Challenges in detecting substances for equine anti-doping.

The artificial increase of the physical capability of horses using drugs is well known in racing and other equine sports. Both illicit and therapeutic substances are regarded as prohibited substances in competition in most countries. Some countries make distinctions for a few, specific drugs which are, however, allowed for use in other countries. The primary objective in the case of doping control is the detection of any trace of drug exposure, either parent drug or any of its metabolites, using the most powerful analytical methods which are generally based on chromatographic/mass spectrometric techniques. Of major concern in horseracing is the absence of a single organization regulating the anti-doping framework; instead of this, individual racing authorities provide rules and regulations often resulting in variations in the applied doping control programmes of different countries. The aim of this paper is to review the recent literature (approximately from 2012 to mid-2016) to highlight the numerous and diverse challenges faced in doping control of racing and equestrian sports, including the detection of designer drugs (anabolic steroids or stimulants) and of other emerging prohibited substances, such as peptides and noble gases in horse urine and plasma. Moreover, the application of 'omics' techniques (especially of metabolomics) deserves attention for establishing possible fingerprints of drug abuse as well as the evolution of instrumental analysis resulting a powerful ally in the fight against doping in equine sports. Copyright © 2017 John Wiley & Sons, Ltd.

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin.

BACKGROUND: The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. METHODS: The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. RESULTS: The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 degrees C or storage at -20 degrees C. CONCLUSIONS: The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.

Stabilization of human urine doping control samples: II. microbial degradation of steroids.

The transportation of urine samples, collected for doping control analysis, does not always meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency (WADA) accredited laboratories. Because sample collection is not conducted under sterile conditions, microbial activity may cause changes to the endogenous steroid profiles of samples. In the current work, funded by WADA, a chemical mixture consisting of antibiotics, antimycotic substances and protease inhibitors was applied in urine aliquots fortified with conjugated and deuterated steroids and inoculated with nine representative microorganisms. Aliquots with and without the chemical mixture were incubated at 37 degrees C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at -20 degrees C as reference. Microbial growth was assessed immediately after inoculation and at the end of the incubation period. Variations in pH and specific gravity values were recorded. Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical stabilization mixture to urine samples inhibited microorganism growth and prevented steroid degradation at 37 degrees C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 degrees C.