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From: "Bruce Heideman" <bheidema@gmp.usyd.edu.au>
Date: Thu, 25 Mar 1999 14:13:54 +1100
Subject: Hunting for unknown molecules
Organization: Graduate Medical Program, University of Sydney, Australia

Hello.

I am hunting for a lipid based molecule in blood plasma. My current plan
is to obtain spectra for the plasma sample when I know the substance is
not released into circulation, then search for the new peak(s) with
plasma samples that must contain the molecule of interest. I am
interested in the best techniques to achieve this aim. The samples are
crude blood plasma, with little known about the molecule in question.

Cheers,

Bruce Heideman

bheidema@gmp.usyd.edu.au


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From: "M Sweeney - MSMS Consulting" <mattsweeney@earthlink.net>
Date: Thu, 25 Mar 1999 06:50:36 -0800
Subject: Re: Hunting for unknown molecules
Organization: EarthLink Network, Inc.

Bruce

Unless this unknown molecule is present in very large concentrations you
will need to do some sample prep.  "Lipids" cover an extremely wide range of
polarities and chemical properties so it is hard to say for sure what to do.
Morris Kates wrote an excellent book called "Techniques of(in?) Lipidology"
that covers just about everything you would want.  I would consider doing
several steps of liquid/liquid partitioning to get a fraction of a more
limited range of polarities.   Then this might be taken to a silica or
sephadex column and further fractionated prior to ultimate analysis by GC/MS
or LC/MS.

SO...
1) partition aq plasma/pentane
2) partition aq from 1) against CH2Cl2
3) partition aq from 2) against EtOAc
4) then concentrate 1-3 and...(do more prep chromatography or just a sep-pak
and..)
5) run pentane,CH2Cl2 by GC/MS
6) run CH2Cl2, EtOAc by LC/MS on an RP-C18 by apci

The pentane fraction will have very volatile non-polar fairly
"unfunctionalized" good GC compounds for the most part.  The CH2Cl2 will be
fairly wide in range of polarities (some overlap with fracs 1 and 3.  Some
will GC and some won't so easily).

You could just try a quick water:Meoh:CHCl3 partition system I think it is
called a Folch wash or something like that.  It is monophasic in its initial
composition and then you adjust it to get a CHCl3 and MeOH/AQ layer.  The
CHCl3 might have your lipid.  Then just concentrate it and dilute in
suitable solvent (meoh?) sep-pak it and run in by APCI on a long C18 with a
slow gradient.  A then process the heck out of the data.

good luck

Matt Sweeney
mattsweeney@earthlink.net
Mass Spec Consulting

Training/Operations/Consulting/Method Development
LC/MS Pharmacokinetics, Peptides, Proteins, Metabolism,
Maintenance Classes, Specialist in Finnigan Equipment and Software