I'm still on the LC-MS learning curve (been working with
an ion trap instrument for a few months). I mostly
analyze nucleoside analogs. My question is that,
despite my best efforts at tuning some analytes seem to
give a number of ions e.g.,[M+H]+, [M+Na]+, [2M+H]+,
[2M+Na]+ and, in the assay I'm working on now, the
most intense ion is the [2M+Na]+. This is true over a
large conc range (100 ug/mL to low ng/mL).

There are many literature methods out there for
nucleosides and their derivitives. No one that I can find
is monitoring clusters. My questions are: 1) Can I do
anything to favor [M+H]+ over the other ions e.g.,
tuning, mobile phase pH (I tried this also at 3 pHs)? 2)
If I have a number of analyte related ions produced and
I only monitor one (I do currently do SRM on the [2M+
H]+ because it gives me the best S/N), am I
risking invalid quantitation i.e. can I assume that the
ratio of analyte-related ions will be constant?

Thanks, in advance, for your help.

What is your buffer?

My buffer is 10 mM ammonium acetate pH 5 with 2%
MeCN (I'm using a polar-encapped C18 column--
Synergi Hydro RP by Phenomenex).
Thanks.

Hi Jeff,

If I remember well I have seen a paper where they use triethylamine in the mobile phase and achive to favor [M+H]+ over the sodium adducts (I think it was for the analysis of nucletides and nucleosides). You may want to give it a try.

Kostas

I have found that for ESI, higher spray chamber temperatures and voltages form more M+H and fewer adducts. The clusters have lower energies than M+H, and can sometimes be broken up to M+H. I have only worked with drugs though; I haven't worked with nucletides. Just be careful that you don't decompose your analyte.

Can you use ammonium formate at pH 3.5 instead of the ammonium acetate? It is difficult to see where the sodium is coming from.

What is a polar endcapped column? What are these "polar endcaps" (vs standard TMS)?

Does anyone actually know what these "polar endcaps" really are?

1. I have tried amm formate at pH=3 with no significant
change in the distribution of ions/adducts. I'm new
enough to LC-MS to be unsure, but I thought that Na+
was ubiquitous in most glass and not easily eliminated
from any aq. mobile phase (pls advice if this isn't the
case).

2. I don't think it is related to my problem (since I can
see the same distribution of ions in direct infusion of my
sample into the flow path--no column), but the "polar
enapping" of the Synergi column is proprietary. It
seems like some of this info (on the polar encapping
groups) may be available in the literature. For
example, Agilent's Zorbax-bonus RP is also listed as
having "proprietary polar encapping," but I recall an
article in LC-GC that identified the encapping as amide
groups (don't recall the exact structure).

Thanks again

Hmm, I would suspect that if the "polar endcaps" are amides or something like that, then the inevitable column bleed would be detectable by MS.

All columns bleed, despite what column manufacturers say. If they did not, they would last indefinitely, barring some type of plugging or mechanical failure. Columns bleed the endcap first, followed by the ligand. Luckily, TMS and C18 ligands are not ionizable, hence they are not seen by MS.

The same bleed from columns that contain embedded polar groups are ionizable and are detected by MS. The embedded polar groups are amides, carbamates, ethers, etc. If these same groups are the endcaps, guess what, they would bleed and it would be seen by MS.

I suspect that these "polar endcapped" columns simply possess silanol activity (silanols are a polar endcap!).