Dear Gerd,
> Dear Wolfgang, are you sure that these methods are suitable to measure arginine. This
> is one of the most critical AA in GC-C-IRMS. Other critical ones are glutamin
> /glutamate and asparagine/aspartate. Has anyone a satisfying derivatisation method to
> get them all. For 15N and 13C.
Thanks for pointing out the error of my ways. Somehow, I managed to
ban arginine completely from my mind. Despite having dealt with AA
from plasma albumin and collagen (both the N- and C-terminal regions
of the latter contain a fair amount of arginine), I have never paid
close attention to this particular AA. We were more interested in
Ala, Gly, Val, Leu, Pro, Phe and Hyp (= OHPro). So, it's a out of
sight out of mind attitude of mine, I suppose. Sorry for that.
In other words, I won't put my hand in the fire for any of the
references quoted in my earlier message as far as arginine is
concerned.
However, as far as glutamate and aspartate are concerned
derivatisation protocols such as N-pivaloyl-isopropylates (Metges),
N-acetyl-propylates (NAP), N-acetyl-methylates (NAM),
N-trifluoroacetyl- propylates (TFAP) and N-trifluoroacetyl-methylates
(TFAM) do work. There is also the ethyl-chlorformate method (Husek)
but I don't know of the top of my head how well this work for Glu and
Asp. Works like a spell for the neutral AA though.
Personally, I prefer NAP, TFAP and TFAM from a chromatogtaphic point
of view (using medium polar stationary phases, i.e. OV1701
and OV225 equivalents such as BP10, BP70X, CP-Sil19CB etc.; as
mentioned by Michael Haisch). From a GC/C-IRMS point of view I am
very sceptical about the TFA derivatives (in spite of some
publications using TFA for AA). In my experience, TFA has a
detrimental (if not to say disastrous) effect on the combustion
mediator (CuO/NiO/Pt) which undermines my faith in measured isotopic
ratios from such compounds.
On the other hand , TFAM is the method of choice if one is interested
in chiral separation on a cyclodextrin/OV1701 stationary phase.
All this would be much easier, d15N of individual AA that is, if we
had a HPLC/C-IRMS system at our disposal. HPLC methods for separation
of underivatized AA do exist and successful attempts of building such
an instrument have been made; and new approaches to tackle this
problem have been floated.
Anyhow, that's my tuppence worth.
Cheers,
Wolfram
********************************************************
Dr. W. Meier-Augenstein, CChem MRSC
Senior Research Fellow
University of Dundee, Dept. of Anatomy & Physiology,
OMS, Small's Wynd, DUNDEE DD1 4HN, United Kingdom
Tel.: +44-(0)1382-34/5124, /4574, /4968
Fax: +44-(0)1382-34/5514
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URL: http://www.dundee.ac.uk/anatphys/wma/wolfram.htm
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