MRM and retention time

[anon2223]

Hello,
I have worked on developing method for a parent compound and its 2 metabolites, all have different molecular weight (M, M-2 and M+14); hence different MRM pairs.
I was able to separate the parent and one metabolite but the second metabolites is eluting at the same retention time of the parent (but in a different MRM channel/transition from parent ) .
Is there a problem with this assay (that I am overlooking)?
Do I have to spend time on method development to make the 2nd metabolite elute at different retention time even though the MRM transition is different.
Any help/advice is appreciated!
Thanks much

[Gaetan Glauser]

If you can ensure that your transitions are selective enough, you don't need to separate them chromatographically. With M-2 and M+14, you should not have big problems, although you may have an interference between the M+2 isotope of the metabolite and the parent compound if product ions are similar. You also have to make sure the resolution of the quadrupoles is set at 1 Da or less, since many triple quads work with a resolution of 3-4 Da by default, to increase sensitivity.

I would thus determine whether the transitions are selective enough, and if it turns out they are not, try to improve chromatography to separate the critical pair.

Hope it helps,
Regards

[anon2223]

Thank you for your response. This does help a lot.
The system I am using is AB Sciex API4000 and using a Unit resolution (0.7 +/- 0.1 amu)
The analytes in question were M and M+14 eluting at same retention time, each having different product ion and the M-2 elutes at a different retention time but has same product ion as the parent M.
So I think I should be fine here, just wanted to clarify my doubts.

Thanks again.