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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.

Besides the identification point system to assure adequate set-up of instrumentation, European Commission Decision 2002/657/EC includes performance criteria regarding relative ion abundances in mass spectrometry and chromatographic retention time. In confirmatory analysis, the relative abundance of two product ions, acquired in selected reaction monitoring mode, the ion ratio should be within certain ranges for confirmation of the identity of a substance. The acceptable tolerance of the ion ratio varies with the relative abundance of the two product ions and for retention time, CD 2002/657/EC allows a tolerance of 5%. Because of rapid technical advances in analytical instruments and new approaches applied in the field of contaminant testing in food products (multi-compound and multi-class methods) a critical assessment of these criteria is justified. In this study a large number of representative, though challenging sample extracts were prepared, including muscle, urine, milk and liver, spiked with 100 registered and banned veterinary drugs at levels ranging from 0.5 to 100 µg/kg. These extracts were analysed using SRM mode using different chromatographic conditions and mass spectrometers from different vendors. In the initial study, robust data was collected using four different instrumental set-ups. Based on a unique and highly relevant data set, consisting of over 39 000 data points, the ion ratio and retention time criteria for applicability in confirmatory analysis were assessed. The outcomes were verified based on a collaborative trial including laboratories from all over the world. It was concluded that the ion ratio deviation is not related to the value of the ion ratio, but rather to the intensity of the lowest product ion. Therefore a fixed ion ratio deviation tolerance of 50% (relative) is proposed, which also is applicable for compounds present at sub-ppb levels or having poor ionisation efficiency. Furthermore, it was observed that retention time shifts, when using gradient elution, as is common practice nowadays, are mainly observed for early eluting compounds. Therefore a maximum retention time deviation of 0.2 min (absolute) is proposed. These findings should serve as input for discussions on the revision of currently applied criteria and the establishment of a new, globally accepted, criterion document for confirmatory analysis. Copyright © 2016 John Wiley & Sons, Ltd.

Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

According to EU legislation a confirmatory method used for residue analysis should be able to confirm the identity of a compound beyond reasonable doubt. To provide an adequate instrumental set-up, Commission Decision 2002/657/EC introduced the concept of "identification points". A second aspect to assure unequivocal confirmation, is the establishment of ion ratio and retention time criteria. Currently, the gold standard for confirmatory analysis of most veterinary drug residues is liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) acquisition mode, isolating one precursor ion and monitoring two a priori selected product ions, yielding 4 identification points. We comprehensively evaluated the use of different low and high resolution LC-MS(/MS) techniques and acquisition modes with respect to the selectivity of 100 veterinary drugs in liver, muscle and urine extracts aiming to critically review the currently established identification points system. A comparison among MS/MS in SRM mode with high resolution mass spectrometry (HRMS) in full scan, all ion fragmentation and targeted MS/MS was made based on a unique inter-laboratory study, which comprises 21 laboratories from four different continents and equipment from all major vendors. In total 186 samples were analysed yielding results for 9282 analyte/matrix combinations. It was observed that the false positive rate approximately doubles if no ion ratio criterion is applied indicating that this criterion is important to prevent false positive results. Full scan HRMS analysis, only monitoring the molecular ion and allowing a ±5 ppm mass tolerance is, in general, less selective than low resolution MS/MS using SRM, and thus full scan alone is considered not sufficient for confirmatory analysis. Furthermore, even though the number of data on all ion fragmentation and targeted MS/MS at high resolution was limited, based on the data obtained, it was observed that the acquisition mode as well as the mass resolution needed, very much depend on the matrix and the compound itself. For complex matrix extracts and non-selective compounds (worst-case situation), only targeted MS/MS, monitoring the precursor ion and a single product ion in HR-MS using a maximum of ±5 ppm mass deviation, leads to comparable selectivity and false positive and negative rate as SRM monitoring two product ions in LR-MS. We conclude that the currently applied identification point system as established in commission decision 2002/657/EC should be revised with respect to the allocation of identification points.

The effect of 17ß-19-nortestosterone (17ßNT) treatment of barrows on residue levels and growth was evaluated. Five barrows were treated three times during the fattening period with 17ßNT phenylpropionate (Nandrosol, nandrolone phenylpropionate 50¿mg/ml,1¿mg/kg body weight). Another five barrows were untreated and five boars (untreated) were kept as positive control. Boars and treated barrows showed a 13 and 9% improvement in growth compared to untreated barrows, with mean final body weights of 121.6, 117.8 and 109.0¿kg, respectively. The bulbourethral glands of the treated barrows were three times heavier than untreated barrows. The histology of the prostate and bulbourethral gland of the treated barrows was comparable to the boars, whereas the control barrows showed atrophic glands. Levels of 17ßNT ester in hair from treated barrows were high, whereas boars and untreated barrows did not show levels above LLQ. It is concluded that analysis of hair can detect illegal treatment with 17ßNT ester in barrows. The size of the bulbourethral gland can also be used for screening in the slaughterhouse.

A new sensitive, fast and robust method for the determination of paraquat and diquat residues in potatoes, cereals and pulses is presented. Different extraction conditions (solvent, time and temperature) have been evaluated using barley grain, potatoes and dry lentils containing incurred residues of diquat and paraquat. The finalised procedure involves extraction with a mixture of methanol/water/hydrochloric acid at 80 °C and analysis by liquid chromatography-tandem mass spectrometry. Diquat D4 and Paraquat D6 internal standards were added to the test portions prior to extraction. A small-scale inter-laboratory validation of the developed method for diquat and paraquat using potato and barley samples was conducted by three laboratories. The precision and accuracy of the method were determined from recovery experiments (five replicates) at 0.01 and 0.1 mg kg(-1). The recoveries obtained (n = 180) were in the range of 92-120 % with associated relative standard deviation (RSD) between 1.4-10 % for all compound/commodity/spiking concentration combinations.