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Separation of metabolites using LC/MS
Posted: Mon Dec 13, 2004 2:48 pm
by 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?
Posted: Mon Dec 13, 2004 4:33 pm
by 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.
Separation of metabolites
Posted: Tue Dec 14, 2004 12:53 am
by 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.
Posted: Tue Dec 14, 2004 5:21 pm
by 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.
The ZIC®-pHILIC column
Posted: Tue Dec 14, 2004 11:08 pm
by 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.
Posted: Tue Dec 28, 2004 8:56 pm
by 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