Chromatography Forum

I am working on separating two amino acids, a tripeptide and their disulfides out of blood samples. Waters has an analysis package "AccQ Tag" for pre-column derivatization of amino acids. The pricing is $428 for 250 analysis. Or I can by fluorescence reagents and develop the derivatization protocol myself, which is less expensive ($180/10mg).

Anyone has ever used "AccQ Tag"? How does it work?

I am working at Waters, and I am currently involved in this type of analysis.

The AccQ-Tag method is very simple, and very sensitive. You mix the analytes with the reagent. The reaction is over in seconds, and the reaction product is stable for hours. You can monitor by UV, or if you need a higher sensitivity or specificity for some reason, by fluorescence.

That is the nice part. You have to realize though that every amino acid reagent will react with every protein, peptide, amino acid or primary amine in your blood sample. Unless you do some sample prep with your blood sample, you will get a big mess. You can reduce the mess by using suitable sample prep procedures. There is an advantage in the fact that a protein has few terminal amino groups and diffuses slowly, which reduces the detector response on a molar basis compared to amino acids and small peptides.

The reaction works by putting a label on the amine function. The reagent works with primary and secondary amines. You can find the details and the entire reaction scheme either on the website or in the catalogue.

Many thanks for your reply.

How is it like working at Waters? Must be thrilling.

There are alternative derivatisation eg using OPA which might be worth looking at. it will certainly be a lot cheaper but could take up more development time. (To find methods use OPA and peptides in a search engine)

Yes, there is an alternative. I analyse Amino Acid content in Solutions for Infusion. Derivatisation reagent that we use are: OPA, FMOC and Borate Buffer.
I am interested about Waters' application too. Can I use the same HPLC system for Amino Acids and for the other Reversed phase Chrom applications? I think these reagent can make a real mess if they are present in the system after AA analysis... Do the Waters solve this problem?

The AccQ-Tag reaction is carried out in the vial. There is no carry over or other silly things that might happen with injectors. You can run the system immediately for other applications.

I use the accQ-tag derivitising reagents and I find it easy to use and less worry regarding the toxicity of OPA. If you are trying to aa in the Blood we use pca first then we add bycarb to nutralize the acid. The supernatant of the sample is what we analize. We run with a gradient and we get nice seperations most of the time. If not the column or the eluents are off. I hope this helps a little.
Tracy

I use AccQtag and it works well for me, but I can't compare it because I haven't tried the alternatives. For me, sample prep is the most difficult part of amino acid analysis, and that will be the same for all methods.

from memory, OPA only derivitises primary aminos, and is less stable, whereas the AccQTag reagent will do secondary as well and stable up to a week. We are using AccQTag for the analysis of amino acoids in natural (river) waters where concentrations are typically nM (Obviously we do some preconcentration but can still get away with less AccQTag reagent giving more samples per kit). analytical frugality.

You can lower the unit cost for the AccQTag by using less of the derivitising reagent, of course this will all depend on the concentration of the aminos in your sample. This will also extend the life of the column (Nova-Pak) that comes certified with the AccQTag kit.

Method development is the key, data validation and quality assurance....


Good luck

peggsy