APPLICATION NOTEBOOK - UNTARGETED METABOLOMICS AND LIPIDOMICS

Significance of the Topic

The integration of advanced chromatography, high-resolution mass spectrometry, and multivariate data analysis has revolutionized metabolomics and lipidomics. These approaches enable comprehensive, untargeted profiling of small molecules and lipids in complex biological and food matrices. By leveraging orthogonal separations (HILIC, reverse-phase, supercritical fluids, and 2D LC) and gas-phase ion mobility, researchers can now resolve thousands of molecular species for biomarker discovery, quality control, and authenticity assessment.

Objectives and Study Overview

Multiple studies demonstrate workflows for:

• Screening polar metabolites in biological samples using HILIC UPLC/QTof MS.
• Separating complex lipid mixtures via UPLC with Charged Surface Hybrid (CSH™) C18 chemistry.
• Performing orthogonal two-dimensional UPLC (HILIC×C18) for high-resolution phospholipid analysis.
• Combining HILIC, ion mobility, and data independent acquisition (HDMSE) for multi-omics profiling (proteins, metabolites, lipids).
• Characterizing biological fluids and tissues in clinical and agricultural settings.
• Applying atmospheric pressure GC-MS E for untargeted metabolomics in plant systems, and high-resolution GC-MS for food authenticity.

Methodology and Instrumentation

Key components across workflows:

• UltraPerformance Liquid Chromatography (UPLC) systems with HILIC, CSH C18, BEH, and UPC2 columns.
• Data independent acquisition: MS E and HDMS E (alternating low/high collision energies for simultaneous precursor and fragment ion collection).
• Ion Mobility Separation (TriWave™) to resolve isobaric species by drift time (collision cross-section).
• Atmospheric Pressure Gas Chromatography (APGC) for soft ionization, preserving molecular ions in GC-MS.
• Waters SYNAPT G2/ G2-Si QTof mass spectrometers for high mass accuracy and resolving power.
• NanoACQUITY UPLC for sensitive proteomics and lipidomics.
• Robust sample preparation: protein digestion, lipid extraction, and solid-phase metabolite cleanup.

Key Results and Discussion

• HILIC-UPLC/QTof assays achieved sub-2 µm peak capacities for polar metabolites, enabling routine untargeted metabolomics.
• CSH C18 UPLC provided superior inter- and intra-class lipid separation, resolving phosphocholines and sphingomyelins, cis/trans isomers, and broad lipid classes in clinical and botanical extracts.
• Orthogonal 2D UPLC (HILIC×C18) dramatically increased peak capacity for phospholipids and minimized isotopic interferences.
• Ion mobility-enabled HDMSE improved specificity and structural elucidation across multi-omics, facilitating the discovery of lipid, metabolite, and protein alterations in biological models (e.g., nephrotic syndrome, CYP2E1-expressing cells).
• APGC-MS E provided abundant molecular ions for derivatized metabolites and enhanced confidence in compound identification, as demonstrated in Arabidopsis fingerprinting.
• High-resolution GC-MS and statistical analysis successfully discriminated basmati rice authenticity and potential adulteration through characteristic volatile profiles.

Benefits and Practical Applications

• High-throughput, untargeted analysis of metabolites and lipids with minimal sample preparation.
• Enhanced confidence in compound identification via orthogonal data: accurate mass, retention time, drift time, and fragmentation.
• Proprietary informatics (Progenesis QI, TransOmics) streamline alignment, peak detection, deconvolution, and statistical modeling.
• Applications in biomarker discovery, plant metabolomics, clinical proteomics, environmental phenotyping, and food authenticity testing.

Future Trends and Possibilities

• Integration of machine learning with multi-dimensional MS data for improved metabolite annotation.
• Expanded use of APGC and supercritical fluid chromatography for non-polar metabolite classes.
• Higher throughput screening in QC labs via transition from QTof to triple quadrupole platforms guided by discovery data.
• Development of robust reference databases incorporating collision cross-section libraries for ion mobility.
• Greater adoption of 2D LC and ion mobility in regulated environments for food fraud detection and clinical diagnostics.

Conclusion

Advancements in chromatographic separation, soft ionization techniques, ion mobility, and high-resolution mass spectrometry, coupled with powerful informatics, have enabled comprehensive and reliable metabolomics, lipidomics, and multi-omics analyses. These workflows provide unparalleled detail in characterizing complex biological and food matrices, driving progress in biomarker discovery, phenotyping, and authenticity testing.

Reference

1. Economic Times, “Basmati Export Adulteration Leaves Bad Taste in Mouth,” 2007.
2. Fletcher A. “Contamination Concerns Force New Basmati Regulations,” FoodNavigator, 2005.
3. Waters SYNAPT G2-Si Product Brochure, 2014.
4. Waters APGC White Paper, 2014.
5. Dunn WB et al. Metabolomics, 2013;9(S1):44–66.
6. Shahaf N et al. Anal. Chim. Acta, 2013;759:20–28.