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.

In November 2004 the Canadian Centre for Ethics in Sport (CCES) has reported an anti-doping rule violation against the Athlete after his sample tested positive for the prohibited substance cannabis at a concentration of 130 ng/mL. The Athlete had used cannabis after the competition and before he provided a sample for drug testing, which explains the reason why his results were so high. Also the Athlete walked away to smoke cannabis with some fans after he was notified by the DCO and Chaperone and finally showed up within the allotted time to provide a sample.

The Athlete filed a statement in his defence and was heard for the SDRCC Doping Tribunal. The Athlete admitted that he had used cannabis and stated that he already had expressed his apologies to the DCO and Chaperone for his behavior after he had returned to provide a sample.
The arbitrator finds that the Athlete had no intention to enhance sport performance and normally only a warning and reprimand would be imposed as first anti-doping violation. However the arbitrator considers the Athlete’s disappearance after notification by the DCO an aggravating factor.

Therefore on 6 May 2005 the SDRCC Doping Tribunal decides to impose a 1 year period of ineligibility on the Athlete, starting on the date of the decision. Any period of provisional suspenstion shall be credited against the total period of ineligibility to be served.

Facts
The Canadian Center for Ethics in Sport (CCES) charges Yvan Darsigny (the athlete) for a violation of the Anti-Doping Rules. The athlete Yvan Darsigny committed a violation by refusing or avoiding the sample collection on January 23, 2005.

History
Due to family matters the applicant didn't treat the Doping Control Officer in a kind way and refused to comply when they came for an unannounced doping test. According to his testimony, the technique of no advance notice testing was unknown to the athlete. The DCO indentified herself correctly and the athlete understood the reason for her visit.

Decision
The period of Ineligibility imposed for this anti-doping rule violation shall be: Two (2) years Ineligibility

Facts
The Canadian Centre for Ethics in Sport (CCES) alleges Ainsly Robinson (the athlete) for a violation of the Canadian Anti-Doping Program (CADP). On the weekend of May 14 and 15, 2005, Mr. Robinson competed at the National Senior Wrestling Championships, which took place in Renfrew, Ontario. On Sunday, May 15th, he was notified that he had been selected for doping control. His sample was positive for the presence of cocaine and metabolites. Cocaine is a prohibited substance on the 2005 World Anti-Doping Agency (WADA) prohibited list.

History
The athlete has a job as security. He confiscated a brown bag with a powdery substance and put it in the bag of his pants. Probably this bag contained cocaine and was absorbed by his skin. He was unaware that this substance could be absorbed by his skin.

Decision
In view of the presence of cocaine and metabolites in his sample, it was acknowledged that an anti-doping rule violation occurred.It cannot be concluded that there are exceptional circumstances which would justify the elimination or reduction of the penalty for such a violation. In accordance with the CADP, the penalty required to impose for a first anti-doping rule violation is a two year period of ineligibility from sport and permanent ineligibility for direct financial support from the Government of Canada. The period of ineligibility shall run from the date of the decision, provided that the period of provisional suspension shall be credited against the total period of ineligibility.

Facts
The Canadian Centre for Ethics in Sport (CCES) alleges Chris McKay (the athlete) for a violation of the Canadian Anti-Doping Program. On September 28, 2005, the Athlete was a member of the Okanagan Suns Junior Football Team. He was randomly selected for out-of-competition doping control at a team practice in Kelowna, British Columbia. The Athlete refused or failed to provide a urine sample for testing.

History
The athlete refused to give an urine sample, returned the sample container and left. He didn't participated in the hearing process; contested the anti-doping rule violation asserted by the CCES or the evidence put forward by the CCES; or offered any explanation for his prima facie refusal to submit to Sample collection.

Decision
The CCES has met the burden of proof that the Athlete refused or failed without compelling justification to submit to Sample collection after authorized notification.
The Athlete's refusal constitutes an anti-doping rule violation as provided in Rule 7.24 of the CADP. This is a first violation.
The sanctions to be imposed on the Athlete are two years' ineligibility and permanent ineligibility for direct financial support from the Government of Canada.

Facts
The Canadian Centre for Ethics in Sport (CCES) alleges Christopher Sheppard for a violation of the Canadian Anti-Doping Program (CADP). The athlete was selected for a no advance notice, out-of-competition doping control in Kamloops, British Columbia.
Pursuant to the rules of CADP the Athlete provided a urine sample for testing on 29 May 2005. His sample tested positive on the prohibited substance recombinant human erythropoietin (rhEPO). Also the B-sample tested positive on this substance.

History
This is the first Canadian doping case to deal with the Prohibited Substance rhEPO.

Decision
It has been established that a Prohibited Substance was present in Mr. Sheppard's urine sample. Therefore, an anti-doping rule violation has occurred under the CADP Rule 7.16. The Athlete raised no issue of no fault or negligence, or, any significant fault or negligence. Therefore, it is no required to examine the elimination or reduction of the sanction. In the circumstances, the imposed sanction for a first anti-doping rule violation of a two year period of ineligibility as provided for by Rule 7.20. CADP Rule 7.37 applies. Mr. Sheppard is from the date of this award permanently ineligible for direct financial support provided by the Government of Canada.
The foregoing period of ineligibility in respect of the sanction and the financial support starts on the date of this decision in accordance with the CADP.

Costs
No submission was made on costs. Unless applied for, no order in respect of the same will be made.

Facts
The Canadian Centre for Ethics in Sport (CCES) Alleges Giulio Zardo (the athlete) for a violation of the Canadian Anti-Doping Program (CADP). On April 21 and 22, 2005, the CCES attempted to conduct no-notice, Out-of-Competition doping control on the Athlete but despite several attempts on these two days, the Doping Control Officer (DCO) was unable to locate the Athlete for the purpose of sample collection. On April 22, 2005, the CCES re-scheduled the Athlete’s no-notice test to an advance notice test. The CCES asserts that the Athlete refused to submit to sample collection on April 23, 2005 after being informed of his selection for doping control and that he has not demonstrated “compelling justification” for his refusal.

History
The athlete was retiring from his sport. In the past he had several Therapeutic Use Exemption (TUEs) he thinks if ask for he would have gotten a TUE again.

Desicion
The required sanction for a first anti-doping rule violation for refusal to submit to doping control, in the circumstances to impose, is a two year period of ineligibility from sport as well as permanent ineligibility from direct Government of Canada funding.
The CADP states that the period of ineligibility shall start on the date of the hearing decision providing for Ineligibility. Any period of Provisional Suspension shall, however, if any, be credited against the total period of Ineligibility to be served.
The period of Ineligibility should start on the date of the hearing decision, namely August 31, 2005.

Costs
Under Rule 7.69 of the CADP, the Doping Tribunal may award costs to any party payable as it directs.

Facts
The Canadian Centre for Ethics in Sport (CCES) alleges Joseph Frans (the athlete) for a violation of the Canadian Anti-Doping Program. Pursuant to the rules of CADP the Athlete provided a urine sample for testing on 8 March 2005. His sample tested positive for cocaine and its metabolites, these substances are on the Prohibited List of the
International Standard issued by World Anti-Doping Agency (WADA).

History
The athlete has no idea how he got contaminated. He does visit parties where he smokes.

Decision
I am satisfied that the Athlete was given every opportunity to participate in a hearing before me. He failed to respond to the various attempts made to acquire his participation in a hearing or waiver of the same. The parties were advised that I was closing the hearing in this matter and that it would proceed merely upon the written record I had received.
The proper selection of the Athlete for testing, the integrity of the sample collection and the chain of custody of the urine sample are all established on the record. The Lab has reported an adverse analytical finding and there is no indication that the chemical analytical process used by the Lab was in any way flawed.
Rule 7.17 and 7.18 make an athlete responsible for any prohibited substance found in the urine sample analysis. As was held in the decision of Arbitrator it is not necessary that intent, fault or knowing 'use* by an athlete be demonstrated to establish this anti-doping rule violation. I am comfortably satisfied that, on a review of all of the evidence before me, an anti-doping rule violation has occulted.
Cocaine is not a "specified substance" identified in Rule 7.7. It is a prohibited substance when it is detected at any level; a point made in the recent cocaine case, supra, by Arbitrator Mew. The failure of the Athlete to participate in these proceedings means that there is no necessity for me to refer to the Exceptional Circumstances Rules 7.38 and 7.39. There must be evidence from the Athlete, or presented on his behalf, that there was either no fault or negligence or no significant fault or negligence. The only evidence before me from the Athlete is the 11 April 2005 explanation sent to the CCES. That statement is one of denial and an inability to explain what has occurred. I take notice of the fact that cocaine cannot enter the human body by the consumption of alcoholic drink or the smoking of tobacco as referred to in the Athlete's explanation. In short, the Athlete has provided no explanation of the adverse analytical finding. In the absence of any satisfactory evidence, by way of explanation from the Athlete, I need not address further the Exceptional circumstances provisions of the Rules. They have no application in this case.
I find that an anti-doping rule violation has occurred under the CADP and the Rules prohibiting the use of a prohibited substance. In the circumstances I have no other choice than to impose the sanction for a first anti-doping rule violation of a two-year period of ineligibility and permanent ineligibility for direct financial support from the Government of Canada. The foregoing period of ineligibility starts on the date of this decision in accordance with the Rules.

Costs
No submission was made on costs. Unless applied for, I make no order in respect of the same.