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Metabolomics - Global Metabolite Profiling (HILIC with +/- ESI HRMS/MSMS)

  • HILIC - hydrophilic interaction chromatography
  • ESI - electro spray ionisation
  • HRMS/MSMS - high-resolution mass spectrometry with tandem mass spectrometry

Hydrophilic interaction chromatography (HILIC) is a separation technique for polar compounds. In the context of HILIC, ESI HRMS/MSMS refers to two mass spectrometry techniques used in combination for enhanced compound identification and quantification:

  1. ESI: Electrospray Ionization is an ionization technique that generates charged droplets of analytes from a liquid solution, facilitating their detection in mass spectrometry. ESI is particularly useful for polar and large biomolecules, making it an ideal ionization method for HILIC-separated compounds.

  2. HRMS/MSMS: High-Resolution Mass Spectrometry (HRMS) combined with Tandem Mass Spectrometry (MSMS) is an analytical technique that provides precise mass measurements and structural information of compounds. In HRMS/MSMS, the first mass analyzer (MS1) isolates a specific mass-to-charge (m/z) ratio, followed by fragmentation of the selected ion in a collision cell. The resulting fragments are then analyzed by a second mass analyzer (MS2) to obtain structural information. This combination helps in the identification and quantification of compounds separated by HILIC with greater confidence.

HILIC + ESI HRMS/MSMS is an analytical approach that combines electrospray ionization and tandem high-resolution mass spectrometry to provide reliable identification and quantification of polar compounds separated using hydrophilic interaction chromatography.


Metabolite profiling aims to measure various metabolites in biological samples using LC-MS technology. It helps reveal insights into biological processes and allows for the quantification of metabolites using MS signal intensity methods for relative comparisons.

The Method

BEH amide-based HILIC is used for profiling, with two SOPs covering a wide range of polar metabolites. The results are semi-quantitative, with identifications of 1000-3000 polar metabolite species on MS1 level and MS2 for several hundred abundant ones. Ion mobility (IM) measurements increase the certainty of identifications. Reference standards are routinely measured as QC.

Typical Metabolite Pathways Covered

  • Carbon metabolism
  • Biosynthesis of amino acids
  • Glycine, serine and threonine metabolism
  • Pyrimidine metabolism
  • Arginine and proline metabolism
  • Cysteine and methionine metabolism
  • Purine metabolism
  • Tryptophan metabolism
  • Others on request

The Workflow

  1. User Meeting, project discussion, and design
  2. Optimized sample extraction (link to Wiki)
  3. LCMS acquisition (or IMMS)
  4. Data processing: Progenesis QI, Cosmiq
  5. Data analysis: Univariate statistics, multivariate data analysis (PCA, BGA), machine learning (PCA, BGA)
  6. Search against metabolite databases (HMDB, LipidMaps) or customized libraries
  7. Pathway analysis MetaboAnalyst

The Output

  1. Primary output: LC-MS raw files, which can be inspected by various software packages.
  2. Final output: A series of tables, including:
    • Peak areas and normalized peak areas for all observed m/z values.
    • Database matches (identification) for all m/z values.
  3. Additional outputs (generated as needed):
    • Multivariate data analysis.
    • Fold changes.
    • ANOVA p-values.
    • Volcano plots.
  4. File formats: Tables and plots are provided in standard formats.
  5. Summary: Key findings are summarized in a Word or PowerPoint document.
  6. Pathway analysis information: The data can be used for pathway analysis with free web-based tools like MetaboAnalyst (


This content is largely based on information from ETH Zurich Functional gnomonics center