Hello,
I am trying to develop a " rapid " HPLC assay for an enzyme that uses alpha-ketoglutarate as cosubstrate and turns it into succinate and CO2.
Starting concentrations of aKG are in the 20 to 300 micromolar range and the reaction should not consume more than 20% of it.
I know that I can inject directly the mixture on a C18 colum (or anion exchange) and use an acidic mobile phase to separate the aKG and succinate and detect them at a low wavelength. (We have a Waters PDA detector)
BUT I would like to improve the sensitivity of the method and thought about derivatization. Either the 2-oxoacid or the acid.
From the litterature it seems that for the aKG dinitrophenylhydrazine would work nicely and for succinic acid, esterification with 4-nitrophenacetyl bromide or benzyl bromide would work too.
My question is, do these derivatization procedures work under aqueous conditions or when some water is present (ie if I dilute the mixture into some organic solvent)
Thanks

What sensitivity are you getting, and what sensitivity do you want to get to? An ELS detector should give you better sensitivity than PDA for these compounds, and you don't have to derivatize

Sensitivity...
I have injected 1 mM standard of aKG and succinic acid (underivatized). It looks like I could perhaps work like that with aKG but I think the precision will be low. It won't work with succinate.
The enzyme assay needs concentrations of aKG ranging from 50 to 200 µM (about 7 to 30 µg/µL)
which should make about 5 to 20 µM of succinate (about 0.5 to 2 ng/µL)
What I want is to measure precisely the amount of these products in various assays within that range of concentrations.
I have considered ELSD but we just don't have the money to buy that type of detector now.
Recently I found in the litterature the method I was looking for, it uses 2-nitrophenylhydrazine and EDC as coupling agent to derivatize acids and it works in ethanolic or methanolic / water solutions.
AD