MS QqQ resolution and response

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

5 posts Page 1 of 1
Hi,

The question is: is the deviation from target mass resolution 0.75da different for different m/z and can this deviation influence the peak area and ratio between peaks in lc-ms significantly?

From what i understand the signal on a qqq become lower the higher you set the resolution. You filter out more of the ion cloud to get better resolution giving less analyte ions but potentially less noise also, in some matrises this give better s/n but Usually optimum is 0.75 da. To achive this the ms has to find the correct voltage settings for each mass to create the target resolution so i gather that even if the target is 0.75 da it may differ a bit over the mass range. I see that my instrument says 0.6-0.9 is acceptable for calibration to pass for calibration compounds when target is 0.75.

Have I undestood correctly?

If i am correct, then it means that the ratio between compounds of different mass is influenced if the lower mass is at 0.6 da resolution and the higher mass at 0.9 da. When recalibrating this can change so the lower is at 0.9 da resolution and the higher at 0.6. And thus the ratio between the two masses change after calibration.

I do relative quantifications and it is important for me to know what can influence the ratios of analytes of different mass. It would imply not to recalibrate within a study set of samples. And to be cautious when comparing datasets run with different instruments.

I have a problem that a few compounds all around 700m/z have lower signal 20% of the expecte and low noise 20% of normal also. Above 720 m/z it looks normal. Instrument is more or less new so i suspect calibration but not sure. It is not sample matrix or method verified on other instruments, ms scans are clean from large contamination. Any suggestions what could cause signal and noise reduction in a certain mass range only?
Some will depend on the accuracy of the mass axis at each mass range. If you have a very narrow mass window for each selected mass (High resolution) and the mass axis drifts then your signal will drop because the mass you are looking for may be partially outside the window in which you are looking.

If you are looking for 701.5m/z in a window covering 700.9-702.1m/z then the target mass can drift quite a bit before most of the peak is not being counted, but if the mass range is 701.3-701.7m/z(high mass resolution) then if the target mass drifts from 701.5 to 701.3m/z, you will be missing half of the mass peak that is now below 701.3m/z. You will have much less noise at the higher resolution but you are looking for your target in a very narrow window and any drift in the mass calibration will cause loss of sensitivity. Even a few degrees room temperature can shift mass calibration 0.1m/z of some instruments.

I have been chasing this on one of my instruments when the room temperature will be 65F one day and 74F the next, the area of my targets will drop over 50% until I recalibrate the mass axis. Also it isn't always a linear relationship, in that all masses drift the same amount, sometimes high mass is off 0.2m/z and low mass may be off 0.5mz or they will shift + at one end of the range and - at the other. If the mass drifts for the precursor, then you will have less ions going through the collision cell to become the product so shifts at higher masses can cause more loss than shifts at lower masses.
The past is there to guide us into the future, not to dwell in.
Thanks for the very clear explanation, much appreciated!
… but be super-careful of relative quantifications of different compounds anyway. They will, of course, have wildly different ionization efficiencies. But after the spray chamber, it's not just the quadrupoles that will have mass-dependent efficiencies. All the ion optics will have an efficiency of ion transfer that varies with mass, and this efficiency will change as the ion optics become more dirty, and it will change every time the instrument is tuned (which it normally will be, at the same time as calibration).
Comparing relative quantification of two ions of different mass this week with the same relative quantification in a couple of weeks' time is probably dangerous anyway. You'd need at least some sort of standard run to check how the instrument is behaving.
Thanks for the advise, I will keep in mind that ESI of ions differ between instruments and can vary also over time. With standards I could confirm that ratios of equimolar concentrations changed with dwell time.

A fault was found on my instrument that explains why my results were changed and this is a new instrument so it is handled under warranty. It was difficult to find as the fault only occurred when dwell/scan times were short and for certain masses. It was a bit tricky to spot as resolution and calibration was spot on when monitoring just a few ions but became incorrect for certain masses at fast scans/short dwell times.

I have actually compared relative quantification on several modern QqQ instruments using a large sample pool prepared for this comparison. The results normalized to area% of major peak area for the lipid class were quite reproducible and similar for all instruments. As long as you compare analytes in the same class that ionize in the same way it seems to work ok. But I understand that that I may not take this for granted, but that was the result from the compared instruments on the studied lipids. When the sample collection gives unknown and variable dilution of your analytes these lipid profiles I think may be the best option for comparing samples. Relative concentrations are not affected by different dilution as long as all analytes are in the linear range, molar concentration are difficult to use when the dilution of the sample is not known ie how much of the biological fluid that was retrieved.

We do lipidomic and use, external calibration curves with IS, IS only and relative quantification within a lipid class. We are not sure what is best for comparing samples as it depends on the circumstances and the lipid but it will be important to have consistent response when we dont have isotope matched IS. Response have usually been very linear and reproducible.

Thanks for the advice I will be carefull with using ratios for quantification as they can change even when using same instrument.
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