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The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes' claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed.

A simple and selective molecularly imprinted matrix solid-phase dispersion (MI-MSPD) method coupled with high performance liquid chromatography (HPLC) ultraviolet detection was developed for rapid screening of clenbuterol hydrochloride (CH) in chicken samples. The new molecularly imprinted microspheres (MIM) were synthesized by using butylamine and chloroaniline as dummy template with aqueous suspension polymerization and revealed good affinity to CH in aqueous solution. The application of the obtained MIM as sorbent of matrix solid-phase dispersion (MSPD) improved the selectivity of extraction procedure and avoided the effect of template leakage on quantitative analysis. Under the optimized conditions, good linearity of CH was obtained in a range of 0.059 18.30 μg mL(-1) with the correlation coefficient (R) of 0.9996. The recoveries of CH at three spiked levels were ranged from 92.0 to 99.1% with the relative standard deviation less than 4.0% (n = 3). The presented MI-MSPD-HPLC method combined the superiority of MIM and MSPD, and therefore could be potentially applied for the determination of CH in complicated biological samples.

Quantum dots (QDs) are semiconductor fluorescent nanoparticles, which can be used for food safety or environmental monitoring with high sensitivity. This work demonstrates the feasibility of detecting clenuterol residue in pig urine using CdSe/CdS quantum dots as fluorescent labels based magnetic core/shell Fe3O4/Au nanoparticles (MCFN) as solid carriers. The detection of clenbuterol is carried out by a fluoroimmunoassay-based biosensor using competitive binding between conjugated clenbuterol antigen-CdSe/CdS QDs and free clenbuterol with immobilized clenbuterol antibodies on MCFN. This assay method allows for clenbuterol determination in a linear working range of 0.5-20000 pg mL(-1). It would provide a simple, rapid, and ultra-sensitive detection method for clenbuterol or other biomolecular analysis.

An emerging trend is recognised in hormone and veterinary drug residue analysis from liquid chromatography tandem mass spectrometry (LC/MS/MS) based screening and confirmation towards accurate mass alternatives such as LC coupled with time-of-flight (TOF), Fourier transform ion cyclotron resonance (FTICR) or Fourier transform orbitrap (FT Orbitrap) MS. In this study, mass resolution and accuracy are discussed for LC/MS screening and confirmation of targeted analytes and for the identification of unknowns using the anabolic steroid stanozolol and the designer beta-agonist "Clenbuterol-R" as model substances. It is shown theoretically and experimentally that mass accuracy criteria without proper mass resolution criteria yield false compliant (false negative) results, both in MS screening and MS/MS confirmation of stanozolol. On the other hand, previous medium resolution accurate mass TOFMS/MS data of the designer beta-agonist were fully confirmed by high resolution FT Orbitrap MS(n) experiments. A discussion is initiated through a proposal for additional criteria for the use of accurate mass LC/MS technologies, to be implemented in Commission Decision 2002/657/EC.

Thirty-six broiler chickens were randomly assigned to .5, 1.0, or 2.0 ppm dietary [14C]clenbuterol HCl for a 2-wk period starting at 5 wk of age. Four birds from each treatment were slaughtered after withdrawal periods of 0, 7, or 14 d. Total radioactive residues (TRR; clenbuterol HCl equivalents) were measured in adipose tissue, kidney, liver, skin with adhering adipose tissue, bile, blood, brain, gastrointestinal tract, heart, lung, spleen, and testes; parent clenbuterol was measured in liver and kidney. In edible tissues, TRR were roughly proportional to dietary [14C]clenbuterol level and inversely proportional to duration of the withdrawal period; kidney TRR ranged from nondetectable (14 d of withdrawal, .5 and 1.0 ppm treatments) to 211.5 ppb for the 2.0 ppm treatment at zero withdrawal. Liver TRR were detectable for all treatment and withdrawal periods. Rapid depletion of TRR from edible tissues occurred during the first 7 d of the withdrawal period, but depletion of TRR was much slower thereafter. Parent clenbuterol was below the limit of detection (1 ppb) or was undetectable in liver and kidney for all dietary levels after 7 and 14 d of withdrawal, but it represented 22 to 48% of the total radioactive residues at 0 withdrawal. The inactive S (+) stereoisomer constituted approximately 73% of the total clenbuterol residue in livers of chickens slaughtered with no withdrawal period, and the active R (-) stereoisomer accounted for the remainder. These data indicate that radioactive residues of clenbuterol were present well after parent clenbuterol had depleted from edible tissues in chickens, and the predominant stereoisomer remaining in livers at slaughter was the inactive isomer.

The development of a supercritical fluid extraction procedure for the extraction of clenbuterol from bovine liver tissue is described. The procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol residues at the low ppb level. Method validation incorporating inter- and intra-assays was carried out on fortified liver tissue and showed good recoveries and low variations (RSD < 15%) for levels of clenbuterol of 0.5, 2 and 5 ppb. The developed procedure was shown to be successful for the determination of clenbuterol in incurred liver tissue. The effects of organic modifiers and of inherent sample moisture on analyte extractability are also presented.

In all EU member states, the use in livestock farming of certain substances having a hormonal action is prohibited. Clenbuterol, the beta-adrenergic agonist, has some growth promoting characteristics. Screening for clenbuterol can be carried out by an immunoassay. Gas chromatography-mass spectrometry (GC-MS) is very valuable for confirmatory purposes. In full scan MS it is impossible to fulfil the EU criteria of four diagnostic ions with one single ionisation mode. Some alternative possibilities are: (1) the use of two different ionisation modes, (2) the use of different derivatization methods or (3) the use of tandem MS. Each derivatisation or ionisation mode on its own did not give a sufficient number of ions. By combining these different possibilities we were able to obtain four ions, fulfilling the EU criteria.

beta-Agonists are substances used in veterinary and human medicine for the treatment of pulmonary disorders. They have found a use as growth promoters to improve meat-to-fat ratios in cattle but they are not authorized for use in the European Union. Due to their presence in trace levels (usually less than 1 microgram/kg), to the diversity of the illegally used compounds and to the complexity of the biological matrices analysed, the detection of these residues requires a very sensitive and specific method of determination. This work describes the strategy of analysis we developed for five beta-agonists in urine and liver. The combination of improved solid- or liquid-phase extraction methods and LC or GC-MS-MS (in the multiple reaction monitoring mode) has shown to provide a system suitable for the control of these substances. The efficiency of extraction and the sensitivity and selectivity allow this multiresidue detection down to, and below, the UK regulatory level of 0.5 microgram/kg. Moreover, the use of LC removes the need for the derivatisation step (cyclic methylboronate derivatives) which is required prior to GC-MS-MS analysis.