Discussion with BustinBilly on matching GCMS with GC-IRMS

In the Saturday Roundup, BustinBilly made a comment:

Don't debate the retention times just look at the damn plots. They are printed @ UsadaAndLandis-FinalAward20-09-07[1].pdf pages 41, 42. They are exactly the same. No doubt.

I used to believe Floyd until I saw the evidence.

To which I replied:

Excellent, not [meant "now"] we can have some useful discussion.

I'm sure you understand that only the F3 fractions are relevant; why the others were included is a mystery.

Your commend say the plots are "exactly the same"

In what ways do you think they are the same? Once you identify some of those we'll have something to talk about.

thanks!
TBV

And BB replied:

The F3 is the 5-alpha-androstandiol-5beta-pregnandiol difference this whole shebang is about. The A sample F3 is @ pages 35, 36. The GC/MS and GC/C/IRMS plots are identical. The two metabolites above and the 5-beta-androstandiol are the 3 peaks in the middle of the GC/C/IRMS plot.

with clarification

I meant the 3 big peaks in the middle.

Because this is important, I've given this discussion it's own post here, and expect to carry on in comments below. Warning: I will be merciless deleting off-topic comments.

---

I believe BB is referring to the the following chromatograms. First the GCMS of the blank, the Landis F3, then the two overlaid to show what Brenna testified, the retention times line up. This was never disputed.

Figure 1:
Blank F3 GCMS plot, labeled by compound; very clean chromatogram.
click for full size
USADA-345

Figure 1a:
Landis F3 GCMS
labeled by compound
click for bigger
USADA-348

Figure 1b:

Blank F3 and Landis F3 GCMS overlaid
Scaling isn't perfect, but the identified compounds line up within TD2003IDCR by time.

(Note the very messy and sloping background in the Landis compared to the blank, and the extra peaks it contains.)

And then the GC-IRMS plot:

Figure 2:
Landis F3 GC-IRMS chromatogram,
labeled by retention time and CIR.
click for full size
USADA 348

To make things clear, below are versions that put the things on top of each other, going along with BB's (and USADA/Majority) claim that the big peaks in the middle are absolutely the ones in question. That is, one is lined up, and the scale adjusted linearly so the other peak lines up as well. This anchors the highest middle peak, and adjusts the scale so the rightmost big peaks line up. These are the "peaks of interest" for the measurements.

Figure 3:
blank F3 GCMS and Landis F3 GC-IRMS
chromatograms overlaid.
click for full size

Figure 3a:
Landis F3 GCMS and GC-IRMS overlaid.
Asserted -28.79/5bA aligned, scale adjusted so 5bPreg matches.
click for bigger.

To make sure I've understood BB's claims, I'll ask now, did I get this right? Please answer in comments below and we'll carry on the dialogue. When needed, we'll add more figures to this post, and if BB wants to add some, we'll include whatever he provides.

Billy writes:

There you have it. The three peaks are the same.

I reply:

OK. Remember what we did, we adjusted the horizontal scale to make those three peaks line up, so we think linear (proportional) adjustment is correct.

Now, look at figure 4, a zoom of the left hand side of figure 3a, where the "anchor" internal standard is supposed to be located.

There doesn't appear to be very good alignment with those peaks is there?

With the linear scaling we used to get the peaks in the middle and right to align, we don't have alignment on the peaks on the left.

How should we try to correct it, by changing the scaling factor, or shifting the left peaks into better alignment?

Figure 4:
zooms in on Figure 3a.
click for bigger

Figure 4a:
Zoom in on figure 3.
Click for bigger.

If these zooms aren't clear, please suggest what I can try to make them better. Alternatively, try these manipulations yourself with Photoshop/Gimp and the previous figures. I do it by making one a background layer, and the other a transparent layer on top, which can be scaled and moved around.

Billy Answers:

There is nothing to correct. It is a qualitative issue.

tbv@trustbut.com said...

By what do you mean "qualitative?"

Should the internal standard align in the GCMS and GC-IRMS the way the analytes of interest line up or not?

BustinBilly said...

It is qualitative. For identification only. Which is different from a quantitative issue, e. g. the T/E quantification that was negated. The identification of the three metabolites is quite obvious. What more is there to say?

docnzym said...

But the internal standards are suppose to be lined up or why would you call them an internal standard (other than for extraction efficiency purposes) By just looking at these three peaks and nothing else then it would seem that you are forcing the answer

BustinBilly said...

What does it matter if the quantified peak-internal standard difference is changed by 0.3-0.4 minutes? The patterns are the same. The peaks were identified in the GC/MS plot.

The plots are from different machines with corresponding columns. A difference is expected.

BustinBilly said...

Look, there are two pertinent points I haven't seen anybody argue away.
1.The substances were identified correctly in the F3 GC/MS plots.
2.The F3 GC/C/IRMS plots for both the A&B samples are nearly identical to the GC/MS plots. The three metabolites of interest are almost exactly identical.

The panel of judges was right when they decided this issue.

TBV adds some figures:

Figure 5:
Lined up on the SI peaks on the left, scaled horizonally so the 5bP lines up.
This is interesting.
Click for bigger.

Figure 6:
Scaling from Figure 4a slid left so SI peak aligns.
5bP on right is way off.
Click for bigger

Figure 7:
Zoom of Figure 5 (not 6) showing 5bPdiol peak alignment
I believe the peaks align.

Figure 8:
Zoom of Figure 5 (not 6) showing 5bPdiol baseline match.
It's not as good as the peak which is why both are shown.

Figure 9:
Zoom of Figure 5 (not 6) showing baseline in the middle.

TBV saaid

[...] responding to BB's 5:45 comment.

1. The GCMS identification isn't in question; that's not in dispute here.

2. This is what we are discussing, and we'll see if it gets argued away or not.

In the comment that Billy started with, he said "They are exactly the same".

We've shown with overlays they are not exactly the same, and Billy has changed to using "nearly identical" for the whole chart and saying the metabolites of interest are "exactly identical".

He says the lack if exact match on the low end near the SI is not relevant. We'll leave this aside for now.

I do want to offer two more versions of overlays for comparison.

Figure 5 is a version of the overlay where the SI is aligned, and the axis linearly expanded so the 5bP aligns. In this, we see a significant left shift of the 5bA and 5bP peaks.

Figure 6 is one where the overlay scaling of Figure 4a is left shifted so the SI seems to align. In this case, it doesn't appear anything on the middle or right aligns, so that might be pointless to pursue.

Figure 7 is a zoom of the 5bP peak [in Fig 5] to show how it was aligned at the top. Figure 8 shows that the bottom alignment might be questioned, but the trailing peaks look OK.

Figure 9 is a zoom [in Fig 5] of what we think is the region of interest. In it, things are not quite as clear to me as they appear to be for BB. Here we see the peak identified as 5bA might align as well with a low GC-IRMS peak as with the one originally claimed, and the one labelled 5aA might align with the GC-IRMS peak that is ostensibly the 5bA.

Are the identifications of the GC-IRMS peaks still as obviously correct on visual examination as they seemed at first?

BustinBilly said...

Yes the identifications are as obviously correct as they seemed at first.

A good analogy would be different sized fonts. If you type the same sentence in the same font in Photoshop, the two sentences will overlay perfectly. Type the same sentence again in the same font-using a slightly larger point. A few letters will line up well on overlay, the rest will not. However it will still be the same sentence.

I hope that analogy helps. I don't see how quibbling over the overlay makes any difference.

tbv@trustbut.com said...

Please be patient while I try to figure out what is obvious to you, and not yet to me.

In Figure 9, we can see that innaccurate horizontal alignment can result in peaks being completely misidentified. The GCMS peak for 5bA is closer to a small GC-IRMS peak just short of 15:00 minutes that has not had it's CIR measured. The GCMS peak identified as the 5aA peak nearly sits on top of the GC-IRMS peak that is measured to be the 5bA peak.

How is that potential misidentification not a problem?

If that misidentification can't happen, what criteria are you using to decide which peak in that region aligns with which peak in the other chromatogram?

[ There's some discussion I'll capture in a bit, but for now, moving on ]

Figure 11:
Landis A sample F2 GCMS
USADA 165
click for bigger

Figure 12:
Landis A sample G2 IRMS
USADA 167
click for bigger

Figure 13:
Figures 11 and 12 overlaid, and scaled so anchor and rightmost labeled peak align.
click for bigger.