Re: Quantitative MS Analysis of Phospholipids

James Kerwin (kerwin@u.washington.edu)
Fri, 2 Oct 1998 15:29:45 -0700 (PDT)

Quantifying phospholipid (PL) classes (as opposed to purified molecular
species of a given class of PL) using any type of mass spectrometry is
not a good idea. Individual classes of PL such as PE and PI, especially
from a source such as pulmonary surfactant, usually consist of a mixture
of 12 to over 30 different combinations of fatty acids (molecular
species). Even if you are using e.g.on-line sample introduction following
a normal phase HPLC separation of individual classes, the simplest PL
class with superb chromatography is going to take 20-120 seconds or more
to elute. Individual species and PL are going to provide widely varying
signal intensities, e.g. phosphtidylcholine (with a quaternary ammonium
moiety) is going to give a very strong signal relative to other classes.
Signal intensity will further vary depending upon the type of
glycerophospholipid linkage (diacyl, alkyl-acyl, alkenyl-acyl) and degree
of unsaturation of fatty acyl groups.

Possible alternatives:

Preliminary separation of PL's from other lipid classes using solid-phase
extraction (Kaluzny et al., 1985, Journal of Lipid Research 26: 135-140)
or thin layer chromatography (CHCl3/MeOH 2/1 v/v w/1%HOAc, silica gel G
plates). Separate PL classes using TLC (Roos and Choppin, 1985, Journal of
Cell BIology 101: 1578-1590; Skipski and Barclay, 1969, Methods in
Enzymology 14: 530-612; Creamer and Bostock, 1986, Physiol. Molec. Plant
Pathol. 28: 215-225) and quantitate total phosphorous using the method of
Mrsny et al (1986, Chem. Phys. Lipids 39: 185-191).

If you have limited sample, resort to normal phase HPLC
(Christie, 1986, Journal of Chromatography 351: 396-399; Dugan et al.,
1986, Journal of Chromatography 378, 317-327; Juaneda et al, 1990, Lipids
25, 756-759). UV quantitation is possible, but not very reliable due to
lack of suitable chromophores of underivatized PL's. A light-scattering
detector (see Juaneda et al ref. above) will give much more reliable
results.

The approach you take will be highly dependent upon the source, complexity
and concentration of your sample, equipment access and expertise. The
least expensive and quickest are TLC-based total phosphorous assays.
HPLC-based experiments are not trivial. Regards,

Jim Kerwin
Botany Box 351330
University of Washington
Seattle, WA 98195 USA

> >Dear collegues!
> >
> >I am trying to quantify phospholipids, espeacially phosphatidylcholin
> >(PC), phosphatidylglycerol (PG), phosphatidylinositol (PI) and
> >phosphatidylethanolamin (PE) from pulmonary surfactant on a Perkin
> >Elmer SCIEX API I MS. So far I am using it as an ESI-MS with
> >an internal standard and than centroiding the peaks for quantification.
> > I am looking for a more sophisticated way to perform this task
> >(with MacQuan or other software). Who is having experiences with
> >and solutions for this problem.
> >
> >Sincerely
> >
> >Gunnar Rau, MD [100.86997@germanynet.de]
> >Medical School Hannover
> >Department of Pulmonary Pediatricc
> >Surfactant Lab
> >30623 Hannover
> >Germany
> >
> >Thank you for help.
> >*************************************************
> >Manfred Raida
> >Niedersaechsisches Institut fuer Peptid-Forschung
> >(IPF)
> >Dep. of Analytical Peptide-Chemistry
> >Feodor-Lynen Strasse 31
> >D-30625 Hannover
> >Germany
> >E-Mail Manfred_Raida@compuserve.com
> >Tel.: +49 511 5466 210
> >Fax: +49 511 5466 102
> >**************************************************
>
>
>