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failed to resolubilize relatively hydrophobic
species such as diacylglycerols, triacylglycerols,
or diacylphospholipids, and the use of acidic (pH
3) and basic (pH 8) reconstitution solvents
enhanced the detection of ionizable compounds.
76
Lyophilization, followed by reconstitution in
puri
metabolomics generally rely on complex multi-
step and multisolvent extraction procedures
(
Figures 2A and 3
) in contrast to simple work-
uids. Rapid rinsing of
solid tissue to remove any blood contamina-
tion
83
and quenching in liquid nitrogen are typi-
cally employed immediately after tissue
harvesting.
59
Tissue homogenization and/or
extraction is also performed at low temperatures
to minimize metabolism.
15,51,55
flows employed for bio
ed water, is also used frequently,
41,80
and
incomplete solubilization of lipids can be miti-
gated by addition of methanol.
44
The main
driving forces behind the inclusion of evapora-
tion/reconstitution step are (1) preconcentra-
tion, (2) change of solvent composition to
ensure good compatibility with LC-MS or
GC-MS analysis, and (3) improved stability of
dried extracts during storage. However, the
bene
Beltran et al.
compared the in
uence of the extraction temper-
ature (
e
20
C, 25
C, and 60
C) on the extraction
of liver tissue and found unacceptable decrease
in precision for glycogen, multiphosphorylated
nucleotides, and ascorbic acid upon increasing
temperature indicative of their degradation/
conversion,
55
while the effect of temperature on
more stable metabolites was minimal.
With respect to homogenization methods, Lin
et al. compared manual grinding using mortar
and pestle versus electric homogenization of wet
tissue and found that the latter method improved
the method reproducibility,
84
whereas the perfor-
mance of manual grinding could be improved
by using lyophilized tissue. A comprehensive
comparison of different tissue disruption tech-
niques for
Caenorhabditis elegans
recommended
the use of bead beating
85
; whereas the rotary
homogenizer method performed the poorest in
terms of precision, metabolite coverage, and
recovery, possibly due to incomplete disruption.
Overall, the selection of extraction solvent was
reported to affect the metabolome coverage
more than the tissue disruption method,
85
so the
majority of studies in the current literature
primarily focus on solvent optimization for
different tissue types.
Boiling water was the preferred method for the
extraction of selected polar metabolites from
muscle tissue, and boiling ethanol and per-
chloric acid showed the poorest overall perfor-
mance in a recent LC-MS comparison.
15
Overall,
methods that can simultaneously extract both
hydrophilic and lipophilic species, such as uni-
phasic and biphasic chloroform/methanol or
ts of preconcentration andwhether it truly
helps to improve metabolite coverage have not
been well established in untargeted studies
except for a recent study of saliva inwhich acidic
and basic hydrolysis with ultrasonication to
disrupt protein binding and fourfold preconcen-
tration were necessary to improve the detection
of low abundance metabolites.
81
Thus, the evap-
oration/reconstitution step may be most favor-
able for low concentration bio
fluids such as
saliva and CSF, while for more complex bio-
fluids such as plasma, the potential losses of
hydrophobic and volatile species and increased
ionization suppression due to preconcentration
may well result in lower metabolite coverage.
Furthermore, the use of higher organic solvent
strengths, shown to improve resolubilization of
hydrophobic metabolites, eliminates the second
advantage, and the long time needed to perform
this step drastically reduces sample throughput.
Therefore, the inclusion of this step should be
carefully evaluated in any developed method.
TISSUE METABOLOMICS
Intact tissues can be analyzed by NMR using
high-resolution magic angle spinning (HR-
MAS) without the need for sample prepara-
tion.
82
However, all other methods in tissue
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