Retention Times II

I'm having problems editing the earlier post, so this is a continuation in a new one.

Figure 5: IRMS chromatograms stacked; Cal mix, Blank F3, Landis F3

Figure 5 does the same thing we showed in the previous post, theoretically in Figure 3, and practically in Figure 4, which showed how the GCMS peaks were correctly identified. Here we've taken the charts from USADA 361, 345 and 349. We see that the cal mix in this case only contains two peaks that match the urine samples -- the 5aA internal reference, and the first metabolite of interest, the 5bA. These matches are outlined in yellow.

There are no standards in the cal mix for the 5aA or 5bP metabolites, thus no positive identification according to a very strict reading ot TD2003IDCR. This is possibly what Dr. DeBoer meant in his B-Sample observer's report when he said peaks were not identified to "minimal WADA criteria."

It is also puzzling, because LNDD had the right cal mix available with the other metabolites. They did, after all, use it in the GCMS runs, leaving no doubt there.

Absent an end-to-end calibration sample match on the peaks of interest, we are left with two options for identification.

1. Assume the unmatched peaks in the blank are the peaks of interest, unproven since there is no calibration to back up the claim;
2. Attempt to match the GCMS that was properly identified to the IRMS that wasn't.

As we've been discussing, there seem to be two ways to do the latter, relying on the "visual gestalt" of the pattern of peaks. This assumes that the peaks are in the same order ("of course" says the Majority), and that no significant peaks appear or disappear between the representations. The panel adopted this approach, even though there is nothing in the technical documents to support it as a methodology. The correct thing would have been to use a cal mix with the right compounds present.

The second method is to attempt to work out the math to map the retention times from the GCMS to the IRMS. The Majority says this can't be done. Herr Doktor Professor is evidently more comfortable with the math and the physics and tried to do this.

Attempting to map the times requires accounting for all the significant differences, and Meier-Augenstein believed he had.

Figure 5: Shimadzu's catalog of relevant factors

Poking at the "details" button on the Shimadzu "relative retention" page, we see why it seemed like a good thing to try:

Figure 6: Shimadzu explains why relative retention is useful

The advantageous point of relative retention is that it depends only on the ratio of distribution coefficients and the effects from some parameters, such as column length and carrier gas flow, are bascially cancelled out.

However, there are some limiations for relative retention. Measurement of errors will increase for target peaks located far from the reference peak.

(emphasis added).

Are relative retention times useful for comparing results across instruments, which the Majority denied?

This journal abstract suggests otherwise:

Using experimental RRT data for 126 PBDE congeners from the literature, predictive regression models were built for seven individual GC capillary columns differing in stationary phases. Each model includes four descriptors which included Wiener index, Randic index, polarity parameter, etc., selected by CODESSA. High predictability was obtained. High multiple correlation coefficients R² indicated that >98.5% (except for stationary phase CP-Sil 19) of the total variation in the predicted RRTs is explained by the fitted models.

Or this one:

Phenolic acids and related compounds were separated by gas chromatography using three separate columns. One of these columns was coupled to a Fourier transform infrared spectrometer. The trimethylsilyl derivatives could be separated and identified by comparing the relative retention times of the three different columns. However, where there was overlap, the accompanying infrared data clearly distinguished between the questionable derivatives, thus enabling characterization of all derivatives.

This one seems particularly apropos:

Since retention times vary with the column length, type of stationary phase and temperature, suitable parameters for comparison include relative retention times and so-called retention indices (RI). Relative retention times are simply the ratios of analyte times to the time of a chosen standard compound.

On values for matches, this EPA lab related document says,

9.8 Qualitative Confirmation- The relative retention time of methyl parathion in samples is tracked by comparing the relative retention time (rrt) of sample to standard within "3 standard deviation. In addition, a second, dissimilar GC column is used to determine if methyl parathion and surrogate were positively identified by both rrts.

The DEA has used relative retention to identify opiates and steroids:

Gas Chromatography (GC): The chromatogram is not shown. Methandrostenolone, dehydrochlormethyltestosterone, and stanozolol eluted at 7.96, 9.32, and 10.29 minutes, respectively. The peak shape for stanozolol was broad in comparison to the other steroids. The mixture in the yellow tablets was not formally quantitated, but was estimated as roughly 100 : 5 : 2.5 dehydrochlormethyltestosterone : methandrostenolone : stanozolol. Table 1 lists the relative retention times for cocaine, heroin, and six other steroids with similar chromatography.

Table 1. GC Relative Retention Times.

Drug (GC)	RRt
Cocaine	0.580
Mesterolone	0.813
Testosterone	0.822
Heroin	0.829
Methyltestosterone	0.836
Methandrostenolone	0.854
Testosterone Acetate	0.882
Fluoxymesterone	0.990
Dehydrochlormethyltestosterone	1.000
Stanozolol	1.103
Testosterone Isocaproate	1.188

It therefore appears that the assertion by the Majority in paragraph 188 that use of relative retention times to provide comparisons across instruments is "unsound and without any reasonable scientific basis" may itself be categorized exactly the same way. It suggests the Panel did not understand the science, or chose not to understand the science, and/or was misinformed by the "independant expert" or Brenna's testimony.

It should be noted that an even more accurate methodology is the use of "Kovats indexes", but those are not appropriate for the chromatograms at issue here. Their use requires a pair of internal standards, one at the beginning, and another at the end, after the target peaks. Since LNDD has not provided a later standard, the only methodology available is the use of relative retention times.

How we got here

1. Brenna, Ayotte and Mongongu testify relative retention time is how peaks are matched between GCMS and IRMS.
   
2. LNDD did not use an IRMS calibration standard to unambiguously identify 5aA and 5bP in samples.
3. LNDD did not use an IRMS calibration standard with a trailing internal standard admitting use of Kovats Indexes.
4. The only quantitative method available to match the GCMS and the IRMS peaks is the relative retention time.
5. Herr Doktor Professor calculated the RRT matches, and they are at least 5%.
6. TD2003IDCR says 1% or 0.2 minutes.
7. Peak identification appears not to be done in an ISL-acceptable manner.
8. Brenna changes story, testifies [absolute] retention times can't work for lots of reasons.
9. Brenna says "relative retention times won't work for reasons I discussed this morning"
10. Brenna says the match is done visually.
11. Panel accepts "visual gestalt" as an acceptable peak identification technique, and lambastes Meieir-Augenstein and Davis for suggesting RRTs are relevant.
    

Is Herr Doktor Professor's math right?

Over at DPF, a number of people who are not in the group generally associated with sympathy towards Landis' arguments have repeated what Meier-Augenstein did, and obtained identical results, down to the 7.2% mismatch of RRTs on the furthest compounds in samples.

[ still working on the math part to get a clear example ]