Chromatography Forum

I would like to set up a LC/MS compatible method for determination of Cystathionine and Serine in biological samples (plasma,tissues,cell cultures). I would prefer a method without derivatization and use of ion pairing reagents (HFBA, TFA - I am afraid that the IP will lower the sensitivity of the method on LC/MS and the concentrations of Cyst in samples is pretty low).
Thanks for suggestions and ideas..

Dear Pistek,
HILIC is a suitable alternative for your application. Earlier this year I set up a similar application for homocystein, methyl malonic acid and succinic acid. (http://lcgceurope.adv100.com/lcgceurope ... ?id=148606)
With the indicated use for biological samples, you will also be able to do very simple matrix elimination, just dilute with acetonitrile (ratio > 1:4 and inject. For most situations this will work nicely. If you have more questions, you can contact me directly.

/Patrik
(PS. I'm associated with the companythat is producing the column)

Pistek

You can use Primesep 100 column for this purpose with TFA, formic acid or ammoniumformate- no IP required. Although I am not sure that formic acid can produce enoughion strength to elute di-aminoacid like cystathionione.
Here are two methods for Serine, alfa methyl-serine, GABA and methionine one UV and another one LC/MS/ELSD compatible)

http://hplcmethods.com/application_114.php

We have separate application for cystine (which is dimeric amino acid resembling your compound (Cystathionine). You can use ammonium formate to substitute sulfuric acid:

http://hplcmethods.com/compound_026.php

I'll see if we have cystine in the lab and can run this application or if you like you can send us a sample of cystathionine.

regards,

Vlad

Thanks for responses. I will consider both methods for eventual use, especially in case if conventional RP methods fail.

Pistek,

You may want to have a look at the publications.

Rapid Communications in mass spectrometry: 2003, 17, 1297-1311 and more importantly 2005, 19, 1587-1602 were it describes the LC-MS-MS method.

In these publications we developed a LC-MS-MS method which analyzed simultaneously 76 amino acids in less than 16 min. The method was developed for the fast screening of multiple inborn errors of metabolism. The method contains HFBA but as you may see we achieve very good LOD for Cystathionine (low fmole level). Also the method will be extremely fast if you need to analyze only these two compounds (Ser and Cystathione as Ser is eluted at 1.6 min and Cystathionine at 3.5 min). You could also try to modify the method to be only isocratic although you would need to flash the column anyway to get rid of the junk...

Hope the above helps...

Thanks Kostas,
I am pleased to read your answer.. which I awaited a little bit, because I know your work on AA from previous discussions on this forum. I look forward to try out your method. Recently, I have purchased a Hypercarb column for determination of some polar analytes, and I will try if it retains Ser and Cysth even without HFBA. I have had a bad experience with HFBA on LCQ ion trap - I lost sensitivity when I tried to retain polar analyte on C18 column using HFBA.

Hi Pistek,

I was pretty busy lately so I was not checking the board regularly, that is why I didn't reply earlier. So you have already purchased an Hypercarb heh? Gly and Ser are the least retained amino acids in that stationary phase.

A suggestion/tip for you: Try ammonium carbonate 10 mM, pH 9.3 for your application. Let me know how that worked if you will try it.

Will do, thanks a lot..