Biology Reference
In-Depth Information
ultra
ltration is biased towards more polar mole-
cules, with signi
effects. Thus, without proper recovery studies
and metabolite identi
cant loss of hydrophobic species
versus solvent precipitation and solid-phase
microextraction techniques.
27,69,70
cation, it is impossible to
delineate which method is better, showing the
need for quantitative recovery studies in the
context of method comparison of different global
metabolomic methods. In addition to the use of
the C
18
sorbent,
42
the combination of C
18
and
polystyrene-divinylbenzene sorbents
72
and the
use of divinylbenzene/n-vinylpyrrolidone copol-
ymer
32,73,74
has been proposed for untargeted
metabolomics. The combination of strong
cation-exchange and anion-exchange sorbents
was also recently employed to improve ionization
suppression effects in plant metabolomics,
75
and
Khin et al. successfully employed ion exchange
SPE to enrich the polar anionic metabolites.
67
Of
course, the main issue in implementing SPE is
the inherent dif
To improve
metabolite
ltration, the
membrane can be extracted with a methanol/
water solvent followed by chloroform extraction
of resulting extracts in order to recover nonpolar
metabolites, but this approach has not yet been
evaluated by LC-MS.
69
The protein content can
alternatively be spectroscopically removed with
a relaxation
coverage
of ultra
filter.71
71
Solid-Phase Extraction (SPE)
Although solid-phase
extraction (SPE)
is
widely used in the
field of targeted bioanalysis,
only a few reports of SPE methods in global
metabolomics have been published to
date.
42,72
e
74
The three main steps of SPE include
analyte partitioning into the sorbent, washing of
the sorbent to remove various interferences that
are less strongly bound to the sorbent than the
analyte of interest, and analyte elution from the
sorbent using a solvent. Michopoulos et al. evalu-
ated the performance of SPE versus precipitation
with methanol and precipitation with acetonitrile
in combination with the reversed-phase ultra
high-performance liquid chromatography
(UHPLC)
e
MS method for global metabolomic
studies on human plasma.
42
Precipitation with
methanol was found to provide best metabolite
coverage, and SPE with a C
18
cartridge was found
to improve method precision over solvent protein
precipitation methods, presumably due to
improved sample cleanup. Interestingly, the
comparison of signal intensity obtained using
three methods for a small subset of selected ions
showed huge differences in SPE versus solvent
precipitation (up to tenfold increases or
decreases), which would indicate metabolite-
dependent incomplete recovery or solubilization,
extraction of only unbound portion of analyte
because no binding disruption step was incorpo-
rated for SPE, or severe ionization suppression
culty of devising general extrac-
tion conditions that will be suitable for exhaustive
extraction of all classes of metabolites in a given
sample, thus introducing analytical bias and
removing the global nature of analysis. However,
the use of several complementary SPE protocols
could be extremely useful in global metabolomic
analysis and the ability to enrich low abundance
metabolites routinely missed by other methodol-
ogies remains an interesting and currently unex-
plored research direction.
Evaporation and Reconstitution Step
Freeze-drying followed by reconstitution in
D
2
O is not recommended for NMR analysis,
due to incomplete redissolution.
33
However,
a notable number of LC-MS studies incorporate
an evaporation/reconstitution step as part of
sample preparation strategy, even though this
step can reduce metabolite coverage due to
poor solubilization of some metabolites.
42,76
e
79
The ability to resolubilize hydrophilic versus
hydrophobic species depends critically on the
composition of the reconstitution solvent, with
reconstitution solvents ranging from highly
aqueous
42,45,76
e
79
to even 80% methanol
53,56
or 100% acetonitrile.
77
Highly aqueous solvents
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