Chromatography Forum

Hello all,

At a previous company I developed a procedure for doing AAA analysis of media using an Agilent 1100 and their Zorbax Eclipse AAA column and reagents (they have the whole method outlined on their site). I'm now at a new company and we contract out our AAA testing but it is very expensive. I'd like to look into bringing this in house using an Agilent 1100 system as I had before but the test samples in this case are more complex than what I had previously tested. Now I'd be teseting vaccines with either aluminum hydroxide adjuvant or detergent and I'm not sure how either of those would react. Any previous experience? Also, I've only done pre-column derivitization... would this work for vaccine samples where the amino acids are not free? Does anyone have a digestion procedure they'd like to share? I can definitely email anyone the procedure I wrote using the agilent system.

Thanks!

I've never done AAA in the presence of aluminum either, but I can say a few things. First, at the pH of the pre-column derivatization (8-10) aluminum hydroxide is insoluble, and is likely to take some of your AA with it. You can try to separate the protein from the aluminum or use a chelating agent to keep it soluble. Talk to your protein chemist and find out if the protein is soluble and stable at a pH that will dissolve the aluminum hydroxide; if you are lucky, you can ultrafilter or dialyze the aluminum out.

The detergents are most likely Tween-80, Triton X100, or SDS. They do not interfere with the derivatization reactions. They will build up on your C18 column so you will need to flush it with 90% MeOH to clean the column.

Most protein hydrolysis is done with refluxing 6N HCl overnight. There are other recipes that various vendors sell. For small samples like yours, a sealed tube method is probably the way to go; Pierce sells a decent setup. Acid hydrolysis destroys tryptophan and depresses serine; you can use alkaline hydrolysis for tryptophan. You may also see some oxidation of methionine to the sulfoxide and sulfone, and cystine to cysteic acid. If those are important, you can quantitatively oxidize sulfur amino acids with ice-cold peroxyacetic acid.

Finally, check out www.abrf.org. They have a section on amino acid analysis that should get you launched.

Hey Mark, thanks for your response, that is very helpful information!