As the world prepares for the upcoming Olympic Games, a new method for detecting doping compounds in urine samples may level the playing field for those attempting to keep athletics clean. Scientists have published a method that uses ion mobility-mass spectrometry to assist regulatory agencies in detecting existing dopants as well as future “designer” compounds.
The researchers will present their findings today at the American Chemical Society’s spring meeting (ACS). The World Anti-Doping Agency (WADA) publishes a list of substances, including steroids, that athletes are not permitted to use each year. However, distinguishing natural or “endogenous” steroids from synthetic “exogenous” steroids used to enhance performance can be difficult.
And regulators face another challenge: “As quickly as we develop methods to detect performance-enhancing drugs, clandestine labs develop new substances that give athletes a competitive advantage,” says the project’s principal investigator, Christopher Chouinard, Ph.D. These designer drugs are undetectable if testing laboratories do not know where to look for their specific chemical structures.
Chouinard’s team at Florida Institute of Technology is attempting to outwit cheaters by developing an assay that can distinguish between endogenous and exogenous steroids and predict the structure of new compounds that may appear in athletes’ urine samples.
As quickly as we develop methods to detect performance-enhancing drugs, clandestine labs develop new substances that give athletes a competitive advantage.Christopher Chouinard
Currently, testing labs analyze samples using tandem mass spectrometry (MS) and gas or liquid chromatography. These approaches break up molecules in the sample and separate the fragments, yielding spectra that can reveal the identity of the original, intact compounds. But it can be tough to differentiate molecules with minor structural differences – including isomers – that distinguish endogenous steroids from exogenous ones, such as the synthetic anabolic steroids athletes take to build muscle.
To accentuate those differences, Chouinard pairs MS with ion mobility (IM) spectrometry, a separation technique he learned as a graduate student with Richard Yost, Ph.D., at the University of Florida. Yost’s team and others found that the differences between isomers could be made even more apparent if the molecules in a sample were modified prior to IM-mass spec analysis by reacting them with other compounds. After Chouinard set up his own lab in 2018, he applied this technique by reacting steroid samples with ozone or acetone in the presence of ultraviolet light – reactions already well-established among researchers who study lipid isomers, but new in the anti-doping arena.
Chouinard’s team reported last year that they had successfully used these reactions in conjunction with IM-MS to improve isomer separation, identification, and quantification of a few steroids in sample solutions. The researchers now report that they have successfully characterized and identified nearly half of the prohibited steroids on WADA’s list using this technique in urine. They also demonstrated that the method can characterize and identify prohibited glucocorticoids like cortisol, which improve athletic performance by suppressing inflammation caused by injuries. The detection limits are less than one nanogram per ml.
In addition to locating known dopants, the team hopes to locate newly created illicit steroids that are not yet known to WADA. They are developing computational modeling and machine learning techniques with collaborators from Florida Institute of Technology, including Roberto Peverati, Ph.D., to try to predict the structure, spectra, and other properties of these molecules.
“If we can develop methods to identify any hypothetical steroids in the future, we could dramatically reduce doping because we would be able to detect these new species immediately, without the lag time associated with anti-doping testing over the last 40 years,” Chouinard says.
Though the assays themselves are quick, simple, and inexpensive, IM instruments are expensive, with prices ranging up to a million dollars, according to Chouinard. However, he adds, with the support of anti-doping funding organizations like the Partnership for Clean Competition (PCC), more labs may be willing to foot the bill, as long as the method offers a significant advantage in detection and deterrence.
Doping by athletes could become tougher to hide with new detection method https://t.co/D4Yc3xszNa— Road Race Management (@RoadRaceMgmt) April 8, 2021