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Chasing down drugged racehorses Investigators developed a method to catch trainers suspected of doping their animals.
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A billion-dollar industry EPO is a protein hormone produced mainly by the human kidneys to stimulate the production of red blood cells. In the 1980s, Amgen, Inc., introduced genetically engineered rhEPO to treat anemia in humans. Later, darbepoetin alpha (DPO) came along as a secondgeneration drug that acts for a longer period of time than rhEPO. Because these drugs stimulate the production of red blood cells and, in turn, bolster the oxygen-carrying capacity of the body, they are abused in horse racing even though they are only approved for human use. (EPO is also abused in human endurance sports.) Trainers inject their animals with the drugs so that the horses develop greater endurance and run faster. “There is no use for [human] EPO in the horse,” says Uboh. “But listen, we’re talking about money. Where money is involved, people will try anything © 2007 AMERICAN CHEMICAL SOCIETY
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yebrows go up when a racehorse that normally lags on the track suddenly beats its competitors by several lengths. Regulators of the horse-racing industry have been suspicious that some animals are being doped with a drug called recombinant human erythropoietin (rhEPO). But without foolproof evidence for the presence of rhEPO, how can they nail the suspects? Cornelius Uboh and colleagues at the Pennsylvania Equine Toxicology and Research Center, West Chester University of Pennsylvania, and the University of Pennsylvania School of Veterinary Medicine have spent 4 years developing an LC/MS/MS method to unequivocally show whether a racehorse is being doped with rhEPO (Anal. Chem. 2007, doi 10.1021/ac070135o). Last year, armed with the method, they were able to help the Ontario Racing Commission in Canada suspend a trainer charged with doping his animals.
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LC/MS/MS chromatograms showing confirmation of rhEPO in horse plasma collected 24 hours after a single injection of rhEPO (4000 IU, which is ~20 µg for a horse that weighs 486 kg).
to win.” The stakes are high—a winning racehorse is a good investment because it can earn a considerable amount of money, both as a racehorse and in breeding. Such a horse is worth anywhere between $1 million and $70 million. In the state of Pennsylvania alone, the value of the horses, combined with the racing industry, is $8–10 billion. Regulators of the industry have been very worried about the abuse of these protein-based drugs. The drugs may harm the animal—rhEPO and DPO are foreign proteins to a horse and, when injected, can produce anti-rhEPO antibodies that may cross-react with the horse’s endogenous EPO. The cross-reaction could inhibit the natural function of EPO and lead to anemia, or even death of the animal. And the horse-racing industry has suffered considerable loss because of the doping. But the lack of a definitive test for rhEPO and DPO has prevented them from penalizing the cheating trainers.
Better detection needed Although some methods have been previously developed to detect rhEPO, they have problems. ELISAs are a fast screening method, but they can’t confirm the presence of rhEPO or DPO in a sample because of possible cross-reactivity with other proteins. Isoelectric fo-
cusing combined with western blotting was developed in 2000 for the detection of rhEPO and DPO in human urine (Nature 2000, 405, 635). The method also works on horse- and dog-urine samples. But on occasion, the technique produces false positives, and it isn’t suitable for detecting rhEPO or DPO in plasma samples. LC/MS and CE/MS methods have also been reported for the detection of rhEPO and DPO, but they aren’t suitable for the real-world samples of racehorse blood and urine. LC/MS detection and confirmation of rhEPO and DPO in plasma are difficult to accomplish. Because the substances are hormone-based drugs, their effective dose and plasma concentration are very low, ~1 ng/mL. And, unlike small-molecule drugs, rhEPO and DPO are difficult to separate from plasma because of the abundant proteins present in the medium. But plasma is still the preferred sample because the concentration of rhEPO or DPO is higher in it than in urine. Despite the challenges, Uboh and colleagues set out to develop a sensitive and reliable LC/MS/MS method for confirmation of rhEPO and DPO at very low concentrations in horse plasma. The one thing the investigators had going for them, unlike for researchers J U LY 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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trying to develop tests for human samples, was that they were looking for foreign substances. “We had the advantage because we were looking for what is not naturally present in the horse,” says Uboh. “That makes our case a little bit less challenging because we’re not looking for equine EPO in the horse. We’re looking for what’s not naturally produced in the horse.”
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doping his racehorses with rhEPO, but they needed conclusive evidence. They had gone into the stables during a period when the animals weren’t competing and collected blood samples. The samples were screened by ELISA and seemed to be suspect, but the data from the ELISA weren’t sufficient to take the trainer to court and penalize him. The Ontario Racing Commission needed absolute confirmation of doping. Development of the “They called us about it, and LC/MS/MS method we said, ‘Sure, you can send us The first step in their method the samples, and we’ll analyze was to separate rhEPO and DPO them by our method,’” says from the abundant proteins in Some racehorses were suspected of being doped with rhEPO Uboh. “The samples turned out horse plasma. They discovered and DPO to increase their endurance. to be positive. So we reported that an immunoaffinity method the results to the racing commisDPO could be detected up to 196 that had been previously described for sion in Ontario.” hours after injection and confirmed up separating human EPO from human The evidence led to the suspension plasma worked just as well for separating to 120 hours, proving that DPO has a of the trainer, Todd Gray, for 10 years more lasting effect in vivo than rhEPO. the hormone-based drugs from horse and a fine of Can$100,000. The three The investigators deliberately gave relaplasma. The investigators compared the horses in his care that had tested positively low doses of the drugs to the effectiveness of monoclonal and polytive for rhEPO/DPO—Lasensa, Rair horses to prove that their LC/MS clonal anti-rhEPO antibodies for the reEarth, and My Wicked Willie—were method was sensitive enough to confirm also suspended from racing. covery and found that the polyclonal trace levels of DPO and rhEPO for anti-rhEPO antibody improved the reProtecting horseracing horse racing. covery of the drugs from plasma nearly Now that Uboh and colleagues have It also turned out that the method twofold. gotten a handle on the detection of Once rhEPO and DPO were separat- was effective in detecting rhEPO long rhEPO and DPO, they would like to past its administration. When Uboh’s ed from plasma, Uboh and colleagues pursue the development of detection group started to develop the method 4 digested the hormones with trypsin. years ago, they had administered 60,000 methods of other protein-based drugs. Out of the handful of peptides that They would also like to further tweak IU of rhEPO to one of their research were produced, the investigators first the LC/MS/MS method so that it can horses, collected samples, and put them chose four peptides as diagnostic markdistinguish between rhEPO and DPO. ers for confirmation of rhEPO/DPO. away in storage because they got busy The distinction isn’t absolutely necesThe four peptides were synthesized and working out the kinks in the technique. sary, because neither rhEPO nor DPO is labeled so that they could be used as in- “Once we had developed the method, naturally produced by the horse—their ternal standards for quantitation. we went back to those samples,” says mere presence in horse plasma is an obHowever, more experiments showed Uboh. “We’re talking about 31⁄2 years of storage—we didn’t think it would take vious violation of the horse-racing rules that two of the four peptides were not us this long, but it did.” They were dein any North American jurisdiction. But detectable when the concentration of lighted to find that they could still conUboh says DPO lasts far longer than rhEPO or DPO was
