Biology Reference
In-Depth Information
metabolomic analysis can be dif
cult to achieve.
The combination of oximation and trimethylsily-
lation is used primarily to reduce the number
of side products and resulting complexity of
GC-MS chromatograms. It has also been success-
fully employed for urine
34,37
and tissue.
58,59
Begley et al. optimized a fast derivatization
protocol including 15 min oximation and 15
min trimethylsilylation at 80
C and showed
that the resulting extracts should be analyzed
within 30 h,
57
while Hong et al. showed
microwave-assisted derivatization can be
achieved in less than 2 and 3 min per step,
respectively.
60
The presence of a trace amount
of water may interfere with reproducible deriva-
tization, so a second evaporation to dryness
using toluene is recommended in some protocols
prior to derivatization.
34
and was found to drastically improve the repro-
ducibility of metabolite features especially for
late-eluting hydrophobic metabolites such as
lipids (2.7- to 3.8-fold improvement in the
number of reproducible features) in comparison
to two protocols: (1) two-step evaporation
followed by reconstitution in ethanol and (2)
solvent precipitation with methanol/ethanol
(1-to-3 plasma-to-precipitant ratio). This app-
roach is bene
cial over the traditional Folch
extraction with chloroform commonly employed
in lipidomics, as the protein layer is precipitated
on the bottom of the vial versus the protein layer
staying between the two phases in the Folch
method, thus not permitting direct LC injections
from the same vial.
Ultra
ltration
Ultra
ltration separates molecules based on
their molecular weight and is a commonly used
method for sample preparation of biological
LiquideLiquid Extraction (LLE)
Approaches
In addition to simple solvent-precipitation
approaches, there is a recent increase in the
development of more selective strategies based
on LLE. For instance, a complex four-step
solvent extraction was successfully used in
a recent study delineating the role of sarcosine
in prostate cancer progression,
61,62
but no
comparisons of this method with more prevalent
solvent precipitation methods have been per-
formed in the literature to date. LLE was also
successfully employed in a recent study of CSF
after solvent precipitation, whereby the samples
were separated into lipid and polar phases using
the combination of water and ethanol/dichloro-
methane followed by GC-MS and LC-MS anal-
ysis of the resulting extracts.
63
Whiley et al.
recently proposed a very promising in-vial
dual extraction method for the global metabolo-
mics of plasma.
64
This newmethod relies on one-
step extraction using methyl-tert-butyl-ether
(after methanol precipitation of proteins) to
separate the metabolites into a hydrophilic
aqueous phase and a hydrophobic organic phase
fluids, especially for NMR analysis.
6,65
e
68
The
use of a 3,000 Da
filter can physically separate
small molecular weight metabolites from
proteins or other macromolecules and is thus
the most common con
guration for metabolo-
mics. For example, for erythrocyte extraction,
Darghouth et al. proposed the use of cellulose
column to separate red blood cells from leuko-
cytes, centrifugation, cell pellet extraction using
a boiling water method for 3 min, followed
by centrifugation and two-step ultra
ltration,
first using a 30,000 Da molecular-weight-cut-off
device followed by a 10,000 Da device for
20 min at room temperature.
68
Ultra
ltration and acetone precipitation were
found to outperform precipitation with per-
chloric acid, acetone, methanol/chloroform, or
acetonitrile in terms of protein removal using
NMR.
69
ltration provided
improved metabolite coverage for polar species
due to better solubilization than acetone precipi-
tation, so it was recommended as the best method
of sample preparation prior to NMR. However,
Furthermore, ultra
Search WWH ::
Custom Search