Does the analysis of the enantiomeric composition of clenbuterol in human urine enable the differentiation of illicit clenbuterol administration from food contamination in sports drug testing?


M. Thevis, Center for Preventive Doping Research – Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.




Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzyl alcohol) is approved for human and veterinary use primarily for the treatment of pulmonary afflictions. Despite the authorized administration in cases of medical indications, the misuse of clenbuterol in animal husbandry as well as elite and amateur sport has frequently been reported, arguably due to growth-promoting properties. Due to various recent incidences of doping control specimens containing clenbuterol, strategies towards the discrimination of a surreptitious application from unintended intake via animal-derived edibles or dietary supplements were required.


The enantiomeric compositions of clenbuterol in human urine samples derived from administration studies with therapeutic amounts of the β2-agonist and authentic doping control specimens were determined. Due to the facts that therapeutic clenbuterol consists of a racemic mixture of (+)- and (−)-stereoisomers and that the first mentioned (dextrorotatory) stereoisomer is retained to a greater extent in edible animal tissue, the differentiation of a recent administration of therapeutic (and thus racemic) clenbuterol from food contamination (stereoisomerically depleted clenbuterol) was considered. Employing deuterated clenbuterol as internal standard, the target analytes were extracted from human urine by means of concerted liquid-liquid and solid-phase extractions and subjected to chiral liquid chromatography hyphenated to high resolution/high accuracy mass spectrometry with electrospray ionization.


Both enantiomers of clenbuterol were baseline separated and relative abundances of corresponding labeled and unlabeled stereoisomers were determined, demonstrating that the therapeutic use of clenbuterol results in racemic mixtures in urine for at least 24 h while adverse analytical findings presumably originating from food contaminations can yield (−)-clenbuterol-depleted pairs of analytes.


The determination of relative abundances of clenbuterol enantiomers can indicate the ingestion of clenbuterol via contaminated food; however, depletion of (−)-clenbuterol in edible animal tissue is time-dependent and thus results can still be inconclusive as to the inadvertent ingestion of clenbuterol when clenbuterol administration to animals was conducted until slaughter. Copyright © 2013 John Wiley & Sons, Ltd.

The sympathomimetic agent clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzyl alcohol, Fig. 1), which exhibits β2-agonist activities, was introduced in 1977 as a therapeutic means for various diseases affecting the respiratory system.[1-3] These health issues include e.g. asthma bronchiale and bronchial hyperreactivity, but also other medicinal applications of clenbuterol have been assessed in the past such as suppression of premature labor (tocolytics),[4, 5] counteracting muscle atrophy (caused by denervation,[6, 7] injury or microgravity[8-10]) or the treatment of heart failure especially in concert with ventricular assist devices.[11, 12] Here, the reported anabolic properties of clenbuterol are of particular relevance, which have also led to a considerable number of cases of misuse in animal husbandry as well as elite and amateur sport.[13-15] While analytical data can unambiguously prove the illicit administration of clenbuterol to domestic livestock (i.e., demonstrating that the drug was provided to animals destined for human consumption), an adverse analytical finding for clenbuterol in a doping control sample represents a complex issue as it might result from the deliberate misuse of clenbuterol (aiming at illicit performance enhancement) or possible ingestion of contaminated food or nutritional supplements.[16-18] Particularly the scenario of the oral intake of clenbuterol via contaminated meat is plausible as demonstrated in a study by Hemmersbach et al. in 1995 where calves received the drug until 1 or 6 days prior to slaughtering and consumers' urine samples were tested 'positive' for clenbuterol with urinary concentrations between 30 and 850 pg/mL.[19] In order to improve anti-doping efforts, the differentiation of the abusive administration of therapeutic clenbuterol preparations by cheating athletes from an inadvertent ingestion via meat products from improper stock farming is of great importance. Here the fact that the drug 'clenbuterol' is composed of a racemic mixture of the two enantiomers (−)- and (+)-clenbuterol (Figs. 1(a) and 1(b), respectively) provides useful information. As shown in earlier studies, therapeutic clenbuterol administration results in the urinary elimination of both stereoisomers in approximately equal amounts as demonstrated for a period of 18 h.[20] In contrast, the accumulation of clenbuterol in edible tissues of swine was found to be enantiomerically enriched towards the dextrorotatory (+)-clenbuterol in a time-dependent manner, i.e., with longer drug cessation periods the (+)-clenbuterol is enriched in tissues while (−)-clenbuterol is depleted.[21, 22] Consequently, ratios of renally eliminated clenbuterol enantiomers might differ in human urine depending on the administered composition, which is racemic in therapeutic clenbuterol and enantiomerically enriched in 'processed' residues found in edible tissues. In the present study, the ratios of enantiomers of urinary clenbuterol found in humans after single therapeutic or sub-therapeutic dosing were determined and compared to compositions of clenbuterol enantiomers in samples collected from elite athletes arguably being subjected to contaminated food.

Figure 1.

Chemical structures of the enantiomers of clenbuterol: (a) R-(−)-clenbuterol (active at the β2-adrenergic receptor) and (b) S-(+)-clenbuterol (inactive at the β2-adrenergic receptor).


Reference materials and chemicals

Clenbuterol (p.a.) was purchased from the National Measurement Institute of Australia (Sydney, Australia) and the deuterated internal standard, 2H9-clenbuterol (p.a.), from LGC Standards GmbH (Wesel, Germany). n-Heptane (HPLC-grade), potassium hydroxide (p.a.), and ammonia (7 M) in methanol were obtained from Sigma (Schnelldorf, Germany), and Strata-X-CW (30 mg, 1 mL) solid-phase extraction (SPE) cartridges were provided by Phenomenex (Aschaffenburg, Germany). Water was of MilliQ grade.

Sample preparation

A volume of 2 mL of urine was enriched with 2 ng of the internal standard (ISTD) 2H9-clenbuterol by adding 20 μL of a working solution containing 100 ng/mL before the addition of 100 μL of 5 M KOH and 5 mL of n-heptane. The sample was shaken for 10 min, centrifuged at 600 g and the organic layer separated and transferred to a fresh test tube. After addition of 1 mL of deionized water, the mixture was again shaken and centrifuged before removing the aqueous layer with a pipette. Finally, 1 mL of 2% acetic acid was used to re-extract the target analyte from the n-heptane layer by shaking (10 min) and centrifugation, followed by discarding the organic phase. The retained aqueous layer was transferred to a X-CW SPE cartridge preconditioned with 1 mL of methanol and 1 mL of water. After the volume of acetic acid had passed the adsorber resin by gravity flow, elution was accomplished with 2 × 250 μL of methanolic ammonia (5%). The combined eluate was collected in an Eppendorf tube and evaporated to dryness under reduced pressure. Subsequently, the dry residue was reconstituted in 100 μL of methanol containing 10 mM ammonium formate, and 20 μL were injected into the LC/MS system.

Liquid chromatography/(tandem) mass spectrometry

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was performed using an Agilent 1260 HPLC system equipped with a Chirobiotic T analytical column (2.1 × 150 mm, particle size 5 µm; Sigma, Taufkirchen, Germany), interfaced to an AB Sciex 5600 QqTOF mass spectrometer. The LC system was operated under isocratic conditions with methanol (containing 10 mM ammonium formate) at a flow rate of 300 μL/min. The effluent was directed via electrospray ionization (ESI) in positive mode into the mass spectrometer, operated with full scan (m/z 100–1100) and product ion scan (precursor ions [M+H]+ at m/z 277 for clenbuterol and [M+H]+ at m/z 286 for the ISTD) experiments. The instrument was set to 'high sensitivity' allowing for mass resolutions of approx. 15 000 (full width at half maximum) and calibrated every 10 injections to ensure high mass accuracy (error <3 ppm).

Assay characterization

The assay characteristics concerning recovery, ion suppression/enhancement, and limit of detection (LOD) were assessed by commonly accepted strategies. In order to determine the recovery, six different urine samples were spiked with 200 pg/mL of clenbuterol before sample preparation and another set was prepared as described and the final solution (prior to LC/MS analysis) was enriched with 400 pg of clenbuterol to represent 100% recovery of the analyte. Peak areas of clenbuterol signals (as generated from extracted ion chromatograms) of both sets of measurements were compared. Since relative abundances of enantiomers are of particular importance in the present study, matrix effects potentially influencing one or both isomers with different impact were evaluated (although ideally compensated by the deuterated analog of clenbuterol used as ISTD). Six different urine samples were tested for interferences and resulting ion suppression or enhancement effects compared to a methanolic solution of clenbuterol. All samples were analyzed in triplicate and peak areas of both enantiomers with and without correction via ISTD were calculated. The LOD (or, more precisely, the capability of the assay to allow for the comparison of peak area ratios of both enantiomers) was estimated from six different urine samples spiked to 50 pg/mL.

Administration study and doping control urine samples

Urine samples from two elimination studies were collected from healthy male individuals (39 and 55 years) having received a single therapeutic dose of clenbuterol (20 µg of clenbuterol hydrochloride, Spiropent tablet, Boehringer Ingelheim, Germany). The study was conducted with ethical approval and written consent was obtained from both participants. Urine was sampled prior to drug administration (blank urine specimen) and up to 162 h post-application. In addition, six doping control urine samples containing clenbuterol between 100 and 300 pg/mL were subjected to the established analytical assay for enantiomeric evaluation. The samples originated from athletes who tested positive in a large series of clenbuterol cases, where food contamination was found to be prevalent in the host country of the competition and warnings were issued beforehand to participants by national anti-doping organizations.[23] Over 100 adolescent football players (younger than 17 years) provided doping control samples containing clenbuterol during this tournament. Triggered by this high number of incidences, food (particularly meat) samples were collected and analyzed, which eventually proved to be contaminated with clenbuterol and, consequently, these residues of the illicitly fed growth promoter were identified as the most likely scenario of how the prohibited substance entered the athletes’ organism.[24]


The detection of clenbuterol in doping control urine samples is nowadays straightforward and sensitive. It happened to be, however, a considerable challenge to determine its origin (i.e. from intended drug abuse or inadvertent ingestion) as several case reports, food chemistry studies and governmental papers documented the issue of the misuse of clenbuterol in animal husbandry.[13, 14, 25, 26] In the present study, the potential utility of the enantiomeric distribution of clenbuterol in urine samples for sports drug testing purposes was investigated, aiming at a means to support the arguably justified 'meat contamination defense' of athletes being confronted with an adverse analytical finding.

Assay characteristics

Clenbuterol was efficiently extracted from urine samples by combined liquid-liquid and weak cation-exchange solid-phase extraction with an average recovery of 52%. By means of chiral HPLC, the enantiomers of clenbuterol were baseline separated under ESI-compatible conditions using methanol/ammonium formate, and the analytes were detected with high resolution/high accuracy tandem mass spectrometry (Fig. 2). Using nine-fold deuterated clenbuterol, adequate sample preparation and analysis were strictly controlled and ion suppression/enhancement effects were accounted for. Indeed, a significant ion suppression of approximately 60% was observed in one out of six spiked blank urine samples particularly affecting the first eluting (−)-clenbuterol enantiomer. Applying peak area ratios of both enantiomers and their corresponding ISTD, a valid comparison of corrected peak areas of the (−)- and (+)-enantiomers was possible. The LOD of the method was estimated with 50 pg/mL via signal-to-noise ratio (>3) combined with absolute signal intensity (a minimum of 100 counts per second was considered mandatory).

Figure 2.

Extracted ion chromatograms with diagnostic precursor-product ion pairs [m/z 277 → 203 (top pane) and m/z 286 → 204 (lower pane) for clenbuterol and the nine-fold deuterated ISTD, respectively] as obtained from high resolution/high accuracy MS/MS experiments. The peaks represent (−)-clenbuterol at 3.7 min and (+)-clenbuterol at 4.3 min. (a) Blank urine sample containing the ISTD only; (b) urine sample spiked with 200 pg/mL of clenbuterol (QC), and (c) authentic doping control urine sample.

Administration study and doping control urine samples

Two healthy male individuals received an orally administered dose of 20 µg of clenbuterol and urine samples were collected and analyzed using the developed methodology. The racemic mixture of the ingested drug was verified by analyzing six replicates yielding an average ratio of (−)-/(+)-clenbuterol of 0.96 (data not shown). As illustrated in Fig. 3(a), the administration studies with oral application of 20 µg of clenbuterol hydrochloride resulted in (−)-/(+)-clenbuterol ratios consistently higher than 1. Following peak values between 1.40 and 1.56 with the first urine samples collected after 2.5 and 2.0 h, respectively, the enantiomer ratios dropped to levels between 1.0 and 1.2 for the first 96 h with a slight but constant increase until the end of the sample collection (at 162.5 h). The curve is in accordance with literature data indicating a more pronounced retention of (+)-clenbuterol in tissue[21, 22] and is consequently consistent with a higher renal elimination of (−)-clenbuterol resulting in (−)-/(+)-clenbuterol ratios higher than 1.

Figure 3.

ISTD-corrected peak area ratios of urinary clenbuterol enantiomers measured after oral administration of 20 µg of clenbuterol hydrochloride (a). At no time point does the (−)-/(+)-clenbuterol ratio fall below 1.0 when therapeutic clenbuterol (i.e., the racemic mixture) is ingested. In the case of 'processed' clenbuterol, which was sufficiently long retained in a living organism, (−)-clenbuterol is depleted and can result in urinary enantiomer ratios considerably below 1.0 as found in one out of six doping control samples (b).

Six doping control urine samples containing clenbuterol presumably originating from contaminated meat were re-analyzed using the above presented approach, yielding the (−)-/(+)-clenbuterol ratios depicted in Fig. 3(b). Except for one sample, all enantiomeric ratios were determined above 1, indicating that in these five cases the ingested clenbuterol was not (yet) enantiomerically depleted. This scenario is expected when clenbuterol abuse in livestock farming is conducted right until slaughtering of the animals (as opposed to cessation of the growth promoter days or weeks prior to the slaughter). Under these circumstances, the tissue will comprise nearly racemic clenbuterol, which incapacitates the test option for the 'processed' drug. However, data as such found with sample number 5 are, with the current knowledge, not consistent with the administration of therapeutic preparations of clenbuterol and would serve as supporting evidence for the inadvertent administration of the prohibited compound via the athlete's diet.


Adverse analytical findings (AAFs) with clenbuterol in doping control samples represent a considerable challenge as to the determination of the drug's origin. Although the regulations of the World Anti-Doping Agency (WADA) generally follow the principle of strict liability (i.e., an anti-doping rule violation is established whenever an athlete's specimen is found positive for a prohibited substance or its metabolites, regardless of an inadvertent or intentional administration), it is of utmost importance to exploit all valid scientific means to protect the innocent athlete from sanctions. The enantiomeric depletion of clenbuterol in animal tissue after ingestion was demonstrated in the past and considered as a potential tool to support anti-doping authorities in deciding whether or not a suspension is required for an athlete whose doping control sample produced an AAF with clenbuterol. If the enantiomeric ratio found in the urine specimen is not consistent with those commonly observed in samples collected after therapeutic use of the drug, reasonable doubt as to the intentional ingestion of the prohibited substance is given and the athlete's guilt questionable. The approach however has proved so far suboptimal as the ratio of clenbuterol enantiomers in edible tissue is substantially influenced by the time course of drug administration to the animal. In other words, an enantiomeric ratio of (−)-/(+)-clenbuterol lower than 1 is definitely inconsistent with a drug administration (if the drug was based on a racemic mixture) and should therefore be considered in favor of the athlete; in contrast, a ratio higher than 1 does not necessarily prove clenbuterol drug abuse as the enantiomeric depletion is time-dependent, i.e., the ingestion of contaminated meat produced until slaughtering under clenbuterol-based growth-promoted farming might contain nearly racemic mixtures of clenbuterol and thus mimics a drug administration in the presented assay.

Nevertheless, the above reported methodology can be considered a starting point for follow-up studies, which should include the analysis of various different clenbuterol products concerning their enantiomeric composition. Moreover, additional administration studies with therapeutic and sub-therapeutic clenbuterol dosages are required to substantiate the pilot data presented in this communication, and further data on clenbuterol disposition in animals over defined periods of time are desirable.


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.