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Sample profiling using untargeted metabolomics is the most efficient technique available to identify unsuspected changes in small molecules and to compare those changes between different experimental conditions. Since this method is an untargeted approach, every ionizable molecule in the sample will be recorded.
Metabolite identification is performed post-acquisition with the mapping of the recorded data on an ion library. Although untargeted workflows are less precise and less sensitive than the targeted approach, it has a very high profiling capacity.
For this reason, this strategy is better suited to explore multiple metabolic pathways simultaneously, rather than analyzing one specific pathway at a time.
The chemical nature of the small molecules that are analyzed in an untargeted metabolomics workflow is incredibly diverse. This heterogeneity means that the molecules will behave differently both on the chromatography column and in the mass spectrometer. We generally perform three different acquisitions, using two different chromatography and two different ionization modes to optimize the number of identification and quantification.
The reversed phase (RP) chromatography separates molecules based on their hydrophobicity whereas the HILIC works the opposite way and separates molecules based on their hydrophilicity. Having two different chromatographic methods allows for a more efficient separation of the molecules, which translates a better identification. In addition, because the molecules can be positively or negatively charged, using a combination of chromatography and two different ionization modes will maximize the number of proper identifications.
Therefore, with a combination of these different methods, we are capable of exploring a bigger portion of the metabolite world.
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