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effects may occur in particular with ESI and
accurate quanti
retention mechanisms are available. Reversed-
phase LC (RPLC) involves typically C18
bonded-silica columns and is able to separate
medium-polarity compounds according to
hydrophobicity. More polar compounds (e.g.,
sugars, amino acids, carboxylic acids, and nucleo-
tides), can be analyzed by techniques such as
hydrophilic interaction LC (HILIC) involving
polar stationary phases (e.g., aminopropyl).
On the other hand, recent developments of
LC
d
including ultra-high-pressure LC (UHPLC),
monolithic columns, and high temperature
d
provided new perspectives regarding chromato-
graphic performance. UHPLC has quickly
become a gold standard in metabolomics, as it
allows well-resolved peaks with narrow peak
width leading to either an increased peak
capacity or a shorter analysis time without the
loss of resolution as compared with conventional
LC. However, these developments make the
chromatographic elution speedmore demanding
for a reliable acquisition by the mass spectrom-
eter. Due to the short peak duration, a high acqui-
sition rate is therefore mandatory to obtain
enough data points per peak. An insuf
cation can be challenging.
5
Matrix-assisted laser desorption/ionization
(MALDI) constitutes another direct MS ioniza-
tion technique for metabolite
fingerprinting.
Although mainly developed for the analysis of
proteins and nucleotides, it is increasingly used
in metabolomics to assess the spatial distribu-
tions of metabolites with the investigation of
tissue sections or speci
c subcellular compo-
nents.
6
Despite serious limitations due to high
background noise of conventional MALDI
matrices in the low mass range, its use is very
promising in the perspective of the spatial reso-
lution of metabolites.
Hyphenated MS Methods
Hyphenated MS
e
based approaches provide
high sensitivity and selectivity and a wide
dynamic range with high-throughput capabil-
ities. Due to the nature of the techniques it
engages, the coupling of GC to MS is the most
straightforward as compared to LC and CE.
The GC-MS technology is mainly devoted to
the analysis of volatile compounds but chemical
derivatization can be applied to extend its applica-
tion domain. Modern capillary GC columns
provide very high peak capacity, allowing the
separation of complex extracts.
7
Typical ionization
techniques include electron ionization (EI), which
produces highly reproducible fragmentation
patterns, and softermethods suchas chemical ioni-
zation.
8
Analytical developments such as com-
prehensive GCxGC-MS further improved the
selectivity and separation ability of the technique.
9
As it is well suited for the analysis of a broad
range of compounds
d
that is, polar and
nonpolar, low and high molecular weight
molecules
d
LC-MS has begun to supplant
GC-MS in several domains where metabolomic
studies are engaged. Sample preparation is often
reduced, derivatization is usually not required,
and chromatographic columns with distinct
cient
number of data points per peak leads to poorer
resolution and sensitivity performance.
10
However, the increasingly high mass-
resolving power and acquisition rate of modern
mass spectrometers such as time-of-
ight (TOF)
and the very high resolution of Orbitrap
-based
technology and Fourier transform platforms
allows the simultaneous determination of
elemental compositions of thousands of
compounds fromaccuratemassmeasurements.
10
The typical bidimensional structure of raw data
obtained from the combination of a separative
method and MS is illustrated in
Figure 1
with
the assessment of a complex sample with
UHPLC-MS.
CE-MS has a signi
cant potential for the
analysis of polar or charged metabolites. As the
separation mechanism is related to the mass-
to-charge ratio of the compounds, CE shows
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