To gain an unfair advantage over competitors, some athletes may resort to sophisticated performance-enhancing drugs that are marketed as “undetectable” in drug tests. However, recent PCC-funded research may soon put the brakes on this behavior with the introduction of methods for anti-doping testing that can detect designer steroids that were previously very difficult or impossible to detect, as well as the development of a database that will help anti-doping laboratories identify novel theoretical steroids that athletes could try to use in the future.
Mass spectrometry has been a staple in anti-doping analyses for decades, often paired with gas chromatography or liquid chromatography, to determine whether a sample contains a substance prohibited in sport by the World Anti-Doping Agency (qualitative analysis) and if so, how much (quantitative analysis). Using these methods, a scientist can determine the retention time of a molecule, which is the time it takes to pass through a chromatography column, as well as the mass to charge ratio, which is the mass of the molecule divided by its charge.
These methods are excellent for detecting many prohibited substances that are distinct, in terms of mass and charge, from substances that occur naturally in our bodies. However, there are limitations when it comes to detecting substances like synthetic steroids that may be isomers of naturally-occurring steroids where they have the same mass and charge, but they differ slightly in terms of their molecular structure.
Dr. Christopher Chouinard, an Assistant Professor in the Department of Chemistry at Clemson University, is using a technique called ion mobility-mass spectrometry (IM-MS) to detect and differentiate similar steroid molecules to improve anti-doping analyses. This work is particularly important in an era where new designer drugs continue to be developed with the goal of evading detection by anti-doping tests and analysis procedures.
In 2020, Dr. Chouinard received a PCC Research Grant to establish a predictive database to facilitate the detection of anabolic androgenic steroids using mass spectrometry methods, and particularly IM-MS. IM-MS provides a measure called collision cross section (CCS) that represents the area of an ion that is colliding with other molecules as it passes through an ion mobility separation cell. The CCS reflects the size and shape of an ion, and even small differences in molecular structure can result in measurable differences in CCS.
In his project, Dr. Chouinard and his team developed a method using liquid chromatography-ion mobility-tandem mass spectrometry that can be run in only two minutes and can detect 40 known prohibited steroids. This method uses the combination of chromatographic retention time and ion mobility CCS to differentiate between isomers or other molecules with similar masses and/or molecular formulas. The data were compiled in a new database that can be used by researchers and labs going forward. They also ran the 40 known steroids through chemical reactions that specifically target particular molecular structures to characterize the unique spectra of the reaction products to provide further data to help definitively differentiate steroid isomers.
In addition to the steroids explicitly banned in sport, the World Anti-Doping Agency also prohibits other substances that have similar chemical structures or biological effects, and this includes the synthetic steroid isomers that have been historically difficult to detect. Dr. Chouinard’s group is developing a predictive database using computational modeling and machine learning to generate data that will allow scientists to more quickly identify novel designer steroids that may become available to athletes in the future. He and his team are also working on developing comprehensive analytical workflows that will enable retroactive data processing that could flag stored samples for reanalysis.
The PCC has been delighted to support this research project, which has the potential to help address the long-standing problem of detecting synthetic steroid use in elite athletes. The IM-MS methods are compatible with gas and liquid chromatography methods commonly used in anti-doping laboratories, making them reasonably practical to implement in routine analysis. Dr. Chouinard has also begun applying these methods to other classes of prohibited substances, including glucocorticoids.
This work is helping bring anti-doping scientists and laboratories one step ahead of those who aim to hinder clean sport. Dissemination of research findings is critical for advancing scientific research as well as anti-doping practice in the field. During the course of this project, Dr. Chouinard and his colleagues published twelve peer-reviewed research articles and shared their work in fifteen conference presentations.
Beyond this specific PCC-funded project, Dr. Chouinard’s research over the years has focused on developing important new technology and methods using ion mobility mass spectrometry, particularly to analyze different types of steroids. His work has broad applications spanning anti-doping, biomedical research, and potential clinical applications. You can learn more about his research in this episode of our Anti-Doping Podcast.
