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of lysophosphatidylinositol in plasma, (2) throm-
boxane B2 uniquely present in serum, (3) an
increase in multicharged ions likely belonging to
peptide species in serum, (4) two metabolites
with formulas C 16 H 29 N 2 O 14 PandC 24 H 28 O 9
uniquely identi
reported a signi
cant loss of features in the region
from 500 to 650 seconds in negative ESI mode in
combination with the heparin anticoagulant. 47
For blood sampling, capillary collection of small
volumes of blood is an attractive option for less-
invasive sample collection, and no signi
ed in plasma, 47 (5) inaccurate
concentrations of sphingosine-1-phosphate and
eicosanoids in serum because the clotting cascade
stimulates blood cell eicosanoid synthesis and
their release fromplatelets, 3,48 (6) increased amino
acid concentrations in serum especially for argi-
nine, 49 serine, phenylalanine, and glycine, 48,49
and (7) increased levels of certain phosphatidyl-
cholines in serum. 48 These recent studies suggest
that both types of bio
cant
differences (fold change
0.05) were found
in endogenous metabolites between capillary
and venipuncture collection, indicating suita-
bility of capillary collection for metabolomic
applications. 45
2, p
>
<
Protein Removal Efficiency and Selection
of Plasma-Precipitant Ratio
To date, the performance of various protein
precipitation methods has been compared exten-
sively in terms of the ef
uid can be successfully
used in metabolomics but also show that care
should be taken when dealing with metabolites
known to change or have high variability in one
matrix versus the other. Overall, plasma is still
generally the preferred bio
ciency of protein removal.
Among the various protein precipitationmethods
(organic solvent, acid, salt, andmetal), acetonitrile,
trichloroacetic acid, and zinc sulfate were found
to provide the best protein removal ef
uid of choice with
about 65% LC-MS studies relying on this sample
type, according to recent estimates. 9
ciencies
as determined using the spectrophotometric
method. 52 However, precipitation with acetoni-
trile was most compatible with subsequent
LC-MS analysis with higher protein removal
ef
Choice of Anticoagulant in Global
Metabolomics
For plasma, the choice of anticoagulant used
during blood collection can in
ciency (93.2%
0.7%) versus ethanol (88.6%
uence the observed
1.1%) for all
plasma species tested. The examination of protein
removal ef
0.5%) and methanol (88.7%
metabolic pro
les with reported differences in
amino acids, lysophosphatidylcholine, and phos-
phatidylcholine. 36,50 For NMR studies, heparin is
the recommended anticoagulant because EDTA
and citrate can give interfering signals. 51 Heparin
is also recommended for LC-MS metabolomics
by several groups, including the Human Serum
Metabolome (HUSERMET) Consortium. 44,50
Denery et al. compared the performance of all
three common anticoagulants in terms of metabo-
lite coverage and precision but did not
ciency by gel electrophoresis also indi-
cated better performance of acetonitrile, acetone,
acetonitrile/acetone, and ethanol/acetonitrile/
acetone versus methanol. 53 However, the number
of metabolites observed for these solvents was
the lowest. The reproducibility of precipitation
methods with pure acetone and acetonitrile
was also worse than for the remaining methods.
Another study using the Bradford assay for esti-
mation of protein concentration found that aceto-
nitrile had the poorest protein removal ef
nd major
les. 45 Heparin
resulted in slightly higher number of features
(3,312 in positive ESI LC-MS) versus EDTA
(2,802) and sodium citrate (2,967), while the
method precision and the number of features in
negative mode ESI were very comparable for all
three anticoagulants. In contrast, Wedge et al.
differences in obtained plasma pro
ciency
(94%) versus pure ethanol (96%) and pure meth-
anol (98%), 54 and similar results were obtained
using NMR. 55 This apparent discrepancy with
previous results could be caused by different
errors inherent in two assays for the estimation
of protein concentration. Heat and acid treatment
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