Choice of volatile buffer and mobile phase in HPLC-MS

[Victor]

I understand that to get the best MS sensitivity you should use in RP-HPLC-MS for basic drugs (I guess most people would use ES for this).

a) A high concentration of organic solvent- I think this helps the spray conditions.

b) formic acid rather than TFA. I think TFA gives ion supression due to ion pair effects with the protonated bases. Are there also other problems with TFA? The manufacturers of some systems seem to suggest that once used, it will remain in the MS for ever! Any comments on these points?

If I want to use formic acid at a higher pH to obtain a different selectivity in my separation, I could adjust the pH with ammonia-still volatile! Assuming my drugs are very basic and this does not affect their protonation state, how will the presence of ammonium formate affect the MS sensitivity? Has anyone done this?

I guess the same things apply if anyone has experience of this question in peptide analysis.

Many thanks for your help

[MG]

(b) You've pretty much described my experience with TFA. To elaborate, in positive mode, TFA will suppress your signal. In negative mode, it will kill your signal.

High amounts of ammonium formate or ammonium acetate, greater than about 20 mM, will cause signal suppression. I don't have a good feeling for whether smaller amounts suppress or enhance signal, versus the acid alone. If you do any experiments, I'd be interested to hear the results.

Some papers on this subject are:
J. Chromatogr. A, 987 (2003) 17-28
J. Chromatogr. A, 937 (2001) 41-47

The first one focuses on chromatography with volatile vs. non-volatile additives. The second compares formic acid/formate buffer to TFA and longer chain fluorinated acids, focusing on signal suppression. As far as I can tell, neither directly address the difference in signal between formic acid and formate buffers.

[Kostas Petritis]

Victor,

In additon to MG's comments. We haven't observe persistent memory effects in negative ion mode (i.e. if you clean your ion-source, you can get rid of the TFA). Of course you shouldn't use it in negative ion mode.

Furthermore, as MG said it is better not to exceed buffers of 20 mM.

Now about different acids and bases that can be used with MS (in terms of volatility) you can see:

K. Petritis et al. Volatility evaluation of mobile phase/electrolyte additives for mass spectrometry LC-GC Europe 15 (2002) 98-102.

You can find it on-line at the link below but I can send you a higher definition version if you are interested:

http://www.lcgceurope.com/lcgceurope/ar ... sp?id=9090

About the sensitivity differences, in general you will see a little bit higher sensitivities with low amounts of formic acid than with small amounts of ammonium formate. While this is the general tred I have seen it always depend on your analytes...