Chromatography Forum

Hi!

I need some advice on acidifying samples. They are all prepared from different biological matrices, some containing a lot proteins/peptides and some less. The compounds I am interested in are pretty nonpolar (steroids).

My setup consists of Oasis HLB in an online cleanup setup coupled with an analytical column and MS/MS.

I have been adviced to acidify my samples. As far I understand this has two effects:
1. Put protein/peptides in a uniformly charged states so that they interfere less with the HLB column.
2. Disrupt any protein binding of my steroids from proteins.

This may give a cleaner signal, decrease ionsuppression and increase recovery.

My question is simple. How strong should this acid be? And how much should I add? (What pH is needed?)

Is 2% Formic acid enough? (that would simplify things for me...)

At least part of the idea to add strong acid to a plasma sample in order to avoid protein binding is to destroy the protein structure. I doubt that 2% formic acid is good enough. The simple answer is to compare the same sample treated one way or the other way.

According to Waters: add 20 µl phosphoric acid to 1 ml of serum.

Regards Bert