65
standard [16]. Resuspension is also a consideration, as the solvent chosen can have
effects on downstream chromatography such as reproducibility of retention times or
peak shape [17].
The optimal extraction method in untargeted approaches depends on the complexity of the sample matrix as well as the class of analytes. Having little to no
information about the metabolites of interest, however, it can be difficult to discern
the optimal extraction protocol. For this reason, straightforward and versatile techniques such as protein precipitation [18], Folch extraction [19], and Bligh-Dyer
extraction [20] involving multiple immiscible solvents are often employed in untargeted metabolomics. Here, different classes of biomolecules are separated into isolated liquid fractions; polar metabolites suspend in the aqueous layer while lipids
separate into a hydrophobic fraction such as chloroform. This phenomenon allows
for simple, broad extractions of metabolites and even lends itself to multiomics
workflows as each different class of biomolecules from a single sample can be easily taken for class-specific sample preparation [21].
The addition of internal standards in untargeted workflows is also common practice [22]. In this context, an internal standard could be used for normalization purposes where each analyte is reported relative to the internal standard, or it could
provide a retention time reference point to provide insight on chromatographic drift
over the course of an experiment [22]. More common, however, is the practice of
sample pooling [23]. This involves pooling equal volumes from each sample for
downstream quality control. This approach operates on the premise that a pooled
sample contains every possible analyte from an entire untargeted experiment in a
single injection and can thus be used to gauge both chromatography and instrumental efficiency. While analytes may in some cases be diluted in the pooled sample,
this methodology works to provide qualitative insight to an experiment. Quality
control of chromatographic and instrumental drift can be determined by periodically injecting the pooled sample mix between samples (after every 10 injections),
over the course of the experiment.
Again, as with targeted metabolomics, the last major consideration for sample
preparation is the composition of the final resuspension solvent. Trying to use
generic solvents which are broadly compatible with any unknown analytes present
in the sample will help to avoid analyte precipitation or having sample conditions
incompatible with chromatography. Some biases can be made however, tailoring the
resuspension solvent to the analytes being measured. For example, if measuring
lipids, a solvent that will minimize lipid precipitation is necessary. Most lipids have
been found to be soluble in chloroform making it an attractive choice for resuspension; however, chloroform would not be compatible with most reversed-phase or
HILIC methods, and therefore cannot be used for resuspension in most applications.
Instead, methanol, which solubilizes most lipids could serve as a substitute resuspension solvent.
4 Fundamentals of Mass Spectrometry-Based Metabolomics
standard [16]. Resuspension is also a consideration, as the solvent chosen can have
effects on downstream chromatography such as reproducibility of retention times or
peak shape [17].
The optimal extraction method in untargeted approaches depends on the complexity of the sample matrix as well as the class of analytes. Having little to no
information about the metabolites of interest, however, it can be difficult to discern
the optimal extraction protocol. For this reason, straightforward and versatile techniques such as protein precipitation [18], Folch extraction [19], and Bligh-Dyer
extraction [20] involving multiple immiscible solvents are often employed in untargeted metabolomics. Here, different classes of biomolecules are separated into isolated liquid fractions; polar metabolites suspend in the aqueous layer while lipids
separate into a hydrophobic fraction such as chloroform. This phenomenon allows
for simple, broad extractions of metabolites and even lends itself to multiomics
workflows as each different class of biomolecules from a single sample can be easily taken for class-specific sample preparation [21].
The addition of internal standards in untargeted workflows is also common practice [22]. In this context, an internal standard could be used for normalization purposes where each analyte is reported relative to the internal standard, or it could
provide a retention time reference point to provide insight on chromatographic drift
over the course of an experiment [22]. More common, however, is the practice of
sample pooling [23]. This involves pooling equal volumes from each sample for
downstream quality control. This approach operates on the premise that a pooled
sample contains every possible analyte from an entire untargeted experiment in a
single injection and can thus be used to gauge both chromatography and instrumental efficiency. While analytes may in some cases be diluted in the pooled sample,
this methodology works to provide qualitative insight to an experiment. Quality
control of chromatographic and instrumental drift can be determined by periodically injecting the pooled sample mix between samples (after every 10 injections),
over the course of the experiment.
Again, as with targeted metabolomics, the last major consideration for sample
preparation is the composition of the final resuspension solvent. Trying to use
generic solvents which are broadly compatible with any unknown analytes present
in the sample will help to avoid analyte precipitation or having sample conditions
incompatible with chromatography. Some biases can be made however, tailoring the
resuspension solvent to the analytes being measured. For example, if measuring
lipids, a solvent that will minimize lipid precipitation is necessary. Most lipids have
been found to be soluble in chloroform making it an attractive choice for resuspension; however, chloroform would not be compatible with most reversed-phase or
HILIC methods, and therefore cannot be used for resuspension in most applications.
Instead, methanol, which solubilizes most lipids could serve as a substitute resuspension solvent.
4 Fundamentals of Mass Spectrometry-Based Metabolomics