I am performing an LC-MS/MS analysis in MRM (Multiple Reaction Monitoring) mode.
I have three different compounds. Each compound has a different Q1 (precursor ion) but produces the same Q3 (product ion) after fragmentation.
Their retention times are also different.
However, when I monitor only one MRM transition (for one compound), I observe signals for all three compounds at their respective retention times.
Could someone please explain why this is happening?
Hi,
it is a phenomenon called cross-talk.
A good explanation can be found at:
https://www.chromforum.org/viewtopic.php?t=38951
With older instruments, this problem could be eliminated by increasing the pause time between the transitions.
Each of the three peaks is separated by 50 seconds in retention time. How could cross-talk happen?
Cross talk can occur, even if compounds are separeted and show different retention times. Lets assume, we have 2 compounds with RT 1 and 2 and transitions e.g. 300->100 and 314->100
At RT 1 300->100 is monitored and gives a signal for compound 1. If there are still ions travelling to the detector, while the instrument changed to transition 314->100, you get a signal for compound 2 at RT 1. Same can occur at RT 2,
a correct signal for compound 2 at RT 2 and an artificial signal for compound 1 at RT 2. In summary, it can be said that 2 signals (at RT 1 and 2) can be seen at both transitions.
Hi Naga,
1st: you have to apply a good chromatographic method to get the three compounds well separated.
2nd: Before running the three compounds, you have to tune each of them first. While individually tunning the three compounds, make sure, that you've selected the highest intensity fragments for each of them.
3rd: If you choose the highest intensities for MRM & you think that they are similar for the different compounds consider the following:
very few compounds might have similar precursors & MRMs, which is very rare to happen. To have this solved:
- If you are analyzing your samples on a Qtrap "QQQ", it's recommended to apply MS3.
- You can also use a high-resolution MS to distinguish the difference.
Hi Dr. Davies,
I am using the Thermo Quantiva QqQ (0.7 FWHM) in negative mode. I have attached the spectrum for review. The arrow indicates cross talk.
I agree with the statement, that is has no neagtive impact on method performance, because all compounds are separated.
But the initial question was, what is the reason for these findings.
A resolution of 0.7 FWHM on a triple quad equals unit resolution and is sufficient to separate ions 2 Da apart (e.g. m/z 223 and 225).
I prefer the explanation that this phenomenon was caused by cross-talk.
It is interesting to see, that in Fig. A, there is no additional signal visible.
Based on the additional signals, visible in the 3 other transitions, I assume the scan order 223->42 (A), 225->42 (C), 231->42 (B) and 237-->42 (D).
Naga Veera Yerra : Is there an additional transition, e.g for an internal standard? This could explain the absence of an additional signal in transition A, if the IS is monitored using another product ion.
Inserting a dummy transition e.g. 500 (or any other number, other than the analytes) ->42 between e.g. transition A and C should eliminate the signal at 2.8 min in transition C.
Andre Stratmann: I’ve added all the transitions in this method. Please take a look. There’s no issue with the method—I’m just curious to know.
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