LC-MS Metabolomics Analysis

Liquid chromatography mass spectrometry (LC-MS) offers the broadest coverage of metabolites due to its ability to work with different column chemistries. Two column chemistry examples include reversed phase liquid chromatography (RPLC) for non-polar to moderately polar metabolites, and hydrophobic interaction liquid chromatography (HILIC) for ionic and polar compounds not retained by RPLC.  

Metabolites separated by either LC or ion chromatography (IC) do not generally need derivatization, nor are they required to be volatile in nature (as in gas chromatography). As a result, LC-MS works with various column chemistries and a broader range of applicable compounds. Due to these qualities, LC-MS has wide applicability in both untargeted and targeted metabolomics analyses.

LC-MS and GC-MS typically use very different mechanisms for sample ionization. With LC-MS, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the two ionization techniques that are most commonly used. ESI is better suited to semipolar and polar compounds, while APCI works well with neutral or less polar compounds. During these ionization processes, anions are formed by a loss of H⁺ and cations are formed by a gain of H⁺, Na⁺, or some other ion such as NH₄⁺ and K⁺.

Unlike GC-MS using electron impact (EI) ionization, the major ion formed in LC-MS does not undergo fragmentation.

Because there is less ion suppression with GC-MS as compared with LC-MS, greater resolution is needed for LC-MS. This is especially true in LC-MS analysis of larger and more complex samples.

Unlike GC-MS, there are no spectral libraries for LC-MS compound identification. However, because the precursor or molecular ion is usually present with LC-MS, it is a crucial part of the metabolite identification strategy and is used to search databases of metabolites, such as the METLIN database  and mzCloud  online fragmentation library. With the recent advances in high resolution accurate mass (HRAM) MS systems (e.g., Orbitrap mass analyzers), it is feasible to calculate empirical formulae from molecular ions.

To truly evaluate unknown metabolites, fragmentation of the precursor or molecular ion is performed. The resultant MS/MS spectrum is then searched against available MS/MS mass spectral libraries for matches. In some cases, this does not lead to a confident identification, and further stages of analysis (MSⁿ) are needed. It is also possible to perform manual de novo interpretation/structural elucidation as part of the metabolite identification process.