Monday, November 12, 2007

Now chasing specificity and methylprednisolone: Mister Idiot, himself

In a comment, the estimable Mr. Idiot writes the following, and we cede the floor to this discussion:

In simplest terms, TbV's and Ali's work has shown that there is some possibility that something, or somethings, other than the right things, have found their way into the peaks of interest. This post is about what that could be.

Exhibit 106 of Floyd's recent document dump gave us some information we were long interested in - the exact substance injected into Floyd's hip on July 8, 2006 (and May 5, 2006 by the way). It was two glucocorticoids called dexamethasone (hereafter dexa') and methylprednisolone (hereafter methyl'). Glucocorticoids (a.k.a. corticosteroids) are a class of non-sex hormone related steroids, but they are still chemically very similar to testosterone.

[MORE]


Both of these substances are banned by WADA, but were covered by a Therapeutic Use Exemption for Floyd. They are both only exogenous. Unlike testosterone your body doesn't make any of this stuff.

Like testosterone, dexa' and methyl' both are metabolized by your body and broken down into other compounds. When you have it injected "intra-articularly" (into a joint) these metabolites can come out in your urine for a long time (weeks at least).

Dexa' comes out in your urine partly intact, and also as four different metabolites. Methyl' comes out partly as itself, and also as sixteen different metabolites.

So, the obvious question is, "Did any of these metabolites co-elute to any degree with the 5aA (or 5bA)?" In other words, “Do any of these metabolites show up as the little peaks, and shoulders, and possible hidden elutes in our peaks of interest?” And if you are an astute Floyd junkie, you will also wonder "If any of these metabolites did co-elute, could that have lead to a strongly negative CIR?" (although as we have seen, the negativity could be increased by a partial co-elute even if the substance was not highly negative, depending on the starts and stops of integration).

So, “Did any of these metabolites co-elute to any degree with 5aA (or 5bA)?” The answer is “maybe.”

Unfortunately, the only way to tell for sure is to have the complete mass spectra data of the relevant peaks. That information is gone.

So, what are we left with? Well, what we would like to have is some evidence that at least one of those 20 metabolites has a habit of eluting at least somewhere near the metabolites of testosterone. The problem is that when something elutes in a GC/MS is not really predictable. In different chromatographic conditions, stuff elutes at different times. And it is particularly hard to predict the retention time when dealing with glucocorticoids like dexa’ and methyl’, because they are highly temperature sensitive, so small changes in the conditions will strongly affect the elution time.

I have not found a single chromatogram available on the internet that has dexa’ or methyl’ along with testosterone (or their metabolites) shown on the same chromatogram.

A further complication is that testing for glucocorticoids is usually done with liquid chromatography / mass spectrometry (LC/MS), rather than GC/MS. This is for several reasons. First GC/MS (at least the way LNDD does it) involves certain steps to prepare the samples to be analyzed. One of the steps is acylation. I don’t get the details of the chemistry, but the effect is if the compound has any hydroxyl (Oxygen and Hydrogen together) groups, it ends up lowering the CIR of the compound. Many of the methyl’ metabolites have numerous hydroxyl groups, so the resulting compound could have seriously negative CIRs.

Another reason LC/MS is used is that glucocorticoids are hard to turn into gases (they have low volatility) and they are very temperature sensitive, making consistent results in GC/MS difficult to achieve. That superiority of LC/MS for analyzing glucocorticoids may be why they aren’t normally shown on the same chromatogram.

So, the summary of all that is that it does not really seem possible to show ahead of time whether these two compounds, dexa’ and methyl’, or and their 20 metabolites, would elute with 5aA or anything else. Too much depends on the particular circumstances of the particular set-up of the particular GC/MS machine using a particular process.

Now, most of you reading this remember the great discussion between Larry and m about whether the demands of TD2003IDCR were met. The most generous take on all that is that LNDD did met TD2003IDCR using a “relaxed relative retention time” standard. The 1% standard stated in the document would need to be relaxed to about 6%.

What that means is that any of these dexa’ and methyl’ metabolites that were within 6% of the peak of interest in the GC/MS have to be considered as possible co-elutes in the IRMS (there may be even more movement of substances than that between the GC/MS and the IRMS, but that’s a conservative, but big enough window).

Remember we are talking two main compounds and 20 metabolites. It seems reasonable to conclude that one or more of them fall within 6% of the 5aA peak in the GC/MS. Looking at the chromatogram, there are some possibilities there. Any one of those could have negatively skewed the CIR in the IRMS.

Of course, the complete mass spectra data could have made all of this moot, because it could have shown the purity of the peaks in the GC/MS and identified everything around it. So we would have known there were possible problems. But, alas, that information was erased.

In the end, this is all about specificity, the way LNDD assures the purity of its peaks. In short, they don't, because they don't have the mass spec data.

And with dexamethasone and methylprednisolone we have compounds that could have caused contamination / interference, and there seems to be no way to show they didn't, and no way to show they did.

So, my guess is that means a big fight over the meaning of the legal requirements of ISL 5.4.4.2.1, and perhaps other documents. The chemistry leads right back to the law. But I won’t go into that right now.

26 comments:

Bill Neumann said...

Your explanation might also explain why Floyd's IRMS results were different from stage to stage. If the testing didn't use exactly the same setup then the dexa' and methyl' compund contributions to the CIR for the compounds of interest might have wandered in and out of the 6% tolerance you've talked about.

wschart said...

I personally am very suspicious of that erased hard drive. The fact that it contained the evidence to show how pure those peaks were leads me to believe that it was erased so that evidence could not be used in the hearing, which then leads to the conclusion that this evidence would have helped Landis. You don't just erase a hard drive by accident, it has to be a deliberate action. Also, normal erasure procedures do not remove data and it often can be recovered by experts. If a more secure from of erasure was used, this is even more suspicious.

the Dragon said...

wschart,

I agree with you. There can be only 2 likely reasons for the erasure, both of which reflect poorly on LNDD.

First, The thoughts you mention. I am a betting man and there are no sure winners, and I would mortgage the house to bet on the proposition that IF the mass spectra contained confirming data supportive of LNDD's findings, it would have been saved.

Second, Inadvertance/incompetance. I guess this is also possible, and would be just more evidence that LNDD is several cut's below on the competance index.

The choice between malfeasance or incompetance, which would you choose?

Regards,

David McGraw

Michael said...

What's worse about the missing hard drive is that the arbs didn't have the guts to stand up and say we can't convict a man if the 'data' can't be delivered. They know what happened to that data and why but were more concerned about their careers than doing the right thing.

Hopefully the arbs at CAS are just waiting to bust this thing wide open. Espcially with the recent revelations that WADA no longer wants to have 'B' sample confirmations.

Unbelievable!

blackmingo said...

Swim,

I knew something was funny about the "cortisone" shots -and I can't believe that it took over a year to clarify that point. Well done.

I think the onus is on them to make sure all those "minor" peaks aren't chock full of carbon13-rich dexamethasone & methylprednisolone metabolites.

Dan

m said...

Mike,

Some comments and errors I noted:

1. OMJ has said there is the very small possibility that some other substance might have eluted at exactly the same time as the 5A, 5B or pregnane in the F3. But the burden was on Landis to show this was likely, e.g. the Cortisone he was taking.

2. The Cortisone metabolites must elute at EXACTLY the same time as the 5A to be hidden under it's GCMS peak. There is no 5 to 6% leeway. We are not comparing GCMS to GC-IRMS here but looking at Landis' F3 GCMS chromatograms.

3. Elution as a close adjoining peak will not affect the 5A carbon measurements. That is what Brenna testified and even Meier.

4. Elution as general noise can, in most cases, be corrected for according to Meier and Brenna. And specifically in the Landis B sample F3 there was no problem with noise according to Brenna.

5. If Landis wants to raise this as a possibility then he needs to pilot a study on his own urine. Do the same or similar separation as was done by LNND and see if any of the cortisone metabolites elute at or near the retention time for the 5A. Look at the mass spectra.

6. The burden is on Landis, not the lab. Despite your claims, and as I pointed out previously, Ayotte's testimony does not constitute an admission that the burden is on the lab to produce the mass spectra or exclude every possibility.

Michael said...

How can Landis prove anything? The lab should be able to produce everything necessary to prove they are correct?

What's your explanation/theory for the deletion of the mass spectra data? Everyone agrees this is very pertinant to the case.

Mike

Mike Solberg said...

Nice response, m. Again, thank you for taking the "pro-Landis" arguments seriously. My response:

1. OMJ also said he would cry foul as loud as anyone if LNDD didn't produce the mass spec data. They didn't. I respect OMJ. He knows a lot more science than I do, but he puts way too much of the burden on Floyd in my opinion.

2. I think you are wrong about that. Say, for example, there was an unidentified little peak within 6% (in seconds) of the 5aA in the GC/MS. When we get to the IRMS, even you admit that the 5aA has moved relative to the internal standard. Well, that little unidentified peak could move too, and not necessarily in the same pattern as the 5aA peak. Especially given that GC is not ideal for working with glucocorticoids, if that unidentified peak was one of the dexa' or methyl' metabolites, it could certainly have moved into an interfering position in the IRMS, with its C12 and C13 added into the mix.

3. I don't have time to look back at the testimony, but surely it would if it had a highly negative CIR, which the metabolites of methyl' may well have, given the acylation of the preparation process and all those hydroxyl groups.

4. I guess my response is that this is "general noise." It is co-elution. I would say they are different problems. That is why ISL 5.4.4.2.1 has both a comment on "specificity" and "matrix interference," different issues.

5. That seems like an obvious thing for Floyd to do, but given the legal restrictions, and the inviable rule that "you can't question the science," I imagine there is a good reason Floyd has not done this to our knowledge. Plus, it would be VERY difficult, if not impossible, to set up test parameters that would match closely enough the circumstances of the TdeF. If nothing else, the hip into which the drugs were injected no longer exists. You couldn't possibly compare how the drugs would metabolize given that Floyd now has an Smith and Nephew hip joint.

6. Well, there is a nice legal argument we need to have, given ISL 5.4.4.2.1 and other documents.

syi

m said...

Mike,

As to 2:

Their is no evidence that the peaks switched places in the GC-IRMS when compared to the GCMS.

There were four central peaks in both the GCMS and the GC-IRMS of the F3. The 5A and 5B, and 2 unidentified peaks. No peaks switched.

The peaks have to match exactly, no 6% leeway.

Larry said...

M -

You say that there's no evidence that peaks switched places from the GC/MS to the IRMS. That's not true - not if you're still arguing that there are reasons in this case for the use of a relaxed peak identification standard like +/- 6%. If there are reasons for relaxing the standard in this manner, then you've admitted the possibility that any peak shown on the GC/MS can appear on the IRMS with a RRT that is +/- 6% from its RRT on the GC/MS. You've admitted that peaks could appear in a different order than the order shown on the GC/MS, or that peaks shown separately in the GC/MS could appear at the same time on the IRMS.

M, this is a necessary consequence of your own argument. The +/- 6% standard gives you the wiggle room you need to identify peaks. But +/- 6% allows for a great deal of wiggle! You're effectively arguing that peaks could "wiggle" their way into some very unexpected positions.

Now, maybe you've dropped the idea that the way to satisfy TD2003IDCR is by dramatically relaxing the standards there. That's OK. I've changed a few of my positions as well. I'd have to hear your new position, and consider the science on which it is based, before determining whether you've now constructed a peak identification argument that would not effectively admit to the possibility of peaks changing positions.

Michael, regarding deletion of the mass spectra data, it would be good to know if this was something LNDD did routinely or whether this is unique to the Landis case. If it's routine, then there may be an explanation for why they did it. For example: I advise clients who have to store medical data, and do so in compliance with medical privacy rules, and as a general matter it's a good idea not to retain this data any longer than is medically necessary.

m said...

Larry,

I'm saying that specifically the peaks did not switch in the Landis B sample F3, not in some theoretical case.

We've been all through this before. Remember my test graphs. Mike agrees with me. There are no unaccounted for peaks in that F3.

tbv@trustbut.com said...

The record on the mass spec data is spread out. I'd expect to find it in the bunch of motions and declarations made about the reprocessing, and possibly Botre's report on the reprocessing, or was that Aguilera?

There didn't appear to be an obvious document retention policy in effect. For reasons that may have been "this disk is getting full", they backed up what the claimed was all the relevant information to a CD-ROM, and wiped the drive for re-use. This was apparently done without discussing the matter with USADA, or any other party. They walked in expecting to be able to get at the data on the drive, and were surprisingly handed CDs instead.

TBV

blackmingo said...

RE: M's comment:
"3. Elution as a close adjoining peak will not affect the 5A carbon measurements. That is what Brenna testified and even Meier."

I've been reading Meier's powerpoint (from TBV's site), and he seems (to me) to suggest that elution close to (separate peaks with tails intersecting) would affect the 5A carbon measurements. I'll try and find the slide number for you to consider.

Dan

Michael said...

Larry,

"Michael, regarding deletion of the mass spectra data, it would be good to know if this was something LNDD did routinely or whether this is unique to the Landis case. If it's routine, then there may be an explanation for why they did it. For example: I advise clients who have to store medical data, and do so in compliance with medical privacy rules, and as a general matter it's a good idea not to retain this data any longer than is medically necessary."

Sounds like an excuse. Would you recommend deletion of medical data in less than a year?

Larry said...

Michael, the answer to your question is "maybe". Not a terribly helpful answer, I'll admit.

I'm only trying to say that maybe LNDD destroyed the data for reasons that had nothing to do with hiding information that might be helpful to FL. They may have had a good reason, they may have thought they had a good reason, they may have had no good reason but no bad intent. Destroying the data out of concerns for medical privacy may have been a good reason, or the reason they thought they had.

Michael said...

Larry,

The only problem I have with that reasoning is that apparently (this is my belief from what I recall) the data was wiped KNOWING that Floyd was contesting the decision.

Knowing that the data was important for explaining something, would you advise a client to erase critical data?

Hopefully CAS will at least address the deletion of Mass Spectra. I wouldn't be surprised if they use this to let Landis off. This seems to me like a much larger issue than the same techs reviewing testing results ie Landaluze.

PEM said...

Larry,

I cannot see how LNDD could have justifiable or believable reasons for destroying data that I would even consider being an innocent mistake, even if it were within their protocol to do so. The Landis affair was not a routine situation. If I was in charge and I had nothing damaging to hide, I would preserve all that I could, especially if I was a body representing fairness.

Exceptions for data preservation could have been made. To me, their action was a blatant violation to preserving evidence. This is not the conduct of a body interested in looking for the truth, but a body covering or hiding their mistakes.

Raw data wiping would be applicable to the routine tests that have negative results and matters not to anyone. All raw data should be preserved for all positive tests that are contested.

Larry said...

Michael and PEM, people erase data, and they dispose of data, and they fail to take care of the data in their possession, and it happens all the time, and the reasons for this are not always nefarious.

How do I advise my clients? It depends on the client and the situation.

But it probably won't surprise you that a company's data retention policy is a very important policy. If a company has a data retention policy, and it's a reasonable policy, and they follow that policy when they dispose of their data, it's going to go a lot better for the company if that data is eventually requested in a court proceeding. There are certainly lawyers out there who advise clients to adopt very aggressive data retention policies, or in other words, to get rid of the data at the earliest possible opportunity. And it's true, one reason for doing so is that some of that data may contain information that is embarassing to the company, or might even subject the company to liability.

Probably the better advice these days (particularly after cases like those involving Enron) is not to be so aggressive. It's become harder to explain why data is being aggressively purged in an age where mass storage is so cheap and where filtering programs can be run to flag sensitive data for long-term storage.

If you'd like to see well-written pieces about data retention policies under U.S. law, see http://www.abanet.org/lpm/lpt/articles/ftr01045.html and http://www.nfib.com/object/IO_21047.html.

However, even the most aggressive data retention policies would require data to be kept if it's reasonable to assume that the data would be relevant in a court proceeding.

The situation gets more complicated with medical records. (I am ignoring for the moment whether the destroyed data in the FL case is properly considered to be a medical record.) U.S. and European law regulate the privacy and security of medical records. All other things being equal, a company would naturally want to limit its exposure under these medical privacy laws, and one way to do so is to get rid of its medical data as soon as it can. U.S. law requires most institutions (such as hospitals, doctors and employers) to hold onto its medical data for relatively long periods (5 years or longer) -- probably anticipating that in the absence of such rules, these institutions would probably seek to get rid of this data when possible at a much earlier stage.

You should consider that long-term storage of sensitive data is a two-edged sword. On the one hand, if a person wants to sue an ex-employer, that person wants the employer to retain all of the relevant information it has about the person, so that person can go through that information and find the stuff needed to win the case. On the other hand, that same person might not want his employer to hold on to sensitive information that might fall into the wrong hands - information like social security numbers ... or failed drug tests!

Consider data retention to be a multi-faceted responsibility. It's not necessarily in anyone's interest to hold on to everything forever. It's certainly a bad idea to destroy data merely because of the potential for liability inherent in holding on to the data. A balance must be struck. This is probably the essence of what I'm saying: in a vacuum, you can't say that the destruction of data is a bad thing. It depends on context and circumstances. And the first piece of context I'd want to see is the relevant data retention policy.

From my perspective as a lawyer, it's difficult for me to speculate on LNDD's destruction of data without seeing their data retention policy (assuming that they had one). I'll admit that there's probably no good explanation for what they did, that the data was probably destroyed carelessly and for no good reason, or because LNDD wanted to eliminate evidence that would help FL. But there ARE other possibilities, however slim they might appear to me or you.

The other factor you have to consider is the ISL and the WADA rules. If the ISL prevented FL from discovering this data, then it wouldn't have mattered much whether LNDD preserved the data. And unfortunately, the majority decision ruled that the ISL did not require this data to be retained.

This is, by the way, another reason to prefer court proceedings over ADA arbitration. In a court, if FL sought discovery of data that had been destroyed under suspicious circumstances, the court might have presumed that the destroyed data contained evidence damaging to USADA's case. I think this is what the arbitrators in the FL case should have assumed.

In any event ... I can't agree that the only possible explanation for LNDD's destruction of data is that they knew the data would help FL's case. But I'll agree with you to this extent: in a rational world far from the governance of the ISL, LNDD and USADA would have faced much more intense scrutiny over this destruction of data.

the Dragon said...

Larry,

There is one very difficult with LNDD using "privacy" as a defense. While I will grant that it is possible LNDD IS NOT the source of constant leaks, anything they want, is a L'Equipe headline in the morning.

Regards,

Larry said...

Dragon, LOL, point well taken.

You know the Las Vegas slogan, "what happens here stays here". At LNDD, not so much.

m said...

Larry,

Great commentary on the balance involved in data destruction vs preservation.

One comment:

"In a court, if FL sought discovery of data that had been destroyed under suspicious circumstances, the court might have presumed that the destroyed data contained evidence damaging to USADA's case."

If a court was bound or had to give deference to WADA's policy of not disclosing lab information beyond the laboratory package (for legitimate proprietary reasons), then the court would be much less likely to draw a negative inference against the lab for destroying the mass spectra data which was not required in the laboratory package.

blackmingo said...

M-

Found those slides from Meier -his powerpoint is in the November 7 major document release:

http://trustbut.blogspot.com/2007/11/major-release-of-hearing-documents.html

In particular, I am taking Meier's slide 13 which is a direct quote from a published article, which reads:
" Overlaping peaks detected by conventional algorithms are systematically distorted in isotope ratio even for closely matched compounds, though high precision is maintained. Further, small trailing peaks can significantly affect the apparent isotope ratio of the major peak."

Later, on slide 36:
"The GC/MS chromatogram of Floyd’s A sample (Fraction 3) is clearly much more complex than the control urine (BLU) let alone the Mix Cal Acetate mix. In the right flank of 5ßA there is clearly a peak not present in the control urine and the peak tail of 5ßA together with the unknown peak tail into 5αA. The peak start of 5αA at 15.4 min is clearly higher than its peak end at 15.6 min. If peak overlap already occurs in GC/MS it will undoubtedly occur in GC/C-IRMS."

My simple impression from these slides:
1) Floyd's sample has overlapping peaks on GC/MS (which could only overlap worse on GC/C/IRMS.
2) Overlapping or trailing peaks significantly affect the estimate of the isotope ratio of the major peak.

Thanks,

Dan

Larry said...

M, thank you. And also, point well taken in your 8:10 pm post. The documents that a party is required to retain depends on the documents that might need to be made available in discovery, and if discovery is limited by the ISL, then a lab like LNDD might legitimately have a more aggressive data retention policy than would otherwise be the norm.

But even in the WADA proceedings, the arbitrators had discretion to order additional discovery. So it's reasonable for WADA labs to expect that they might be called on to provide documents in addition to those contained in the standard ISL package. If data ends up being relevant in a particular case, and if the lab has destroyed the data, then woe to the lab! Particularly after the Enron and Arthur Anderson cases.

I think that LNDD dodged a bullet on the business of data destruction.

PEM said...

Larry and M:

Thank-you for your comments on data preservation.

If the law says you cannot enter my property without a search warrant, then I can flush it or burn it before you get one. All circumstances cannot be anticipated. These are the rules, they must be followed.

If the rules say you cannot look at it, then the point is moot if I destroy it. The only thing to chase is did you follow your protocol concerning how long you must retain it? Does one exist or is it justifiably long enough? Remember, the system is set up to protect sports and athletes and to look for truth. In my opinion, there should be a long retention protocol for all adverse results.

I see lawyers wanting historical information destroyed as they are potential liabilities. Engineers and scientists want to keep the information as it shows pasts operations, what mistakes were made, things to learn from and to prevent from reoccurring.

Should you keep damming information that you could learn from? For self preservation, the answer is an obvious no. This is why regulations dictate retention requirements for 5, 10 or even 25 years. WADA must do the same for the labs.

m said...

pem,

"Engineers and scientists want to keep the information as it shows pasts operations, what mistakes were made, things to learn from and to prevent from reoccurring."

That's how they caught Lance's use of EPO in the 1999 TDF, retrospective testing on old samples to perfect their testing.

! -)

the Dragon said...

m,

"That's how they caught Lance's use of EPO in the 1999 TDF, retrospective testing on old samples to perfect their testing."

Interesting you bring that up. Didn't Dick Pound receive some form of sanction for that adventure?

Also, if memory serves, Ms. Ayotte (sp) stated that any testing was unreliable due to sample stability issues. Or was her statement just "creative"?

I keep waiting for WADA to show some modest level of "ethical" behavior.

I presume doping is bad, not from anything WADA has done, rather from BALCO & OP. I believe athletes cheat, yet IMHO, WADA's motto should be "We Cheat better than Athletes".

Regards,

David McGraw