Athletes looking to cheat by using performance-enhancing drugs will be bringing their best doping strategies to the 2016 Olympic Games, which begin 5 August in Rio de Janeiro. But the Brazilian Doping Control Laboratory is ready to catch them. Outfitted with the newest analytical machines, the lab will be detecting doping substances at the lowest concentrations in the history of the games.
Despite its shiny new equipment, the lab and its staff very nearly spent the games sitting on the sidelines. The World Anti-Doping Agency (WADA) suspended it last month for failing to meet international standards. On Wednesday, 16 days before the opening ceremony, WADA announced it had reinstated the lab. Neither WADA nor Brazilian officials revealed what prompted the suspension.
The Brazilian lab owes its superior doping-detection capabilities in part to vast improvements in mass spectrometry, or mass spec—a 100-year-old technique for identifying molecules based on their masses and the trajectories they take through an electric or magnetic field. The newest technologies allow researchers to more accurately determine the mass of a compound and to differentiate among compounds of the same mass.
That kind of precision enables drug labs to confidently identify minute concentrations of performance-enhancing drugs, sometimes weeks after they are taken. “Instrumentation has gotten so much better,” says Anthony Butch, director of the Olympic Analytical Laboratory at the University of California, Los Angeles. “The lower you can go in detecting something, the longer you’re going to be able to detect it in someone’s urine.”
That, coupled with advances in our understanding of how drugs are broken down in the body, “has made a huge difference for us,” says Butch. Investigators look not only for the prohibited drug compounds but also the metabolites that are produced by the body as the drug is processed; these can be detected in the urine for a long time, he says.
How significant are the advances in mass spec? Since the International Olympic Committee last year ordered the reanalysis of hundreds of samples saved from previous Summer Olympics, 98 additional cheaters from multiple countries were caught—60 from the Beijing games in 2008 and 38 from the London games in 2012. The IOC said Friday that testing of more samples from those years would continue. Samples saved from the 2014 winter games in Sochi, Russia, will also be retested due to evidence that Russia has been sponsoring its athletes’ entrenched doping practices and covering it up by tampering with urine samples at its now-disgraced antidoping lab in Moscow.
During the 2016 Summer Olympics, the Rio lab, located at the Universidade Federal do Rio de Janeiro, will test more than 6,000 blood and urine samples, producing results within 24 hours, says Francisco Radler de Aquino Neto, a chemical scientist and the director of the facility. Lab personnel will be looking for more than 500 different substances on WADA’s prohibited list. Among the molecules they’ll screen for are those that indicate the presence of substances such as anabolic steroids, stimulants, masking agents, and hormones.
WADA had suspended the Rio lab before, in 2013. It took three years and roughly 200 million Brazilian real (US $60 million) from the federal government to whip the lab into shape. That investment bought a dazzling array of mass-spec equipment and other instrumentation and over 500 days of training by international specialists, bringing the lab back into good standing with WADA. About 30 labs around the world can currently tout accreditation by the agency.
WADA sets limits of detection and other criteria that force accredited labs to continually upgrade their equipment, particularly their mass-spec analyzers. In a typical mass-spec system, molecules of interest are given a charge, or ionized, causing some to fragment into pieces. The ionized molecules or their fragments are subjected to various combinations of electric and magnetic fields, causing them to take different trajectories depending on their mass-to-charge ratios. Ions of interest reach a detector, which records their abundance in a mass spectrum. Tracking that data yields information about the particles’ mass, which ultimately helps identify the molecule.
Many molecules or fragments will have the same mass, making it difficult to tell whether one of interest is part of a doping substance or some other molecule in the sample. Newer technologies address that by putting compounds through multiple, sequential rounds of mass spectrometry, called tandem mass spectrometry. That system takes the charged molecules or fragments from the first round and shatters them again, making smaller pieces. The likelihood of a molecule of interest shattering the same way another compound shatters is low.
The Rio lab is equipped with several tandem systems, including one that utilizes quadrupoles—groups of four cylindrical metal rods. A voltage consisting of radio frequency and direct current can be applied to the rods in a manner that works like a filter. By adjusting the voltage, investigators can select for ions based on their mass-to-charge ratios. Only those ions will be able to follow a stable trajectory through the rods to reach the next step. The compounds are put through a series of three quadrupoles that filter ionized molecules or their fragments, shatter them, and filter them again.
Sometimes differentiating among even these tiny fragments can be tricky. Two pieces may have different atomic structures but similar mass. For that, the Rio lab turns to instruments with even higher resolution. One of those is the orbitrap—a barrel-shaped mass analyzer that forces charged fragments to cycle around an inner spindle. The fragments’ mass-to-charge ratios can be determined by tracking their elliptical trajectory. Another high-res tool in the lab’s arsenal is a time-of-flight mass spectrometer. It determines a particle’s mass-to-charge ratio by measuring the time it takes for the particle to travel a known distance. Both the orbitrap and the time-of-flight instrument determine the masses of particles with more precision than tandem systems alone.
As drug testing improves, so do the sophistication and adventurousness of athletes’ performance-enhancing drug regimens. “They used to take a big whopping dose, and it was easier to detect,” says Butch. Now they take smaller doses more often, and try things that nobody expects people to willingly put in their bodies—like experimental or veterinary drugs that aren’t approved for humans. “Right now the big thing is peptide hormones and growth factors,” he says. Those weren’t placed on WADA’s prohibited list until the mid-2000s, says Butch, because “nobody thought anybody would ever use something like that.” Thanks to tandem mass spectrometry machines, we can catch those cheaters too.