Biology Reference
In-Depth Information
the linearity of the predetermined calibration
curves can no longer be valid.
analyze. To account for these differences, use of
representative compounds from all
three groups as internal references has been
proposed.
26
Nevertheless, with proper care regarding both
the sample processing and experimental condi-
tions, precise results can be obtained to compare
relative metabolite concentrations. In general,
quantitative methods for GC-MS are less popular
than for LC-MS. External calibration methods are
sometimes used for quantitation; however,
literature-reported applications of SILIS to
GC-MS are quite limited. In contrast, in vivo
e
labeling methods attract more attention. One
study reported the use of
13
C-labeled yeast
cell extracts as a source for internal standards
to quantify intracellular metabolites in
S. cerevisiae cells. This study focused on measuring
nonoxidative pentose phosphate pathway inter-
mediates using GC-MS.
53
More recently, a simpli-
Gas ChromatographyeResolved MS
(GC-MS) Methods
GC-MS is a robust method for metabolomics
applications. It combines high separation effi-
-
ciency and sensitive detection following electron
ionization.
27,48
In particular, it is the preferred
method for the analysis of metabolites with low
molecular weight, as they can be quite effectively
analyzed with good sensitivity and reproduc-
ibility. Although the volatile, low-molecular-
weight metabolites can be directly analyzed,
nonvolatile, polar metabolites require chemical
derivatization
d
often silylation
d
before analysis.
There is a large literature on derivatization
methods in GC-MS.
49
To improve resolution in
GC-MS, the development of two-column
approaches (2D GC-MS) offers further enhance-
ment and is well suited for the analysis of
complex biological mixtures.
50
e
52
The quantitative accuracy obtainable from
GC-MS critically depends on numerous factors
including sample collection, storage, extraction,
derivatization, stability, and analysis. By
comparison, instrumental stability reproduc-
ibility is much less problematic. In addition,
the ionization method of choice
d
electron
ionization
d
avoids complications such as ion
suppression. Factors that are of most concern
for GC-MS are the ef
fied GC-MS method for absolute quantitation of
metabolites using commercially available
13
C-labeled algal extract for use as internal stan-
dards has been reported.
54
13
C isotope labeling
was also used to estimate metabolic
uxes and
concentrations in mammalian (hepatic) cells using
a combination of GC-MS and LC-MSmethods.
45,55
Results of these studies promise more widespread
use of in vivo
e
labeledmetabolites as internal stan-
dards
for
routine quantitative metabolomics
applications.
ciency of derivatization
and the stability of derivatized metabolites.
Regarding the derivatization process, metabo-
lites can be broadly grouped into three classes:
Class 1 metabolites, which contain hydroxyl or
carboxyl groups, exhibit the highest ef
NMR SPECTROSCOPY
NMR spectroscopy is one of
the most
information-rich techniques in the
field of metab-
olomics. It exhibits a number of important charac-
teristics for both identi
ciency
towards derivatization and stability of the deriv-
atized metabolite; Class 2 metabolites contain
amine or phosphoric groups and are very
sensitive to experimental conditions and are
measured with intermediate precision; and Class
3 compounds, with amide, thiol, or sulfonic func-
tional groups, are more dif
cation and quantitative
analysis of metabolites in complex biological
samples. In particular, the high resolution and
superb reproducibility enable access to a large
number of metabolites and their accurate concen-
trations. Unlike MS, NMR allows structural veri-
cult to derivatize and
cation/identi
cation of both known and
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