Doping detection via isotope ratios

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One of the most active areas of current security research is performance-enhancing drugs, both on the the detection and the evasion side. Testosterone is particularly difficult to detect because there's basically no chemical difference between the exogenous and endogenous testosterone (EPO is a somewhat easier case because the glycosylation is different) and you can't rely on testosterone levels because there's a lot of natural variation. The standard test relies on the testosterone/epitestosterone ratio, but there's a lot of variation there too and athletes can evade it by taking both testosterone and epistestosterone.

One clever technique, suggested by Southan et al. relies on the isotope ratio of carbon 12 (C-12) and carbon 13 (C-13). It turns out that the isotope ratio is determined by diet and therefore somewhat different in each individual. So, by comparing the C-12/C-13 ratio in testosterone to that of other precursors (e.g., cholesterol), you can determine whether the extra testosterone is exogenous. In order to make this technique work you first separate out the various compounds using gas chromatography (GC). You then use mass spectrometry (A MS to determine the isotope ratios of the various fractions. Unfortunately, the reactive groups on the steroids tend to react with the GC column (used to separate the fractions), which gives you lousy results. It's tricky to protect the groups because techniques that involve adding extra carbon atoms change the isotope ratios.

The Nov 30 issue of Rapid Communications in Mass Spectrometry contains a paper by Sephton et al. describing a technique (hydropyrolysis) for protecting these reactive groups without changing the carbon ratios, potentially rendering this a viable detection technique.

For discussion: How would you counter this detection technique?

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Since cholesterol is absorbed from the diet (along with other precursors) it should be possible to manipulate the observed C12/C13 ratios by consuming properly C13 "doped" cholesterol (or whatever) before tests. One could also manufacture steriods with differing C12/C13 ratios. In general, this seems like a very expensive way to force people to use more expensive evasion techniques. The arms race will inevitably continue.

BTW, I'm rather surprised by this problem with GC. The material in the column is the so-called "stationary phase", and it is straightforward to get columns with custom stationary phases that would not react with a given steroid. I imagine that the issue is that conventional chromatography usually uses a polar stationary phase and a less polar mobile phase, and a polar phase might do bad things to steriods, but one could just do so-called "reverse phase" chromatography if really needed.

How is testosterone for doping applications typically manufactured? Could you harvest it from a collaborator being fed a similar diet? Alternatively, could you synthesise the stuff from things in your diet (or even from cholesterol from your own bloodstream)? Manipulating the C12/C13 ratio in the stuff sounds expensive....

I don't have ready access to the paper but I wouldn't think that the isotope ratios would vary all that much so you'd need very accurate (expensive) data to prove anything. Presumably that makes the chromatography more demanding.

Deuterated testosterone is commercially available, but I didn't see any C-13 labeled in the Sigma catalog, at least. C13 cholesterol is available. If you had to push the ratio the other way, there is the possibility of synthesizing C13-depleted compounds. All plenty expensive, of course. It also depends on what exactly they are measuring. If they look at intact testosterone, you could label any position with anything (but watch for deuterium shifting the elution from the GC), if they are fragmenting it, you'd need to be more careful.

On the cheaper end you could try to manipulate the diet by choosing C13 rich/poor foods to match your testosterone, assuming that is actually practical, but you still would run up big mass spectrometry bills trying to get that right. Measuring isotope ratios accurately is not run-of-the-mill MS work.

If you can manufacture two grades of testosterone, one at the high end of observed isotope ratios and one at the low end, individuals can mix the two proportionally to obtain any ratio in between.

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