25th International Meeting on Organic Geochemistry IMOG 2011

P-020
Improvement of HPLC-protocols for intact polar lipid analysis
Jan Schröder, Julius Lipp, Lars Wörmer, Kai-Uwe Hinrichs
MARUM Center for Marine Environmental Sciences, University of Bremen, Organic Geochemistry Group, D-
28359 Bremen, Germany (corresponding author:jschroeder@marum.de)
Intact polar lipids (IPL) are the building blocks of
microbial membranes and have been successfully
applied as biomarkers in a variety of environmental
samples. Due to their taxonomic specificity and
property to select for live biomass, IPLs offer a
detailed view of abundance and composition of
microbial communities [1,2].
The analysis of IPLs relies on traditional protocols
for solvent extraction and chromatographic separation
by high-performance liquid chromatography coupled
to mass spectrometry (HPLC-MS) [1,3]. However, the
underlying analytical procedures have remained
virtually unchanged for several years and recent
technological advancements have not yet been
implemented. Only recently, Huguet et al. compared
different extraction methods for quantitative analysis
of IPLs and showed significant differences in
extraction yields also differing between the different
IPL classes [4].
Most environmental samples comprise a very
complex mixture of organic compounds and pose a
challenge to separation and identification. Typical
analytical problems are low IPL concentrations
combined with a high background of sample matrix.
These issues can be overcome by efficient extract
clean-up and sample pre-concentration followed by
improved HPLC-MS methods.
The established chromatographical protocol uses a
column with diol packing material and normal-phase
eluents for chromatographical separation according to
the polarity of the polar head group [1]. We have
tested columns with different packing materials for
their potential for IPL analysis. Additionally, we
evaluated the effect of eluent system, buffer
concentration and composition, and injection volume
on the chromatographic resolution and selectivity. In a
first step, we implemented an analytical protocol on a
reversed phase column where IPLs are separated by
polarity of their apolar core lipid, i.e. side chain length.
Besides type and purity of the reversed-phase
packing material, the buffer concentration was found
to have a dramatic effect on peak shape and peak
tailing (Fig. 1).
We will present a comprehensive overview of
current analytical problems and show results of newly
developed extract clean-up protocols and further
optimization of existing extraction methods of samples
covering a variety of environmental settings.
Additionally, we will compare different HPLC methods
and highlight their specific advantages and
disadvantages for IPL separation and mass
spectrometric characterization.
Fig. 1 Mass chromatograms of an IPL standard mixture
analysed by reversed phase HPLC-MS using (A) an
unoptimized method adapted from the established normal
phase method, and (B) an improved separation with a
modified buffer system. Peaks: (1) C16phosphoethanolamine-DAG,
(2) C16-phosphocholine-DAG,
(3) C16-phosphocholine-DEG, (4) C16-plasmalogenphosphocholine-DAG,
(5) phosphocholine-archaeol, (6) C21phosphocholine
DAG.
References
[1] Sturt, H.F. et al. (2004) Rapid Commun. Mass
Spectrom. 18, 617-628.
[2] Biddle, J. F. et al. (2006) Proc. Natl. Acad. Sci. USA
103, 3846-3851.
[3] Rütters, H. et al. (2002) J. Microbiol.: Meth. 28, 149-
160.
[4] Huguet, C. et al. (2010) Limnol. Oceanogr.: Methods 8,
127-145.
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