Citrulline, Ornithine and gamma-aminobutyric acid

[Heikki]

I was wondering and trying to search for an answer, but I came across none. Is it possible to separate citrulline, ornithine and gaba with UV-wavelength using PITC? I mean does it work in a matrix where there most propably are other aminoacids present as well?

[Sniff]

There's a great volume of "Methods in Molecular Biology" entitled "Amino Acid Analysis Protocols" (volume 59) that basically gives step by step instructions on how to separate pretty much every PITC derivitized amino acid in biological sample matrices. For more info on this volume, check out:

http://www.amazon.com/Analysis-Protocol ... 930&sr=8-1

If you have access to phthaldialdehyde (OPA), I highly recommend using that with either sodium sulfite or N-acetyl cysteine. The reaction is complete in seconds (even at dilute concentrations) and it gives a very strong and distinct absorbance at 330nm. I've separated every amino acid I've tested so far (about 15 of them, including GABA) in under 5 minutes using a Zorbax Eclipse XDB C-18 column (4.6x75mm, 3.5um particle size). The only downside of this reaction is the products are unstable, but I've gotten around this by being consistent in the time between mixing the OPA with sample (30 seconds) and have consistently gotten CV's less than 1% with standards. Other people have had good success using anion exchange columns (using sodium sulfite in the reaction). That volume of "Methods in Molecular Biology" also has a good protocol for OPA derivitization. Good luck!

sniff

[Mark Tracy]

The OPA/thiol chemistry can be automated with many of the popular autosamplers. The derivative is also fluorescent ex.335 em.445.

FMOC is another alternative. The derivative absorbs at 265, and is also fluorescent at 315. The derivative is quite stable, but making it relies on rapid and repeatable mixing of the reagents, so automation is trickier.

[Kostas Petritis]

As discussion have deviated from the original derivatization agent used, have in mind that you can separate (and analyze) these amino acids in their underivatized form.

Under the conditions described Citrualline, GABA and Ornithine are eluted at 2.6, 9.5 and 12.1 minutes respectively...

Have a look at:

Piraud M, Vianey-Saban C, Petritis K, et al.
Ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometric analysis of 76 underivatized amino acids of biological interest: a new tool for the diagnosis of inherited disorders of amino acid metabolism
RAPID COMMUNICATIONS IN MASS SPECTROMETRY 19 (12): 1587-1602 2005

[Heikki]

The OPA/thiol chemistry can be automated with many of the popular autosamplers. The derivative is also fluorescent ex.335 em.445.

Yes, but my problem is that I don't have a fluorescence detector available. That is why I'm currently only interested in derivates which express themselves at UV-spectrum.

[HW Mueller]

Heikki, all fluorescing substances have to absorb light first. Chances are that FMOC derivatives absorb more strongly than PITC derivatives.

[Sniff]

Using our UV detector, monitoring at 330nm, I can detect down to about 12.5pmol. Currently, I have the autosampler adding 90uL of 20mM OPA to 10uL of our samples, mixing 2x, and then injecting on a 2.5uL loop. From the first contact of reagent to sample until injection, 18 seconds elapses. If I let the reactions go longer, there is no increase in the product peak and, ulike the many reports by Molnar-Perl, I'm not seeing significant side-products, even after a few hours at room temperature.

[Mark Tracy]

If you are limited to UV-VIS, another popular derivative is DABSYL; it absorbs strongly at 425 and is free from many interferences. The reaction is not difficult, but it is a bit slow.