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?

Abstract

RATIONALE: Clenbuterol (4-amino-a-[(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. METHODS: The enantiomeric compositions of clenbuterol in human urine samples derived from administration studies with therapeutic amounts of the b 2 -agonist and authentic doping control specimens were determined. Due to the facts that therapeutic clenbuterol consists of a racemic mixture of (+)-and (À)-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. RESULTS: 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 (À)-clenbuterol-depleted pairs of analytes. CONCLUSIONS: The determination of relative abundances of clenbuterol enantiomers can indicate the ingestion of clenbuterol via contaminated food; however, depletion of (À)-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.

Anabolic agents: recent strategies for their detection and protection from inadvertent doping

“…The antidoping research, which also includes investigations to protect athletes from inadvertent doping, is now focused on the development of methods to differentiate between clenbuterol originating from medication and from contaminated meat. Promising results are provided by studies of pharmacokinetics and metabolism of clenbuterol 54 55. A further tool for the differentiation of inadvertent and intentional ingestion of doping agents maybe hair analysis 56…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…By means of single quadrupole GC-MS systems, LODs of 2 ng/mL were accomplished, just meeting the MRPL of 2012; however, in 2013, the MRPL for clenbuterol was lowered to 0.2 ng/mL [79] thus necessitating assays with substantially better sensitivities. [82] By means of chiral liquid chromatography and high resolution/high accuracy tandem mass spectrometry, doping control as well as elimination study urine samples containing clenbuterol were analyzed, demonstrating that such enantiomeric analyses can support the interpretation of clenbuterol findings; however, inconclusive results were also obtained, demonstrating the need for further research. LC-MS(/MS) however outperformed GC-MS-based assays as shown by Nicoli et al, reporting on a methodology enabling the quantification of clenbuterol in urine at a lower limit of quantification (LOQ) of 5 pg/mL.…”

Effects of clenbuterol enantiomers on growth of young male rats

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