APPLICATION NOTEBOOK - STRUCTURAL ELUCIDATION

Importance of the topic

The structural complexity and extensive co-elution of metabolites and lipids in biological and plant extracts pose significant challenges for confident identification. Traditional LC/MS and MS/MS techniques often yield mixed fragment spectra and limited specificity, which can lead to false positives or negatives. Incorporating ion mobility separation (IMS) and collision cross-section (CCS) measurements into high-resolution, accurate-mass UPLC/MS workflows provides an orthogonal dimension of gas-phase separation based on three-dimensional ion conformation. This additional selectivity helps resolve isomers, reduce spectral interferences, and enhance structural elucidation in metabolomics, lipidomics, drug metabolism, and natural-product analysis.

Objectives and Overview

This Application Notebook on Metabolomics and Lipidomics #720005245EN presents multiple strategies to improve structural elucidation and compound identification by combining UPLC, high-definition mass spectrometry, ion mobility, and advanced acquisition methods:

• Demonstrate improved spectral cleanliness and specificity for metabolites and lipids by HDMS E (IMS-MS E).
• Showcase time-aligned parallel (TAP) fragmentation for detailed MS3-like structural information of complex natural products.
• Explore the benefits of CCS measurements as an orthogonal identifier in residue screening studies.
• Illustrate separation and CCS determination of isomeric amino acids using travelling-wave IMS.
• Apply IMS and MS E to differentiate flavonoid isomers in complex plant extracts.
• Highlight IMS-MSE for resolving co-eluting metabolites in drug-metabolism matrices.

Methodology and Instrumentation

The following instrumentation and methods were employed across studies:

• ACQUITY UPLC Systems with HSS T3 columns (2.1 × 100 mm, 1.7–1.8 µm) and rapid gradients (5–20 min).
• SYNAPT HDMS and SYNAPT G2/ G2-Si High Definition Mass Spectrometers featuring TriWave™ IMS (pre- and post-collision T-Waves) and TOF analyzers.
• Ionization by ESI in positive and negative modes; gas-phase conditions with nitrogen and helium drift gases.
• Data-independent acquisition (MS E) coupled with HDMS for parallel low-energy precursor and high-energy fragment spectra.
• Time-aligned parallel (TAP) fragmentation: quadrupole selection → CID in first cell → IMS separation → CID in second cell (pseudo-MS3).
• DriftScope™ Informatics for automated CCS calibration, drift-time visualization, and four-dimensional peak picking.
• MassFragment™ and MarkerLynx/MetaboLynx application managers for structural annotation and multivariate profiling.

Main Results and Discussion

• HDMS E provided cleaner product-ion spectra by separating co-eluting precursors along drift time, enhancing lipid and metabolite identification specificity.
• TAP fragmentation of Ginsenoside Rb1 in Chinese Ginseng enabled sequential loss of sugar moieties and pseudo-MS3 driftograms, facilitating detailed structure assignment in a single run.
• Measured CCS values (±2%) proved robust across solvent standards and complex matrices, improving pesticide screening by reducing false positives/negatives when combined with mass and retention-time filters.
• Travelling-wave IMS on SYNAPT G2 resolved leucine and isoleucine isomers (ΔCCS ~2.8 Å2) and enabled automated CCS calculation via DriftScope calibration.
• Flavonoid C-glycoside isomers (vitexin vs. isovitexin; orientin vs. isoorientin) were baseline-separated by UPLC and drift time, while HDMS E cleaned up co-eluting interferences, yielding unambiguous fragmentation profiles.
• In drug-metabolism studies, IMS-MSE resolved busespirone metabolites (+32 Da variants) and removed matrix interferences from fragment spectra, enabling straightforward peak picking in four dimensions (RT, m/z, drift time, intensity).

Benefits and Practical Applications

• Orthogonal IMS separation multiplies chromatographic and mass resolution, increasing peak capacity and confidence in complex-mixture analysis.
• CCS serves as a robust gas-phase identifier unaffected by matrix, improving screening selectivity in environmental, pharmaceutical, and QA/QC workflows.
• TAP fragmentation integrates MS3-like experiments in a single analytical step, reducing sample handling and analysis time.
• IMS-MSE simplifies spectral interpretation by decluttering fragment ion spectra and isolating true product ions from co-eluting species.
• Applications span metabolomics, lipidomics, traditional-medicine profiling, pesticide residue analysis, drug-metabolism, proteomics, and MALDI imaging.

Future Trends and Opportunities

• Expansion of CCS libraries for broader compound classes and integration with computational modeling for structural prediction.
• Development of higher-resolution IMS platforms and novel drift gases to enhance isomeric separations.
• Real-time IMS-MS workflows for rapid screening in clinical, food-safety, and environmental monitoring.
• Integration of IMS-MS with ion-mobility imaging and multidimensional separation techniques (e.g., LC×IMS×MS).
• Automation and machine-learning-driven four-dimensional data analysis for high-throughput metabolomics and QA/QC.

Conclusion

Combining UPLC with high-definition ion mobility mass spectrometry and advanced acquisition modes such as MS E and TAP fragmentation delivers unprecedented selectivity and structural insight in complex biological and natural-product analyses. The added drift-time dimension and CCS measurement significantly enhance isomer resolution, spectral clarity, and identification confidence, positioning HDMS as a critical tool for next-generation metabolomics, lipidomics, and compound-profiling applications.

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