Adverse analytical findings with clenbuterol among U17 soccer

Drug Testing
and Analysis
Short communication
Received: 1 February 2013
Revised: 13 February 2013
Accepted: 16 February 2013
Published online in Wiley Online Library
( DOI 10.1002/dta.1471
Adverse analytical findings with clenbuterol
among U-17 soccer players attributed to food
contamination issues
Mario Thevis,a,b* Lina Geyer,g Hans Geyer,a Sven Guddat,a Jiri Dvorak,c,d
Anthony Butch,e Saskia S. Sterkf and Wilhelm Schänzera
The illicit use of growth promoters in animal husbandry has frequently been reported in the past. Among the drugs misused to
illegally increase the benefit of stock farming, clenbuterol has held a unique position due to the substance’s composition,
mechanism of action, metabolism, and disposition. Particularly clenbuterol’s disposition in animals’ edible tissues destined
for food production can cause considerable issues on consumption by elite athletes registered in national and international
doping control systems as demonstrated in this case-related study. Triggered by five adverse analytical findings with
clenbuterol among the Mexican national soccer team in out-of-competition controls in May 2011, the Fédération
Internationale de Football Association (FIFA) initiated an inquest into a potential food contamination (and thus sports drug
testing) problem in Mexico, the host country of the FIFA U-17 World Cup 2011. Besides 208 regular doping control samples,
which were subjected to highly sensitive mass spectrometric test methods for anabolic agents, 47 meat samples were
collected in team hotels during the period of the tournament and forwarded to Institute of Food Safety, RIKILT.
In 14 out of 47 meat samples (30%), clenbuterol was detected at concentrations between 0.06 and 11 mg/kg. A total of
109 urine samples out of 208 doping control specimens (52%) yielded clenbuterol findings at concentrations ranging from
1–1556 pg/ml, and only 5 out of 24 teams provided urine samples that did not contain clenbuterol. At least one of these
teams was on a strict ‘no-meat’ diet reportedly due to the known issue of clenbuterol contamination in Mexico. Eventually, owing
to the extensive evidence indicating meat contamination as the most plausible reason for the extraordinary high prevalence of
clenbuterol findings, none of the soccer players were sanctioned. However, elite athletes have to face severe consequences when
testing positive for a prohibited anabolic agent and sufficient supporting information corroborating the scenario of inadvertent
ingestion are required to be acquitted from anti-doping rule violations. Hence, governmental contribution is urgently needed to
combat the illegal use of clenbuterol in stock breading. Copyright © 2013 John Wiley & Sons, Ltd.
Keywords: sport; doping; mass spectrometry; meat; residue; anabolic agents
The therapeutic agent clenbuterol [(RS)-1-(4-Amino-3,5-dichlorphenyl)2-(tert-butylamino)ethanol, Figure 1) is an approved bronchodilator
and tocolytic agent, which has been further investigated as a potential
means in therapy concerning various different conditions including, for example, amyotrophic lateral sclerosis,[1,2] muscle atrophy,[3]
Pompe disease,[4] and heart failure.[5–8] However, the particular
growth-promoting properties of clenbuterol, whose mechanism(s)
of action have recently been further clarified,[9,10] have also been
a temptation to abuse as proven, for example, in stock farming/
meat production[11–16] and elite sport,[17] which resulted in the
prohibition of the drug in animal husbandry[18,19] and sport.[20]
The issue of illicit administration of clenbuterol to animals destined
for food production has evidently resulted in several cases and
epidemics of poisoning,[13,21–24] stressing particularly the corresponding health problems. In addition, the contamination of
dietary products with clenbuterol can affect an athlete’s integrity
and career when the drug is inadvertently ingested,[25] resulting
in an adverse analytical finding during doping controls.[26–28]
Despite the continuously improving analytical options and
methods in sports drug testing programmes especially by
means of liquid chromatography/(tandem) mass spectrometry
Drug Test. Analysis (2013)
[LC-MS(/MS)],[26,29,30] the differentiation of therapeutic/deliberately
administered clenbuterol from ingested unintentionally residues
remains a complex challenge.[31] Consequently, any adverse
* Correspondence to: Mario Thevis, Institute of Biochemistry - Center for
Preventive Doping Research, German Sport University Cologne, Am Sportpark
Müngersdorf 6 50933 Cologne, Germany. E-mail: [email protected]
a Institute of Biochemistry - Center for Preventive Doping Research, German Sport
University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany
b European Monitoring Center for Emerging Doping Agents, Cologne/Bonn,
c Schulthess Klinik, Lengghalde 2, CH-8008 Zurich, Switzerland
d Fédération Internationale de Football Association (FIFA), Zurich, Switzerland
e Olympic Analytical Laboratory, Department of Pathology and Laboratory
Medicine, University of California Los Angeles, CA, 90025-6106, USA
f RIKILT Wageningen UR- Institute for Food Safety, Wageningen, the
g Institute of Cardiovascular Research and Sport Medicine – Department of Molecular
and Cellular Sport Medicine, German Sport University Cologne, Germany
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Testing
and Analysis
M. Thevis et al.
clenbuterol[26] was employed to probe for the prevalence of the
prohibited compound.
Analysis of meat samples
Figure 1. Structure formulae of clenbuterol with both enantiomers: a)
R-( )-clenbuterol and b) S-(+)-clenbuterol.
analytical finding (AAF) necessitates careful investigation and evaluation of circumstantial evidence that might prove the athlete’s innocence.[32] In the present communication, the inquest into a food
contamination issue concerning elite soccer players who competed
in Mexico in 2011 is presented. Induced by five AAFs of the Mexican
seniors’ national soccer team with clenbuterol in May of 2011, the
Fédération Internationale de Football Association (FIFA) recognized
the need for a thorough investigation of the circumstances of these
and potential future anti-doping rule violations, particularly in the
light of the FIFA U-17 World Cup 2011 to be held between 18 June
and 10 July 2011 in Mexico. Hence, during the period of this U-17
competition, samples from meals containing meat were collected
at team-hosting hotels concomitantly to doping control specimens
routinely sampled at competition sites from randomly selected
players. While urine samples were subjected to regular doping controls, meat specimens underwent dedicated clenbuterol analyses.
Both food/meat samples and doping control urine specimens
were sampled in seven Mexican cities hosting the FIFA U-17
World Cup 2011, namely Guadalajara, Mexico City, Torreón,
Morelia, Monterrey, Querétaro, and Pachuca. Their geographical
locations are depicted in Figure 2.
A total of 47 food samples were collected from meals served in
the restaurants catering the competing teams. The samples
varied in type from lasagna sauce containing meat, to tenderloin
steak, veal, chicken, turkey, salmon, and catfish. Specimens
were sent out for clenbuterol analysis to the RIKILT Wageningen
UR – Institute for Food Safety employing accordingly validated
and accredited methods. A validated method using LC-MS/MS was
employed based on a RIKILT standard operating procedure.[33–35]
The decision limit (CCalfa), the limit at and above which it can be
concluded with an a-error probability of < 1% that a sample is
non-compliant[36] is 0.01 microgram/kg.
Results and discussion
The use of clenbuterol in animal husbandry is prohibited in
the entire European Union (EU) and, consequently, this ban
(indirectly) extends to all meat-exporting countries aiming at
selling their meat produce to EU member states. The risk of
inadvertent ingestion of compounds such as clenbuterol via
contaminated food has been reported for various countries in
the past, among which Mexico has recently hosted great sporting
events such as the FIFA U-17 World Cup 2011 discussed herein.
Being aware that illicit methods in stock farming and meat
production might result in issues for athletes undergoing doping
control tests,[37,38] FIFA decided to initiate and support the
inquest into clenbuterol findings obtained before and during
the tournament; in addition to the regularly collected doping
control specimens, it organized the sampling and analysis of
food specimens obtained from meals served at the team hotels
in Mexico.
Analysis of doping control urine specimens
Two hundred and eight (208) doping control urine samples were
collected during the tournament (32 in Guadalajara, 8 in Mexico
City, 32 in Torreon, 32 in Morelia, 32 in Monterey, 36 in Queretaro,
and 36 in Pachuca) according to official guidelines and shipped
to the assigned World Anti-Doping Agency (WADA) accredited
laboratories for analysis. A validated sensitive LC-MS/MS-based
detection method allowing for detection limits of 1 pg/ml for
Analysis of doping control urine specimens
Employing highly sensitive analytical methods for the detection
of clenbuterol in urine samples, a total of 109 out of 208 drug test
specimens (52%) were shown to contain the prohibited drug. The
observed concentrations ranged from 1.3 to 1556 pg/ml
(corrected to a specific density of 1.020) with an average value
of 105 pg/ml (standard deviation 273 pg/ml), and a median of
28 pg/ml (Table 1). The simplified distribution of clenbuterol
concentrations found in sports drug test samples is illustrated
in Figure 3, showing the majority of findings (72) with less than
50 pg/ml while 4 samples contained more than 500 pg/ml of
clenbuterol. Since clenbuterol is not a threshold substance, any
finding that is confirmed according to the requirements of WADA
represents an AAF that entails respective consequences. The
code numbers of doping control samples were assigned through
FIFA to the host cities of the athletes, resulting in data presented
Table 1. Summary of adverse analytical findings in urine samples –
descriptive statistics
Number of
findings [n]
Figure 2. Geographical location of the Mexican cities hosting the FIFA U-17
World Cup 2011.
conc. [pg/ml] [pg/ml]
Copyright © 2013 John Wiley & Sons, Ltd.
Drug Test. Analysis (2013)
Drug Testing
and Analysis
Clenbuterol findings among U17 soccer players attributed to food contamination issues
Table 3. Presentation of clenbuterol findings per team. Dark-grey
colouring indicates teams where all collected samples contained
clenbuterol and light-grey colouring is used for squads tested entirely
Doping control samples [n]
Figure 3. Clenbuterol concentrations observed in 109 out of 208 doping
control specimens collected during the tournament.
in Table 2, which demonstrate that AAFs originated from all
competition locations; however, it cannot be excluded that an
athlete ingested contaminated produce in one city before being
transferred to another city where a test sample might have been
collected. The ten samples yielding the highest clenbuterol
concentrations were collected in Queretaro (5 findings between
209 and 1556 pg/ml), Monterrey (4 findings between 389 and
1500 pg/ml), and Guadalajara (1 finding at 265 pg/ml), while
samples collected in Torreon did not exceed levels higher than
18 pg/ml.
In total, 24 teams participated in the FIFA U-17 World Cup 2011
and only 5 did not produce any AAF for clenbuterol. The teams 5,
14, 17, 21, and 24 (Table 3, grey background) provided the sum
of 42 doping control urine samples that all returned negative
test results. One of these squads declared to have followed
a strict ‘no-meat’ diet during the tournament due to the
public warning[11,37] issued concerning meat contamination
problems related to illicitly used growth promoters in Mexico
(Prof. J. Dvorak, pers. comm.). In contrast, urine specimens from
teams 4, 10, 16, and 20 (Table 3, black background) were all
found to contain clenbuterol, and the remaining 15 participating
squads provided at least one AAF during their presence at the
World Championships.
Analysis of meat samples
By means of specialized detection assays, 47 meat samples were
qualitatively and quantitatively tested for clenbuterol. In 14
specimens (30%), clenbuterol was detected at 0.06 – 11 mg/kg
Positive [n (%)]
6 (75)
5 (62)
13 (93)
6 (100)
0 (0)
5 (62)
3 (37)
1 (17)
5 (83)
8 (100)
6 (60)
9 (90)
11 (79)
0 (0)
5 (50)
6 (100)
0 (0)
1 (17)
1 (12)
8 (100)
0 (0)
9 (64)
1 (12)
0 (0)
with an average value of 2.5 mg/kg and a median of 0.6 mg/kg
(Table 4). The simplified distribution of clenbuterol in the
different meat samples is illustrated in Figure 4. The majority of
the findings (9) were below 2 mg/kg while 3 samples showed
high concentrations of 6, 10, and 11 mg/kg clenbuterol. The
highest clenbuterol concentration of 11 mg/kg was observed in
a specimen declared as beef/chicken.
These reported analytical results corroborate the concerns
raised regarding inadvertent ingestion of clenbuterol, potentially
as well as evidently leading to adverse analytical findings in
doping controls. In agreement with earlier studies,[11,22,38] meat
contamination was proven in selected cases of meal samples
collected at hotels accommodating the participating teams,
although much effort was put into enforcing the ban of
clenbuterol misuse in Mexico by respective authorities, which
led to a reduction of observed contamination incidences from
Table 2. Compilation of specifics of adverse analytical finding in relation to the host cities
City of competition
Samples collected [n]
Mexico City
Drug Test. Analysis (2013)
findings [n (%)]
24 (75)
5 (63)
16 (50)
16 (50)
11 (31)
32 (89)
5 (16)
Average conc.
SD [pg/ml]
Copyright © 2013 John Wiley & Sons, Ltd.
Median [pg/ml]
Min [pg/ml
Max [pg/ml]
Drug Testing
and Analysis
M. Thevis et al.
Table 4. Summary of adverse analytical findings in meat samples –
descriptive statistics
Number of average conc.
findings [n]
[mg/kg] [mg/kg]
Figure 4. Clenbuterol concentrations observed in 14 out of 47 meat
samples collected during the tournament.
555 (in 2005) to 89 (in 2010). Nevertheless, the problem persisted.
Based on this reported data, which corroborated the scenario of
unintentional clenbuterol intake by over 50% of the athletes
attending the FIFA U-17 World Cup 2011, all players were
acquitted from anti-doping rule violations per decision of FIFA
and WADA and no sanctions were awarded[39] as the ingestion
of as little as 1 mg of clenbuterol can lead to the urinary concentrations reported herein.[40] The dimension of the Mexican food
contamination issue, which has affected public health as well as
the world of sport, became eventually even more evident when
Mexican government inspectors shut down another 14 livestock
markets due to the detection of clenbuterol traces in meat
samples later in 2011.
The study was supported by the Federal Ministry of the Interior of
the Federal Republic of Germany, the Fédération Internationale
de Football Association (FIFA, Zurich), and the Manfred-Donike
Institute for Doping Analysis (Cologne), Germany.
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