acid in lc-esi-ms

[skunny]

Hi

I was wondering if an expert could help me.

I quantify tenuazonic acid with lc-ms/ms with the quechers method. in the original paper they use 0,1 % formic acid in water + 10 ml of acetonitrile. When I use 0,1 % formic acid my recovery rate ist about 0. So i tested different concentrations of acid: 1 , 2, 10 and 15, and it turns out that 1 and 2 % work best and the recovery rate gets worse again with 10 and 15 % of FA. What could be the reason? the higher the acid concentration the better the ionization , is that right?

I use the negative mode, i thought acid would help the protonation, but in the negative mode i have deprotonation..

maybe someone has an idea, thank you so much!

[James_Ball]

In negative mode should remove protons and positive mode should add them, so I believe you are seeing the correct results.

The acid concentration needed for optimum sensitivity will be different for each analyte and each source design so you have to adjust until you find the best combination.

[lmh]

(1) recovery isn't the same thing as signal-strength. If your problem is that you've got a low recovery of the analyte (you extract it but it isn't being extracted) then that has nothing to do with the LC-MS. It's a really good idea to divide up the big overall problem ("I can't find analyte") into the two separate smaller problems ("I can't get the analyte out of the sample and into a vial" and "once I've got it in a vial, my signal strength is low"; in fact this second question is also two separate mini-problems: "my chromatography is poor" and "my MS detection is poor" but they're often interrelated)

(2) Solution acid-base chemistry applies only to the extraction, not to the electrospray. Having formic acid in the running buffer doesn't harm ionisation in negative mode. Electrospray isn't just about drawing out the ions that happen to be in solution, so choosing a pH where there are more ions in solution doesn't necessarily make it better. It's an electrochemical process. Inside the spray needle, formic acid is dissociating to formate and H+ ions. If you're in negative mode your needle is negative and you're using a positive voltage outside the needle to draw out droplets with negative charge. The positive H+ ions have to go somewhere too, and they're going back to the needle, where they're being reduced. The ability of formate to dissociate and H+ ions to get reduced is part of what's happening, and if it doesn't, the whole process stops working; there are no more formate ions going the other way, and sharing their negative charge with the things you're trying to ionise. So if, for example, in a well-meant attempt to make your compound ionise better in negative mode you sling a load of some friendly alkaline buffer in there, you may well find that your positive large amine that you're using as a buffer is rubbish at getting reduced, and the ionisation efficiency plummets through the floor even though your analyte ought to be deliciously ionised.

(3) But in any case, your analyte is a secondary amine with other functional groups too, and will ionise positively in solution at acidic pH. But by the time you're using 10%, the formic acid is no longer merely an acidifying additive. At that concentration, it's a solvent. And who knows whether your analyte is stable in that much formic acid anyway... it's rather a lot! If your analyte is poorly retained, then injecting it in 10% formic acid may also influence its chromatography, and that of potential coeluting competitors for ionisation.

[H.Thomas]

You're talking about a QuEChERS-extraction. 1% formic acid is the standard concentration for extracting acidic analytes. The partitioning between water and acetonitrile is influenced by pH, at lower pH your acid will be more hydrophophic, so recoveries will be better.

You didn't mention your matrix - if it is some kind of wet material like apple, cucumber etc., you simply extract 10 g of produce with 10 mL acetonitrile containing 1% formic acid. Add water if you have dry material. Use a non-buffered salt (MgSO4 + NaCl, 4:1). You may need to do some clean-up of the extracts.

This does not mean that you should use 1% formic acid in your HPLC eluent - this ist far too much. For positive mode use 0.1% or less, for negative mode usually sensitivity will be better with 0.05% acetic acid.