Losing Molecular Ion LCMSMS

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

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I am looking at Herbicides using EPA 8321B HPLC/MS/MS. Using Sciex API3200. Analytes of interest are 2,4,5-TP(Silvex), 2,4-D and using 2,4-D-d3 as internal standard and 2,4-DB-d3 as a check on extraction(surrogate).

I had trouble setting up the MRM for 2,4-DB-d3 as I could not get the molecular ion in any abundance. Normal transition would be 250/164, but the 250 or any other mass around it was not present, but the 164 was, even at infusion. I build a method using 164 as the parent then breakdown masses from that in the transition. It holds very stable throughout all my calibration and sample runs.

I had built the MRM for 2,4-D-d3 using 222/164 and it was working well. Now when performing analysis the response will hold through the curve then drop to half as the run progresses, if I schedule it to run again back to back with the first run, the response will begin where it was at the end of the first pass, then fall about half after the second run of the calibration curve.

Now I am noticing that there is also a peak for the same transition as the 2,4-DB-d3 based on 164 as parent stacked on top of the 222/164 transition. It is as if I am losing molecular ion and only getting the major daughter ion. The longer the instrument runs, the worse it gets.

Has anyone ever experienced something like this? The mobile phase is water and methanol with acetic acid as modifier. Just not sure why the loss of molecular ion as the run proceeds. Chemically I would not think it is lost while the sample sits on the autosampler. Matrix is 50/50 water methanol with a small amount of formic to keep it acidic. Would there be something about the TurboV source in ESI mode changing to cause it?

The target analytes seem to follow the same process as the recoveries fall near 100% as the run progresses, yet the 2,4-DB-d3 recovery will increase to over 200% as the internal standard falls to 1/2 its initial response, which is expected as it does not lose sensitivity like the rest.
The past is there to guide us into the future, not to dwell in.
can't really comment, but check what ions you're looking at and getting: some of those masses looked like they were derived from the average molecular weight rather than a particular isotope peak. It's difficult working on such heavily chlorinated things.

My other tentative thing is that polychlorinated phenols (which is what your fragment is) are really weird; I've seen, a long time ago, one of them run as a radical ion. I'm not sure if the balance of radicals versus normal pseudomolecular ions might change through a period of using an instrument??

Sounds like a tricky one.
I do usually get a pair of molecular ions because of the chlorine and have to pick one, or sometimes both and use two MRMs. Just seemed strange that during setup all but the surrogate gave decent molecular ions while optimizing using infusion, the surrogate I never saw more than a trace of molecular ion.

I noticed something else that if I vary the starting aqueous concentration in the mobile phase sometimes I get better molecular ion response. I am beginning to wonder if it has something to do with pH. That would seem strange but then with these fairly labile analytes who knows.
The past is there to guide us into the future, not to dwell in.
I made another run last night, actually ran the entire sequence four times back to back. The internal standard responses stabilized somewhat and by choosing a different product ion for the surrogate that now is more reliable.

What I do notice is that with the internal standard being 2,4-D-d3 and a target of 2,4-D, the more of the target there is, the higher the response of the internal standard, so the native is contributing somewhat to the response of the deuterated analog. There is also a 2,4-DB native in the standard mixture though it is not a target for the analysis and it seems to have the opposite affect in that the higher the standard the lower the response of the deuterated analog. The 2,4-D pair I know co-elutes so I imagine the 2,4-DB does also. Not sure if I can achieve separation of the peaks, without making the run an hour long, but will see if that will help.

Still seems to be a problem keeping the molecular ion though and not sure how to keep that from breaking down in the source.
The past is there to guide us into the future, not to dwell in.
I am just confirming you are using negative mode and only acetic acid, no ammonium acetate (either in the mobile phase or post-column)? Some ammonium acetate mixed in would be common.

I am not an expert in 8321B, but the lack of a surrogate parent ion during development seems suspect and a good place to revisit. A Q1 scan of what masses are available as parents/pseudo-parent masses for your source conditions. Relying on unintentional, in-source fragmentation may be unstable over operating time/conditions.

If you find the solution, please let us know.
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