Chromatography Forum

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

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

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.