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Clenbuterol testing in doping control samples: drug abuse or food contamination?

Clenbuterol testing in doping control samples: drug abuse or food contamination?

Meat as a Doping Trap

Clenbuterol, a ß2-sympathomimetic drug, has been on the list of ­substances prohibited in sport for over 2 decades. ­Due to its putative performance-­enhancing properties, urine samples are routinely tested for its presence during doping control: by using modern liquid chromatography-mass spectrometry instru- ments, detection limits at the level of just a few picograms per millilitre are possible. This sensitivity not only makes ­detection possible long after use of the ­illegal ­substance ­has been discontinued but, ­for a ­number of regions outside Europe, has also ­uncovered a food contamination problem that could make life very difficult for professional ­athletes.

Clenbuterol is a racemic mixture of two enantiomers (Fig.1). Banned in sport since 1992, its presence is usually tested for in doping control with the aid of liquid chromatography/(tandem)-mass spectrometry. On account of its anabolic properties, clenbuterol is not listed in sport as a ß2 agonist, but in the category S1.2 (”Other Anabolic Agents“) and is thus prohibited at all times, i.e. both during the sporting event itself and at any other time [1]. There is no threshold limit for clenbuterol: the detection of the analyte in doping control samples immediately results in an ”adverse analytical finding“ (AAF). To maximise the potential time frame for detection following discontinuation of a (presumably illegally) consumed substance, instrumental analysis has been pursuing significant improvements over the last few years. This has resulted in very low detection limits and, as a consequence, athletes’ consumption of clenbuterol has been proven in a great many cases. Figure 2 gives a typical example of analytical clenbuterol testing, on a urine sample containing approx. 3pg/ml clenbuterol. The method used here consists of a liquid-liquid extraction from urine into methyl tert-butyl ether with subsequent re-extraction into aqueous hydrochloric acid (0.06 M) and LC-MS/MS analysis.


Fig. 1 Clenbuterol enantiomer structures: a) (-) clenbuterol (active isomer) and b) (+) clenbuterol (inactive isomer)

Clenbuterol in foodstuffs

The fact that clenbuterol ­exhibits anabolic effects – especially at high doses of the drug – has also led to abuses of the substance in the area of meat production. As a result, this industry has banned its use in food production at an international level [2]. Yet in 2010 and 2011, athletes tested positive for clenbuterol after participating in overseas sporting events outside the European Union (in China and Mexico). The results are traceable back to contaminated foodstuffs. In 2010, routine doping control of a German team returning from a tournament in China found low – yet clearly detectable – concentrations of clenbuterol in the urine of every single member of the squad [3]. A follow-up study, conducted with people residential in China and with tourists staying in China for various lengths of time and in various locations, further illustrated the problem of illegal use of clenbuterol in animal feed: based on current anti-doping regulations, no fewer than 22 of the 28 volunteers tested would have returned ”positive“ test results. As a number of anti-doping organisations were aware of this problem in advance of the Olympic Games in 2008, advisory notices warning against eating meat in China were circulated to athletes, so as to guard against inadvertent consumption of clenbuterol and falling into this ”doping trap“. After all: differentiating between a drug abuse incident sometime in the past – and thus relevant for doping control – and a low dose as consumed in contaminated food is, in analytical terms, a highly sophisticated task.

In Mexico, a similar situation can be found: here, too, a series of results from food testing have uncovered the unlawful use of clenbuterol in animal feed over the last few years [4, 5]. The potential repercussions for professional athletes were highlighted first in May 2011, when five players of the Mexican national squad (men’s football) tested positive for the b2 agonist [6]. This finding proved to be especially problematic, since the U-17 World Cup for junior football teams was scheduled to take place the following month. As a result, soccer governing body FIFA organised a comprehensive programme of food quality testing alongside routine doping controls during the tournament. All in all, athletes submitted 208 urine samples for doping control testing. The analytical findings revealed the presence of clenbuterol in 109 cases, with concentrations of up to 1500 pg/ml urine [7]. The food samples taken in parallel were tested in a suitably-equipped laboratory in the Netherlands, with 14 of the 47 samples containing – in some cases considerable – quantities of clenbuterol. Viewed as sufficient to account for the drug’s presence in the doping control samples, no player was sanctioned for breaching the anti-doping rules. A breakdown of the samples by tournament location revealed no clear trend: the matches had been played in Guadalajara, Mexico City, Monterrey, Morelia, Pachuca, Querétaro and Torréon, with 8 to 36 players being tested at each location. Since some teams played matches at several locations, no conclusions can be drawn from urine samples about the place where contaminated food was eaten. One interesting aspect may be noted, however: of the 24 teams taking part, five teams returned consistently negative sets of doping control results. Of these, at least one had heeded the advisory notice and refrained from consuming meat for the entire competition.


Fig. 2 Extracted-ion chromatograms of a urine sample with approx. 3pg/ml clenbuterol (left), compared to a blank urine sample. The enantiomers remain unseparated by conventional liquid chromatography.


Fig. 3 Extracted-ion chromatogram of a urine sample with approx. 200pg/ml clenbuterol (A) with separation of the (-) and (+) enantiomers at 3.7 and 4.3 min. The upper patterns represent clenbuterol, while the lower patterns depict the 9x(2)H-labelled internal standard, also present as a racemate. As a comparison, (B) shows a blank urine sample.

Challenges for analytical testing

These examples clarify the analytical difficulties surrounding clenbuterol in the context of doping control testing. Accordingly, a number of approaches have been taken to generating analytical methods capable of differentiating between foodstuff contamination and previous (non-recent) deliberate consumption of the drug. One of the more recent strategies adopted relies on the fact – as mentioned above – that clenbuterol is a racemate (see Fig.1) in its drug formulation. After administering clenbuterol, a pig study has shown that enrichment in edible tissue (e.g. muscle) is considerably higher for the therapeutically inactive (+) stereoisomer than for the (-) stereoisomer. Accordingly, once clenbuterol treatment is discontinued, a significant disparity in concentration between the two components will develop over time [8].

Enantiomer analysis offers new potential

This would mean that there is a functional difference between a drug product dose and food contamination: a difference that is detectable insofar as the (-) stereoisomer complement of clenbuterol has undergone sufficient depletion. To test this hypothesis, a method for enantiomer separation with subsequent isotope dilution analysis using LC-MS/MS was developed, with the aim of determining the enantiomeric ratio and thus potentially identifying either a case of therapeutic drug dosage or a case of contamination. Figure 3 presents a chromatogram of a urine sample with clenbuterol following enantiomer separation: baseline separation is clearly visible here. Two excretory studies with simple therapeutic doses of clenbuterol were conducted. Results were as expected: the (-)/(+) enantiomer ratio in urine never fell below 1 at any point during the 160 hours of testing. This is due to the fact that the (+) stereoisomer also exhibits elevated tissue retention in such cases, meaning that the (-) stereoisomer is excreted to a significantly greater degree [9]. Following the oral intake of a clenbuterol enantiomer mixture with the (-) stereoisomer component already depleted (as might occur in cases where contaminated meat is consumed), the ratio may show a decremented value that cannot be successfully reconciled with therapeutic administration of the substance. While the authors are not aware of clearance studies involving enantiomer-depleted clenbuterol mixtures, results from athletes’ urine samples have nonetheless shown that (-)/(+) ratios significantly lower than 1 have been determined in a number of cases. In consideration of the fact that a value greater than 1 cannot prove doping took place, yet a value less than 1 is, on the basis of current scientific knowledge, inconsistent with the therapeutic administration of clenbuterol as a drug in humans, the determination of the enantiomer ratio for clenbuterol findings is certain to contribute some useful data. While other – potentially wide-ranging – studies are required to evaluate the potency of enantiomer analysis, one possible approach seems nonetheless practicable.

Bibliography
[1] World Anti-Doping Agency. The 2013 Prohibited List. 2013, http://www.wada-ama.org/en/World-Anti-Doping-Program/Sports-and-Anti-Doping-Organizations/International-Standards/Prohibited-List/ (07-01-2013)
[2] SGS. Clenbuterol – contaminated meat hits China, Mexico, and sports. 2012, http://www.industrial-newsroomcom/news-detail/browse/1/t/meat-contaminated-with-clenbuterol-in-china-mexico-and-spain/?tx_ttnews[backPid]=104&cHash=cb9204c612 (16-01-2013)
[3] S. Guddat et al. (2012), Drug Test. Anal. 4: 534
[4] M. C. Estrada-Montoya et. al (2008), Ciencia Y Tecnologia Alimentaria. 6: 130
[5] ProMED. Clenbuterol food poisoning, meat – Mexico. 2010, (30-10-2012).
[6] Reuters, Five Mexico internationals at Gold Cup test positive for clenbuterol. In: The Guardian 2011
[7] M. Thevis et al. (2013), Drug Test. Anal.; in press: 10.1002/dta.1471
[8] D. J. Smith (2000), J. Agric. Food Chem. 48: 6036
[9] M. Thevis et al. (2013), Rapid Commun. Mass Spectrom. 2013; 27: 507

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L&M orient 1 / 2014

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