An article "M" sent in word format, that we hope not to have butchered in transformation. F3 Mix Cal Bl. Urine GC-MS IS 640.2 642 641.4 5B 908.4 911.4 911.4 5A 930 934.8 932.4 P 1147.2 1152.6 1150.2 GC-IRMS IS 872 870.5 872 5B 1318 1316 1323 5A 1352 1354 P 1671 1674 SOURCES:
Was the 5A Androstanedial properly identified in the GC-IRMS chromatogram?
Landis was found to have doped with synthetic testosterone. This finding was based on identifying some metabolites of testosterone by gas chromatography (GCMS), in particular the 5A, and then analyzing the carbon content of those metabolites by GC-IRMS to determine whether the testosterone was synthetic.
What I want to establish with my graphs is that the metabolites, in particular the 5A, was accurately identified.
Principals of Identification:
In gas chromatography substances (analytes) pass through a GC column and are recorded as a pattern of peaks, with the size of the peak proportional to the amount of the analyte. Analytes are identified using 2 methods. Each analyte takes a unique time to pass through (elute) the GC column. This is called its retention time (rt), often measured relative to some known substance, called the internal standard. One method is to compare the rt of a sample substance with the rt of a known substance in a “reference material”; if there is a match within a certain degree, here 1%, then one can be sure that the substance is identified. The second method and most reliable method is to analyze the mass spectra of the substance as it exits the GC column with a mass spectrometer. This process is called GC-MS. However, in the GC-IRMS the substance is combusted to carbon atoms after it exits the GC column, so no mass spectra can be analyzed and identification is based on rt. Typically a substance will take longer to pass through the GC-IRMS because of the combustion phase and the exact rt will vary depending on the GC settings used.
“Generally chromatographic data is presented as a graph of detector response (y-axis) against retention time (x-axis). This provides a spectrum of peaks for a sample representing the analytes present in a sample eluting from the column at different times. Retention time can be used to identify analytes if the method conditions are constant. Also, the pattern of peaks will be constant for a sample under constant conditions and can identify complex mixtures of analytes. In most modern applications however the GC is connected to a mass spectrometer or similar detector that is capable of identifying the analytes represented by the peaks.
The area under a peak is proportional to the amount of analyte present.”
Three metabolites of testosterone are 5A, 5B and P. These occur naturally in urine. Brenna testified that one can compare the pattern of peaks (and corresponding rts) in the GCMS with the pattern of peaks in the GC-IRMS.
With that let us look at the graphs.
I have relabeled the graphs as follows.
I = Internal Standard; B = 5B androstandial; A = 5A androstandial; P = pregnane: a,b,c,d = small unidentified peaks; X = large unidentified peak
ISSUE: WAS THE A IN THE GC-IRMS THE SAME A AS IN THE GCMS.
IDENTIFICATION IN THE F3 SAMPLE:
The I, B, A, and P in the F3 sample are properly identified in the GCMS because their retention times match those of the Mix Cal Acetate, which is the reference standard here. Further it appears that the mass spectra for each also verify the identity of each analyte. We should also note that the analytes in the blank urine are also properly identified because their retention times match those in the Mix Cal, and that a sequence of peaks has been established based on retention times. Since the analytes in the blank urine match the Mix Cal, the blank urine can also be used as a reference standard.
The question is whether the analytes in the GC-IRMS are properly identified. In particular is the peak identified as A (the 5A) in the GC-IRMS the same peak properly identified as the A (the 5A) in the GCMS.
When we compare the retention times of the IRMS analytes to the those in GCMS Mix Cal Acetate we find that they are off by about 4-6%, but follow the same pattern. Based on the Meier and Brenna testimony we know that the retention times and relative retention times of the analytes in the GC-IRMS cannot match those in the GCMS within a 1% standard because of the delay caused by the combustion time and the general difficulty of matching conditions on different machines. In addition the temperature ramps, and possibly even the columns were different in this case. Meier conceded that one can often only achieve a 2-3% match under ideal matching conditions. Nevertheless the retention times/relative retention times here matched within 4-6% and the pattern of peaks (retention times) also matched.
Since the same substances are in both the GCMS and IRMS F3 sample, we know that all the major (and even minor) peaks that appear in the GCMS must also appear in the GC-IRMS, unless the peak disappeared (contained no carbon).
In the GCMS the A followed the B by 21.6 seconds, which is quite close together. Indeed 20 seconds is the outer limit for matching retention times specified by WADA 2003IDCR. In the IRMS the A followed the B by 34 seconds. So there was some further peak separation in the IRMS of the A and B. The B followed the IS by 268 seconds in the GCMS, and by 446 seconds in the IRMS, a significant separation in the IRMS which explains why the retention times and relative retention times are so far off. So it appears that the peaks separated but did not change their sequence.
SHIFTING OF PEAKS?
Could the B or A peak in the GCMS have shifted to some other position in the IRMS rather than in the 4 central peaks? This is highly unlikely given the pattern of peaks and retention time matching. And for the B we know that this is impossible, because it’s retention time matches that of the B in the Mix Cal. TBV and others claim the A could have shifted to the peak at b. But in that case what happened to the b peak, it must be accounted for? The only way for the A to have shifted to b, is for both of those peaks to have switched positions. Moreover, somehow the peak size of the b must increase to look like the A, and the peak size of the A must shrink to look like to b. The probability of this happening is close to zero.
USE OF BLANK URINE AS REFERENCE STANDARD
One other corroborative fact is that the retention times of the I, B, A, and P in the IRMS F3 match those in the IRMS Blank Urine, and the I and B match those in the Mix Cal. Recall that the retention times of all of the analytes in the GCMS Blank Urine matched those in the GCMS F3 and Mix Cal. This shows that in this case the retention times of the analytes in the Blank Urine and any reference standard like the Mix Cal should be identical. That is the Blank Urine can be used as a reference standard in the IRMS, and as we have seen all the retention times match with those in the F3, including the 5A. Now it’s true that if the 5A moved in the IRMS Mix Cal then it would move in the Blank Urine also. But we have seen from the Mix Cal retention time that the 5B did not move relative to its position in the 4 central peaks. The internal consistency is just too strong. The 5A did not move, and follows the 5B in the IRMS just as it did in the GCMS. Its retention time matches the Blank Urine.
The fact that the Blank Urine can be used as the Mix Cal in the IRMS suggests that even if the Mix Cal had contained the 5A it would not have provided any more information. If the Mix Cal had contained the I, B, A, and P, and all the retention times had matched would we be any more sure that the A was properly identified than using the Blank Urine. Not really, because if the A had shifted in the Blank Urine, it would have also shifted in the Mix Cal.
DIFFERENT COLUMN AND RAMP CONDITIONS IN GCMS
The claim that the 5A peak shifted or switched in the GC-IRMS because the chromatographic conditions were different than in the GCMS can be easily tested. Simply duplicate those conditions and run a new GCMS and see whether the peaks shifted. It wouldn’t cost that much. Landis doesn’t do that because he knows it won’t change anything. I note that Shackleton’s published study at figure 3, shows the same sequence of 4 central peaks with the 5B closely followed by the 5A using the same chromatography column as used by LNND. Even if a different column was used for the GCMS as some are claiming the sequence of peaks did not change.
So we have 2 propositions:
1. The A in the IRMS is the A in the GCMS.
2. The A in the IRMS is some other peak in the GCMS, the b.
I challenge those who question the identification to assign a probability to each statement, and back it up with analysis. I assign a probability of 95% to 1, and 5% to 2.
GCMS F3 retention times: USADA 324
GCMS Mix Cal Acetate retention times: USADA 309
GCMS F3 Blank Urine retention times: USADA 324
GC-IRMS F3 retention times: USADA 351
GC-IRMS F3 Blank Urine retention times: USADA 351
GC-IRMS Mix Cal Acetate: USADA 362