Biology Reference
In-Depth Information
ranges. Due to the nature of GC, the analytes of
interest must be suf
the large number of metabolites in biological
matrices, chromatograms are complex and long
analysis time is often required for satisfactory
peak separation. Column properties (column
length, stationary phase, internal diameter),
carrier gas velocity, carrier gas types, and oven
temperature program are important factors that
in
ciently volatile and ther-
mally stable. Although GC is known to be suit-
able for separation of analytes that are
relatively hydrophobic, some interesting classes
of polar metabolites such as sugars, nucleotides,
and amino acids are not amenable for direct GC
analysis due to their poor volatility and interac-
tion with active sites present in the GC system.
23
As such, samples are usually subjected to chem-
ical derivatization to reduce the polarity of the
metabolites and increase their volatility and
thermal stability prior to GC/MS analysis so as
to extend the metabolic coverage.
7,23
The most
commonly used derivatization protocol involves
a two-stage process of oximation followed by tri-
methylsilyation. Oximation using methoxyl-
amine hydrochloride converts ketone groups to
oximes; this step prevents enolization reactions,
which can introduce multiple products.
24
Mono-
saccharides exist in the form of cyclic and open-
ring structures. Oximation inhibits cyclization
and results in fewer derivatized peaks per sugar,
thereby reducing the chromatographic com-
plexity.
4
In silyation reaction, trimethylsilyl
groups replace the active hydrogens of func-
tional groups in compounds such as alcohol,
carboxylic acids, thiols, and amines.
25
Silyation
predominates in metabonomics compared to
acylation and alkylation due to its universality
in chemical group coverage, ease of procedure,
and highly volatile nature of the by-products of
reagent.
21,23
However, its main disadvantages
are the requirement for samples to be dry and
derivatized samples are sensitive to the hydro-
lytic effects of moisture in the air.
uence chromatographic separation.
4
Time-
of-
ight (TOF) and quadrupole mass analyzers
are most commonly used in GC/MS-based
metabonomics.
19
Quadrupole mass analyzers
have high sensitivity and good dynamic range
but slow scan rates compared to TOF. On the
other hand, TOF instruments provide high
acquisition rates (meaning that more data points
and spectra can be acquired across the peak, up
to 500 spectra/second). These rates render high
chromatographic resolution and facilitate peak
deconvolution.
26
EI is the most common ioniza-
tion technique used in metabonomic studies.
The standardized EI energy of 70e V gives rise
to a highly reproducible fragmentation pattern
characteristic of the analyte with minimal vari-
ability between instruments. This characteristic
fragmentation pattern facilitates construction of
EI mass spectral libraries for compound identifi-
-
cation through mass spectrum matching.
Quality control (QC) samples, usually prepared
from pooling aliquots of the biological samples,
are commonly used in GC/MS-based metabo-
nomics and are randomized for analysis among
the study samples to provide quality assurance
for data acquisition and analysis. Further details
on QC samples will be discussed in a subsequent
section of the chapter.
Data Analysis
Due to the complexity of biological samples,
metabonomic investigations generate a plethora
of data, and their meaningful interpretation
requires the use of appropriate statistical tools
to convert the data to a workable format. Data
analysis in metabonomic studies can be divided
into three stages: data preprocessing, processing,
and model validation.
20
GC/MS Data Acquisition
Following derivatization, a small volume
(0.5
e
2
L) of the derivatized sample is injected
into the GC/MS system.
4
The sample is volatil-
ized in the heated injector and the carrier gas
(helium or hydrogen) aids the transfer of the
analytes to the column for chromatographic
separation and MS for detection.
5
m
Due to
In data preprocessing,
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