LCMSMS - Different Precursor Ion but Same Product Ion?

[wan]

Hi, I was hoping that someone can enlighten me on quantitation of a compound, when the native and labelled has the same product ions.

E.g. Native has precursor ion of m/z 179 and labelled has precursor of m/z 181. Both fragments to give product ions of m/z 105 and 120. The only difference is m/z 179 fragments to m/z 161 and m/z 181 fragments to m/z 163 as well.

How can quantitation be possible, using m/z 120? The scientist was telling me he uses m/z 120 as it has the highest intensity.

[Jetjamnong]

Use the m/z 161 for the targets compound instead m/z 120 for do quantitation or decrease the collition energy to change the intensity ratios of m/z 179:161:120.

[wan]

The problem is he told me he is using m/z 120 for quantitation and I wondered how that is possible.

[Jetjamnong]

What's about the RT of target compound and it analogue (I assumed this compound is M-d2 used for IS), if present with the same period of RT this compound can not using the m/z 120 for quantitative work becuase such ion has interference with the m/z 120 of labelled compound. maybe you should try to use lower flow to separate enough.

[MaryCarson]

It is quite possible, because he is not using m/z 120, he is using m/z 179>120 and m/z 181>120. Provided resolution on Q1 is sufficiently tight, there should be little cross-talk between these two ions. This can be tested by simply analyzing solutions with only the analyte or only the IS using the acquisition parameters that look for both.

[bisnettrj2]

I agree with Mary. Sufficient specificity in Q1 should allow only m/z 179 through for the specified dwell time, which should then go through Q2 to be dissociated and filtered to 120 in Q3. After that specified dwell time, only m/z 181 is allowed through for the specified dwell time, which then goes through Q2 to be dissociated and filtered to 120 in Q3. I'm currently working on a method with three compounds transitioning to the same product mass in the same monitoring segment - the only difference being the parent mass. Narrowing my Q1 width is the only way I can get accurate signals for each of these analytes.

The question should be - does he detect anything in a blank with IS when monitoring m/z 179-->120? If he does, then check the Q1 settings - is his Q1 mass filter range too wide (more than 1 amu)? If yes, then he's not seeing 179-->120, he's seeing a partial signal from 181-->120. If not, then everything should be fine.

[lmh]

Also relevant to check whether there's an apparent IS signal when you inject compound without internal standard. There may be, if the natural compound has an appreciable +2 isotope peak. Are these real masses, by the way, or ones made up to illustrate the problem? if they're real, I assume that the internal standard is fluorinated?? If they're made-up ions, then if you really have two fragments from the heavy IS separated by 2 mass units, and only 1 of these fragments in the actual compound you're analysing, it may mean that some of the heaviness is natural +2 not caused by the position the label is in? Does that make sense? Sorry, in a rush!

[Gaetan Glauser]

I agree with the 2 last posts. Cross-talks are very unlikely with modern instrument and a resolution of <1 Da on the first quad should work fine. However, be aware that the contribution of the 13C2 peak of m/z 179 may contribute to the 181->120 transition. Without having the molecular formula, it can't be calculated precisely but it should be about 1.5-2% of the 12C peak.

[Gaetan Glauser]

lmh, you were quicker than me

[lmh]

GaetanGlauser, I was quick but stupid! I got worried about the mass loss of 20, which of course in unlabelled things is usually HF, but I suppose this is probably an 18O-labelled internal standard, and the losses of 18 and 20 are just labelled- and unlabelled H2O. This means that the fact the internal standard has both losses doesn't mean that there is unlabelled material present; it just means that it has several groups able to leave as water. Sorrreeeee! Next time I will try to think before I write.

[Camisotro]

There are also frequently situations where a higher mass analogue or substituted derivative (e.g. metabolites) can fragment in-source to the same mass as the lighter molecule, and then also fragment in the MS/MS to the same fragment. Probably not going to happen very often with a difference of only 2 amu but something to watch out for in method validation.

I was once working with cyclophosphamide, and the labelled internal standard we had was 4D on the chloroethyl chains - I forget if it was all on one chain or 2 per chain. So of course, we couldn't use the lowest [M + H]+ peak for the internal standard because it had a significant contribution from the [37Cl]2 variety of the main compound. Moving two mass units higher gave us a more unambiguous ion.