Amino Acids with EZfaast by Phenomenex

[rb6banjo]

Have any of you ever used this product? What is your opinion of it?

[mattmullaney]

Works reasonably well, data compare well to Waters AccQTag and Thermo Dionex AAA using IPAD detection. My samples were typically fermentation broth and mammalian cell culture media (both "spent"). Data agreed with YSI instrumentation for Glutamine, as I recall.

EZ:faast is a bit more work than AccQTag, much more work than Thermo Dionex AAA (virtually none, no derivatization). I used NPD for GC detection.

Does this help?

[rb6banjo]

Yes. Thank you. I appreciate your insight.

[MSCHemist]

I have a similar method that is a bit of a rip off from Phenomenex that I use for amino acids. It works well on my GC-FID. I use ethyl chloroformate and ethanol the kit uses propyl chloroformate and n-propanol. I can see amino acids into the single ppm range doing splitless. The only amino acid I can't do is Arginine which does not react with alkyl chloroformates. I'd be happy to share my method.

[rb6banjo]

MSChemist--

In their product brochure, they talk of a catalyst to help with the reaction. Do you merely mix the AA's with the reagent or is there a catalyst?

What is a good solvent for isolating the derivatives for analysis?
What column do you typically use for the separation?

Thanks for your input.

[MSCHemist]

The main catalyst is pyridine, picoline also works.

My procedure is pretty simple I do the reaction in 1 autosampler tube and then transfer to another with a microinsert.

*100ul of sample in 0.1N HCl
*20ul of 1000ppm l-Norvaline ITSD
*125ul of Ethanol:Pyridine (4:1) [picoline works as well]
*55ul of 0.5N NaOH (critical for good glutamate response)
*add 50ul of ethyl chloroformate:Chloroform (5:3)
shake it dip it into a sonicator bath just mix it a bit allow the reaction to happen. It is done within a minute.
*add 250ul of chloroform with 1% ethyl chloroformate and mix well [this second dose enhances a few AA's. I tried extracting with isooctane but had no recovery of glutamine and reduced recovery of a few others)
*Transfer the chloroform layer to a tube with microinsert.

I use a db-5MS column with a glass wool Agilent 4647 split liner. 1701 Column works better and I think you miss serine and theronine on the db5 but my main interest in glutamate and glutamine.
I inject 10:1 split because I don't need the sensitivity my samples are as high as 10% MSG.

I buy the AA18 standard from Sigma and my calibration runs from 100ul of straight AA18 (~200ppm) to 1/20 of it. I also make separate solutions of glutamine and I beleive cysteine because the AA18 standard is missing a few.

Conditions
injector 240 10:1 split
oven
80 deg 2min
+30deg to 150
+10deg 280 hold 2.67min
FID at 280

You can substitute n-propanol and propyl chloroformate for ethanol and ethyl chloroformate like Phenomenex does and picoline and pyridine both work. I've also tried isobutyl chloroformate with methanol or ethanol. The sensitivity was significantly higher on the FID but the chromatograms weren't as clean and they eluted later so the GC program was longer.

Sample prep I just dissolve in 0.1N HCl typically for me 0.1-0.2g to 50ml. I don't do any cleanup but you can use an SCX column and elute with NH4OH.

BTW do not use reagent or other denatured alcohol. They contain 1% IPA or methanol that will cause side reaction. I just use 96% ethanol from the liquor store (Everclear or generic).

[rb6banjo]

Thank you so much! I'll give this a shot. I really appreciate your willingness to share this.

[MSCHemist]

No problem. The NaOH took me months to figure out why I wasn't geting good glutamate response. The method was originally developed by P Hušek and improved by others then Husek got hired by Phenomenex to perfect it for their kit. He is working on some exotic fluouroalkylated chloroformates for chiral analysis but good old ethyl chlroformate or propyl should work for you.

Here are some references for you
http://www.ncbi.nlm.nih.gov/pubmed/14516081
http://epub.uni-regensburg.de/12318/1/H ... 090719.pdf

[MSCHemist]

BTW the method is also reported to work well with several organic acids like succinate, fumarate, malate etc. I am trying that now. It be great if it could also do guanylate and inosinate (It should react with the hydroxyls and amines I'm not sure about the phosphate oxygens though though) but it has not been reported to.

http://libres.uncg.edu/ir/uncg/f/w_jia_ ... l_2007.pdf

Edit never mind in order to do organic acids the derivitization has to be done in several steps with addition of several extra solutions or it forms cyclic compounds. BSTFA is probably easier.

[rb6banjo]

Thanks. I have another application where I'm looking for isovaleric acid in a sample. This might be helpful information down the road.

[MSCHemist]

I am looking at various protocols over the weekend. The di and tricarboxylic acids can aparently form cyclic poducts so it is very sensitive to the reaction mixture. I saw succinic and malic fine on Thursday but Fumaric I was getting a mix of the proper trans and the cis isomer (diethyl maleate). It looks like I need to add the ECF in two main doses and use some bicarbonate in the mixture. I am planning to try a few conditions next week. I also found a few articles doing NADH and NADPH so I should be able to do nucleotides like disodium guanylate and inosinate but I was unsucessful trying it so far.

http://www.mdpi.com/2218-1989/1/1/3/pdf

though I've never had trouble seeing valeric, isovaleric, and pretty much every single carboxylic acid through steeric on the GC/MS without derivitization

[MSCHemist]

Fumaric acid is driving me nuts. I can get the other acids with most conditions as long as there is a fair ammount of NaOH in the media.

Fumaric I finally found one condition that doesn't cause significant cis isomerization and fumaric acid is the only analyte that derivitizes well.

100ul sample in water
100ul ethanol
200ul acetonitrile
40ul pyridine
10ul 5% (0.6N) HCl
20 ul Ethyl Chloroformate
250 ul Chloroform to extract
500 ul of 1N NaHCO3/Na2CO3 (2:1)

[MSCHemist]

So far the best protocol for TCA acids and Amino acids is the one in this thesis
http://theses.cz/id/sd5ijp/Michal_Kamen ... S_2012.pdf
they comment on the trouble with fumaric
100ul samp
75ul ethanol pyridine (4:1)
75ul chloroform:ethyl chloroformate (9:1) vortex
75ul 1.5N NaOH vortex to neurilize first portion ECF
75ul chloroform:ethyl chloroformate (9:1) vortex
75ul 3N HCl

There are several metabolomics protocols out there using methyl chloroformate which might be more reactive. I can't get the article but the
url is

http://www.nature.com/nprot/journal/v5/ ... 0.108.html

[lad]

Dear All

This was very interesting threat! I was planning to place an order of EZFaast next week, but now I think I will try MSCHemist protocol first.

But I don't have a GG, so I will have to do use LC-MS, does anyone have a idea of the LC-ms conditions? I have look into the thesis by Hannelore Kaspar (linked to earlier in the discussion) but the use an ion pairing reagent which I would like to avoid and it look to me as there are some inconsistencies in the methods sections regarding which column they have used.

All help is highly appreciated!

Best
Lars

[MSCHemist]

The Hannelor dissertation is a slight modification of Ezfaast. Ezfaast uses propyl chloroformate, n-propanol and pyridine and a mix of isooctane and chloroform for extraction. I mainly use ethyl chloroformate. I mainly use the dissertation protocol in my last post with some modifications. No one mix is best for all analytes. I've had to play with it for optimal so I can screen flavors for nonvolatiles.

200ul total aqueous (with 0.1N bicarbonate (helps with acids) and 33ul of 7N NaOH)
150ul ethanol pyridine (4:1)
100ul 20% Ethyl Chloroformate in Chloroform
cap and vortex immediately upon adding for at least 10 seconds min then unscrew cap to allow gas to escape
100ul 20% Ethyl Chloroformate in Chloroform (second dose helps caboxlic acids)
200ul of 5N HCl (not sure how essential it is but it seems to help clean the extract)
inject chloroform layer

I've been able to use this for carnosic acid and propyl gallate as well. Sadly I couldn't do inosinate or guanyate with it.

I've compared methyl, ethyl, and isobutyl chlroformates. Methyl gives a lower response but with faster chromatography
ethyl seems the best compromise plus I have the option of dissolving the sample in ethanol rather than water for oil based samples
Isobutyl chloroformate doesn't work well with isobutanol due to it being insoluble with water it works better with methanol and gives a higher response than ethyl chlroformate/ethanol but the chromatography is longer as it takes higher temp to elute.

Propyl Chloroformate n-propanol I haven't tried. I understand it works well as it is the highest chain chloroformate with a water soluble alcohol. The alcohol is important because with carboxylates initially an anhydride is formed and the alcohol attacks the anhydride forming the ester. A mismatch between chloroformate and ethanol R can lead to a small ammount of side products because there is always some of the chloroformate alchol present due to degradation of the chloroformate.

A possible benefit of propyl chloroformate is the higher signal and the fact the derivitve is nonpolar may make it possible to replace the chloroform with more isooctane or hexane. That makes it more amenable to automation having a robot sample from the top layer.

For HPLC I thought FMOC or other chloroformates are more typically used as volatility is not a concern.
http://www.chem.agilent.com/Library/chr ... 801193.pdf

I have the opposite issue. Lots of GC capacity but no HPLC. I work in flavors where GC is king. That is why I dabble a lot with derivitizations.