Causes for drifting signal intensity in LC/MS over time?

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

6 posts Page 1 of 1
Hi everyone,

Long time lurker, great people here. I hope someone can drop me some knowledge! I've recently had to deal with an issue that I don't usually need to, as I am attempting a quantitation by LC/MS without the use of an isotopically labeled internal standard. I am using external calibration with the same, unlabeled analyte (despite informing my client that I'm not crazy about this approach).

However, this is becoming a problem, as I am unable to get reproducible peak areas over time. Specifically, this is not random but drifts in a general direction over the course of hours, but immediate effects can be seem in back to back injections. Sometimes, the drift goes toward increased peak area and some days the trend is reduced peak area. Still other days, the precision is very tight, ~ 1% RSD. Area of the final injection can be 200% or more of the initial injection, happening over the course of several hours. I have noticed drift in peak areas in the past in unrelated assays with entirely different chromatographic setup while using internal standards, but this was never an issue I had to deal with since the area ratios remained tight.

So, this issue seems independent of my current method, but the method causing me to beat my head against the wall utilizes HILIC chromatography with an ammonium formate (pH 3.0) buffer. The column was flushed with strong mobile phase for a long time and the ion source cleaned. I am having this issue with only 50 ng/mL purified standards before even trying samples, so matrix suppression seems unlikely to me (but who knows?).

I was thinking that maybe it has to do with an unstable electrospray, but as I can tell the capillary looks like it is good shape, and indeed the instrument was requalified by the vendor recently.

So, I guess I'm just wondering from your experience if this kind of signal drift is pretty typical with LC/MS, or if I might have a real problem here. Any advice on where to look to improve it or an explanation of the mechanism of this would be very helpful. I am confident that this is not the result of injector precision, sample instability, or carryover.

Thank you!
dja
One more piece of crucial information: I am using single quad Agilent 6150, 6120, and 6130 mass specs. The issue has occurred on all three instruments!

Thanks again!
dja
The first causes that I would suspect are the ones you ruled out at the end of your post. Are you sure that the injected volume is appropriate for your system (older LC systems can't inject 1µL in a reproducable way, for example).

For other guesses I think you need to give some more information. What is (the nature of) the analyte? Isocratic or gradient? Which composition of mobile phase? Concentration of buffer? ...

Is there any chance to try RPLC or is your analyte too polar? It would be interesting to see if you have the same problems by switching to a different LC mode on the same system.
I always try to keep an eye on the analyzer vacuum over the course of a run. I have noticed on one of my tests that if the vacuum changes from 3.3x10^-5 to 3.2x10^-5 torr I see a drastic shift in area counts, simply because the orifice is starting to pick up a little crud on it.
The past is there to guide us into the future, not to dwell in.
How stable is the temperature in your lab? Try monitoring with a logger ( some systems also record room temperature). Due to a bug in the control software of building management system temperature conteol of out lab was shutdown after working hours. It took us a long time to discover that was the couse of the strange signal changes we saw
Hey everyone,

Thanks for the feedback. It sounds like at least a couple others have had similar issues.

Unfortunately, the lab temperature is not very well controlled. Typical variations throughout the day can be up to 5C in one lab (my issue has been observed on three different instruments in two different labs). I did not expect this to be much of an issue since the source and quadrupole are kept at such a higher temperature than the lab. I never stop finding things that surprise me though. I wonder if it has to do with the electronics/transducers?

I have not kept an eye on the vacuum stability, but it looks like I can save that data with my run so I should be able to check this out. Currently it's a nice vacuum at about 7.2 x 10^-6 torr on the instrument I'm currently set up on.

Again, I very much appreciate the help! I'll report back.

-dja
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