Chromatography Forum

So doing drug analysis, I know that given a compound and it's deuterated analogue, the deuterated analogue will always have an RT before the drug of interest due to the added mass.

Does this also apply for fragments of the parent compound?

I.e. if I have THCA, and THCA-d3, THCA should always come out after the THCA-d3 ion. But will

a) the fragments of THCA-d3 also always come out after the parent (I am seeing this is not the case).

b) will fragments of THCA always come out after the associated THCA-d3 fragments?

I am presuming that you are doing GC-MS/MS - if you are doing HPLC then molecular mass per se has no influence on retention.

In GC, all else being equal, larger molecules elute after smaller ones, so adding deuterium to a molecule makes it elute later, not earlier. This also applies to breakdown products, metabolites etc.

The ionized fragments that you see in the mass spectrum were created in the ion source of the MS (or in the collision cell), they did not come through the column and so do not have retention times that can be affected by their mass.

You might be confusing the elution time on the X-axis of a chromatogram with fragment mas on the X-axis of a mass spectrum.

Peter

My question pertains to any quadrupole application but generally I'm referring to LC/MS/MS in this case. The retention time on column will be the same for the deuterated analogue as well as the analyte, but the RT given in the results will differ due to the difference in time spent in the quadrupole. A heavier ion will reach the detector before a lighter ion all other things being equal, so the deuterated analyte will of course be first due to the higher mass. I'm talking of RT differences of 0.01 or so, but a difference nonetheless.

What I'm seeing however, is say for THC-d3, I have a RT of 1.22, THC is at 1.23, but my THC transition ion is at 1.22, and my THC-d3 transition ion might be at 1.22 as well. And I'm wondering why that is. I would assume the smaller, lighter fragment should reach the detector last.

I would be surprised if the residence time of the ions in the MS was anywhere near long enough to give a difference in retention time measured in minutes of seconds as is usual in chromatography. You give no units so it is impossible to properly interpret the numbers, but I do note that your difference is 1 unit at the last decimal - so it is very likely to be simply a readability error.

How repeatable is this difference ? - how many times have you done the measurement ?

Peter

In GC, all else being equal, larger molecules elute after smaller ones, so adding deuterium to a molecule makes it elute later, not earlier. This also applies to breakdown products, metabolites etc.

Peter, I have to correct you on this one. I can show plenty of examples where the deuterated molecule elutes earlier than the non-deuterated component in GCMS.

It would indeed seem logical that the increased molecular mass makes it elute later. But deuterated compounds, compared to the same non-deuterated molecules show less affinity towards the stationary phase. If you combine both effects, it elutes earlier

In GC, all else being equal, larger molecules elute after smaller ones, so adding deuterium to a molecule makes it elute later, not earlier. This also applies to breakdown products, metabolites etc.

Peter, I have to correct you on this one. I can show plenty of examples where the deuterated molecule elutes earlier than the non-deuterated component in GCMS.

It would indeed seem logical that the increased molecular mass makes it elute later. But deuterated compounds, compared to the same non-deuterated molecules show less affinity towards the stationary phase. If you combine both effects, it elutes earlier

I'm intrigued. Can you give one example - what kind of compound and the analytical conditions.

Peter

Peter, I have to correct you on this one. I can show plenty of examples where the deuterated molecule elutes earlier than the non-deuterated component in GCMS.

It would indeed seem logical that the increased molecular mass makes it elute later. But deuterated compounds, compared to the same non-deuterated molecules show less affinity towards the stationary phase. If you combine both effects, it elutes earlier

The deuterated molecule will ALWAYS reach the detector first in a quadrupole.

Peter, the units are minutes. So in my example, THC-d3 has an RT of 1.22 minutes, THC is at 1.23 minutes, but my THC-d3 2 ion might be 1.22 minutes and my THC 2 ion might also be 1.22 minutes. The XIC shows all of the peaks essentially on top of one another but there is a slight difference.

I know that the deuterated analyte will always come first due to reaching the detector first (this is always true in a quadrupole, for GC/MS and LC/MS/MS). It's one of our fundamental criteria for calling a positive result.

I'll see if I can redact a chromatogram and upload a picture to better see what I'm talking about.

I would be surprised if the residence time of the ions in the MS was anywhere near long enough to give a difference in retention time measured in minutes of seconds as is usual in chromatography. You give no units so it is impossible to properly interpret the numbers, but I do note that your difference is 1 unit at the last decimal - so it is very likely to be simply a readability error.

How repeatable is this difference ? - how many times have you done the measurement ?

Peter

Another possible explanation is the time the MS takes to scan - depending on which direction the scan goes (high mass to low or low mass to high) it will get to the higher or lower masses earlier or later. This effect is enough to make different ions in a GC-MS scan hit chromatographic peak maximum at different times. The difference is almost always only one or two scans, the difference you see is 0.6 s, which seems long for scan delay between two similar masses, and so I still think that it is a readability error.

The repeatability is still an important question.

Peter

Here is an image of one of our calibrators for our THC assay.

You can see that the THCCOOH parent ion (THCA 1) RT is 1.760, and the THCA-d3 1 is 1.758. This is what I would expect, the deuterated ion to come before the non-deuterated. It's not as pronounced here as it is in GC/MS, but still follows the same principle.

But you can also see the transition ions we are measuring, THCA 2 is 1.759, which is before the parent ion, albeit by not much. THC-d3 2 is 1.760.

What I'm wondering is how the speed of the fragments pertains to the speed of the parent inside the quadrupole. I would expect a fragment to come out after the parent ion. If at the time of fragmentation the parent and the fragment are at the same position (as they must be?), why doesn't the smaller fragment take a longer time to reach the detector given the smaller m/z?

Another possible explanation is the time the MS takes to scan - depending on which direction the scan goes (high mass to low or low mass to high) it will get to the higher or lower masses earlier or later. This effect is enough to make different ions in a GC-MS scan hit chromatographic peak maximum at different times. The difference is almost always only one or two scans, the difference you see is 0.6 s, which seems long for scan delay between two similar masses, and so I still think that it is a readability error.

The repeatability is still an important question.

Peter

The repeatability is constant. Our GC/MS and LC/MS/MS results always show the deuterated analog before the non-deuterated analyte. I've encountered this in every test I've ever run on quadrupole mass specs, be it GC/MS, LC/MS/MS, or LDTD (laser diode thermal desorption)-MS/MS for the past 6 years.

It's one of our fundamental criteria for calling a positive result that the analyte cannot have an RT value less than the corresponding deuterated I.S., given that the deuterated analyte is the same compound (i.e. Morphine and Morphine-d6). Given that deuterated compounds should show the same bonding affinity and chemical properties, I can only attribute this to the residence time in the quadrupole. Since they have the same charge, it makes sense that the heavier molecule should reach the detector first.

If the difference is repeatable then it is not a readability error, which leaves scan skewing as a well known phenomenon in quadrupole MS. In principal the residence times for ions of different masses will be different; this is the basis for TOF-MS but in TOF-MS the heavy ions go more slowly, which is opposite to what you see, and flight times are in microseconds on a path at least ten times longer than the ion path in a quadrupole. Your time differences are hundreds of milliseconds, five or six orders of magnitude too large for differences in residence time.

Peter

Yeah, I am strictly talking about quadrupole and triple quad MS here.

As I've always understood it, chromatographic separation should be the same for any analyte and it's deuterated analogue because they chemical properties of the two molecules are the same, they only differ in mass.

This would (theoretically) only effect the residence time in the quadrupole, as the heavier ion will have a lower residence time (move faster) due to the lessened effect of the electromagnetic field because of the higher mass.

The way I had it explained to me back in the day in my Instrumental Chem class was with a bowling ball versus a basketball. If you roll them across the room, you would expect the basketball to get there first because it's lighter and faster (like in TOF). But if someone was to kick the ball perpendicular to the path of travel, the basketball would be deflected much farther than the bowling ball, which creates a longer path across the room, so the bowling ball will actually reach the end of the room first despite being heavier.

I've always accepted that as the reasoning to why the I.S. always comes out first in GC/MS and LC/MS/MS. Our GC/MS chromatograms exhibit the same thing, I'm looking at one now in which Morphine-d6 has an RT of 9.69, and morphine has a reported RT of 9.71. This roughly 0.01-0.02 time difference is fairly routine for all of our compounds of interest when compared to the deuterated standard.

Which direction does your instrument scan - light to heavy or heavy to light ?

Scan skewing is a problem for deconvoluting total ion signals in GC-MS, I daresay if you google it you will find it explained much better than I could.

We need an MS expert to jump in here.

Peter

Which direction does your instrument scan - light to heavy or heavy to light ?

I didn't even know this was a thing...

It certainly is a thing - it could explain the apparent differences in retention you see. No other explanation that I can think of makes sense.

Deconvolution software asks which direction the MS scans so that it can make appropriate corrections for scan skewing.

Peter