by
cc21609 » Mon Apr 09, 2018 6:32 pm
Some more musings.........
Your molecule is presumably of the form, ignoring the exocyclic DB, CH3(CH2)n(OH)2NH2 where n ~17,18, 19 etc.
I have written -NH2 as a terminal group just for convention.
You may be able to use the NH2 as a marker, since we would generally expect loss of NH3 as well as H2O.
Depending on the resolution settings of your instrument, you may be able to see loss of 17 amu AND 18 amu from the molecular ion.
Assigning the fragments derived solely from the
[MH-NH3]+ ion may help; comparing these with those from the metabolized [MH-NH3]+ may further help to define location of modifications.
Regards
The issue we have is the order of events I think. If we lose >1 H2O before the backbone fragments, then we basically have no idea which -OH group it was from. If we can control it to 1 H2O, then look at the fragments, we can identify which quadrant it is from and potentially use that to reference the metabolite.
Just some thoughts from the overcautious.... Be careful, there are so many things that can make life difficult!
Firstly, intramolecular rearrangements are very favoured in CID in LC-MS, but this also depends on your instrument (they often require the ion to be in a particular conformation so the reactive groups are approaching one another in the correct way; if you're using an ion-trap with a 30msec collision time, there is all the time in the world for the ion to find itself in the correct configuration).
The rearrangements may be quite extreme (and also highly unlikely-looking in solution chemistry; gas phase chemistry in a vacuum can do odd things).
Secondly, unless you have accurate mass, be cautious about mass differences. For example, if you're already looking at a sodium adduct, you can also gain mass 16 by looking at a potassium adduct instead.
Thirdly, remember that it is surprisingly difficult to tell the difference between an ion with a very labile hydroxyl group, easily lost as water, and an ion that can easily form a cluster-ion with water in the spray chamber. Making conditions in the spray chamber cooler and lower-energy will favour the +18 in both cases. There is a similar problem with ammonium adducts and compounds with very labile amine groups.
Basically when you think you've identified the product, comparing retention time and fragmentation to an authentic standard is essential if you want to be sure it's the right identification.
If you have source fragmentation, it is probably the hydrogen adduct that is fragmenting? If you can find the sodium adduct instead, it probably isn't fragmenting in the source (they're tougher); this can not only check that you've got the correct precursor mass (because you will have fragments that are hydrogen adducts, but the sodium peak only for the parent ion), but also give you something else for which to collect fragments (it is reasonably likely that the sodium and hydrogen adducts will undergo different fragmentations, though in some instruments sodium just gives terrible results!).
It looks like it is the M+H fragmenting. There is very minor fragmentation from the source since M+H is intact. However, if we ramp up the collision energy it just starts to lose the -OH groups as water. I totally understand how difficult it is to favor a less energetically favorable backbone breakage vs. the rearrangement. We generally do not see the sodium adduct in the mass spec experiments that we did. And we do plan to spike to check the retention time once we have a general idea which compound it is to tackle.