MRM and doing what I've been told not to do!

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

10 posts Page 1 of 1
Hi All,

In recent days I've been playing a little more with both SIM and MRM methods for the detection of a set of specific chemical compounds at trace levels using an Agilent GC-QqQ instrument. Traditionally, SIM and Scan methods have always been used for the detection of these compounds, but I wanted to give MRM a try, partly as a learning opportunity (I'm curious) and partly so I can see for myself the relative sensitivities of MRM v SIM methods. I also like the novelty of baselines of nearly zero.

One thing that has always been drummed into me about MRM is that "thou shalt not select product ions which are also present in your precursor ion list", presumably to eliminate interferences with near identical m/z to the precursor, although any further explanation of this would be much appreciated.

This is all every well in theory, but in practice the smallish (~170-240 m/z) molecules I'm looking at have a tendency to fragment into very specific ions many which are common to both the precursor and product ion list which is a bit of a problem as it leaves me with a much smaller list of potential product ions many of which are not terribly abundant.

Because of what I'm doing, I'm trying to maximise sensitivity and to be quite honest, the selectivity which MRM brings to the mix is not terribly important as after all, I'm comparing the method directly against SIM where the interferences alluded to in texts on the subject are 'par for the course' and thus can't be more of a problem with MRM than they are already.

As people with far more experience of this than me, is including ions in the product ion list which are also precursor ions that big of a problem? I would love to know your thoughts?

Kind Regards

TD2
you can do this of course (monitor 240 > 240, for example) but in most cases the background will be very high so you won't like it (at least I didn't, when I tried)

my experience is it only works well when your precursor can withstand collision energies up to 50V (at least 40V) without too much fragmentation; in this case, you will get nice and clean 240 > 240 peak with very low background
I interpret your question a bit differently from trozen, let me know if I am wrong. For a multiple set of compounds within a single MRM method, you are "worried" to select product ions for certain compounds which have identical m/z to the precursors of other compounds. In that case, you might only face selectivity issues if these precursors do not fragment entirely (residual precursor ion in the "fragmentation spectrum"), and if retention times are similar.
Hi,

Many thanks for your replies on this. Whilst both responses are much appreciated (and help to fill gaps in my knowledge), I think that perhaps Gaetan's interpretation was closest to the mark - Apologies for any confusion caused by my somewhat imprecise question.

To clarify, I would like to monitor several precursor ions and a select number of their product ions. As a hypothetical example, in my list of ions for one of my compounds I have 240>134 and also 134>77.

According to a more experienced colleague (who I hope isn't a forum member!), I shouldn't do this, but he's not able to articulate why and can only state that's how he was trained, that this was backed up by an engineer and something having read an application note about eliminating interferences. Generally speaking, he is hugely knowledgeable and knows his stuff, so I'm at a bit of a loss on this one as it seems a bit illogical to me!

Perhaps I lack knowledge and imagination, but I can't see why having the 134 ion as a precursor and product ion for different transitions is an issue. To my understanding, when the instrument is set up for the 240>134 transition, only the 240 ion will pass MS1 and will undergo fragmentation and produce the 134 fragment which will pass MS2 and hit the detector. When MS1 subsequently switches to the 134 ion, the 240 ion will not pass and hence will not undergo fragmentation to form the 134 or 77 ion, the latter of which would pass MS2.

Am I missing something really obvious here? Is selecting the example transitions mentioned above somehow wrong or odd?

Kind Regards

TD2
Travisdog2,

your assumption is absolutely correct; triple quad scans its precursor > product pairs sequentially and they do not affect each other as long as you have sufficient interchannel delay, which I think most instruments set automatically to avoid any crosstalk
Hi,

As trozen mentioned, no crosstalk anymore in modern triple quadrupoles. Maybe your colleague is a bit old school and was trained in these days where crosstalks between MRM transitions were common.

I think I made a mistake in my previous post. After careful thinking I can see only one case where a selectivity issue might occur with the example you provided: if the m/z 240 ion experiences in-source fragmentation into m/z 134, and this m/z 134 fragment further breaks into m/z 77. Then there would be additional signal from the molecule 240>134 into the 134>77 channel.
Hi,

Thank you both for your help on this and your comprehensive responses. I think I now have a slightly better grasp on MRM and have a bit more confidence in explaining to my colleague (who I suspect was using MRM many moons ago when crosstalk was a problem) why what I was planning isn't completely mad!

Thank you both :)

Kind Regards

TD2
Travisdog2 wrote:
Hi,

Many thanks for your replies on this. Whilst both responses are much appreciated (and help to fill gaps in my knowledge), I think that perhaps Gaetan's interpretation was closest to the mark - Apologies for any confusion caused by my somewhat imprecise question.

To clarify, I would like to monitor several precursor ions and a select number of their product ions. As a hypothetical example, in my list of ions for one of my compounds I have 240>134 and also 134>77.

According to a more experienced colleague (who I hope isn't a forum member!), I shouldn't do this, but he's not able to articulate why and can only state that's how he was trained, that this was backed up by an engineer and something having read an application note about eliminating interferences. Generally speaking, he is hugely knowledgeable and knows his stuff, so I'm at a bit of a loss on this one as it seems a bit illogical to me!

Perhaps I lack knowledge and imagination, but I can't see why having the 134 ion as a precursor and product ion for different transitions is an issue. To my understanding, when the instrument is set up for the 240>134 transition, only the 240 ion will pass MS1 and will undergo fragmentation and produce the 134 fragment which will pass MS2 and hit the detector. When MS1 subsequently switches to the 134 ion, the 240 ion will not pass and hence will not undergo fragmentation to form the 134 or 77 ion, the latter of which would pass MS2.

Am I missing something really obvious here? Is selecting the example transitions mentioned above somehow wrong or odd?

Kind Regards

TD2

From my take on this, you are looking at one analyte using two different Precursor ions?

This would not be a problem unless another compound present has the same precursor that could fragment into the same daughter ion. This would only be a problem if you are not separating these chromatographically, but if you lack the separation then you have to look carefully at the relative response of the two results to eliminate the second transition being from a different molecule. As mentioned above, if you are getting two precursors for the same analyte, then there is in-source fragmentation happening on your analyte instead of all the fragmentation happening in the collision cell.
The past is there to guide us into the future, not to dwell in.
Thankyou James,

As always a succinct response that gets to the heart of the matter.

I am using 2 precursors ions and whilst another compound present does share some of the same daughter ions (the compounds are chemical cousins), from a chromatographic standpoint they are separated by about 2 minutes, so this shouldn't be a problem. I think that I see your point regarding in source fragmentation, but wouldn't this be accounted for in the calibration?

Kind Regards

TD2
Travisdog2 wrote:
Thankyou James,

As always a succinct response that gets to the heart of the matter.

I am using 2 precursors ions and whilst another compound present does share some of the same daughter ions (the compounds are chemical cousins), from a chromatographic standpoint they are separated by about 2 minutes, so this shouldn't be a problem. I think that I see your point regarding in source fragmentation, but wouldn't this be accounted for in the calibration?

Kind Regards

TD2



It will as long as the in source fragmentation is well controlled and reproducible.
The past is there to guide us into the future, not to dwell in.
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