Chromatography Forum

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.

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.

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

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.

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.

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.

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!

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.

lmh, you were quicker than me

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.

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.