66
4.3 Separations
Analytical separation of metabolites prior to mass analysis provide a means for
more comprehensive analysis metabolites, enabling greater depth of coverage.
There are analytical tasks that do not require separations for the analysis of metabolites; however, these approaches sacrifice broad metabolite coverage in favor of
other important performance characteristics of the assay. For example, directinfusion high-resolution MS (DI-HRMS) allows for the analysis of metabolites
without the need for chromatographic alignment and extensive sample preparation
[24]. In addition, direct-infusion methods also allow for maximum sample throughput [2]. Many imaging mass spectrometry techniques also do not use any chromatographic separations. However, these technologies allow for the unique ability to
spatially localize specific m/z to regions of a sample, which can be of unique importance in clinical applications [25]. Although separation-free techniques can be used
for metabolomics analysis, isomeric compounds cannot be separated and ion suppression effects must be mitigated [2]. To address these challenges, typically liquid
chromatography, gas chromatography, capillary electrophoresis, and ion mobility
are used.
4.3.1 Liquid Chromatography
One of the first widely accepted types of liquid chromatography in a column format
was normal-phase chromatography which was derived from thin layer chromatography (TLC) [26]. Normal phase separations employ a polar stationary phase, often
consisting of silica [26, 27]. This polar stationary phase is ideal for retaining and
separating polar molecules in highly nonpolar solvents such as hexanes, which can
be incompatible with downstream components and not provide the necessary polarity for efficient electrospray ionization [28]. While normal phase has lost much of
its popularity due to its major limitations, it is still used in limited capacities due to
its effective class separations of analytes such as lipids, as well as its compatibility
with organic solvents which are necessary for the stability of some molecules [29].
In contrast to normal-phased chromatography, reversed-phase chromatography
is defined by a nonpolar stationary phase which retains and separates nonpolar,
hydrophobic analytes very effectively [30]. Historically, reversed-phase chromatography has been the gold standard in LC-MS, which has percolated into LC-MS
based metabolomics [31]. Reversed-phase chromatography offers versatility in
mobile phase/sample composition and can be used in flow regimes from nanoflow
(< 1 μL/min) to analytical flow (>100 μL/min < 1 mL/min). Furthermore, reversedphase chromatography produces highly reproducible retention times and peak
shapes [32, 33]. One large hurdle associated with the use of reversed-phase chromatography for metabolites, however, is the inherently polar properties of the majority
of endogenous small molecules. As discussed above, this problem has led to the
E. S. Rivera et al.
4.3 Separations
Analytical separation of metabolites prior to mass analysis provide a means for
more comprehensive analysis metabolites, enabling greater depth of coverage.
There are analytical tasks that do not require separations for the analysis of metabolites; however, these approaches sacrifice broad metabolite coverage in favor of
other important performance characteristics of the assay. For example, directinfusion high-resolution MS (DI-HRMS) allows for the analysis of metabolites
without the need for chromatographic alignment and extensive sample preparation
[24]. In addition, direct-infusion methods also allow for maximum sample throughput [2]. Many imaging mass spectrometry techniques also do not use any chromatographic separations. However, these technologies allow for the unique ability to
spatially localize specific m/z to regions of a sample, which can be of unique importance in clinical applications [25]. Although separation-free techniques can be used
for metabolomics analysis, isomeric compounds cannot be separated and ion suppression effects must be mitigated [2]. To address these challenges, typically liquid
chromatography, gas chromatography, capillary electrophoresis, and ion mobility
are used.
4.3.1 Liquid Chromatography
One of the first widely accepted types of liquid chromatography in a column format
was normal-phase chromatography which was derived from thin layer chromatography (TLC) [26]. Normal phase separations employ a polar stationary phase, often
consisting of silica [26, 27]. This polar stationary phase is ideal for retaining and
separating polar molecules in highly nonpolar solvents such as hexanes, which can
be incompatible with downstream components and not provide the necessary polarity for efficient electrospray ionization [28]. While normal phase has lost much of
its popularity due to its major limitations, it is still used in limited capacities due to
its effective class separations of analytes such as lipids, as well as its compatibility
with organic solvents which are necessary for the stability of some molecules [29].
In contrast to normal-phased chromatography, reversed-phase chromatography
is defined by a nonpolar stationary phase which retains and separates nonpolar,
hydrophobic analytes very effectively [30]. Historically, reversed-phase chromatography has been the gold standard in LC-MS, which has percolated into LC-MS
based metabolomics [31]. Reversed-phase chromatography offers versatility in
mobile phase/sample composition and can be used in flow regimes from nanoflow
(< 1 μL/min) to analytical flow (>100 μL/min < 1 mL/min). Furthermore, reversedphase chromatography produces highly reproducible retention times and peak
shapes [32, 33]. One large hurdle associated with the use of reversed-phase chromatography for metabolites, however, is the inherently polar properties of the majority
of endogenous small molecules. As discussed above, this problem has led to the
E. S. Rivera et al.