TABLE 2.

Most common analytical methodologies for mass spectrometry-based bulk metabolomics

Method	Description	Advantages	Limitations
Direct infusion (shotgun) MS	• Direct infusion of sample extracts to the MS detector • Mainly used as a fingerprinting method • Relative quantification • Used with low-resolution, high-resolution MS, or tandem MS instruments	• Increased analytical throughput • Increased intersample reproducibility	• Matrix-dependent signal suppression or enhancement in the analysis of complex samples; ion suppression may lead to decreased sensitivity, especially of less abundant species • Difficulties in resolving isobaric metabolites (compounds with the same nominal mass) in low resolution MS • No resolution of isomeric compounds
LC-high resolution MS	• LC interfaced with the MS detector: metabolites are separated before their detection • Nontarget analysis or broad profiling of aqueous and lipid metabolites, typically covering 400–2000 compounds	• High sensitivity • Broad coverage of metabolites or tailored analysis associated with specific sample preparation, separation method • Ideally suited to lipidomics • Possible combination of relative (semiquantitative) and quantitative analysis of some selected metabolites • Separation of isomeric compounds	• Usually semiquantitative; full quantification of all detected compounds is not possible • Matrix effect and ion suppression, although reduced compared with shotgun-MS • Generation of large amounts of data
Targeted LC-MS	• Typically, LC coupled with a triple quadrupole MS detector for the analysis of preselected metabolites	• High selectivity and sensitivity • Approach of choice for quantitative analysis • Suitable for the analysis of metabolites at low concentration	• Information of preselected compounds only • Specific sample preparation focused on the selected metabolites

Abbreviations: LC, liquid chromatography; MS, mass spectrometry.