Sports Doping: Analytical Chemistry is Not Magic

Jul 15 2008 Published by under Cycling, Doping

The world's greatest sporting event is on at the moment; I'm sure you are all glued to the live feed from every morning (in the US, whatever time wherever you may be) [Go Vande Velde!!!!!]. You may possibly have missed the expected, i.e., the first doping positive- Beltrán was booted for suspicion of EPO doping. And you know we just love the doping stories around here. [Update 7/16/08: Another EPO positive non-negative "A" sample]
One of the larger ones of recent interest was the Landis debacle. The debacle in which the accused rider's defense rested in no small part on the probity of the testing lab and the validity of the analyses which provided evidence of his testosterone doping.

This brings me to a fantastic and highly relevant post that is up on the 49 percent blog. In a recent entry, Samia details some of the difficulties of producing ready-for-the-courtroom analytical results. [Update: On re-reading, I didn't really make it clear that Samia is not writing specifically about sports-doping but rather about analysis of soil and water samples. The essential principles having to do with experimental / analytical design and interpretation of results are generalizable to many other types of chemical analysis]

Because this kind of analysis involves rather small minimum reporting limits and use of very sensitive instruments (GC/ECD), there is a lot of quality control involved. This data has to be defensible in court if required, and auditors are very picky about how things are done (with good reason). You can't just stick the samples on the GC and report the raw data without comparing it to controls. Examples of possible variables are things like matrix interference, column contamination, inconsistences in sampling methods, possible contamination of reagents or glassware, etc. So there are all kinds of what we call "blanks" to control for this interference and help nail down problems if we start getting weird numbers somewhere. We spike these samples with known amounts of the analyte of interest, as well as another compound called a surrogate that can (again) help weed out where problems are occurring. It works like this: if you know how much spike solution or surrogate you injected into a blank, and you get some weird percent recovery, it's time to investigate possible lab contamination. There are also bottle blanks where the engineers in the field rinse their glassware and send the liquid rinse as a separate sample-- this is a control measure that evaluates possible field contamination.

She is much more capable than I of describing the complications, of course. My experience with analytical chemistry is mostly through collaborations. Nevertheless I learned very quickly that my view of "Gee, can't I just hand you a bunch of samples and you can give me nice reliable, repeatable levels of exogenous drugs, neurotransmitters, their respective metabolites? Three different tissues from two different species? All by next Thursday? ..thx k bai!" was......naive.
Even before you hand off experimental tissues (blood, brain regions, etc) to the chemist types you have to deal with control issues. You can't just analyze the raw blood (or even plasma) or throw a few mg of brain tissue into the machine you know. There will be some so-called extraction steps at the bench to clean up your samples, removing a bunch of other proteins, molecules and gunk that isn't of primary interest. Since it is in many cases uncertain what effects this may have on your quantification of, say, methamphetamine or dopamine or 5-HIAA, you have to include parallel control samples in which you have spiked the sample with a known quantity of what you are trying to measure. This allows you to correct for the extraction (and storage, and...) procedure in your test samples by reference to what changes were produced in the known sample. If you have multiple compounds you want to quantify? Well, depending on the situation, you may want to have multiple controls. Finally, in some cases you may want to spike the sample itself with a similar compound that will resolve as clearly analytically distinct so that you have the best possible control comparison.
And as many scientists can attest from their diverse experiences.....stuff happens. Analyses get blown, weird results are obtained, troubleshooting is required. In most scientific situations, we are not dealing with absolutely irreplaceable samples; after all the experiment can be re-run if necessary. In sports doping cases? Not so much. And therein lies the problem for any sports fans that have even a nodding acquaintance with analytical chemistry and the limitations detailed by Samia. I, for one, am under no delusions that the laboratory analyses of athletes' blood or urine samples are perfect and magical. So when there is any evidence of sloppiness, a lack of confirmation from multiple aliquots of the same sample, any evidence of contamination, etc....well, confidence in the verdict is not. very. high.

11 responses so far

  • leigh says:

    i never did envy the analytical chemist. some people enjoy the challenge, and i'm glad they exist.
    by the nature of my path in life, i have associated more with synthetic chemists, who know exactly what has been added to their reaction. on the other hand, analysis of biological samples is quite the opposite- you know nothing without scores of controls!
    the pressure must be very high in these types of cases.

  • juniorprof says:

    She is much more capable than I of describing the complications, of course. My experience with analytical chemistry is mostly through collaborations. Nevertheless I learned very quickly that my view of "Gee, can't I just hand you a bunch of samples and you can give me nice reliable, repeatable levels of exogenous drugs, neurotransmitters, their respective metabolites? Three different tissues from two different species? All by next Thursday? ..thx k bai!" was......naive.
    I once pulled this stunt for some endocannabinoid measurements. The look on the chemist's face was priceless. Needless to say he set me straight (appropriately). Unfortunately the project never really got off the ground. Now would be a good time to rethink that one while its fresh in the old hopper.

  • leigh says:

    measuring ec concentrations came up in a recent brainstorming session- trying to come up with a way to finish off my thesis. definitely not the answer at this stage of my studies, it got shot down immediately.

  • anon says:

    leigh, you have a wrong view of synthetic chemistry. I'm going to limit it to methodology development. Let's not get into a pissing match of whether analytical or organic is tougher. Just want to set the record straight that it's not that simple. Often you don't know what the hell you got in the end and you have to figure out the mechanism and optimize the route even when you do know.

  • DrugMonkey says:

    There's chemistry smack? Dude, there's chemistry smack, who knew....
    /pops corn

  • Samia says:

    I'd like to clarify that the only matrices I work with are waters and soils-- the tissue stuff is handled by a different branch entirely, and Volatiles takes care of the airs (the only thing I've really done in that lab are some preliminary water screenings). I'm not exactly sure what kinds of controls the tissues people use, but now I have a good excuse to poke around there and ask some questions... 🙂 My guess is that since AFAIK they are working with mostly fish tissues, they are probably looking for rather low-weight organic compounds and not giant molecules like antibodies or proteins. I have no idea what kinds of controls are involved in doping cases.

  • DrugMonkey says:

    My point is that the structural limitations in the approach are quite similar. It takes very little imagination to translate what you wrote to other analytic chemistry settings. It isn't about a specific assay for testosterone or EPO or whatnot, the design and interpretive issues are quite similar.
    you may or may not have noticed but I get kinda jazzed about areas where broader scientific understanding can make things better. for example, the sports doping entities might realize that the relatively minor marginal cost of having multiple independent lab determinations and expectation of a legal-standard of practice would head off a lot of these post-decision lawsuits/hearings and fan bad feelings all around.

  • leigh says:

    i have no first-hand knowledge of synthetic chemistry myself, and i hope i didn't come off trying to imply that i did. i have associated with folks doing synthesis in a training program. their complaints can basically be summed up as such: "biology makes things so messy, in chemistry we know exactly what we put in there, wtf." i took them seriously, maybe they were just looking to piss and moan.

  • anon says:

    That's comparing organic synthesis to biology and not to analytical chemistry. Comparing analytical chemistry to biology will bring up the same problem.
    Oftentimes you don't know what the hell the function of the stuff you put in is. Or even if you think you do, you're wrong and it turns out to re-arrange into some small nano-structure in solution that is the super catalyst and the only way to tell is to do rigorous kinetics. There is still a lot of fundamental knowledge lacking in often used catalytic coupling reactions. It's a black box that works and most people don't think of it too much beyond that. Improving outcomes becomes empirical with some intelligent guesses thrown in.

  • The Journal of Applied Physiology has a couple of articles in press right now on this topic written by a couple of very prestigious physiologists. The long and short of them is that EPO testing is unreliable.

  • Kerry Maxwell says:

    As a cycling enthusiast, I want to believe these results are the the product of shoddy testing. But I've been burned too many times (Tyler Hamilton, Floyd Landis, Michael Rasmussen, etc). Landis and Rasmussen revealed their true colors as not really nice people, liars who had no qualms about hurting the people around them. My first impulse is to question the testing, but I can't say time has been kind to those who claim innocence.

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