September 1, 2012 (Vol. 32, No. 15)

Mass Spec and Imaging Are Among the Tools Antidrug Testers Use to Catch Up with the Cheaters

Bad enough Nicholas Delpopolo finished seventh in the 73 kg judo competition at the London 2012 Summer Olympics, losing to Nyam-Ochir Sainjargal of Mongolia in the repechage stage.

Worse, Delpopolo was the first London 2012 athlete barred from the Games of the XXX Olympiad after testing positive in-competition for a banned substance—a metabolite of cannabis, 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid. In a statement, Delpopolo blamed the result on “inadvertent consumption of food that I did not realize had been baked with marijuana.”

The substance is among cannabinoids banned by the World Anti-Doping Agency (WADA). Back in May, WADA president John Fahey said the agency may relax its ban on cannabinoids so their presence alone won’t disqualify athletes—unless substances resulted in enhanced performance. Any such change is unlikely to take effect until the 2014 Winter Olympics in Sochi, Russia.

Cannabinoids weren’t the most abused drug category last year. That distinction, says WADA, is held by testosterone; six of 13 test failures or “adverse analytical findings” resulted from elevated testosterone-to-epitestosterone (T/E) ratios.

The normal T/E ratio is 1:1. If the reading reaches 4, a carbon isotope ratio test is performed to determine if the testosterone is natural or store-bought.

“If it’s less than four, they call it negative, even though it may not be negative. But it’s too expensive to do carbon isotope ratio in every case. That’s a bit of a long-standing problem,” Don Catlin, M.D., a member of the International Olympic Committee (IOC) Medical Commission, told GEN from London.

Another four failures last year resulted from anabolic steroids, followed by one each from “peptide hormones, growth factor, and related substances,” and from beta-2 antagonists. The thirteenth failure was a reported case of tampering.

It’s unrealistic to expect a cheat-free Olympics anytime soon, but drug testing labs are committed to ensuring that medalists win fair and square. [sav_a/Fotolia]

Commonly Abused Drugs

Other commonly abused drugs include diuretics, which athletes use to meet weight in sports like judo and boxing—but which dilutes urine enough to complicate detection of other drugs—and erythropoietin (EPO), a hormone that boosts red blood cells, enabling improved oxygen flow. EPO boosts endurance, which is key for sports such as track & field and cycling, while cheating athletes more interested in strength are likelier to use anabolic steroids.

“The athletes like [EPO] because it really is very strong, and improves their performance. But because it’s difficult to test for, they take low doses and stay below the radar,” adds Dr. Catlin, who founded the University of California, Los Angeles Olympic Analytical Laboratory, the nation’s first antidoping lab and the largest one accredited by WADA.

The UCLA lab’s current director, Anthony Butch, Ph.D., told GEN the challenge for antidope testers is catching up to the cheaters.

“It’s easy to change the structure of something and still have its activity. It’s harder to find it. In some instances, you really need to know what you’re looking for. So, unfortunately, the testing in some instances lags behind the use of these things. And we know that,” Dr. Butch said. “The validated methods to identify these compounds seem to take time because they have to be scientifically sound. That’s not really a limitation of these games. That’s just simply a limitation of the business we’re in.”

He and Dr. Catlin agree there’s reason for hope in London 2012’s most extensive-ever Olympic crackdown on doping agents. Every medalist, and about half of all other Olympic athletes, must give random blood and urine samples for antidrug testing.

Drug Testing

For this sport, so to speak, the venue is not Olympic Stadium or the Aquatics Centre in London, but a drug-testing lab in suburban Harlow that accounts for most of GlaxoSmithKline’s £20 million ($31.2 million) Olympics contribution. The lab tests about 5,000 samples (up from 4,770 at Beijing 2008), and another 1,250 from athletes with disabilities at the companion Paralympics.

Some 150 scientists led by David Cowan, Ph.D., co-founder and director of the Drug Control Centre at King’s College London, are testing samples for some 240 banned substances at the lab, which operates 24/7 during the Games and can turn around most tests in 24 hours. That requires completing analysis in eight hours, so that the remaining time can be used for possible retesting and confirming results.

Most banned drugs are screened through mass spectrometry—either gas chromatography–mass spectrometry (GC/MS) or liquid chromatography–mass spectrometry (LC/MS). Dr. Cowan’s team uses Waters ACQUITY UPLC®/tandem mass specs; Waters has no GC/MS tools.

Also at the lab are GE Healthcare ImageQuant™ LAS4000 digital imagers for detecting EPO and other biomolecules. Proteins are enriched, separated using isoelectric focusing, then transferred via Western blot to filter membranes and probed with antibody conjugates, resulting in emission of light by the imagers. Those tests take 48 hours, compared with one day for GC/MS or LC/MS tests.

“Endogenous EPO and recombinant EPO differ slightly in the overall charge of the molecule. Using the highly sensitive imager, you can see the bands that relate to naturally occurring and recombinant EPO,” Pilar Anton Serrano, global communications manager for GE Healthcare’s Detection & Guidance Solutions business, told GEN.

After the Olympics, the testing lab will see much of its equipment replaced with tools better suited for its future function as site of the MRC-NIHR Phenome Centre. Rohit Khanna, Ph.D., Waters’ VP, worldwide marketing, told GEN of one example: Waters will replace the lab’s mass specs with its Xevo and SYNAPT mass specs designed for phenotypic testing.

“We will share what we have learned in setting up the lab for London with the IOC and hope that we can deliver a ‘blueprint’ for future Games. We have already shared lessons learnt in setting up the lab for London 2012 with colleagues in Rio (de Janeiro),” site of the 2016 Summer Olympics, Glaxo spokeswoman Sarah Alspach told GEN.

Those lessons should include improving on London 2012 by random-testing all Olympic athletes at Rio. Before Delpopolo’s expulsion, five athletes were ejected by IOC, and numerous others by national sports bodies, for testing positive before events. They include Albanian weightlifter Hysen Pulaku (the anabolic steroid stanozolol) and Uzbek gymnast Luiza Galiulina (the diuretic furosemide).

“If you win gold, that’s what it’s all about. And some people are willing to take the risk of doping because they feel that winning is more important than cheating,” Dr. Butch said.

While today’s testing makes it unrealistic to expect a cheat-free Olympics anytime soon, that shouldn’t stop IOC and national bodies from showing the same commitment to stepping up drug testing that manufacturers show toward improving the equipment used for catching the cheaters.

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