Difference of 1 Da in the m/z of protonated molecules

[Lelo Richard]

Hello all,
I don't have enough experience in LC/MS and I would like to figure out about 2 compounds, which have a difference of 1 Da in the m/z of protonated molecules (m/z 849.4 and m/z 850.5), but yield exactely the same fragments in differents MS^n experiments. Can they be configurational isomers? If so, why a difference of 1 Da for those compounds considering their respective most abundant isotope?
My work is about impurity characterization. I study the fragmentation of available reference substances and use these data to elucidate the structures of unknown related substances by comparison. I use LC to separate different compounds present in sample and by coupling to MS (experiments are done up to MS^5), I try to identify or elucidate the structures of those compounds. The compounds for which I want to figure out the difference of 1 Da are very well separate with the LC conditions used. The MS experiments were mainly performed using an ESI source in the + ion mode and were also confirmed with APCI+.
Any help will be really appreciated

[Kostas Petritis]

Richard,

I do not know exactly what kind of compounds you have there but if you have peptide like compounds or compounds that contain amines then you can have +/-1 in your molecular weight by the following modifications:

-1 Allysine (from Lysine)
-1 Amide formation (C terminus)
-1 Oxidation of lysine (to aminoadipic semialdehyde)
1 Deamidation of Asparagine and Glutamine to Aspartate and Glutamate
1 Citruline (from Arginine)

Such modifications could lead to products that can be separated by LC and give almost identical fragmentations.

Hope the above helps,

Kostas

[Lelo Richard]

Kostas,
I thank u for ur comments.
I should have said that I'm analysing macrolide antibiotics (containing an aglycone and 2 sugars moieties: Desosamine and cladinose). Different combinations of atomes leading in a difference of 1 Da for the protonated molecules are not in agreements with the data of the fragmentation which are fully identical for both peaks.

[Kostas Petritis]

It would maybe possible if you had a neutral loss where the two molecule differ. Maybe identifying this neutral loss could give more insights of what the difference between the two molecules (i.e. +/- OH, +/- NH2)

[Lelo Richard]

[quote="Kostas Petritis"]It would maybe possible if you had a neutral loss where the two molecule differ. Maybe identifying this neutral loss could give more insights of what the difference between the two molecules (i.e. +/- OH, +/- NH2)[/quote]

Those are actually the kinds of combinations to which I refered to in my last post but which didn't give any insights.

[Kostas Petritis]

That was the most likely possibility (i.e. the two molecules differ by C-X1 and C-X2 where X1=X2+1. The first fragmentation for both is a neutral loss of the respective parts and every further fragmentation comes from these fragments which would have given identical fragmentation).

I find the possibility of configurational isomers unlike due to the 1 Da difference.

There is another (unlike) possibility. Although with very similar structures, for one of them you see the M+ while for the other the [M+H]+. Any quaternary amines? This is in more in the sphere of imagination...

Have you tried accurate mass measurements of your compounds?

[Lelo Richard]

Kostas,
I thank u very much for ur constructive comments. I thoughly agree with what u say, especially if the difference is proven to be really 1 Da. However, I didn't manage to find X1 a nd X2 groups from the initial loss(es).

[quote="Kostas Petritis"]
Have you tried accurate mass measurements of your compounds?[/quote]

I was already thinking about the precision of the measurements. I will try accurate mass measurements though I don't have previous experience with these measurements. On the other hand, there is a pair of configurational isomers for which I have reference substances. This pair is well separated and identical molecular ion masses are observed.
What would be ur point if the difference happen to be less than 1 Da but more than 0.1 or 0.2 Da.
Thank u before hand.

[Kostas Petritis]

I do not think that a ToF or FTICR will change the measurements from 1 Da down to 0.1 or 0.2 Da. Even if your ion-trap was not correctly calibrated it would still give accurate mass differences so I believe that your molecules really differ by 1 Da.

My idea was that with accurate measurements (several digits after the point) and with the structure knowledge that you already have for your compounds it would be easier to come with the right molecule. Even if it only gives you the emperical formula (or a number of emperical formulas) you would be able to say what is right and what is wrong...

However, I didn't manage to find X1 a nd X2 groups from the initial loss(es).

I just want to be sure that we talk of the same thing. We agree that if you have a neutral lose you wouldn't be able to see these X1 and X2 in your mass spectrum... but you would be able to see the major fragment from which every other fragmentation will begin. The difference between your molecular ions and that particular fragment should give you the X1 and X2 masses...

[Lelo Richard]

Kostas,
I thank you so much for all your comments.
For further discussion I wanted to post a typical structure of the references I used. However, I was not able to do it and thus, I sent it to you by e-mail with some comments. I hope it will help for further discussion.
Once more, thank you so so much for all your contributions

[Kostas Petritis]

Hi Richard,

Thank you for the molecule structure and the additional information you send me by e-mail. You eliminated several other possibilities with the additional information you gave me.

There is another possibility and that is the formation of a dimer of your molecule. The double charged ion of your dimer will give you an m/z with a -1 difference of your monomer. Furthermore, it is very likely to start seeing double charged ions at these masses (close to m/z=1700) and your molecule has enough nitrogens to create these double charged ions. Furthermore this is in accordance with your statements (in the e-mail you sent me) that: "It is not clear for me whether the fragmentation becomes already identical after the loss of the first sugar (the lower one (cladinose), containing methoxy moiety, ). It seems that after that loss, there is still a difference of 0.5 Da. However, after losing the second sugar (desosamine: containing dimethylamine moiety) all the fragments are clearly identical."

Finally, I would guess that the monomer and dimer would be very well separated by LC... does the ion with -1 m/z (your dimer) is eluted after your monomer?

Does your ion-trap has enough resolution to give you the charge of your molecule (traps like the Agilent XCT have higher resolution than the Thermo-Finigans LCQ so you are able to say in general their charge)? Otherwise, accurate mass measurements will for sure give you the isotopic distribution resolution you need to say if it is a single or double charged ion.

Finally, if you haven't look at higher m/z already, have a look if you ever you can see the single ion of your dimer. If you haven't look higher of your molecules MW, you might see more fragments of higher molecular weight than 850 -and less than 1700-.

Hope the above helps,

Kostas

[Lelo Richard]

Kostas,
I thank u for ur e-mail and for keeping giving comments about my thread.
It's the unknown structure (Dimer?) which shows 1 Da more than the reference.
The unknown (dimer?) is well eluted after the known (monomer?)
However, about the assumption of dimer: Those dimers should be observed for almost all the related substances present in the sample. On the other hand, as the dimers will have MW around 1600, shouldn't they be eluted later than all the monomers (MW around 800)?
I use an ion trap from Thermo Finnigan.
I will try to look for the single charged dimers in the higher mass region.

[Kostas Petritis]

Hi Richard,

Actually I didn't know which has 1 Da more (if it is the reference or the unknown). The dimer should have been eluted later than the monomer (which is the case?).

Maybe you could first eliminate this possibility (by looking in the higher mass region) before I give more thought on this.

[Lelo Richard]

Hi Kostas,
You are right I'd rather look at the higher mass region fisrt in order to make it clear about the possibility of dimer.
Thanks for all