By Anonymous on Thursday, October 16, 2003 - 07:29 am:
I am about to embark on amino acid analysis by LC-MS and am swamped with information! Some researchers do not derivatise, some use alkyl chloroformates, and many other derivatising agents are mentioned. Can anyone advide me on a reliable and sensitive method?
By MG on Thursday, October 16, 2003 - 08:55 am:
I've never analyzed these, but a colleague of mine was and here's my recollection of what he was doing:
Ionization is not a problem for the underivatized forms, it's chromatographic retention that's the problem. This person was using either TFA or HFBA as an ion pair reagent and was getting decent retention and separation of the underivatized forms on a reverse phase column this way (he needed separation as he was using a single-quad). Such reagents as TFA and HFBA will suppress ionization in ESI (both polarities). This person was using APCI(+), which is affected the least by TFA and HFBA. In my experience with polar basic compounds using TFA for retention, TFA did not suppress the signal at all in APCI(+). Be aware that either of these reagents will hang around in your LC system, and will take alot of flushing to remove. They may hang around in your column forever. They will suppress ionization in ESI(+), and completely kill it in ESI(-) and APCI(-). But they are volatile and won't leave deposits in your ion source.
Whether this will give you a reliable and sensitive method, I can't say. I am trying to move away from using things like TFA, and would probably first try HILIC if ordinary reverse phase provided no retention, although I don't know if it would work for these compounds. Maybe someone can tell us whether ion exchange with volatile buffers would be possible for amino acids?
By Einar Pontén - SeQuant AB on Thursday, October 16, 2003 - 02:54 pm:
Why make things difficult?
Try ZIC™-HILIC for a straightforward separation using conditions suitable for LC-MS.
By Kostas Petritis on Thursday, October 23, 2003 - 05:39 pm:
Hi Anonymous:
There are similar discussions in the past... see: http://www.lcresources.com/discus/messages/3282/3143.html?MondayApril1420030826pm
Except from the articles mentioned there about underivatized amino acid analysis you may also see: Rapid Commun. Mass Spectrom. 17 (12), 1297-1311, 2003.
This is the first of two papers discussing the ESI-MS-MS analysis of underivatized amino acids for the diagnosis of inherited disorders of amino acid metabolism. The forthcoming paper will be published in J. Chromatogr. B.
Hope that the above helps,
Kostas