Isotope Fingerprints in Sports Doping

The use of performance-enhancing anabolic substances has been banned since the 1970s in competitive sports. However, substances, especially steroids, are still frequently detected and misused in sports. Doping is now a global problem that follows international sporting events worldwide.

About 300,000 doping tests are performed annually, with on average 1,800 observed violations. The majority can be attributed to athletics, bodybuilding and cycling. The athletes are more aware than ever of anti-doping policies, rules and regulations. Therefore, authorities depend on accurate analysis without exceptions because doping control test results can mean the difference between competing and being banned. In this blog article, I will give you a brief overview of how isotopes and isotope fingerprints can be used to determine the source of origin of steroids in sports doping.

How are steroids examined?

The most common understanding of steroids is that they are drug supplements that enable athletes’ enhanced performance. Anti-doping laboratories examine samples such as urine to screen, confirm and quantify banned drugs. However, steroids, like testosterone, are in many ways an analytical challenge since synthetic copies of endogenous steroids are chemically and pharmacologically identical to their endogenous analogs. Consequently, simple analyte detection and quantitative determination of these steroids in urine cannot be used as proof of administration of performance-enhancing drugs. Here, analyzing stable isotopes to provide characteristic isotope fingerprints of urinary steroids offers an alternative solution. To establish this fingerprint, isotope ratio mass spectrometry (IRMS) is used, measuring the stable isotopes of sample compounds. The application of isotope fingerprints in doping control investigations has become mandatory in doping laboratories worldwide for final confirmation in adverse analytical findings.

How are isotope fingerprints used for doping control?

Variations in the natural abundance of carbon isotopes (12C and 13C) are defined through biological or chemical processes. Consequently, so-called isotopic fractionation occurs, resulting in a slight variation of 13C isotopes. Since pharmaceutically produced anabolic-androgenic steroids are predominantly derived from C3-plant material, botanical processes (C3 photosynthesis) define their carbon isotope composition. Using this source material for the production of steroids results in a distinct isotope fingerprint for the synthetic steroids compared to our naturally produced endogenous steroids.

Watch this video to learn how isotopes are used for doping control and get a great overview of how stable isotope analysis works:

How is isotope analysis regulated in sports doping?

Organizations, including the International Olympic Committee (IOC) and the World Anti-Doping Agency (WADA), established gas chromatography coupled with isotope ratio mass spectrometry as a routine analytical technique used to combat the misuse of endogenous anabolic steroids. Doping control laboratories accredited by WADA must use methods for the determination of the stable carbon isotope ratios (¹³C/¹²C) of steroids in urine.

Samples showing suspicious steroid profile parameters are isotopically characterized in order to prove the origin of the steroids. Administration of synthetic steroid copies reveals depleted carbon isotope ratios of the excreted compound and its metabolites. Endogenous reference compounds are not affected and therefore selected for an individual internal standardization of the isotope ratios.

How do we measure stable isotopes?

The technique used for stable isotope analysis is IRMS. In contrast with standard mass spectrometry, as in quadrupole MS or Orbitrap MS, IRMS is capable of measuring the smallest differences in the isotope composition with high accuracy, which is quite important as differences in isotope ratio occur in the per mill range. Therefore, these are too small to detect with a standard mass spectrometer (i.e., quadrupole MS).

For the compound-specific stable isotope analysis, this instrumentation can also be coupled with chromatographic separation units such as GC-IRMS or LC-IRMS. But there are also hyphenated systems that combine various mass spectrometers.

How does compound identification work with GC-IRMS?

Unfortunately, GC-IRMS solely supplies information on the isotopic composition of a compound of interest. Due to the destructive nature of the methodology, all analytes are converted to CO2 as analyte gas prior to the analysis in IRMS. However, it is possible to couple GC-IRMS with an organic MS system. This setup provides the isotopic compositions and the qualitative and quantitative compound detection from a single run. Concomitant data are critical to qualify the true identity of a compound.

Another creative solution is coupling GC-IRMS with high-resolution mass spectrometry for final confirmation in sports drug testing. Prior to routine anti-doping control and analysis, it is essential to understand the metabolic fate of drugs, since knowing metabolites accelerates the drug discovery and method development process. A hyphenated system combining the power of IRMS and high-resolution accurate mass (HRAM) Orbitrap GC-MS provides detailed structural information of metabolites, while stable isotope composition is measured simultaneously. Learn more about the final confirmation in sports drug testing here.

When sports doping control test results can mean the difference between competing or being banned, authorities depend on accurate analysis without exception. There are various reliable, cost-effective mass spectrometry–based solutions to help reveal the truth hidden in virtually any sample, because when fair competition is on the line, so is your reputation. Find out more here.