67
development of many derivatizing strategies for small molecules to make them less
polar and aid in reversed-phase retention.
Hydrophilic interaction chromatography (HILIC) is a relatively new chromatography technique which is a variation of normal phase chromatography [34]. Briefly,
HILIC relies on a thin layer of water which surrounds the polar stationary phase,
allowing for analytes to interact with the water layer rather than the stationary phase
directly [34]. This interaction with water lends to the retention of polar, hydrophilic
molecules without the need for mobile phases which are incompatible with mass
spectrometry. This normal-phase variant has increased opportunities for performing
metabolomics without concern for analyte hydrophobicity. In addition, HILIC provides a method of separation capable of retaining and effectively resolving polar
metabolites without the need for derivatization as with reversed-phase chromatography or incompatible solvents like normal-phase. Despite its clear advantages over
reversed-phase and normal-phase in the context of metabolites, it does have limitations. Retention time and peak shape have been observed to be less robust than
reversed-phase requiring a great deal of care in buffering of mobile phases as well
as long re-equilibration periods between injections [35]. All of these factors and
others have led to hesitance in the field towards adopting HILIC, with some asserting that a new method of separating polar molecules is still needed [36].
Because reversed-phase and HILIC techniques offer complementary coverage of
the metabolome, they are often used together to provide a more comprehensive
analysis of sample analytes [21]. Many common extraction methods such as the
Folch extraction or the Bligh-Dyer extraction afford separations of metabolite
classes into distinct sample fractions [19, 20]. This fractionation allows for nonpolar to be analyzed by downstream reversed-phase, and polar metabolites from the
same sample to be analyzed by HILIC [21]. While this approach can significantly
increase analysis time, it provides a much more comprehensive view of the metabolites in a given sample set.
4.3.2 Gas Chromatography
Gas chromatography (GC) has also been shown to provide a high degree of sensitivity and reproducibility for volatile analytes. Rather than using changes in solvent
composition to separate analytes as in LC, GC takes advantage of analytes having
different boiling points by ramping temperature [37]. When coupled to a mass spectrometer, GC offers reliable platform for metabolomics [38]. One consideration
when integrating these techniques is an ionization source. In most GC experiments,
electron-impact (EI) or chemical ionization (CI) are used for ionization before mass
analysis [39]. Much like LC-MS, GC-MS can be used to effectively separate and
analyze complex mixtures and is effective in both targeted and untargeted experiments. However, there are certain limitations associated with GC-based metabolomics. As GC relies on analyte volatility, it is vital that analytes be volatile enough to
transition into the gas phase easily in order for GC-based methods to be effective
4 Fundamentals of Mass Spectrometry-Based Metabolomics
development of many derivatizing strategies for small molecules to make them less
polar and aid in reversed-phase retention.
Hydrophilic interaction chromatography (HILIC) is a relatively new chromatography technique which is a variation of normal phase chromatography [34]. Briefly,
HILIC relies on a thin layer of water which surrounds the polar stationary phase,
allowing for analytes to interact with the water layer rather than the stationary phase
directly [34]. This interaction with water lends to the retention of polar, hydrophilic
molecules without the need for mobile phases which are incompatible with mass
spectrometry. This normal-phase variant has increased opportunities for performing
metabolomics without concern for analyte hydrophobicity. In addition, HILIC provides a method of separation capable of retaining and effectively resolving polar
metabolites without the need for derivatization as with reversed-phase chromatography or incompatible solvents like normal-phase. Despite its clear advantages over
reversed-phase and normal-phase in the context of metabolites, it does have limitations. Retention time and peak shape have been observed to be less robust than
reversed-phase requiring a great deal of care in buffering of mobile phases as well
as long re-equilibration periods between injections [35]. All of these factors and
others have led to hesitance in the field towards adopting HILIC, with some asserting that a new method of separating polar molecules is still needed [36].
Because reversed-phase and HILIC techniques offer complementary coverage of
the metabolome, they are often used together to provide a more comprehensive
analysis of sample analytes [21]. Many common extraction methods such as the
Folch extraction or the Bligh-Dyer extraction afford separations of metabolite
classes into distinct sample fractions [19, 20]. This fractionation allows for nonpolar to be analyzed by downstream reversed-phase, and polar metabolites from the
same sample to be analyzed by HILIC [21]. While this approach can significantly
increase analysis time, it provides a much more comprehensive view of the metabolites in a given sample set.
4.3.2 Gas Chromatography
Gas chromatography (GC) has also been shown to provide a high degree of sensitivity and reproducibility for volatile analytes. Rather than using changes in solvent
composition to separate analytes as in LC, GC takes advantage of analytes having
different boiling points by ramping temperature [37]. When coupled to a mass spectrometer, GC offers reliable platform for metabolomics [38]. One consideration
when integrating these techniques is an ionization source. In most GC experiments,
electron-impact (EI) or chemical ionization (CI) are used for ionization before mass
analysis [39]. Much like LC-MS, GC-MS can be used to effectively separate and
analyze complex mixtures and is effective in both targeted and untargeted experiments. However, there are certain limitations associated with GC-based metabolomics. As GC relies on analyte volatility, it is vital that analytes be volatile enough to
transition into the gas phase easily in order for GC-based methods to be effective
4 Fundamentals of Mass Spectrometry-Based Metabolomics