[LC-MS/MS] Analyte response increases with methanol

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

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Hi All,

I have an LC-MS/MS method for a compound we have been using for the past 4 years. Recently we have had a problem where we observe a peak with the same retention time and same MRM as our compound in blanks.

I can only assume it is some form of carry-over or contamination. However, this has not been the case for the last 4 years. Cleaning the column and system did not seem to help. Perhaps the cleaning procedure was not strong enough. A new column (and on a second system) did help, with no signal at all for the MRM in blank methanol injection after 200 samples.

The problem has resurfaced when a user installed the column backwards (i.e. column then guard, in the opposite to normal flow). Now there is a peak for our MRM again.

Why I haven't been able to fix this problem: Following a brief clean with 70% isopropyl alcohol, the peak was gone. Multiple runs through the gradient with no injection show no response. All is good. Then, a fresh methanol solution was injected (10uL) and the peak is back. Subsequent injections of methanol only increase the MRM peak height.

I am very confused. Anyone with more experience have any idea what is going on?

Details:
Mobile phase typically 70%acetonitrile:water, with 0.1% formic acid.
Column is Zorbax SB-C18 2.1x50 mm 5-Micron.
System is Agilent 1200 HPLC with Agilent 6410 QQQ
Compound MRM is 602>>418 m/z



TL;DR: Stable, validated method now has response to analyte increasing with each injection of methanol (blank).


Cheers,
Matt
- Is your system flow-through or fixed loop injection?

The key is to use a wash solvent that dissolves your compound perfectly. For instance, if you analyze very polar compounds you do not want to use 100% MeOH as a wash solvent but something like 90/10 water/MeOH. And the other way around.

You should increase the volume of wash solvent it uses between every injection. It would help us if you post a screenshot of these settings.

- Did you rule out the possibility of contamination of the MeOH itself?

Inject another solvent. Or just inject mobile phase. Try to isolate the contamination to the system (needle, seat, loop, column,..) or the blank being injected.
If using the Agilent 1200 autosampler, it will be rinsing the loop with the mobile phase since it is a flow through design. It can however have something contaminating the switching valve that could be causing the carryover, might want to swap that out for a new one.

Since it appeared on the second instrument when the column was reversed, it may be something that is very highly retained by the column and takes days or weeks to pass completely through. That would be why it appeared so quickly when the column was reversed.
The past is there to guide us into the future, not to dwell in.
Thanks for your comments.

I can 'clean' the column where it doesnt show the peak when I run the method without injection. A single injection of fresh methanol will result in a peak. Any injection (methanol, mobile phase, ACN) or no-injection run after that will result in a peak too.
MBull wrote:
Thanks for your comments.

I can 'clean' the column where it doesnt show the peak when I run the method without injection. A single injection of fresh methanol will result in a peak. Any injection (methanol, mobile phase, ACN) or no-injection run after that will result in a peak too.


If you remove the column and do an injection do you get a peak for the same compound? If so, then it will probably be contamination in the autosampler, probably the rotor in the switching valve.
The past is there to guide us into the future, not to dwell in.
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