Artifacts in amino acid derivatization with BSTFA...help

[Cat5Hurricane]

Derivatizing "theoretically pure" amino acids to confirm they are in fact free of contaminants by GC/MS (TOF). Usually I derivatize with propyl chloroformate and that works for most amino acids. I didn't get a signal for arginine so I switched to BSTFA:TMCS (99:1). I derivatized 0.5-1 mg in 500 uL of BSTFA:TMCS at 80 degrees C overnight. I noticed on a test run, the presence of ornithine so I prepared a sample of that as well. On three different lots of arginine, I got a peak (equivalent in area roughly as the analyte peak) that was identified as ornithine. It was within 5mDa by accurate mass and the fragmentation pattern was identical to ornithine. Moreover, the retention time to that of ornithine standard was exact. I also detected 3-amino-2-piperidone (gamma lactum of ornithine...think ornithine cyclizing on itself...see images below). In one of the three lots, I detected lysine and confirmed it by accurate mass as well (didn't run standard because I didn't expect it).

Since I know ornithine and lysine can be detected by propyl chloroformate, I derivatized a sample of arginine and detected no ornithine or lysine. This brings me to the conclusion that ornithine (and lysine in one of the lots) is being produced either as a mechanism through the derivatization or forming when it hits the injection port (250C) and is not present in the arginine (as would be expected). Does anyone have any thoughts on why or how these artifacts are forming? I have attached the structures below so that you can see how similar they are to one another. Thanks!

[Suresh S]

Hello,

The problem is not with the injector, but with the BSFTA:1%TMCS silylating agent. It has been known that this silylating agent can convert Arginine, glutamine and asparagine to ornithine, glutamic acid and aspartic acid respectively. I found this information in "A Handbook of Derivatives for Mass Spectrometry". I believe Arginine is converted to the silylated ornithine in your case.

Arginine undergoes both alkali and acid hydrolysis to give ornithine. I am not sure, but i think hydrolysis of arginine to ornithine happens in the human body catalysed by enzymes. Hope this helps.

Best,
Suresh Seethapathy

[Cat5Hurricane]

Suresh

MUCH THANKS!!! I don't have the book you referenced but was able to refine a google search from what you told me and keep clicking through multiple references until I found the original reference:

Leimer KR, Rice RH, Gehrke CW.1977. Complete mass spectra of the per-trimethylsilylated amino acids. Journal of Chromatography A141,355–375.

Thanks again for your help!!!