol. http://jat.oxfordjournals.org/content/early/2014/08/07/jat.bku098.abstract

A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds.

Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL.

In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, suggesting that only a small number of common steroids are likely to occur in a forensic context. The steroids were often in high concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse were seen in the majority of cases.

The method presented serves as a fast and automated screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids.

 

[Michael Scally MD]

Wang J, Yang R, Yang W, Liu X, Xing Y. Impact of progesterone administration on doping test of endogenous steroids. Anal Bioanal Chem. http://link.springer.com/article/10.1007/s00216-014-8027-x

Progesterone (PROG) is a naturally occurring progestagen, which has been used to prevent preterm birth, control persistent anovulatory bleeding, and treat premenstrual syndrome in clinical practices.

Studies on the metabolism of PROG have demonstrated that PROG is the precursor of other steroids such as 5beta-pregnane-3alpha,20alpha-diol (PD), testosterone (T), and 17-hydroxyprogesterone. PD is the most commonly used endogenous reference compound (ERC) in the isotope ratio mass spectrometry (IRMS) analysis for doping control.

It is expected that the PROG administration could affect the carbon isotope ratios (13C/12C, expressed as delta 13C-value) of PD and T metabolites, and lead to the false-negative or false-positive results in doping test. The influences of oral and intramuscular administration of PROG on the urinary steroid profile and carbon isotope ratios of steroids were investigated in this study.

It was demonstrated that the urine concentrations and the delta 13C-values of PD were affected obviously. The depleted delta 13C-values of PD could be used to suggest PROG administration. Using PD as ERC may result in the distorted evaluation for suspicious urine sample in IRMS analysis when PROG is ingested. The 5alpha-androst-16-en-3alpha-ol and 11beta-hydroxyandrosterone could be used as the alternative ERCs in case of PROG administration.

The carbon isotope ratios of androsterone (An) and etiocholanolone (Etio), two T metabolites, remained unchanged throughout the excretion study, which suggested that the delta values of An and Etio could still be used as the urinary markers of T administration even when PROG was administrated.

 

[Michael Scally MD]

Stable Carbon Isotope Ratio Profiling Of Illicit Testosterone Preparations
http://onlinelibrary.wiley.com/doi/10.1002/dta.1533/abstract

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport.

It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products.

Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. δ13C ≥ −25.8 ‰).

In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of ‘enriched’ substrates – which if administered to human subjects would be considered problematic for the use of current GC-C-IRMS methodologies for the doping control of testosterone in sport.

The distribution of δ13C values for this illicit testosterone sample population (n = 283) ranged from −23.4 ‰ to −32.9 ‰ with mean and median of −28.6 ‰ – comparable to previous work.

However, only 13 out of 283 testosterone samples (4.6 %) were found to display δ13C values ≥ −25.8 ‰, confirming that in the vast majority of cases of illicit testosterone administration, current GC-C-IRMS doping control procedures would be capable of confirming misuse.

 

[jesse]

very interesting im a new b and happy to get good info aney time thanks

 

[Michael Scally MD]

Krug O, Thomas A, Walpurgis K, et al. Identification of black market products and potential doping agents in Germany 2010-2013. Eur J Clin Pharmacol. http://link.springer.com/article/10.1007/s00228-014-1743-5

PURPOSE: The desire to increase the athletic performance, to 'optimize' an individual's appearance, and to complement but also to arguably substitute exercise by means of drugs and drug candidates has generated a considerable (illicit) market for compounds such as anabolic-androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely developed for therapeutic use, their abuse/misuse generates enormous health risks, which has necessitated comprehensive controls of compound trafficking by customs and anti-doping authorities.

METHODS: From 2012 to 2013, the Bureau of Customs Investigation confiscated products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants (13 kg), selective estrogen receptor modulators (SERMs; 24 kg), and human growth hormone (hGH; 3500 ampules). In cooperation with the Bureau and under the umbrella of the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne Anti-Doping Laboratory analyzed an additional 337 (black market) products between 2010 and 2013, allowing to monitor developments in drug use and, hence, the anticipation of new challenges in sports drug testing. Main tools utilized in characterizing confiscated materials were liquid chromatography-high resolution mass spectrometry (LC-HRMS), gas chromatography-high resolution mass spectrometry (GC-HRMS), and polyacrylamide gel electrophoresis (PAGE) with subsequent bottom-up identification of peptidic compounds using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS).

RESULTS: Among the 337 substances analyzed in the doping control laboratory in Cologne, 67 active ingredients were found, 49 of which being categorized as doping agents by the World Anti-Doping Agency (WADA). A total of 83.7 % accounted for steroidal substances (predominantly testosterone, trenbolone, and nandrolone and corresponding esters), 12.8 % accounted for peptide hormones and growth factors (predominantly hGH and growth hormone releasing peptides (GHRPs)), 3.2 % of the products contained hormones and metabolic modulators, and 0.3 % accounted for diuretic agents. Outstanding findings were the detection of the selective androgen receptor modulator (SARM) LGD-4033, the thymic hormone thymosin beta4, and a fusion protein of unknown biological activity.

CONCLUSIONS: Trafficking of considerable amounts of arguably performance and/or body-enhancing compounds has been observed during the past 4 years, the majority of which is categorized as relevant to sports drug testing. Several substances are of fake/non-approved nature and represent enormous health risks to the 'customer'.

 

[jesse]

thanks for the info im new at all this and the more i know the better resulte ill be getting thanks again

 

[Michael Scally MD]

Brooker L, Cawley A, Drury J, Edey C, Hasick N, Goebel C. Stable carbon isotope ratio profiling of illicit testosterone preparations - domestic and international seizures. Drug Test Anal. http://onlinelibrary.wiley.com/doi/10.1002/dta.1533/abstract

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport.

It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products.

Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. delta13 C >/= -25.8 per thousand).

In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of 'enriched' substrates - which if administered to human subjects would be considered problematic for the use of current GC-C-IRMS methodologies for the doping control of testosterone in sport.

The distribution of delta13 C values for this illicit testosterone sample population (n = 283) ranged from -23.4 per thousand to -32.9 per thousand with mean and median of -28.6 per thousand - comparable to previous work.

However, only 13 out of 283 testosterone samples (4.6 %) were found to display delta13 C values >/= -25.8 per thousand, confirming that in the vast majority of cases of illicit testosterone administration, current GC-C-IRMS doping control procedures would be capable of confirming misuse.

 

[Millard]

Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. delta13 C >/= -25.8 per thousand).

No doubt some athletes have found chemists to synthesize testosterone preparations to trick CIR testing. If they also manage T:E ratios, their testosterone could still go undetected. Reports that CIR testing has closed the "testosterone loophole" were premature.

 

[jesse]

Brooker L, Cawley A, Drury J, Edey C, Hasick N, Goebel C. Stable carbon isotope ratio profiling of illicit testosterone preparations - domestic and international seizures. Drug Test Anal. http://onlinelibrary.wiley.com/doi/10.1002/dta.1533/abstract

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport.

It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products.

Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. delta13 C >/= -25.8 per thousand).

In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of 'enriched' substrates - which if administered to human subjects would be considered problematic for the use of current GC-C-IRMS methodologies for the doping control of testosterone in sport.

The distribution of delta13 C values for this illicit testosterone sample population (n = 283) ranged from -23.4 per thousand to -32.9 per thousand with mean and median of -28.6 per thousand - comparable to previous work.

However, only 13 out of 283 testosterone samples (4.6 %) were found to display delta13 C values >/= -25.8 per thousand, confirming that in the vast majority of cases of illicit testosterone administration, current GC-C-IRMS doping control procedures would be capable of confirming misuse.

thanks for the grate info happy to have people who know what thair talking about.

 

[Michael Scally MD]

Esposito S, Deventer K, Van Eenoo P. Identification of the growth hormone-releasing hormone analogue [Pro1, Val14]-hGHRH with an incomplete C-term amidation in a confiscated product. Drug Testing and Analysis. http://onlinelibrary.wiley.com/doi/10.1002/dta.1730/abstract

In this work, a modified version of the 44 amino acid human growth hormone-releasing hormone (hGHRH(1-44)) containing an N-terminal proline extension, a valine residue in position 14, and a C-terminus amidation (sequence: PYADAIFTNSYRKVVLGQLSARKLLQDIMSRQQGESNQERGARARL-NH2) has been identified in a confiscated product by liquid chromatography-high resolution mass spectrometry (LC-HRMS).

Investigation of the product suggests also an incomplete C-term amidation. Similarly to other hGHRH analogues, available in black markets, this peptide can potentially be used as performance-enhancing drug due to its growth hormone releasing activity and therefore it should be considered as a prohibited substance in sport.

Additionally, the presence of partially amidated molecule reveals the poor pharmaceutical quality of the preparation, an aspect which represents a big concern for public health as well.

 

[jesse]

thanks for the info

 

[Michael Scally MD]

'Living In The Dark Ages': Why Scientists Say The NFL'S hGH Test Is BS.
https://sports.vice.com/article/living-in-the-dark-ages-why-scientists-say-the-nfls-hgh-test-is-bs

Since its inception in 2004, hGH testing in sports has been an abject failure, dogged by ongoing scientific dispute and a near-total inability to identify doping athletes.

Roughly 15,000 athletes have been subjected to hGH blood tests over the last decade; only 10 reportedly have been suspended for positive results, and two of those have been overturned through science-based legal appeals.

 

[Michael Scally MD]

Failed Doping Test by Rita Jeptoo, Kenyan Marathon Champion, Is Confirmed
http://www.nytimes.com/2014/12/20/s...oo-kenyan-marathon-champion-is-confirmed.html

David Okeyo and Jackson Tuwei, who are vice presidents of Athletics Kenya, said in November that Jeptoo’s A sample, taken in an out-of-competition test in Kenya on Sept. 25, showed traces of the blood booster EPO.

 

[Michael Scally MD]

Thevis M, Kuuranne T, Geyer H, Schanzer W. Annual banned-substance review: analytical approaches in human sports drug testing. Drug Test Anal. http://onlinelibrary.wiley.com/doi/10.1002/dta.1441/abstract

Within the mosaic display of international anti-doping efforts, analytical strategies based on up-to-date instrumentation as well as most recent information about physiology, pharmacology, metabolism, etc., of prohibited substances and methods of doping are indispensable.

The continuous emergence of new chemical entities and the identification of arguably beneficial effects of established or even obsolete drugs on endurance, strength, and regeneration, necessitate frequent and adequate adaptations of sports drug testing procedures.

These largely rely on exploiting new technologies, extending the substance coverage of existing test protocols, and generating new insights into metabolism, distribution, and elimination of compounds prohibited by the World Anti-Doping Agency (WADA).

In reference of the content of the 2014 Prohibited List, literature concerning human sports drug testing that was published between October 2013 and September 2014 is summarized and reviewed in this annual banned-substance review, with particular emphasis on analytical approaches and their contribution to enhanced doping controls.

 

[Michael Scally MD]

What are the 19 known methods of cheating to pass performance enhancing drug tests?
http://sportsanddrugs.procon.org/view.resource.php?resourceID=002706

Nathan Jendrick, fitness writer, in his 2006 book titled Dunks, Doubles, Doping: How Steroids Are Killing American Athletics, wrote the following:

"The following are methods that are currently used, were used at some point in history, or can supposedly be used to beat steroid tests in athletics...

 

[Michael Scally MD]

"Ben Johnson" steroid still responsible for more doping cases than any other.
http://www.insidethegames.biz/news/...ponsible-for-more-doping-cases-than-any-other

More than a quarter of a century after the Seoul Olympic Games, stanozolol, the anabolic steroid that triggered Ben Johnson's positive test, was responsible for more doping cases in 2014 than any other single cause.

According to listings compiled by The Anti-Doping Database, a subscription-based information resource, stanozolol last year accounted for 22 athlete suspensions, compared to 15 attributable to methylhexaneamine, the second most common prohibited substance. http://www.dopinglist.com/

 

[Michael Scally MD]

Cadwallader AB, Murray B. Performance-Enhancing Drugs I: Understanding the Basics of Testing for Banned Substances. Int J Sport Nutr Exerc Metab. http://journals.humankinetics.com/ijsnem-in-press/ijsnem-in-press/performance-enhancing-drugs-i-understanding-the-basics-of-testing-for-banned-substances

Whenever athletes willfully or accidentally ingest performance-enhancing drugs or other banned substances (such as drugs of abuse), markers of those drugs can be detected in biological samples (e.g., biofluids: urine, saliva, blood); in the case of some drugs, that evidence can be apparent for many weeks following the last exposure to the drug. In addition to the willful use of prohibited drugs, athletes can accidentally ingest banned substances in contaminated dietary supplements or foods and inadvertently fail a drug test that could mean the end of an athletic career and the loss of a good reputation. The proliferation of performance-enhancing drugs and methods has required a corresponding increase in the analytical tools and methods required to identify the presence of banned substances in biofluids. Even though extraordinary steps have been taken by organizations such as the World Anti-Doping Agency to limit the use of prohibited substances and methods by athletes willing to cheat, it is apparent that some athletes continue to avoid detection by using alternative doping regimens or taking advantage of the limitations in testing methodologies. This article reviews the testing standards and analytical techniques underlying the procedures used to identify banned substances in biological samples, setting the stage for future summaries of the testing required to establish the use of steroids, stimulants, diuretics, and other prohibited substances.

 

[Michael Scally MD]

U.F.C. Hires Investigator as Antidoping Chief
http://www.nytimes.com/2015/04/07/sports/ufc-hires-investigator-as-antidoping-chief.html

Two months after promising to ramp up the drug testing of its more than 500 fighters, U.F.C. hired the federal investigator Jeff Novitzky to spearhead its antidoping initiative. Novitzky will begin serving as U.F.C.’s vice president of health and performance later this month when he leaves his job as a special agent for the United States Food and Drug Administration. Novitzky came to national prominence during the Balco investigation that implicated Barry Bonds and Marion Jones, among others, and also led a doping inquiry of Lance Armstrong.

 

[Michael Scally MD]

Lobigs LM, Knight EJ, Schumacher YO, Gore CJ. Within-subject haemoglobin variation in elite athletes: a longitudinal investigation of 13 887 haemoglobin concentration readings. Drug Testing and Analysis. http://onlinelibrary.wiley.com/doi/10.1002/dta.1809/abstract

The Athlete Biological Passport (ABP) estimates individualized reference ranges for key blood markers, such as haemoglobin concentration ([Hb]), using predetermined population mean, between- and within-subject variances.

Here, we aim to reassess previously published estimates for within-subject [Hb] variance and determine whether sex-, analyzer-, sport-, or season-specific values are required.

Our reference population contains 7723 male (mean ± SD, 22.3 ± 4.6 years of age) and 6164 female (21.6 ± 4.3) athlete observations from 49 sports. [Hb] was calculated using one of three cytometers; Bayer-H3 (1997–1999, n = 4554), ADVIA-120 (1999–2010, n = 8636) and Sysmex XT-2000i (2010–2012, n = 697).

The final model was a linear mixed model for [Hb] with analyzer (H3, ADVIA, Sysmex), sex (male, female), sport (power-endurance, endurance, skill, team, disabled and non-athletes), season (summer, winter), and the interaction between sex and sport as fixed effects and athlete as a random effect. The model included an exponential correlation structure to allow for within-subject autocorrelation, and allowed different within-subject variances for each sport.

Within-subject [Hb] variance (g2/L2) was significantly less for power endurance (35.09, 95% CI 33.50 to 36.76), disabled (25.82, 95% CI 21.71 to 35.28) and non-athletes (34.30, 95% CI 28.53 to 35.87) than for endurance (40.35, 95% CI 39.62 to 47.22) and team sports (38.70, 95% CI 37.68 to 39.76) athletes.

No new evidence was found to justify adjusting the current within-subject [Hb] variance estimate.