News Release

Doping by athletes could become tougher to hide with new detection method

Peer-Reviewed Publication

American Chemical Society

WASHINGTON, April 5, 2021 -- As the world awaits the upcoming Olympic games, a new method for detecting doping compounds in urine samples could level the playing field for those trying to keep athletics clean. Today, scientists report an approach using ion mobility-mass spectrometry to help regulatory agencies detect existing dopants and future "designer" compounds.

The researchers will present their results today at the spring meeting of the American Chemical Society (ACS). ACS Spring 2021 is being held online April 5-30. Live sessions will be hosted April 5-16, and on-demand and networking content will continue through April 30. The meeting features nearly 9,000 presentations on a wide range of science topics.

Each year, the World Anti-Doping Agency (WADA) publishes a list of substances, including steroids, that athletes are prohibited from using. However, it can be difficult to distinguish an athlete's natural or "endogenous" steroids from synthetic "exogenous" ones administered to boost performance.

And regulatory bodies face another challenge: "As quickly as we develop methods to look for performance-enhancing drugs, clandestine labs develop new substances that give athletes a competitive advantage," says Christopher Chouinard, Ph.D., the project's principal investigator. Those designer drugs evade detection if testing labs don't know to look for their specific chemical structures.

Chouinard's team at Florida Institute of Technology is trying to outsmart cheaters with an assay that can differentiate endogenous and exogenous steroids and can also anticipate the structure of new compounds that might show up in athletes' urine samples.

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.

Last year, Chouinard's team reported they had successfully used these reactions with IM-MS to improve isomer separation, identification and quantification for a few steroids in sample solutions. Now, the researchers report they have tested this technique in urine against nearly half the prohibited steroids on WADA's list and have shown it can successfully characterize and identify these compounds. They also showed the method can characterize and identify banned glucocorticoids, such as cortisone, that improve athletic performance by suppressing inflammation from injuries. Detection limits are below one nanogram per ml.

In addition to tracking down known dopants, the team wants to be able to find newly created illicit steroids not yet known to WADA. With Florida Institute of Technology collaborators including Roberto Peverati, Ph.D., they are developing computational modeling and machine learning techniques to try to predict the structure, spectra and other characteristics of these molecules. "If we can develop methods to identify any theoretical steroids in the future, we could dramatically reduce doping because we would be able to detect these new species immediately, without the lag time that's been associated with anti-doping testing over the last 40 years," Chouinard says.

Though the assays themselves are quick, simple and inexpensive, IM instruments are costly, with a price ranging up to roughly a million dollars, Chouinard notes. However, he adds, with the support of anti-doping funding organizations like the Partnership for Clean Competition (PCC), more labs might be willing to foot that bill, so long as the method offers a significant advantage in detection and deterrence.


A press conference on this topic will be held Monday, April 5, at 10 a.m. Eastern time online at

The researchers acknowledge funding from PCC, an anti-doping research organization founded by the U.S. Olympic and Paralympic Committee, the National Football League, Major League Baseball and the U.S. Anti-Doping Agency.

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Paternò-Büchi reaction and its utility in improving ion mobility-mass spectrometry identification of performance enhancing steroids


Mass-Spectrometry-based methods have become the gold standard for quantification of prohibited anabolic steroids. However, despite the advantages of high resolution and tandem MS, definitive identification of potentially novel, or 'designer', compounds remain challenging due to their structural similarity to other steroids and low concentrations. Our group has recently investigated selective reactions that can potentially provide more structural information about steroids. One promising example is the Paternò-Büchi (PB) reaction, a photochemical reaction where a radical carbonyl compound (formed by exposure to UV radiation) attacks a C=C double bond to form an oxetane ring derived from cycloaddition. This reaction is commonly used with tandem mass spectrometry to locate the C=C double bond in unsaturated lipids. During this research, we initially investigate the use of the Paternò-Büchi reaction with the simplest carbonyl, acetone, for the separation of steroid isomers using ion mobility-mass spectrometry (IM-MS). This research focuses on isomers of both endogenous steroids and anabolic androgenic steroids prohibited by the World Anti-Doping Agency (WADA). One important isomer pair is testosterone/epitestosterone, which displays nearly identical MS/MS fragmentation pattern and collision cross section (CCS). However, after undergoing the PB reaction (at nearly 50% efficiency) testosterone adopts a new CCS of 205.3 Å2, while epitestosterone presents three unique drift peaks at CCS of 181.9, 190.4, and 202.2 Å2. Several endogenous pairs, including aldosterone/cortisone and 17-hydroxyprogesterone/21-hydroxyprogesterone, were also shown to demonstrate unique ion mobility fingerprint patterns. Studies are currently underway to investigate the patterns observed for PB reaction products of over two dozen other WADA-prohibited anabolic steroids. Additionally, we will also investigate the use of other carbonyl compounds (e.g., butanone, pentanone, etc.). The overall goal of the research is to gain more confidence in the identification and resolution of steroid isomers in a variety of applications.


Ion mobility-mass spectrometry characterization of WADA- prohibited anabolic androgenic steroids


Advancements in analysis of compounds relevant to doping in sport are critically important. Emerging doping agents, including novel anabolic androgenic steroids (AAS), are constantly developed, and failure to establish reliable testing methods will result in the rampant use of illicit drugs without penalty. Ion mobility-mass spectrometry (IM-MS) provides high sensitivity, specificity, and analysis times on a millisecond timescale, as opposed to previous methods (i.e., chromatography) that take several minutes to hours. Ions are separated and characterized based on their gas-phase transport in an electric field. The differences in size, shape, and charge are collectively referred to as the rotationally averaged collision cross section (CCS). This is a key feature that we can take advantage of to successfully isolate and identify compounds of interest. Herein, IM-MS is used as a tool to characterize CCS for AAS listed on the World Anti-Doping-Agency (WADA) 2021 Prohibited List. Characterization was performed using an Agilent 6560 IM-QTOF, a drift tube-IMS (DTIMS) instrument that allows for direct measurement of CCS using the multi-step method. Furthermore, the single-field method was evaluated for its ability to accurately measure CCS on a chromatographic timescale. We have demonstrated that the error for these values, in comparison with their accepted multi-field values, is <1%. We evaluated several isomer groups, including testosterone, and epitestosterone which yielded CCS for their [M+H]+ ions of 173.9 ± 0.1 Å2 and 174.2 ± 0.1 Å2 respectively. CCS for their [M+Na]+ ions yielded 198.7 ± 0.1 Å2 for testosterone, 199.6 ± 0.1 Å2 for epitestosterone and 198.5 ± 0.1 Å2 for prasterone. Isomer pairs aldosterone and cortisone, yielded CCS values of 187.6 ± 0.1 Å and 189.7 ± 0.1 for their [M+H]+ ions, and 199.4 ± 0.1 Å2 and 215.23 ± 0.1 Å2 for their [M+Na]+ ions respectively.

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