Amino Acids with EZfaast by Phenomenex

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

21 posts Page 2 of 2
Thanks MSchmist for sharing all you insight on this - Highly appreciated.

I currently use FMOC derivatization with FL detection, but I would like to use a internal standard and I have been able to find an appropriate one. With MS detection I could just use an isotope labeled AA. But thinking of it, I might jusut see if i can get the FMOC AA's to fly and then use that.

BTW we I am also moving into flavor, next item after the AA's are the nucleoside monophosphates (but that is for another post)
There are several ITSD's possible
Norvaline
Norleucine
p-chlorophenylalanine to name 3

I am trying the IMP+GMP as well. I tried the chloroformates (ECF&MCF) and I also tried using phenylboronic acid followed by ECF and MCF. Nothing

I tried phenyltrimethylammonium hydroxe + sample in injector at 300C from Knapp's book. Nothing

I have tried several different conditions and several different silylating reagents. Knapp's Handbook of analytical derivitizations says pyrdine:BSTFA:TMCS 30:30:1 and RT or heat to 150. I tried that as well as MSTFA and acetonitrile. I am thinking I may try using DMSO which is the most polar aprotic solvent there is with trisil (BSA/TSIM/TMCS) the most powerful silyating reagent there is. So far all I have is empty chromatgrams with solvent peaks and trace silyated impurities.
I have tried several ISTD but the problem is that I used Fl detections and most ISTD overlap with soem of the other peaks in the chromatogram, which is why I would like to move to MS detection.

For the nucleotide, I don't have any input here as I will try with HPLC and i don't need derivatization for that.

For the AA, I will try the propyl derivatization when I get all the chemicals i need (some of them are on backorder right now ). For the actually chromatography I will look close on this: http://www.ncbi.nlm.nih.gov/pubmed/19166726

I will poste and update when I get it to work.

Lars
I took a quick look arround to see what could be done for Arginine since it does not react with chloroformates enough for GC (the guanadino group does not derivatize).

One report recommended treatment with arginase and analysis as ornithine. It sounds like it would be expensive to buy the isolated enzyme.

The tradiotional method is to react the guanidino group with pentaflouropropyl anhdride. I assume this is a water sensitive rxn unlike the chlroformate one. This might be a possibility if the arginine is extracted in the organic layer. What I've read indicates it derivitzes enough to end up in the organic layer but it just adsorbs to the GC column. Therefore the possbility is to do the chloroformate extraction, extract it with chloroform then do the PFPA treament to derivitize the side chain.
[25] D. Tsikas, B. Schubert, F.M. Gutzki, J. Sandmann, J.C. Fr¨olich, J. Chromatogr.
B 798 (2003) 87.
Arginine came up as part of a full amino acid profile. Treatment with 50% aqueous hydrazine hydrate at 70 deg for 3 hours leads to complete conversion to ornithine which is then doable by chloroformate derivatization.

I am first trying MSTFA TMCS acetonitrile to convert to TMS ornithine because I have the materials in house.
A bunch of papers came out in the last year using trifluoroacetylacetone as a first step prior to ethyl or isobutyl chloroformate derivatization.

http://www.omicsonline.org/open-access/ ... 000248.pdf
**this one even does arginine which is possible. Guanidino compounds condense with 2,4 diones to form pyrimidyl derivatives*

http://link.springer.com/article/10.100 ... 011-1957-y

http://pubs.rsc.org/en/content/articlel ... c4ay03098b

I have been trying to repeat their protocols with no success. I've tried different buffers and pH's altered the solvent ration more in line with other papers (their mix for the chloroformate derivatization is way high aqueous and low pyridine compared to others with no success. I can get chloroformate derivatives that I can identify especially when I match the chloroformate and alcohol (methyl chloroformate with methanol and ethyl chloroformate with ethanol). It is quite frustrating as they can use an FID and simple db-5 column and their chromatograms are beautiful.

I'm skeptical though. Acetylacetone reacts with primary amines so how do they get proline right in line with the others? Also why use ammonium acetate buffer when the reagents react with both primary amines and carboxylic acids. I tried phosphate instead. I wonder if they are simply getting methyl esters from the methanol/chloroformate and ethyl or isobutyl carbamates from the chloroformate rather than schiff base esters.

If anyone gets it to work let me know.
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