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of plasma using methanol/ethanol. The metabo-
lite coverage of SPME can be further augmented
by adding additional coatings such as a phenyl-
boronic acid coating and a polar-modi
flow conditions, while small molecular weight
metabolites are retained using a polystyrene
divinylbenzene stationary phase. These metabo-
lites are then eluted onto the analytical column
for analysis using column-switching. The utility
of TFC for global metabolite pro
ed poly-
styrene divinylbenzene coating to the generic
in vivo SPME work
ow. 70 The technique is
highly complementary to solvent precipitation
(1) to identify unstable metabolites whose levels
in vivo are not accurately captured by traditional
blood withdrawal and solvent precipitation
techniques and (2) to estimate biological avail-
ability of metabolites by comparing free
(detected by SPME) and total metabolite concen-
trations (detected by solvent precipitation after
solvent disruption). The former advantage is
illustrated by the fact that more than 100 features
in positive and negative ESI mode could only be
detected by in vivo SPME and could not be
detected at all
ling was
first time. 105 The
comparison of this new method after 1/1 dilu-
tion of plasma with water versus standard
approach of plasma precipitation with cold
methanol yielded similar numbers of features
(
recently examined for the
2,900) with slightly poorer repeatability for
TFC method. The signal intensity for TFC was
generally lower than for solvent-precipitated
plasma, with the authors reporting an approxi-
mately threefold decrease in response even after
the correction for differences in the injection
volume between the two methods, possibly indi-
cating that bound lipids and other hydrophobic
compounds are washed away with proteins.
TFC also reduced the presence of phospholipids
by 10- to 60-fold, which can be important from
the ionization suppression perspective. The
main potential advantages of TFC are high
throughput, high degree of automation, and
minimal sample handling, which should mini-
mize the introduction of extraneous contami-
nants throughout the sample handling process
and inadvertent sample losses. One disadvan-
tage of the technique is that untreated samples
can reside different amounts of time in the refrig-
erated autosampler, which has the potential to
introduce higher variability and systematic bias
for metabolites that can undergo enzymatic
conversion or degradation. This issue merits
investigation in the context of untargeted
pro
w
in blood samples after with-
drawal. 27
cant changes in
other metabolites such as adenosine monophos-
phate and glutathione ratios were observed in
plasma, showing that plasma metabolome is
not completely representative of metabolome at
the time of sampling. Thus, in vivo SPME
provides a novel method for biomarker
discovery of unstable or labile metabolites that
cannot easily be examined by other global
methods and a specimen-free method permitting
long-term temporal studies in small animals
such as mice and/or precious genetically modi-
Furthermore, signi
fied strains of animals to monitor various biolog-
ical processes in multiple compartments without
need for animal sacri
ce.
Turbulent Flow Chromatography (TFC)
Turbulent
ling studies.
ow chromatography (TFC)
permits direct online injection of untreated
serum or plasma into LC-MS system. The sample
is injected using a high
Dried Blood (or Bio
uid) Spot Analysis
One emerging technique in the
flow rate onto large-
field of bio-
analysis and drug discovery is the dried blood
spot (DBS) analysis, which is also successfully
used for neonatal screening of inborn errors of
metabolism using targeted metabolomics d one
particle (25 e 50
m) small i.d. (0.5 e 1.0 mm)
TFC column. This column does not retain large
macromolecules, so they are washed away to
waste with a mobile phase under turbulent
m
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