LC-MS assay for polar compounds

[sunny12]

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!

[gapeev]

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

[sunny12]

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.

[lmh]

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.

[sunny12]

Thanks for your input. See how it goes

[Vlad Orlovsky]

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.