Persistent carry over peaks on UHPLC-MSMS

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

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Dear all, good day ~

I would like to ask for advise on a persistent carry over issues that I am currently experiencing.

We are working on multiresidue pesticides analysis using Querchers method on a Sciex 4500QTrap unit with Sciex Exion LC.

I have set an external and internal rinsing of the autosampler after every injection. I have tried numerous wash solvent which according to some journals and papers are effective as wash solvents for pesticide analysis.
The solvents that I have tried are: Methanol only, Methanol:Water (1:1), Acetonitrile only, Acetonitrile with 0.1% formic acid, Methanol:Acetone (80:20), the magic solution of ACN:IPA:Methanol:H20 (1:1:1:1).

Injection of blanks give high intensity of a few pesticides (especially TPP as we are using TPP as our 'internal std').

I tried to isolate the carry over/ghost peak issue and the brief is as below:

i) Only use the MS and do an infusion of all the solvents used to check if its solvent contamination issue or MS issue

ii) No high signal was observed - all suspected carry over 'peak' MRM transition was less than 5k cps intensity - so I think it's not an MS or buffer/solvent issue
iii) Next, I have purge and flush the system (both pump A and B) without column with the following solvent at high flow rate (2mL/min) for app 15min each
-first with water
-then with methanol
-next with magic solution of ACN:IPA:Methanol:H20
-then with acetonitrile

iii) After this, I tried to inject a high load (max 50uL injection) without column of the blank solvents - no ghost peaks were observed

iv) After numerous high load 50uL injection (to clean the sample loop as well) with methanol, IPA, water, acetonitrile - all with no peaks observed

At this point, I assumed that the carry over or contamination does not come from the MS or the LC.

v) I connected the column, did a flushing/conditioned on the column (basically, 30min 98% A, then 60min 98% B and 10min 95% A (my run initial gradient)

A - Water with 0.1% formic acid and 5mm amm formate
B - Methanol with 0.1% formic acid and 5mm amm formate

vi) After doing the above, I test run the blank injections and ghost/carry over peaks were observed (same RT of std run on those 'detected analytes')

vii) Suspecting it to be column issue, I changed the column to a brand new column and to my disappointment, the ghost peaks were observed (at an even higher intensity)

I have not passivate my LC yet.

Should I do that as my last resort, or is there any other advice from the kind experts here? Did I miss out any troubleshooting steps?

Hope there is someone kind to guide and give some advise.

Thanks a lot in advance !

Sincerely,
Ken
Pesticides can be lipophilic and are solubilized by a stronger solvent than just Methanol or ACN. Thus, I would try IPA as a needle wash.

In addition, I would also have some IPA in your mobile phase to ensure that TPP is 'booted' off your column (and thus cannot accumulate).
Hi HPLC Chemist

Thanks for the reply.

I did tried to purge and flush the system with IPA.
I have also injected IPA as solvent blank (to try to remove anything from the injection loop)
I have also tried my rinsing and wash part with 100% IPA

- unfortunately, I still have the TPP peak observed (and a few other pesticides with smaller peaks) in my blanks.

I have not tried having IPA in my gradient buffer ~

I read some paper that mentioned using 50:50 methanol: water or 75:25: methanol: water as their wash solvent and there is no carry over observed (even with using TPP as the IS) ~

Any further kind feedback from you?

Thanks !
IPA is a 'stronger organic' than Methanol or ACN, so you will need less (Hexane is stronger but then you are into 'normal phase'). TPP may be left over from previous injections and completely retained on the column (by water) until it is 'dumped' even into a 'blank' injection.

I would use a 100% organic mobile phase B in a gradient (which contains some IPA).
HPLC Chemist -

Thank you so much sir for taking the time to feedback.

Noted on your suggestion.

I have actually tried a new column and the TPP is still there in blank injections.

Guess that either I need to re-flush the whole system with IPA for a longer period of time, and multiple full load injection of IPA to try and clear the loop.

Else, do you think I should passivate the system?

Thank you once again ~
Hello everybody,

I would like to follow up on this question since it seems unresolved..

I am having the same problem with unexplained carryover that happened with three unrelated analyses (different mobile phases, different analytes). Does anybody have positive experience with this? I would be happy to share the details then :))
When we face carry over issues, we usually put a mobile phase and a few injection vials both composed of H20:MeOH:ACN:IPA:formic acid (25:25:25:25:1 or even 2), disconnect the MS (but not the column) and run 200 injections using a fast program (e.g. an injection every 2 min). Then switch back to normal conditions and check for carry-over. If still present but reduced run another 200 injections. This procedure has often been successful for us, but it certainly depends on the analyte properties.
2-3 times, I have come up to "carryover" issues in LC-MS/MS that turned out to be mobile phase contamination. Personally, I would do the following: In no-injection mode, execute 3 consecutive runs, then leave mobile phase flowing for 30-40min, then 1 injection.

If your peak area keeps dropping during the first 3 injections, and then rises significantly after equilibration for 30min, your mobile phase is contaminated. If this is the case, the first suspect is your balance.

Hope this helps :)
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