When I analyze 20 amino acids with GCMS, I use MSTFA to react with sample, but there are some combined peaks cannot be separate. Like proline-Isoleucine, and i cannot detect tryptophan and glutamine, arginine, I don't know why. And there are always some cell debris cannot dissolved in MSTFA only.
Dear Xiaofang
In respect to cell debris, I would check extraction procedure as a separate step. I do not know what kind of method You use, but in general after chloroform/methanol and drying You should not have visible debris.
In respect to separation, if You do not use already well established method and working on method development, I would suggest to select problematic pairs of AAs, for example Pro-Ile and include two standard vials, one - consists of Pro only, and 2nd - Ile only. Based on this You should be able to separate them within Your samples.
Arg and Gln are not easy to detect on GCMS in my experience. I also would add to this list Asn, and sulfur-AA. I am not saying that this is impossible, but Your method has to be really good. And an abundance of these AAs in Your samples should be above noise.
Use standards, check also column, extraction procedure and keep trying, I believe You will solve this.
I see two possible problems: incomplete derivatization; incomplete GC separation.
Are you sure that all the 20 amino acids are fully derivatized under your conditions ?
I still have a very old literature source Alan E. Pierce, Silylation of Organic compounds (1968), chapter 8: Amino Acids and Related Compounds, showing that the problem is not a new one. Arginine is already described as difficult to silylate, and silylated derivatives, in general, hydrolyse easily and should be protected from moisture.The carboxyl group is silylated more easily than the amino group
Concerning the GC separation, not described in your question, have you checked that the 20 amino acids can be fully separated under your conditions; based on previous literature sources ?
Best regards
Patrick Arpino
Dear Xiaofang
In respect to cell debris, I would check extraction procedure as a separate step. I do not know what kind of method You use, but in general after chloroform/methanol and drying You should not have visible debris.
In respect to separation, if You do not use already well established method and working on method development, I would suggest to select problematic pairs of AAs, for example Pro-Ile and include two standard vials, one - consists of Pro only, and 2nd - Ile only. Based on this You should be able to separate them within Your samples.
Arg and Gln are not easy to detect on GCMS in my experience. I also would add to this list Asn, and sulfur-AA. I am not saying that this is impossible, but Your method has to be really good. And an abundance of these AAs in Your samples should be above noise.
Use standards, check also column, extraction procedure and keep trying, I believe You will solve this.
Thank you for your reply. I read some reference and they said arginine will convert to orithnine and citrulline, but many people can detect glutamine. I will try again. Thank you.
Glutamine and Glutamic acid are both probably being converted to a pyroglutamic acid derivative and being detected together as a single peak. Arginine typically gave a weak signal that corresponded to its conversion to ornithine during derivative formation. It seemed Arg readily formed several unstable derivatives that appeared as small peaks in the run. Try forming the derivatives of Arg, Glu, Gln and Trp separately and injecting them to see where their MSTFA derivatives elute. Tryptophan was very hard to get because it oxidized easily to other compounds in the presence of any residual oxygen especially in the presence of light and acid. Proline and Isoleucine can be detected by selected ion monitoring (SIM) even though they co-elute. You should be able to adjust the chromatographic conditions (shallower thermal gradient) to allow for a better separation of these 2 amino acids.
Thank you for your answer. I can detect glutamic acids but the library regard it as glutamine. I will try SIM mode to separate proline-isoleucine peak. As tryptophan, I can detect standard tryptophan but cannot detect it in yeast cell. I don't know if it is oxidized by oxygen.
Try using the EZ:Faast kit from Phenomenex. However I find the GC column they recommend has a rather short life and the MS detector needs regular cleaning.
see https://www.phenomenex.com/Info/WebDocumentServe/ezfaast_user_guide.pdf
Thank you, sir. I have see it at some papers but not know what is it. Thank you for your attachment.
hhI Hi 112
Hi Xiaofang Yuan,
You can read papers
* Gas Chromatography-Mass Spectrometry (GCMS) of Amino Acids
* GC-MS METHODS FOR AMINO ACIDS DETERMINATION IN
DIFFERENT BIOLOGICAL EXTRACTS
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=0ahUKEwjYhLTM_ubQAhXMjiwKHT6rDYIQFggsMAI&url=http%3A%2F%2Fwww.uvm.edu%2F~dmatthew%2Fchem201%2Fchem201_GCMS_lab.pdf&usg=AFQjCNHj6qbzwM3mFckVqA5iNdWFekrcpg&sig2=q62g3J2WF71ixGCFsZtRig&cad=rja
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&ved=0ahUKEwjYhLTM_ubQAhXMjiwKHT6rDYIQFgg_MAU&url=http%3A%2F%2Fchem.ubbcluj.ro%2F~studiachemia%2Fissues%2Fchemia2016_1%2F20Culea_etal_213_222.pdf&usg=AFQjCNEDMC4s3i1QRuS4SVHR5s9Sm5tbPw&sig2=3kXBeclJNG4WMkqttK4QnA&cad=rja
MTBSTFA derivatization will provide better resolution for amino acids. MTBSTFA produces dimethyl-tert-butylsilyl (TBDMS) derivatives, which are higher molecular weight compared with the trimethylsilyl (TMS) derivatives produced by MSTFA.
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