Sunday, June 3, 2007

My Research, and Why Floyd Landis is an Ass





The Good: J.T. Brenna, master of the chemical universe, testifying at the Floyd Landis trial a couple weeks ago.




The Bad: Landis attorney Maurice Suh.






The Ugly: Floyd Landis, doped Tour de France winner and Lance wannabe.



And here by “my research” I mean my professor's research, and by “Floyd Landis” I mean his lawyer, who tried to argue against my professor. This isn’t strictly about Australia, but as my professor’s research is the real reason I’m here in Melbourne, I thought I might explain the principles behind gas chromatography and how they relate to the Floyd Landis Trial.

It’s always exciting when you find that your academic research has practical applications, so imagine the rush (yeah, I said rush) you get when you read about your research professor testifying at one of the biggest trials of the year. My professor back in Ithaca, Dr. Brenna, was in LA just a couple weeks ago, testifying twice as an expert witness for the United States Anti-Doping Agency (USADA) against last year’s Tour de France winner Floyd Landis. He specializes in some separation sciences, one of which is gas chromatography.

Chemists are control freaks who love manipulating substances. Separation science is the art of separating different chemicals. Sometimes you can use simple techniques to separate different materials—draining water from sand, for example, or letting the water evaporate, or boiling it. Separation becomes more difficult with chemicals of similar characteristics.

A GC with some attachments. This is what I've been spending my time with, instead of friends, for the past couple months.


Gas chromatography is able to separate very similar chemicals by sending them through a long, heated column that contains some stationary substance. Depending on that stationary substance, your chemicals will be attracted, repelled, or indifferent to it. If you were then to send a gas through the column and slowly raise the temperature to push the chemicals out, they would individually exit the column, depending on their attraction to the stationary substance. A “gas chromatograph” or “GC” is also equipped with a detector and timer that record the relative amount and time of these exiting chemicals via a series of peaks. Below is a sample GC readout, which shows a number of peaks, each corresponding to a different chemical. Those further to the right have exited the column later; those to the left exited the column sooner. Larger peaks indicate a larger amount of a chemical.


For a given column, stationary substance, and temperature program, you’ll find that each chemical takes a characteristic amount of time to travel through the column (“retention time”). You can put one chemical in the GC at a time, and determine its characteristic retention time. Later, when you have a sample of many unknown chemicals, you can put them in the GC and determine the chemical by its characteristic retention time.

Sounds pretty logical, right? Well, there’s a complication to this—sometimes completely different chemicals have identical retention times. Or sometimes their retention times overlap. In these cases, you haven’t separated your substances at all! You don’t know what chemical you’re looking at, or how much of the chemical your sample had. You won’t be able to convict Floyd Landis with this shoddy data!

That’s where two dimensional GC (“GCxGC”) can be useful. GCxGC uses TWO columns, one right after the other. The different columns contain different stationary substances, so they separate chemicals in different ways. If your chemicals have identical retention times with one column, they should be separated by the second column. Below is a sample readout from a GCxGC. Now, instead of looking straight at the peaks, we’re looking above them. The two axes correspond to the retention times on the two different columns.

Look at the peaks numbered 8 to 12. They would have been very close together, if not completely unseparated, on the first column. Fortunately for us the second column has separated them. Good thing, because now we have separated each chemical, and know how much of each we have.

Dr. Brenna wants to use a GCxGC to separate steroids. Last semester, he won a $1.3M grant from USADA to develop GCxGC steroid separation. My research professor in Melbourne, Dr. Marriott, is an expert on GCxGC, and is collaborating with Dr. Brenna on this steroid project. Their lab teams are currently looking at 27 steroids on the GCxGC and determining the correct column and temperature program to individually identify the steroids. They will create a library of characteristic retention times, and eventually be able to take urine samples, put them through the GCxGC, and determine both the identity and amount of these 27 steroids in a single sample of urine. Not only that, they’ll be able to do it much more precisely than anyone has done it before.

Think of how many times Lance Armstrong was accused of using steroids by a science lab, only to have the accusation retracted, based on insufficient evidence. Think of the current Landis trial. Hopefully Dr. Brenna will be able to provide the United States Anti-Doping Agency with a superior steroid testing protocol for the future.

What exactly were the separation scientists looking for in Floyd Landis’ samples? They were looking at his level of testosterone (a steroid). Everyone has some testosterone in their body naturally, and some have much more than others. The scientists look at the amount of testosterone relative to another steroid. They have found that this ratio has an upper limit naturally, and that this limit is only exceeded when one takes extra testosterone, which is precisely what happened in the case of Floyd Landis. Sorry, Floyd. You’re out. USADA says so, and J.T. Brenna agrees.

If you’re interested in a general article recapping the Landis trial, look here.

There are some articles that discuss Dr. Brenna, part in the trial, and his financial connection to USADA. Here and here.

If you’d like to read more about gas chromatography, you’re crazy. But here, here, and here you go.

11 comments:

DBrower said...

Hi, we linked this into the roundup of Landis news at trust but verify. Your explanation of 2d GC is usefull, and we'd agree it will help dope testing.

Unfortunately, you seemed to have missed some of the problems with the testing in the Landis case. Since you seem informed, your analysis as someone knowledgeable and attempting to argue against the Landis claims would be interesting.

We covered the hearing in detail, and focused on the below in our post-hearing post about the fatally flawed LNDD protocol.

To our knowledge, none of the USADA witnesses, including Dr. Brenna, contradicted the points that Landis (and the bad Mr. Suh) made.

I. The substances in the T/E GC were not properly identified by three diagnostic ions, as required by the WADA test requirements. There is therefore no way of knowing if there was coelution in the urine matrix. LNDD seemed to have collected the data, but not looked at it, and before it was to be re-examined, erased it.

II. First, there was nothing in the protocol to properly identify the peaks in the IRMS testing. While Dr. Brenna showed that the calibration standards had matching retention times with samples in the GC part of the IRMS, there was no cal mix identified in the IRMS portion anywhere near the required times per the WADA spec. Times differed in the IRMS from the GC by around 6 minutes. This is way outside the WADA requirement that the times be within 1% or .2 minutes (12s) whichever is smaller.

Second, the cal mixture used did not contain the actual metabolites being measured as out-of-legal-values in the test, being the 5a and 5b.

Third, the reported ranges for the 5a and 5b had a mutual variance that is far outside the ranges between the two that have ever been seen in the literature, even when doing analysis of known-doping subjects.

A reasonable conclusion is that the IRMS peaks were misidentified, and the values reported are of different substances, and not the 5a and 5b that are claimed.

It is known that Landis was taking cortisone, and if one of the misidentified peaks had a substantial cortisone component, that would account for it's strikingly negative c12-c13 isotope ratios.

To date, we haven't heard anything like a scientific refutation of these Landis points.

Can you offer any counter-arguments?

thanks,

TBV

GMR said...

Dear Adam

Does the 1D ion trace pictured above acquire three ions? The 1D trace shows no sloping baselines. Question was there manual subtraction of background noise? I was about to state that it had no co-eluting peaks but on closer inspection I do detect shoulders. I am not certain how the two pictures between the 1D and the 2D GC relate. Is the peak at 18:00 475 the same peak in the 2D graph as number 15?

If any other accredited lab other than the LNDD had analyzed Floyd's B samples or even Dr. Brenna's lab, Floyd would be riding in the 2007 Tour de France starting on Saturday. However the LNDD personnel used 20 year old equipment, without the use of user manual, ran the equipment outside of manufacturer's operating parameters, literally "cooking" the results: sloping baselines, co-eluting peaks, using fewer ions than required, using manual background subtraction and constructing what ever the results they would like via software manipulation. Now imagine the world judging you on the sloppiness of this work.

Mainstream media reporters understand less about the science and more about the emotion. You may actually see the video of your professor testifying as this was the first open arbitration hearing. Use the http links for online viewing. The best analysis and coverage of the hearing was done by Trust But Verify.There is also an e-book by Arnie Baker The Wiki Defense. How the French Lab (LNDD) & US Anti-Doping Agency Botched Floyd's Test that covers the numerous lab errors.

The GC 2014 by Shimadzu looks state of the art. The Isoprime 1 at the LNDD was state of the art in 1987. Imagine twenty years from now how overworked technicians could operate the GC 2014 in a manner that the two columns are out of sync, running in a non-linear fashion and create results that condemn an athlete's career.

Look forward to the explanation of linking the 1D and 2D graphs!

Adam said...

Hey, thanks for the two comments there and the other links!

To be honest, I haven't followed the trail as closely as you have, and only intended for the entry to serve as an explanation for potential GC and GC/GC uses for the uninitiated. I do appreciate your comments and hope that you have found satisfactory responses from sources more knowledgeable about the trial than myself.

Adam said...

The 1D GC and 2D GC in my entry are example GCs. They do not relate to the trial, or each other. They are merely intended to show how a second column can represent the data better and reveal coelusion on the first column.

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