Yesterday we discovered that Paul Pogba, the Juventus midfielder was provisionally suspended after drugs test found “non-endogenous testosterone metabolites” in a urine sample. The sample was collected on 20th August after Pogba was an unused substitute in a game in Italy. Pogba can face a ban of four years if the listed offences are proven. The Italian National Anti Doping Agency (NADO) reported he is charged under the WADA code for: Violation 2.1 – the presence of prohibited substance or metabolites in a urine samples and Violation 2.2 the use of attempted use of a prohibited substance.
This made me look back on the blog and I realised I hadn’t discussed the detection of testosterone in the past. This surprised me as I spent a substantial time during my PhD doing exactly that!
Testosterone is listed as a prohibited substance by WADA under S1: Anabolic Agents (commonly referred to as anabolic steroids, WADA prohibited list). It is the third most common finding in the category, after stanzolol and drostanolone (WADA 2020 report). Steroids are by far the most commonly detected group of substances.
What is non-endogenous testosterone?
Non-endogenous means ‘coming from outside the body’. Endogenous means naturally produced. Some people prefer the term exogenous over non-endogenous (again meaning foreign or externally produced). Testosterone is produced by everybody and present in all urine samples. This is endogenous. If someone uses prescription testosterone (or illicit testosterone) they will also have non-endogenous/exogenous testosterone in their urine samples. The use of testosterone would benefit sports people mainly by increasing strength through muscle building, also potentially for recovery from injury, but I won’t go into those in detail here.
How do we test for testosterone?
Using standard testing methods, if we detect an exogenous substance that should be enough, as it shouldn’t be there at all! Most labs use gas chromatography or liquid chromatography coupled with mass spectrometry analysis techniques for exogenous substances. However, as testosterone should be there in every sample so how do we tell if it is endogenous or exogenous? Normal or boosted? We have a few tools to use.
Traditionally the urine would be analysed for testosterone concentrations (above a certain amount is highly suspicious), the ratio of testosterone to epitestosterone (a very similar naturally occurring compound) is also monitored (T/E ratio) and reported in each sample. In the vast majority of people the T/E ratio is around 1:1. Statistical analysis shows that anyone who has a T/E ratio of 4:1 or more is highly unusual, usually an indication of doping. The T/E ratio and concentrations of metabolites of testosterone and analysed in each urine sample and plugged into what is known as the athlete biological passport, steroidal module (ABP).
I have written previously about the ABP, but for blood samples. For example this one on Sky cyclist Jonathan Tiernan-Locke . The steroidal module works in a similar principle to the blood module but based on the analysis of steroids present in the urine sample. Over time your values shouldn’t fluctuate too widely and big fluctuations are then flagged as suspicious.
The next tool at our disposal is gas chromatography coupled with combustion then isotope ratio mass spectrometry (GC/C/IRMS). This technique is used when the urine sample of an athlete is suspicious through the ABP, or as a request when the testing authority has suspicions based on other information. The use of the technique is effective, with over 2% of samples analysed testing positive (as opposed to 0.77% in all samples combined). However only around 4 000 samples are analysed this way out of a total of nearly 250 000 urine samples collected each year. Cost is one reason for this low uptake. It is a highly specialised method that not all laboratories can do. In fact the Bloemfontein lab in South Africa were suspended from using this technique earlier this year, presumably for errors in sample analysis or technical difficulties.
The GC/C/IRMS method is based on the different isotopes of carbon that are present in samples. Carbon is made up of three isotopes, carbon 12 (majority), carbon 13 (around 1%) and carbon 14 (very small amount, lets ignore carbon 14). This method is based on the relative difference between the 13C/12C isotope ratio. The ratio found in synthetic testosterone (pharmaceutical) differs from that found in the endogenous steroid. Only by a very small amount, but enough to be measurable.
The results from this test are reported in the WADA statistics as “the GC/C/IRMS result is consistent with an exogenous origin”. They found 91 such positive cases this way in 2021 (WADA stats). In my PhD work I analysed samples following an administration study and found that using GC/C/IRMS vastly increased the time you could detect exogenous steroid administration over the use of T/E.
In summary, Pogba has been caught with a suspicious GC/C/IRMS profile of testosterone and/or metabolites. It was reported initially that this was ‘elevated testosterone’, which was not what the NADO said. Elevated would mean high testosterone levels, which could be measured using standard GC-MS, however the presence of ‘non-endogenous’ would need to be measured by GC-C-IRMS and would not necessarily mean high levels of T were there.
Football doesn’t have many high profile doping stories and Paul Pogba is about as high profile as you can get. It has been pointed out several times online, however, that Juventus is no stranger to doping scandals… We’ll keep an eye on this story.
Only a few are caught as masking drugs hide the real doping, Alan Moore wrote thishttps://backpagefootball.com/sure-theyre-all-at-it-are-you-entertained-part-2/127087/