Race for the prize

Whether it is sports doping, or food traceability issues – detection and quantification of Clenbuterol is becoming vital. Here we discover that LC-MS is more than up to the task at hand

Clenbuterol is a sympathomimetic amine (beta-2 agonist) used in the treatment of breathing disorders, as it relaxes the smooth muscles in the airways acting as a bronchodilator. A common side effect is due to its thermogenic properties, which increase basal metabolism rates, blood pressure, and oxygen transport helping build muscle mass, while reducing body-fat. These combined properties – characteristic of a beta-2 agonist with thermogenic side-effects – resulted in Clenbuterol being labeled as a banned substance in Olympic athletes and for animals used for human consumption. Recently, a case study using a liquid chromatography triple quadrupole mass spectrometer (LC-TQ) demonstrated excellent accuracy and precision results for the low level quantification of Clenbuterol in equine plasma for the purposes of routine screening.

Clenbuterol is routinely administered to horses as a bronchodilator and to fight lung infections by veterinarians and trainers, however, its use in horseracing is controversial, with many US states banning the substance. The Association of Racing Commissioners International (ARCI) imposes a minimum fine of $1000 for a trainer’s first offence; while it is banned in Canada in any Equine Canada sanctioned competitions. Its human use has also received attention with professional cyclist Alberto Contador being banned for two years after testing positive for Clenbuterol and his victory revoked in the 2010 Tour de France1.  Celebrities have abused Clenbuterol because of its repartitioning effect of promoting muscle formation over the deposition of fat2  and in some countries, like the United Kingdom, Clenbuterol is banned as a Class C drug, with a maximum penalty of fourteen years’ imprisonment for possession with intent to supply³. There has also been evidence that meat contaminated with the drug is being produced and sold on the global market, despite it being banned from the human food chain in both the USA and the EU. During the recent horsemeat scandal in Europe, several products labeled as beef were found to contain horsemeat from animals treated with the drug. There were also instances in China where Clenbuterol-tainted pork caused hundreds of people to become sick⁴.

Racing associations restrict the use of Clenbuterol in the period preceding competitions, because of the effect that it can have on athletic performance. The drug can have a short-term stimulatory impact on the oxygen consumption and cardiovascular function of a healthy horse, providing an unfair advantage during races5. There is currently significant debate in the USA over regulation of Clenbuterol, as US state associations set their limits individually. In recent years, the trend has been towards more strict regulation. Two years ago, the American Quarter Horse Association (AQHA) officially advanced a proposal for a 30 day withdrawal of Clenbuterol prior to a horse competing in a race, and established threshold levels of 200pg/ml or less in urine and 1-2pg/ml in blood6. Increased withdrawal periods were subsequently adopted by associations in several states, including New Mexico, California and Oklahoma⁷. However, following extensive consultations with racing bodies, veterinarians and industry chemists and pharmacologists, the regulators of eight US states have committed to implement the Mid Atlantic Uniform Medication Program⁸. This agreement sets a lower withdrawal period for Clenbuterol of 14 days prior to racing. This lower limit reflects advances in the detection and quantification of Clenbuterol in horses’ systems, in the highly sensitive testing of horse serum with LC-MS systems.

Because of the low concentrations found in horse serum, regulators previously relied on urine samples in testing for Clenbuterol. However, urine possesses several unsatisfactory characteristics as a forensic sample. Urine may test positive for Clenbuterol over four weeks after a prolonged course of therapy, making it difficult to distinguish between legitimate treatment and illegal administrations before a race⁹. This is reflected in the AQHA’s 30-day withdrawal guideline for competitions. Moreover, horse samples may oscillate between positive and negative, making it difficult to be certain of a definitive result¹⁰.

However, it is now possible to make use of highly sensitive liquid chromatography mass spectrometry (LC-MS) for the unequivocal identification and quantification of Clenbuterol in equine plasma. Unlike urine samples, results from plasma do not oscillate between positive and negative11. Testing serum also allows the immediate detection of Clenbuterol administered by the intra-tracheal route or nebulization, which would be the likely strategy for any pre-race doping¹². Precise LC-MS quantification can distinguish between residual traces from treatment and administrations designed to enhance short-term performance, particularly as there is good correlation between concentrations of Clenbuterol in serum and its pharmacological effects¹³. This allows regulators to set a lower withdrawal period, while maintaining complete confidence that no pharmacologically significant residue of the medication will be present in the horse during a race.

Case study Over 48 hours, 400 matrix samples of Clenbuterol in crashed equine plasma were injected by the dilute-and-shoot technique on the Bruker EVOQ LC-TQ, without the use of an in-line divert valve. Equine plasma was crashed using cold acidified acetonitrile (3:1 v/v). The samples were centrifuged for 10 minutes and the supernatant diluted 1:1 v/v with mobile phase A. Four calibration curves were generated, and between each two adjacent calibrations, 100 matrix samples were injected. The goal was to maintain a consistent response factor (<10% RSD) for all calibration curves, demonstrating superior ion source robustness and sustained high sensitivity performance.

The response factor is the area ration (calibrant/IS) divided by the concentration of the calibration range. The RSD% of response factor is a stringent measure of sensitivity because it takes into account every calibration point on the curve. Any decrease in sensitivity will be reflected by a higher %RSD response factor. The %RSD of the response factors of the four calibration curves was less than 5%, which is well within the acceptable criteria of validated bioanalytical methods. Figure 1 shows the overlaid first and fourth calibration curves, indicating almost no divergence across the calibration range, indicating that all sensitivity accuracy and precision criteria were met. Tables 1 and 2 indicate the sustained high sensitivity performance of the ion source after repeated exposure to the plasma samples, a key requirement for the harsh environment in a routine bioanalytical laboratory. Figure 2 shows the chromatograms for the first calibration level (5ppt) for all four calibration curves. Clenbuterol is a key part of the legitimate treatment of many racehorses, but is also a banned substance for competitive racing in both human and equine athletes. Increasingly sensitive and robust methods for Clenbuterol screening during horseracing competitions are available, such as that demonstrated on the Bruker EVOQ LC-MS. Consequently, regulators can reduce withdrawal periods while maintaining complete confidence that any illicit use of the drug will be detected. The method is equally applicable to food testing applications.

[caption id="attachment_34356" align="aligncenter" width="348" caption="Figure 1: Overlay of curve 1 and curve 4, indicating sustained high sensitivity performance over the course of 400 plasma injections."][/caption]

[caption id="attachment_34358" align="aligncenter" width="320" caption="Figure 2: Representative MRM chromatograms of the 5 ppt Clenbuterol in equine plasma (150 fg on-column) from the first to fourth calibrations, illustrating consistent peak areas, and thus response factors."][/caption]