Techniques and Challenges of Doping Control

This is the first in a series of three articles designed to answer what I felt were the primary questions coming out of the reader forum after Ironman announced the sanctions of Beth Gerdes and Lauren Barnett.

The first question was, "How does this process of saying 'the lab found X' actually work? How reliable is it? Can we trust it?" Basically, there seemed to be a real disconnect in terms of understanding how we go from an athlete peeing in a cup to Ironman giving a sanction. That's what this article is designed to answer. This is also designed as a foundation to understand some of the more specific questions that will follow. You cannot understand, for example, whether or not a contaminated or tainted supplement is a likely cause of an AAF (Adverse Analytical Finding) without actually understanding how a lab analyzes a sample.

By The Numbers: The Science of Drug Testing

The key analytical tool in anti-doping efforts is mass spectrometry: High-Performance Liquid Chromatography (HPLC) Mass spectrometry (MS) is an analytical technique that separates and ionizes chemical species then detects the present ions based on their mass-to-charge ratio. In simpler terms, a mass spectrum measures the masses within a sample." HPLC refers to, "a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture."

HPLC/MS is well established science, and it can be very precise. However, precision comes at a cost. From an in-depth article in The British Journal of Sports Medicine, "Anabolic agents: recent strategies for their detection and protection from inadvertent doping ", "The price to pay for comprehensiveness, however, is the methods' sensitivity. While targeted analyses for selected steroids allow for detection limits as low as 5 pg/mL non-targeted approaches have to compromise sensitivity and typically higher detection limits of screening protocols at approximately 10–20ng/mL." Initial sweeps are broad - looking for "red" as opposed to looking, specifically, for "pink" or "magenta," because of realistic constraints of time and money.

Chris Ganter, a fellow pro triathlete and - in his former life - analytical chemist for 10 years for Johnson & Johnson's pharmaceutical division and an incredible resource for this article elaborated: "The lab should have a method that serves as a cursory sweep for a breadth of compounds of interest with similar chemical structures or physical properties (e.g. all SARMS (Selective Androgen Receptor Modulators) with the same solubility and HPLC stationary phase interactions). We would have called it a 'purity (impurity) method.' It would just save time and money if nothing is detected."

Chris continued, "If something is there at the right level, you get a response. That would be a suspect OOL (Out Of Limit) or AAF. But the method might not be sensitive or selective enough for quantitation purposes. So the lab might have an assay method for that specific compound on hand to back it up. So you would have better methods available if you had a suspect AAF that you needed to confirm and/or quantitate. Like using a ruler and then moving to a micrometer. But you can’t just turn up the reso on the ruler, you need a micrometer on hand."

Challenge/Technique #1. Screening/Casting Wide Net. There are a lot of banned substances on the list. And a lab has to screen for all of them, at least during in-competition; certain substances are allowed out of competition, like psuedoephedrine, a common decongestant, which is subject to a threshold limit for IC (In Competition) testing, meaning WADA (World Anti-Doping Agency) labs only need to care if there is more than DLng/ml, where DL is the "Decision Limit." The current threshold list is extremely interesting, because it's one of WADA's more technical publications and actually offers a rather clear insight into the hard science - confidence intervals, for example - used to make these determinations. While only certain substances have decision limits, all substances have limits of detection (LOD), which is how precisely a given method can reliably (99.9% reliably according to WADA) detect the quantity of a given substance that is present. The LOD is typically at least an order of magnitude lower (eg 2.0 v 0.1-2ngml) than the decision limit, and in some cases may even be much lower. The reader can take confidence that the reliability of this technique for confirming a substance is present - and present in the stated amount - is very high.

Challenge/Technique #2 Naturally Occurring Substances. The most contentious substance subject to a decision limit is Growth Hormone (hGH), because it's a substance your body produces naturally and in levels that fluctuate throughout the day. The problem with some of those who take aim at the hGH test is that it's much more of an outlier than the norm. There are only a handful of endogenous substances (things your body makes naturally) that are banned - testosterone is another - and the nuances of the tests for these substances often focus on both levels and also on trying to identify synthetic vs natural substances through analysis of the carbon atoms. As is often the case, the criticisms imply that a specific flaw is indicative of a general flaw, something which I do not believe to be the case. The issues with mass spec as a tool for detecting abuse of hGH simply don't apply to most of the other substances on the banned list.

Challenge #3 Wide Range. While certain compounds are more likely in one sport than another, we've also seen those lines being blurred as it's clear that, for all athletes, speed of recovery is essential, especially as improved detection methods have pushed athletes more towards micro-dosing. To this end, a "speed" athlete like a sprinter might take EPO just as an "endurance" athlete like a cyclist or triathlete might take an anabolic. The idea is to enhance the body's ability to recover.

I recall (I cannot find the original source, apologies) one of the sprinters from Trevor Graham's group that was busted by USADA (United States Anti-Doping Agency) who then cooperated with their investigation talking about training on THG - a designer anabolic steroid. She said, essentially, "the workouts didn't really change; it was how often we could do them." Labs have to look for everything for every athlete. Plus they need to look for other substances that aren't PEDs (Performance Enhancing Drugs) themselves, but which are illegal for other reasons - say, diuretics, which dilute your urine in order to mask the presence of PEDs.

Challenge/Technique #4. Pharmacokinetics. Virtually any substance you put into your body is metabolized. And you pee out not just a compound itself - because almost nothing is metabolized 100% - but also its various metabolites. The timeline of how something is metabolized - which falls into a field called pharmacokinetics - is really valuable. Drug metabolism is governed by half-lives, which means it is logarithmic. That means the fall off is really rapid at the outset and then, as you get out along the asymptote, it's very slow. This has been demonstrated to be relevant in several clenbuterol AAFs that were deemed to be no fault, because subsequent drug tests revealed that that the clenbuterol was no longer detectable, meaning it was probable that this was a case of recent ingestion of a very small amount - tainted meat - as opposed to long term usage of a larger amount for performance enhancement at a much earlier date. This is one reason why pre-competition testing is so valuable; two data points are more than twice as valuable as one.

However, metabolism is not an "even" process. Different metabolites can be formed at different times. And the presence - or absence - of certain metabolites can help to paint a picture of when something was ingested. There was a rash of recent AAFs in MLB during the pre-season for stanozolol, which is an "old school" (Ivan Drago-esque) anabolic steroid. What happened? The testing labs developed better tests for so called LTMs (Long Term Metabolites). "Long term" refers to how long the body produces this metabolite. LTMs are substances which your body produces long after the initial (or final) dose of a given drug.

In other cases, information comes from shorter term metabolites. Some metabolites are processed very quickly. In this case, if someone is attempting to blame a tainted supplement or nutrition product that they took before/during the race, the absence of these metabolites can undercut the credibility of that story. The more that is known about how a drug metabolizes, the more information anti-doping labs have to work with.

The Importance of Skill: The Art of Drug Testing

Challenge/Technique #5. Additional Testing Fundamentally, mass spectrometry is a tool. It requires a human operator. The skill of the lab manager comes overwhelming from knowing what to specific molecules to look for and what targeted tests to run based on the various information initial analyses - and prior tests - contain.

The BJSM article describes, "the need to complement target-oriented analytical methods as usually employed in routine sports drug testing by non-targeted approaches had become evident. Consequently, strategies to combat the purported as well as proven abuse of designer steroids have been evaluated, two of which were particularly promising: the non-targeted approach focusing on the detection of characteristic and common fragment/product ions derived from conserved nuclei of AAS, and the indirect approach based on monitoring the biological effect of AAS on the profile of endogenous steroids."

Challenge/Technique #6. Indirect detection This approach is a mix of science looking for more/different metabolites and some medical detective work. This detective work is based around the fundamentals of endocrinology. Hormones exist in a feedback loop.

"The indirect approach for the detection of designer steroids is based on endocrinological feedback mechanisms and the effect of these steroids on the profile of urinary endogenous steroids (steroid profile), which is monitored for each athlete within the Athlete's Biological Passport (ABP, vide infra). It is well known that the administration of AAS leads to a suppression of the excretion of endogenous steroids. Therefore, suppressed endogenous steroid concentrations may trigger a search for unknown designer steroids. This strategy was successfully applied in the detection of the misuse of norbolethone in 2002 where unusual steroid profiles suggested the misuse of a yet unknown (or at least not screened) AAS. However, the determination of arguably steroid abuse-influenced urinary steroid profiles does not allow for reporting an AAF. The administered drug still needs to be identified; nevertheless, the suspicion resulting from steroid profile analyses is of great importance in target testing of athletes and further investigations of the collected doping control urine samples."

You can't give an AAF for a "suspicious" sample, but suspicious samples can help guide the testing process to find something that you can get an AAF for. Note that cases of accidental ingestion of a truly contaminated supplement are less likely cause the sorts of significant long-term endocrinological changes that would raise suspicion.

Challenge/Technique #7. Data sharing. The results of drug tests are saved and shared within the WADA community through ADAMS (Anti-Doping Administration & Management System). The BJSM article continues, "To globalise [the indirect and non-targeted approach] strategy, that is, to be able to compare steroid profiles analysed at different locations, WADA and the accredited doping control laboratories harmonised the employed analytical methods. Additionally the ADAMS was further developed to enable combining the collected steroid profiles of all doping control samples and the direct comparison with individual and population based reference ranges."

The ABP - and prior tests - help form a track record of both suspicious circumstances and the absence of them. While the Blood Passport is a unique case - because you can be handed an ADRV (Anti-Doping Rule Violation) for blood passport values, in all cases, your test history forms a sort of larger "biological" passport that plays an increasingly important role.

For those wanting more, you can learn a lot through the various scientific papers published on anti-doping science and also reading through many of the Arbitration Decisions that are a matter of public record. USADA has a helpful list of CAS decisions

A Bias Against False Positives

Certainly there is some truth that presenting too much information could empower athletes to circumvent the system. Yet it's clear that athletes circumvent the system regardless. I definitely sense that there is a closed mentality within the WADA community that has negatively impacted public perception. I've seen repeated accusations that the process is biased against the athletes. By the time an athlete is served with notice of an AAF, there's a very clear reason that the assumption is that they are guilty. But until that point, the system is overwhelmingly biased against false positives. The degree of certainty required is extremely high - 99.9%. This mandate against false positives are overwhelmingly the single biggest obstacle that WADA signatories face in the fight against doping.

Torben Pottgiesser did a study, "Detection of autologous blood doping with adaptively evaluated biomarkers of doping: a longitudinal blinded study", further discussed by Ross Tucker. At a certainty of 99%, 8 of 11 athletes who were "cheating" were flagged by the ABP but there was one false positive; at 99.9%, it was 5 of 11. WADA mandates 99.9%.

The TL;DR summary is this. Science works. HPLC/MS provides confirmation of a substance's presence in a given quantity with near absolute certainty. It also provides a great deal of information as to probable "hows" and "whys" of that presence as well.