Chromatography Forum

Dear colleagues,

Perhaps, I am asking for something obvious, but the following question is not clear to me.

We have a system of HPLC + MS triple quad with ESI source. When we develop a MRM method for a new compound, at first we perform MS1 to determine M+H ion and then we perform MS/MS to get a fragment spectra for this parent ion. For both these experiments we use 10000-100000 ng/ml solution to get enough sensitivity.

After that we make an MRM method using the determined parent mass ([M+H]) and, for example, 4 daughter masses (F1, F2, F3, F4). And we inject solutions in concentration range 5-1000 ng/ml to see that this method works well and to find out the LOD and etc.

The question is:
Why the sensitivities we have for these 4 pairs [M+H]/F in MRM mode do not always correspond to the intensities of respective peaks in MS/MS?
I mean that why, for example, we can have in MS/MS
I(F1)>I(F2)>I(F3)>I(F4)
and in MRM
S([M+H]/F2)<S([M+H]/F1)<S([M+H]/F4)<S([M+H]/F3)
??? (S is an area of a respective peak on MRM-chromatogram)
Dwell times are equal for all MRM pairs.

Thank you for your comments in advance.

Best regards,
Elena

If all voltages, collision energies etc were the same, you should have gotten exactly the same fragment ratios. If not, as (I think) you infuse in one case and do LC-MS/MS in the other case, you might have isobaric impurities that interfere with your compound of interest (in the case of infusion)...

If all voltages, collision energies etc were the same, you should have gotten exactly the same fragment ratios. If not, as (I think) you infuse in one case and do LC-MS/MS in the other case, you might have isobaric impurities that interfere with your compound of interest (in the case of infusion)...

In all cases we do LC-MS/MS...

Just an idea...

Perhaps some kind of saturation, since 10000-100000 ng/ml solutions seem very high concentrations to inject on LC-MS/MS to me...

What happens if you inject the same concentration and compare the intensities seen in product scan and MRM?

To get the full spectra you only use one collision energy, gas pressure, etc. Many instruments optimize each product ion CE, etc., individually in MRM mode, so it is entirely possible, even likely, that the ratios won't match those generated by the full scan product ion spectra.