Scientific Opinion on the Regulatory Status of 1,3-Dimethylamylamine (DMAA) / Bastiaan J. Venhuis, Dries de Kaste. – (European Journal of Food Research & Review 2 (2012) 4 (November 26) : p. 93-100)

Content:
- Introduction
- Nomenclature
- Pharmacology
- Intranasal Application
- Oral Application
- Discussion
• Effect on the heart
• Effect on the Blood Pressure
• Effect on the Lungs and the Nose
- Conclusion

1,3-Dimethylamylamine (DMAA) is a pressor amine often found in food supplements for athletes at dosages of 25-65 mg. Historically, the compound has been used as a nasal decongestant but its oral application is largely unstudied leaving the regulatory status of such food supplements as unlicensed medicines undetermined. We therefore reviewed the literature on DMAA and similar amines in order to deduce an effective oral dosage. Based on our findings we conclude that oral preparations with >4 mg DMAA per dose unit should be considered as effective as a bronchodilator. Food supplements that exceed that limit are in fact subject to the Medicines Act and require licensing. Dosages higher than 100-200 mg are expected to cause serious adverse events.

Screening for 2-quinolinone-derived selective androgen receptor agonists in doping control analysis / Mario Thevis, Maxie Kohler, Joachim Maurer, Nils Schlörer, Matthias Kamber, Wilhelm Schänzer. - (Rapid Communicaton in Mass Spectrometry 21 (2007) 21 (15 November); p. 3477-3486)

• PMID: 17985352
• DOI: 10.1002/rcm.3247

Abstract

Selective androgen receptor modulators (SARMs) represent a class of emerging drugs with high potential for misuse in sports, and therefore members of this group are banned as anabolic agents by the World Anti-Doping Agency. Preventive approaches to restrict their use include early implementation of target analytes into doping control screening assays and evaluation of the mass spectrometric behavior of these drugs to allow their unequivocal identification as well as the characterization of structurally related compounds and metabolic products. Four model SARMs with the 6-alkylamino-2-quinolinone structure, including the advanced drug candidate LGD-2226, were synthesized. Fragmentation pathways after positive electrospray ionization and collision-induced dissociation were studied using an LTQ Orbitrap mass analyzer, and diagnostic product ions and common dissociation pathways were employed to establish a screening procedure targeting intact quinolinone-based SARMs as well as putative metabolic products such as dealkylated analogues. Therefore, features of a triple quadrupole mass analyzer such as multiple reaction monitoring and precursor ion scanning were utilized. Sample preparation based on commonly employed liquid-liquid extraction and subsequent liquid chromatographic/tandem mass spectrometric measurement allowed for detection limits of 0.01-0.2 ng/mL, and intra- and interday precisions between 3.2 and 8.5% and between 6.3 and 16.6%, respectively. Recoveries varied from 81 to 98%, and tests for ion suppression or enhancement effects were negative for all analytes.

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.

Screening for gene doping transgenes in horses via the use of massively parallel sequencing / Jillian Maniego, Bogusia Pesko, Jocelyn Habershon-Butcher, Jim Huggett, Polly Taylor, James Scarth, Edward Ryder. - (Gene Therapy (2021) 19 July)

• PMID: 34276046
• DOI: 10.1038/s41434-021-00279-1

Abstract

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.

Thevis M, Kamber M, Schänzer W. Screening for metabolically stable aryl-propionamide-derived selective androgen receptor modulators for doping control purposes. Rapid Commun Mass Spectrom. 2006;20(5):870-6.

Screening for performance enhancing substances and quantification of ethanol in different Arishta manufactured in Sri Lanka / Punchividanelage Nilu Jayashika Fernando, Shehani Pigera, Suraweera Arachchilage Nimesha Rashani, Ravindra Fernando, Pabasara Weerasinghe, Tharaka Deepal Godakumbura, Madunil Anuk Niriella, Seevali Jayawickreme, Arjuna Priyadarshana de Silva

• Ceylon Medical Journal 65 (2020) 4 (31 December); p. 112-117
• PMID: 34825559
• DOI: 10.4038/cmj.v65i4.9282

Abstract

Background: Arishta have been used in Ayurveda medicine for over thousands of years in Sri Lanka to treat various diseases. Ashwagandharishta, Balarishta and Dashamoolarishta are usually prescribed to obtain an anabolic effect, and Ashwagandharishta and Dashamoolarishta for androgenic effect in males. Thus, these arishta have been shown to have similar effect as anabolic androgenic steroids and stimulants in Western medicine. Therefore, arishta could potentially be used by athletes to improve their performance in sports leading to unintentional doping. Additionally, ethanol develops in-source during arista fermentation, which can affect athletes health.

Objective: The aim of this study is to investigate whether the anabolic androgenic steroids or stimulants banned by World Anti-Doping Agency are present in these arishta, and to determine their ethanol content.

Methods: Methanol extractions of Ashwagandarishta, Balarishta, Dashamoolarishta from four different manufacturers were screened for 21 stimulant and 22 anabolic androgenic steroids banned by World Anti-Doping Agency, using Gas Chromatography Mass Spectrometer. Ethanol content of the twelve Arishta samples were also measured.

Results: Anabolic androgenic steroids or stimulants were not present in the tested Arishta samples, and percentage volume / volume ethanol content of all Arishta was between (5.80-8.35) ±0.5.

Conclusion: The tested brands of Ashwagandharishta, Balarishta and Dashamoolarishta did not contain stimulants or anabolic androgenic steroids banned by World Anti-Doping Agency.

Screening for the calstabin-ryanodine receptor complex stabilizers JTV-519 and S-107 in doping control analysis / Mario Thevis, Simon Beuck, Andreas Thomas, Maxie Kohler, Nils Schlörer, Ileana Vajiala, Wilhelm Schänzer. - (Drug Testing and Analysis 1 (2009) 1 (January); p. 32-42)

• PMID: 20355157
• DOI: 10.1002/dta.13

Abstract

Recent studies outlined the influence of exercise on the stability of the skeletal muscle calstabin1-ryanodine receptor1-complex, which represents a major Ca(2+) release channel. The progressive modification of the type-1 skeletal muscle ryanodine receptor (RyR1) combined with reduced levels of calstabin1 and phosphodiesterase PDE4D3 resulted in a Ca(2+) leak that has been a suggested cause of muscle damage and impaired exercise capacity. The use of 1,4-benzothiazepine derivatives such as the drug candidates JTV-519 and S-107 enhanced rebinding of calstabin1 to RyR1 and resulted in significantly improved skeletal muscle function and exercise performance in rodents. Due to the fact that the mechanism of RyR1 remodelling under exercise conditions were proven to be similar in mice and humans, a comparable effect of JTV-519 and S-107 on trained athletes is expected, making the compounds relevant for doping controls. After synthesis of JTV-519, S-107, and a putative desmethylated metabolite of S-107, target compounds were characterized using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-high-resolution/high-accuracy Orbitrap mass spectrometry. Collision-induced dissociation pathways were suggested based on the determination of elemental compositions of product ions and H/D-exchange experiments. The most diagnostic product ion of JTV-519 was found at m/z 188 (representing the 4-benzyl-1-methyl piperidine residue), and S-107 as well as its desmethylated analog yielded characteristic fragments at m/z 153 and 138 (accounting for 1-methoxy-4-methylsulfanyl-benzene and 4-methoxy-benzenethiol residues, respectively). The analytes were implemented in existing doping control screening procedures based on liquid chromatography, multiple reaction monitoring and simultaneous precursor ion scanning modes using a triple quadrupole mass spectrometer. Validation items such as specificity, recovery (68-92%), lower limit of detection (0.1-0.2 ng/mL), intraday (5.2-18.5%) and interday (8.7-18.8%) precision as well as ion suppression/enhancement effects were determined.

Screening of anabolic steroids in horse urine by liquid chromatography-tandem mass spectrometry / Nola H. Yu, Emmie N.M. Ho, David K.K. Leung, Terence S.M. Wan

• Journal of Pharmaceutical and Biomedical Analysis 37 (2005) 5 (29 April), p. 1031-1038
• PMID: 15862683
• DOI: 10.1016/j.jpba.2004.08.041

Abstract

Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography-mass spectrometry (GC-MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method for the detection of anabolic steroids that are poorly covered by GC-MS. Enzyme-treated urine was processed by solid-phase extraction (SPE) using a Bond Elut Certify cartridge, followed by a base wash for further cleanup. Separation of the steroids was carried out on a reversed-phase DB-8 column using 0.1% acetic acid and methanol as the mobile phase in a gradient elution programme. The mass spectrometer for the detection of the steroids was operated in the positive electrospray ionisation (ESI) mode with multiple reaction monitoring (MRM). Urine samples fortified with 15 anabolic steroids (namely, androstadienone, 1-androstenedione, bolasterone, boldione, 4-estrenedione, gestrinone, methandrostenolone, methenolone, 17alpha-methyltestosterone, norbolethone, normethandrolone, oxandrolone, stenbolone, trenbolone and turinabol) at low ng/mL levels were consistently detected. No significant matrix interference was observed at the retention times of the targeted ion masses in blank urine samples. The method specificity, sensitivity, precision, recoveries, and the performance of the enzyme hydrolysis step were evaluated. The successful application of the method to analyse methenolone acetate administration urine samples demonstrated that the method could be effective in detecting anabolic steroids and their metabolites in horse urine.

In December 2004 the Canadian Centre for Ethics in Sport (CCES) has reported an anti-doping rule violation against the Athlete after his A and B samples tested positive for the prohibited substances marijuana (cannabis) and cocaine.

After notification a provisional suspension was ordered. The Athlete filed a statement, sustained with evidence, in his defence and was heard for the Tribunal.
The Athlete admitted the use of marijuana for his medical condition (haemophilia) without a TUE. He denied the use of cocaine and argued that tampering, contamination or a false positive as possibility for the test results.

The Tribunal did not accept the Athlete's arguments and evidence and finds that Athlete used marijuana without medical advice and without a TUE. With no evidence of tampering or a false positive and with marijuana and cocaine metabolites found in his samples the Tribunal concludes that the Athlete commited an anti-doping rule violation.
Without exceptional circumstances the Tribunal decides on 7 February 2005 to impose a 2 year period of ineligibility on the Athlete starting on the date of the provisional suspension, i.e. on 5 January 2005.

Facts
The Canadian Centre for Ethics in Sport (CCES) alleges Roland Green (the athlete) for a violation of the Canadian Anti-Doping Program. The athlete provided a urine sample pursuant to the Anti-Doping Regulations following the UCI sanctioned "UCI MTB World Cup" in Houffalize Belgium on 30 May 2004. His sample indicated the presence of prednisolone a metabolite of Budesonide.

History
The athlete in his submission states that he finished 21st in the race in Belgium despite feeling unwell and was selected for random testing. He admitted that on the race morning he took 2 to 3 puffs of Symbicort-200. He made no declaration to that effect on his anti-doping control form provided at the time of giving the urine sample. He states that he knew he was not cleared to use the inhaler but did so anyway. He states that he did this because he hoped that the testing would miss these medications and I would avoid a huge problem.

Decision
1. The Definition of doping in Article 4(2) has been established. A First Doping Offence has occurred under Article 130(1). The Doping Offence involved the use of a Prohibited Substance.
2. The athlete is disqualified from the UCI MTB World Cup event held on 30 May 2004 for having committed a Doping Offence during competition.
3. Under Article 130 as modified by the principle of lex mitior and upon a finding of the application of the principles of Article 124 a period of suspension for six months is to be served.
4. Under Article 150 and 134 and having regard to the principles of Article 124 the suspension commenced on 5 July 2004 and will end on 4 April 2005 having taken account of the period of inactivity set out in Article 152(b).

Fine
Under Article 128(2) the athlete, being a licence-holder in the mountain bike trade team, must pay an obligatory fine. In accordance with Article 128(4) the fine is set at
CHF 2000.