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.

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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.