Chromatography Forum

Hi,

I'm in the process of setting up an energetics LC-MS assay to detect ATP & Phosphocreatine. So far, I've tried a Hypercarb column from Thermo (150x2.1mm; 5um) using pH 10.0 Water (with Ammonium acetate) as mobile phase A and pH 10.0 Acetonitrile (with Ammonium acetate). I'm now facing Ion Suppression, especially for the Phosphocreatine and trying to shift the gradient out and reduce the flow rate (from 500ul/min to 250ul/min) resulted in a total loss of peaks and now I think the column is stuffed. Even trying to re-set the column by flushing through THF was not successful.

So, my first question would be if anyone has had any experience with very polar compounds using a Hypercarb column and could give me any advise on this?

I'm now thinking of changing to a C18 and using an ion-pair reagent (DMHA). I saw in the literature that people are using DMHA formate with pH adjusted to 6.8 with formic acid. Does DMHA format means I make it up in Water an adjust the pH with formic acid and therefore it's now called a 'formate'?


I'd appreciate any help on this from anyone who has done similiar things before!

What are your MS parameters (voltages, polarity, current readout)?

I'm performing ESI ionization in the negative ion mode. Capillary temperature is set at 400C. Curtain Gas: -10, Collisio Gas: 6, Ion Spray Voltage: m-4500V, Ion Source Gas 1: 70, Ion Source Gas 2: 70.

PCr was eluting first at around 1min and ATP at around 3.5-4mins. I also had internal standards, Cyclocreatine and 13CATP.

The only thing I'd mention is that if you do choose to use the ion pair reagent, you may find it hard to get rid of:
viewtopic.php?f=3&t=15613
That might not be a serious problem to you though. Good luck.

Thanks for your input. See how it goes

Try something like this:
http://www.sielc.com/Compound-Alendronic-Acid.html

if you use a shorter column you might get away with lower concentration of buffer for better sensitivity.