Chromatography Forum

Dear all,

at the moment I am trying to analyse glyphosate, AMPA and glufosinate by the well known derivatization with FMOC-Cl. This reaction is done at pH 9 in a sodium borate buffer solution (7 mM). Unfortunately the reaction is only working well for glyphosate. For glufosinate I get a yield of around 40 % and AMPA is not derivatized at all (at least I see only the underivatized molecule in the mass spectra when tuning for the compounds).
I have to say that this reaction was working before in our institute.
Do you have any idea what could be the reason and what I can try to solve this problem?
Thank you all in advance.

I'm not an expert in this analysis but have you tried to increase the temperature? e.g. 45°C 1hour.

Yes I tried higher temperature and also longer reaction time (overnight). Whats making me crazy is that it worked before using a time of 4 hours at room temperature.
Is there a possibility that is hydrolyzed when entering the ESI or when it is mixed with an organic solvent? I try to tune them by mixing the reaction mixture with acetonitrile before entering the ESI.

I have encountered journals and published methods that suggest these compounds may interact with glass.

I have encountered journals and published methods that suggest these compounds may interact with glass.

I concur. Deactivated glass might work.

Isn't this derivatization normally used to give the molecules a chromaphore for UV or FLD detection?

There are methods for doing these by LC with MS detection underivatized.

http://sciex.com/Documents/tech%20notes ... 280%29.pdf

I know that when doing the OPA derivative with sodium borate buffer you need the pH to be near 10 before it works well but you also have to first oxidize with a sodium hypochlorite solution. (EPA 547 post column HPLC/FLD)