Separation of metabolites using LC/MS

[corrine]

I'm currently working on the separation of metabolites and would like to separate ATP, ADP and AMP using LC/MS. I have tried various buffers like ammonium acetate, formic acid..etc but could not get a good separation esp for ATP, that does not seems to bind to my reversed phase colunm. So is there any recommendations for buffers which i can use or columns for separation?

[MG]

This looks like it might be a good candidate for HILIC, although I have never run HILIC on these compounds. If you're wondering what HILIC is, just search this board, or search the literature for Hydrophilic Interaction Chromatography.

If you're stuck with a reverse-phase column and you're running positive ionization, you could try adding TFA (0.01% to 0.2% by volume) to your mobile phase. Use the smallest amount that gives you the retention and separation you need. If you must use negative ionization for these compounds, don't use TFA.

[corrine]

Thanks for ur reply. I understand that using HILIC will be under normal phased LC. I would like to also separate both polar and non-polar compounds such as ATP and adenosine at the same time. The column that I'm currently using is actually Waters ATlantis dC18 & also Waters Symmetry 300 C18.

[MG]

Adenosine is still quite polar. I think it would be retained in HILIC.

HILIC is a cousin of normal phase, but usually uses water (+ ammonium acetate or formate) and acetonitrile as mobile phases.

[Einar Ponten]

Yes, the polymeric ZIC®-pHILIC column is indeed suitable for this application and LC/MS!

We have data showing nice separations of these in both standards and crude extracts. A manuscript is currently in preparation.

Please, send us an e-mail and we will support you.

[Kostas Petritis]

Corrine,

There is an already developed LC-MS method for ATP, ADP and AMP that uses dimethylhexylamine as ion-pairing reagent and high pH for their separation. They detect them in positive ion mode (they select either the [M+H]+ or the [M+DMHA+H]+). You have to use a high pH resistant column for this (they are using the Waters XTerra for this).

For more you may see:

Title: Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography-electrospray ionization mass spectrometry
Author(s): Qian TX, Cai ZW, Yang MS
Source: ANALYTICAL BIOCHEMISTRY 325 (1): 77-84 FEB 1 2004