[AgentCooper]

Hi, i'm a biologist with a modest amount of chem and analytical chem knowledge.

I'm following a recent publication method for extracting some metabolites from cells using an acidic organic solution, neutralizing it, then analyzing by LC-MS. The extraction solution is 20% 0.1M formic acid (FA) 80% MeOH, and the neutralization solution is 15% ammonium bicarbonate (ABC). Neutralization is essential because the metabolites cannot be extracted completely without acidic extraction but are unstable in acidic solution and interconvert to each other within minutes, giving inaccurate measurements and ratios. There are a few ambiguous parts of this method and I was wondering if I could get some thoughts.

1) The 15% ABC produces a lot of effervescence, which I assume is CO₂. Should this solution be degassed either by filtering or by leaving to stand for a couple hours? Should it be made up fresh each time?

2) The method says that the extraction mix is neutralized by addition of 10% v/v of the neutralization solution. However, when I check the pH of neutralized extract buffer it just comes up as ~ pH 4.0 instead of pH 2.1 for the pre-neutralized acidic extraction solution. I've checked pH with paper and probe. I know the organic may be obfuscating accurate detection of pH, but if I swap out the 80% MeOH for more H₂O (so moles of FA are the same) I get the same result. In this case is it likely that "neutralize" doesn't mean "change to pH 7.5ish" but rather "no longer super acidic"? Or am I not neutralizing correctly somehow? Perhaps because my ABC isn't the correct formulation?

Any thoughts?

Thanks!