dopingPerformance-enhancing drugs are substances that have the capability to improve performance in humans. In sports those substances are sometimes referred to endogenic aids and one highly popular reference is anabolic steroids or their precursors with the colloquial term “steroids.”

Administration of performance enhancing drugs has certainly occurred since the time of the original Olympic Games. Back then the ancient Olympic champions were professionals often competing for olive wreaths and huge cash prizes. The athletes often experimented with herbal medication, drank wine potions, used hallucinogens and ate animal hearts or testicles to enhance their performance.1, 2 Many things have changed since then. However, the administration of performance-enhancing drugs remains common in our modern society.

In our recent history, amphetamines were the first ‘effective’ performance enhancing drugs, which were used widely by soldiers in World War II. Soon after, amphetamine abuse crossed over into sports in the early 1950s. These drugs minimized the uncomfortable sensations of fatigue during exercise and were therefore especially useful for endurance sports.3 Within the same decade Russian athletes began to experiment and apply testosterone, while the first synthetic steroid was created soon after in the U.S. and released by Ciba Pharmaceuticals with FDA approval. Back then doping wasn’t prohibited and was applied for performance enhancement by many athletes, since some athletes are willing to win at any cost. Consequently, the first causalities of cyclists occurred a few years later. For instance, Knut Jensen and Tommy Simpson both died in the 1960s due to dehydration during a race while using performance enhancing drugs. Performance enhancing drugs have been banned for decades by the International Olympic Committee and the World Anti-Doping Agency. But doping administration remained common. The urge of winning seems to be too strong…Or are the tools for drug analysis not advanced enough?

It is indeed not that simple, since steroids are very common hormones in our bodies, it may be challenging to distinguish between their origins. For instance, steroids help build muscle tissue and increase body mass by acting like the body’s natural hormone “testosterone.” But how can we distinguish naturally-produced steroids in our bodies from their synthetic analogues? Both are chemically identical, meaning that natural and synthetic analogues cannot be distinguished with a simple LC-MS or GC-MS analysis. We can measure the presence of testosterone (T) and another hormone called epitestosterone (E), a natural product of steroid metabolism that provides no benefit to the athlete. The ratio between T/E may tell us if additional testosterone was taken. But athletes found way to manipulate the epitestosterone levels. So, drug abuse cannot be proven with concentration analysis or structural identification alone.

There is, however, another way to distinguish the origin of chemically identical compounds. But this information is hidden within the atoms themselves. The answer is – isotopes!

Most people relate isotopes to radioactivity or contamination. But there are two types of isotopes around us: the radioactive isotopes (non-stable) and stable isotopes. The word “stable” already indicates stable isotopes do not decay. So, no radioactivity and no threat. Looking at stable isotopes can tell us a story about a chemical compound, for instance, how or where was it formed. Isotopes can therefore be used in various disciplines — such as geology, forensics, environment or food authentication — giving us a lesson about the origin, the path and the fate of substances that we would like to investigate.

So how can we distinguish between naturally produced steroids and synthetic analogues? With their isotope fingerprint. Most of the synthetic steroids are produced from C-3 plant material, for instance wheat, soybeans or barley. This plant material consists of a distinctive isotope signature. Our naturally produced endogenous steroid in our bodies, however, have another isotope fingerprint. The origin of testosterone can be distinguished by measuring the mean value of stable isotope composition of steroids in our bodies and comparing this value to the testosterone. If the athlete is clean, stable isotope composition should remain identical between testosterone and other endogenous steroids in his body. However, if the athlete was taking synthetic testosterone, we will be able to see a difference in stable isotope composition for testosterone compared to the body mean value. –  Got you!

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The technique that is used for stable isotope analysis is the isotope-ratio mass spectrometry (IRMS). For the compound-specific stable isotope analysis this instrumentation can also couple with chromatographic separation unit as GC-IRMS or LC-IRMS. But there are also hyphenated systems that combine different mass spectrometers, like GC-MS-IRMS, for simultaneous isotope analysis and structural identification. A great overview of various techniques and methods is available here. Check back for future blog posts on more technological possibilities!

Additional resources:

Article: High Tech Doping: Building Better Testing “Mouse Traps”

Application note: GC-MS-IRMS: Undisputable results by coupling of GC-IRMS with high-resolution mass spectrometry (AN30405)

Info graphic: Isotope Fingerprints Doping

References:

  1. Larry D. Bowers, PhD “Athletic Drug Testing,” Clinics in Sports Medicine, Apr. 1, 1998 https://www.ncbi.nlm.nih.gov/pubmed/9580843
  2. Sally Jenkins  “Winning, Cheating Have Ancient Roots,” Washington Post, Aug. 3, 2007
  3. Timothy Noakes, MD, DSc  “Tainted Glory – Doping and Athletic Performance,” New England Journal of Medicine, Aug. 26, 2004