APPLICATION NOTEBOOK - TARGETED METABOLOMICS AND LIPIDOMICS

Importance of the Topic

Targeted metabolomics and lipidomics are essential for validating the biological significance of discovered metabolic changes and for routine clinical or research analyses. Hypothesis-driven quantitative assays focus on defined sets of metabolites or lipids, enabling sensitive, reproducible measurement even in complex biological matrices.

Objectives and Overview of the Study

The Waters Metabolomics and Lipidomics Application Notebook assembles robust workflows for targeted analysis of key endogenous compound classes, including amino acids, biogenic amines, glycerophospholipids, sphingolipids, acylcarnitines, and oxylipins. It also illustrates how these workflows support discovering and validating biomarkers in diverse applications such as toxicology, clinical research, and cancer progression studies.

Methodology and Instrumentation

Sample preparation varies by analyte class: protein precipitation and derivatization for amino acids; mixed-mode SPE for catecholamines; kit-based extraction for phospholipids; and EPC-SPE or lipid enrichment for lipidomics. Chromatographic separations employ either UPLC, UPC2, or specialized columns (HSS T3, BEH HILIC, UPC2 Trefoil, ACQUITY CSH, or Oasis µElution SPE), coupled to Waters Xevo TQ-series or SYNAPT Q-ToF mass spectrometers using MRM, MS^E, or full-scan acquisition. Data analysis uses MassLynx, UNIFI, TargetLynx, MarkerLynx, or TransOmics software.

Main Results and Discussion

Fast, reliable quantification of twenty amino acids in urine achieved 7.5-minute runs, LOQs to 0.2 μM, and FDA-compliant accuracy and precision. Targeted analysis of 183 metabolites on the AbsoluteIDQ p180 Kit provided high-throughput coverage of six biochemical classes in mouse serum with UPLC/MS and standardized data processing. Oxylipin profiling separated over 100 oxygenated fatty acids in under 10 minutes by UPLC-MS/MS, revealing both enzymatic and non-enzymatic products. Phospholipid lipidomics used mixed-mode SPE and HILIC-UPLC/MS/MS to quantify 215 lipids from plasma in a single run. Catecholamines and metanephrines were measured in plasma by mixed-mode SPE and HILIC chromatography with LC-MS/MS in 4-minute assays with LOQs to 0.29 pg/mL. Bile acids were profiled by UPLC-MS^E in five minutes, resolving isomeric conjugates, and applied to hepatotoxicity studies. The ionKey/MS System achieved 10x sensitivity gains for lipidomics and steroids in sub-microliter injections by microflow UPLC-MS. UPC^2-MS enabled rapid free fatty acid profiling without derivatization and minimal sample prep, resolving C8–C36 species in 3 minutes. Convergence chromatography with TOF or MRM detection separated nine steroids in two minutes without derivatization. Chiral UPC^2 on Trefoil AMY1 and CEL1 columns resolved fragrance enantiomers in 2-3 minutes versus 15-50 minute GC runs.

Benefits and Practical Applications of the Method

• High throughput and short run times (2–10 minutes) accelerate large-scale studies.
• Minimal sample preparation (protein crash, SPE, or kit-based) reduces hands-on time and artifact formation.
• Sub-microliter to low microliter injection volumes reduce biological sample volume requirements.
• Enhanced sensitivity and dynamic range facilitate detection of low-abundance metabolites and lipids.
• Robust, reproducible separations of isomers, isobars, and enantiomers support accurate identification and quantification.
• Orthogonal chromatography (HILIC, UPC^2, RP-UPLC) paired with MS/MS or MS^E offers comprehensive metabolome coverage.

Future Trends and Opportunities

• Automation and high-density plate formats will further increase throughput for large cohort studies.
• Integration with ion mobility separation (HDMS) will enhance resolution of structural isomers and conformers.
• Expansion of predefined kits and standardized software workflows will streamline adoption and ensure data consistency across laboratories.
• Future stationary phases with sub-2-µm chiral particles will enable even faster enantiomeric analyses by UPC^2 and UPLC.
• Combined lipidomic and metabolomic panels will support deeper systems biology and precision medicine applications.

Conclusion

Waters’ suite of chromatographic and mass spectrometric solutions—including UPLC, UPC^2, SPE workflows, and ionKey/MS—provides rapid, sensitive, and robust analytical platforms for targeted metabolomics and lipidomics. These high-throughput assays enable accurate quantification of diverse small molecules (amino acids, biogenic amines, bile acids, steroids, free fatty acids, phospholipids, oxylipins, and more) with minimal sample preparation, supporting both discovery and routine analyses in toxicology, clinical research, and biomarker validation.

References

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