Sports Doping: Closing Pandora’s Box

The athletes competing at the 2022 Winter Olympics have had their three-week shot at fame and glory in Beijing. Those who made the podium received their medals. Anthems were played, parties were enjoyed. But we don’t yet know for certain who really deserves the accolades. Because analytical chemists have another 10 years to catch athletes cheating with performance enhancing drugs. There may be athletes at this year’s games using substances that cannot yet be detected – so the race is on to develop and validate new methods before the decade is out.

The story behind this unexpectedly troubling scenario started to unfold over a century ago, with athletes, classical chemists, and pharmacists all playing their part. Today, Pandora’s Box is well and truly open – and analytical chemists have the daunting responsibility of helping the authorities close it again.

The alkaloid arrival    
 

Institutionalized anti-doping analysis started in the 1960s, despite the fact that the use of drugs to enhance sport performance began much earlier. For example, an endurance walker in Britain said in 1807 that he had used laudanum (which contains opiates) to keep him awake during a race (1). Also in the 19th century, pure alkaloids were isolated from plants by classical chemists – morphine (1803), strychnine (1818), caffeine (1819), and cocaine (1859) – piquing the interest of people seeking to apply the effects of alkaloids outside of treating medical problems. One of those non-medical uses was in Coca-Cola, which contained cocaine and caffeine when launched in 1886. It became popular with some athletes, including a group of French cyclists and a champion lacrosse team (2). At the time, the application of alkaloids in human sports was not considered to be a problem, despite deaths surely having occurred as a result (few cases were confirmed). The Box had been cracked open, but nobody was interested in closing it – yet. 

In the 1930s, true synthetic compounds like amphetamines were developed (though they were invented much earlier). These psychostimulants were marketed initially as inhalers for congestion or as energy boosters – and, during World War II, as compounds to improve the performance of soldiers. During and after the War, soldiers were thought to have introduced amphetamines into sports, which caused several deaths in the 1950s and 1960s. This toll was deemed unacceptable to the public and sport authorities had to react. The age of anti-doping analysis had begun in earnest.

Enter anabolics

Pandora’s Box also released the phenomenon of anabolic-androgenic steroids (AASs) into sport after World War II. German soldiers had used endogenous AAS testosterone to increase their aggressiveness and to improve their physical strength; and, in the 1950s, Russian athletes were thought to have used testosterone to improve their sports performance. US athletes responded in the 1960s by applying methandienone, an exogenous AAS. Methandienone was developed to treat muscle dystrophy in the elderly, but was withdrawn from the market because of health risks. Athletes, however, had already tasted the forbidden fruit…

Attempts in the 1970s to unambiguously detect the use of exogenous AAS by immunoassays failed, but GC-MS, introduced in the 1980s, made it possible to identify metabolites of exogenous AAS in urine sample of athletes in the range of 100 nmol/L without reasonable doubt. GC was combined with tandem MS (MS/MS) in the 1990s and 2000s to further push the limits of detection down to the lower range of 1 nmol/L. Although GC-MS significantly expanded the identification power by MS, one hurdle was the need for chemical derivatization to increase volatility and stability. The application of liquid chromatography MS/MS in AAS metabolite detection leaped that hurdle in the 2010s, making it possible to detect AAS under non-volatile and less thermo-labile conditions. Nowadays, using LC-Ion Trap MS/MS, detection limits can reach as low as 10 pmol/L. The high sensitivity and the presence of so-called slow excretion metabolites results in a long detection window that makes it very difficult for athletes to continue abusing exogenous AAS.

To detect the abuse of pharmaceutically applied endogenous AAS (testosterone, androstenedione, dehydroepiandrosterone, and so) steroid profiling was initiated based on the ratio between the steroid’s testosterone and epitestosterone. However, again due to a lack of analytical specificity, some athletes were falsely accused of doping offenses. GC combined with combustion/isotope ratio MS (C/IRMS) presented a solution in the 2000s and is now the go-to tool for detection of endogenous AAS abuse.

So, have we closed Pandora’s Box? 

I would argue that the Box is open wider than ever before. Though several challenges have been overcome in terms of expanding detection limits for alkaloids, psychostimulants, and AAS metabolites, the question of where the substances came from – and thus the athlete’s guilt – isn’t always as easy to answer. Banned substances can originate from contaminated supplements, regular medicines, or even in common foods – meat from animals treated with certain agents to increase meat production can be the source of low concentrations of AAS metabolites or other agents. 

But not all athletes are innocent; some want to enhance their performance by any means necessary, and so new compounds or methods continue to be found. In the 1980s, athletes became interested in peptide hormones, such as human growth hormone (hGH) and human chorionic gonadotrophin (hCG). The abuse of hGH was initially hampered by limited availability of safe pharmaceutical preparations, but it has become more prevalent with the availability of recombinant hGH. The detection of peptide hormones remained a challenge for decades – clinically adequate immunoassays were able to detect hormones in the 1980s, but identification in the context of anti-doping analysis requires a higher level of legal certainty. Since 2010, identification of hGH abuse has been based on the differential detection of hGH isoforms using specific immunoassays.

In the 1990s, athletes discovered the benefits of using recombinant human erythropoietins or epoetins (EPOs), often referred to as erythropoiesis-stimulating agents (ESAs). These compounds are highly active protein hormones that stimulate the production of erythrocytes, which improves delivery of oxygen from the lungs to the working muscles.

Today, the direct identification of the abuse of ESAs is based on the analysis of blood or urine using a combination of electrophoresis (for example, isoelectric focusing or sodium dodecyl sulfate- or sarcosysl-polyacrylamide gel electrophoresis) with double Western blotting and immunochemical detection. Indirect identification is tackled by a so-called hematological module of the Athlete Biological Passport (ABP), which, by detecting hematological parameters in blood, applies a Bayesian approach to determine the probability that an athlete’s hematological variation might be due to the abuse of ESAs. Also, a growth hormone module of the ABP based on hGH biomarkers has been considered and investigated since the 2000s.

Within the anti-doping community, such approaches to identify protein hormones are considered reliable and robust – though there are doubters (usually outside the analytical chemistry community). A broad scientific discussion may be needed to improve the analytical sensitivity and specificity of the test protocols. As for the classical doping agents, MS-based identification might end this discussion, but continuous development of analytical technology remains essential. In the future, LC-MS/MS and capillary electrophoresis (CE)-MS/MS may be key to unambiguously detecting the abuse of protein hormones. 

Who’s ahead in the race?

Sports doping is a continuous battle between authorities and athletes. Some athletes – no doubt aided by doping chemists – find new ways to illegally enhance their performance, while analytical chemists work hard to find methods of detection. Who has the advantage in this race? It can take years to adequately validate new analytical tools after the introduction of a doping trend, and that’s why anti-doping authorities made it legal to re-analyze biological samples for up to 10 years after collection. But these rules mean that the final winner of a 2022 Olympic medal only will be known in 2032… 

Pandora’s Box will remain open for the foreseeable future. Moreover, the current anti-doping policy shifts the problem of drugs and methods in sports toward other doping agents and methods instead of closing the Box. For example, it recently became clear that the incidence of the use of thyroid hormones in certain sport populations is significantly higher compared with the prevalence of relevant thyroid diseases in reference populations. Not currently on the World Anti-Doping Agency’s list of prohibited substances and methods, the use of thyroid hormones is raising new medico-ethical questions and of great concern to sport authorities. Manipulation of athletes’ genes is another serious issue; in fact, genetic doping has been investigated by official judicial authorities and is an officially forbidden method. 

Get in the game

I believe analytical scientists must play a key role in anti-doping discussions. After all, we are the driving force behind the technologies applied in anti-doping analysis. Moreover, the development of sophisticated analytical tools for anti-doping analysis can have an important impact on wider clinical chemistry and thus medicine in general. For example, the ABP module approach could be applied in the pursuit of precision medicine, where individualized approaches are needed to predict and monitor treatments instead of relying on a one-size-fits-all approach. 

At the same time, analytical chemists must try to avoid the perils of tunnel vision; for example, only seeing the advantages of an ultra-sensitive method or taking pride in scientific advances without considering the impact on others. Remember that any development in this field can have ethical repercussions; we should carefully weigh the pros and cons in every instance and from multiple angles.

In the context of WADA’s list of prohibited substances and methods, my view is that analytical chemists should look beyond the development or continuous improvement of analytical technology, and start asking new questions; for example, what role can analytical science play in determining the origin of a doping agent? And how can we build new ABP biomarker knowledge?

If we continue down the current path of increasingly sophisticated and sensitive technologies that can detect and identify substances beyond current lower detection limits of 10 pmol/L, we may begin detecting pharmaceutical substances that originated from drinking water (residues of certain medicines are already commonly detected in waste water and subject to environmental investigation). 

We should not be solely focused on the goal of catching as many doping offenders as possible, but instead making anti-doping activities as robust as possible – and that will almost certainly mean cooperating with other scientific disciplines. If we do not, innocent athletes may be crushed by attempts to close the “lid.” With each passing decade, the sanctions faced by potential doping offenders are getting increasingly severe – so we cannot get it wrong.

In criminal law, the presumption of innocence is essential. And any accusing authority must supply a court with as much evidence as possible. Subsequently, any proven violation leads to an appropriate sanction. In sport law, the presumption of innocence seems to function differently. The accusing authority supplies a certain minimal amount of evidence and the accused athlete must prove her or his innocence. Put another way, any accused athlete is guilty unless proven innocent. When it comes to anti-doping analytical procedures, the minimal amount of evidence is well defined in the respective regulations; however, authorities may not always reveal the full details of the analysis – mainly to prevent others using the information to circumvent regulations or develop new methods of doping. But this lack of transparency is arguably unfair to innocent athletes. Without all the details, how can athletes adequately defend themselves? As pressure to reinforce sanctions increases, so too should the pressure to release more analytical details.

As sport organizations and politicians work together to find the right balance, we analytical chemists will continue working behind the scenes, playing our part in the war to close Pandora’s Box. But we are not mere foot soldiers – we must also do our utmost to ensure the Box is closed in a fair way.