Precursor and Product Ion Mobility and Collision Cross Section Determination by Travelling Wave Cyclic Ion Mobility – Mass Spectrometry

Importance of the topic

Ion mobility–mass spectrometry (IM-MS) provides an orthogonal separation dimension that is highly valuable for structural characterization of small molecules and isomers in pharmaceutical, environmental, natural product and toxicology applications. Travelling-wave cyclic ion mobility (cIM) extends separation pathlength and resolution, enabling measurement of collision cross section (CCS) for both precursor and product ions. Combining CCS measurements with chromatographic retention, high-resolution mass-to-charge (m/z) and in-silico fragmentation enhances confidence in compound identification where conventional MS/MS alone cannot resolve positional or structural isomers.

Objectives and overview of the study

The study evaluated the capabilities of a travelling-wave cyclic ion mobility–mass spectrometry platform for:

• Measuring CCS of intact precursor ions and of product (fragment) ions.
• Resolving and discriminating isomeric compounds (e.g., linear vs branched PFOS isomers; flavonoid positional isomers).
• Developing workflows for processing and reviewing multidimensional datasets that combine m/z, retention time, CCS and intensity.
• Demonstrating integration with in-silico fragmentation prediction to annotate product ions and generate CCS fingerprints for small molecules relevant to pharmaceuticals and environmental analysis.

Methodology

Samples and applications investigated:

• Natural-product flavonoid isomers (isoorientin, isovitexin, orientin, vitexin).
• System suitability mixture including common MS standards (acetaminophen, caffeine, sulfaguanidine, sulfadimethoxine, Val-Tyr-Val, verapamil, reserpine, terfenadine, Leu-enkephalin).
• Pharmaceutical compounds (betaxolol, ciprofloxacin).
• Per- and polyfluoroalkyl substances (PFAS) including PFOS isomers.

Chromatography and ionization:

• UPLC: ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.8 μm), 35 °C, 0.3 mL/min, 10 μL injection.
• Mobile phases: A = 95% H2O/5% MeOH with 2 mM ammonium acetate; B = MeOH with 2 mM ammonium acetate. Gradient durations: 22 min for PFOS isomers, 5 min for standards.
• Electrospray ionization performed in positive and negative modes; typical capillary voltages 0.3–0.5 kV; source temp ~100 °C; desolvation temp 550 °C and gas flow ~1000 L/hr.

Mass spectrometry and acquisition:

• Travelling-wave cyclic ion mobility–MS platform (SELECT SERIES Cyclic IMS) configured to obtain extended ion mobility separations including product ion mobility measurements.
• Acquisition strategies included HDMSE (ion-mobility-enabled data-independent acquisition) and HDMS (IM-MS) with and without quadrupole isolation.

Data processing and in-silico annotation:

• Data acquisition and review: MassLynx and DriftScope.
• Peak detection and initial processing: ApexRT (peak detection), CompareCSV (multicolumn matching with tolerances).
• In-silico fragmentation and spectrum prediction: CFM-ID v2.4.
• Custom lightweight tools for visualization, CCS calculation and review built with Python libraries (Streamlit, plotly, pandas, numpy, matplotlib).
• Workflow steps: multidimensional peak detection/centroiding, CCS calibration, matching experimental precursor and fragment m/z and CCS to in-silico predictions, and generation of CCS-annotated identifications.

Used instrumentation

• SELECT SERIES Cyclic IMS (travelling-wave cyclic ion mobility platform).
• ACQUITY UPLC system with BEH C18 column (100 × 2.1 mm, 1.8 μm).
• MassLynx and DriftScope software for acquisition and drift analysis.
• Auxiliary software tools: ApexRT, CompareCSV, CFM-ID v2.4; Python stack (Streamlit, plotly, pandas, numpy, matplotlib) for visualization and CCS calculations.

Main results and discussion

Key findings:

• Precursor and product-ion CCS values provided diagnostic information that discriminated linear versus branched PFOS isomers; separation of m/z 379 product ions showed distinct drift-time profiles for linear PFOS and a branched isomer (P5MPHpS).
• Drift time and measured CCS differentiated isomeric flavonoid precursors and their product ions (e.g., orientin/isoorientin and vitexin/isovitexin), enabling structural assignment not possible by MS/MS alone.
• Small-molecule product ions produced reproducible and specific CCS fingerprints; combining experimentally measured CCS for products with in-silico fragmentation predictions improved annotation confidence for pharmaceutical-relevant compounds.
• Workflow examples demonstrated sequential processing steps: peak detection, CCS calibration, centroiding, matching experimental features to predicted fragment m/z and CCS, and annotation of precursor/product pairs. A summary table of precursor and product m/z and CCS values was generated for environmental, natural product and pharmaceutical compounds.
• Lightweight application development accelerated data review and allowed rapid interrogation of multidimensional results (m/z, retention time, CCS, intensity).

Benefits and practical applications

Practical advantages demonstrated:

• Enhanced isomer resolution for environmental monitoring (PFAS) and natural products, improving identification where m/z and MS/MS are ambiguous.
• Product-ion CCS measurements add a new identification parameter for spectral libraries and retrospective data mining.
• Combined CCS + in-silico fragmentation increases specificity for metabolite and impurity ID in pharmaceutical and toxicology studies.
• Workflow and software integration show feasibility for routine implementation in labs seeking multidimensional confirmation (RT, m/z, CCS, fragmentation).

Future trends and potential uses

Projected developments and opportunities:

• Expansion of CCS libraries to include product-ion CCS values alongside precursor CCS to support broader library searching and automated annotation.
• Deeper integration of in-silico fragmentation tools and machine-learning models to predict CCS and fragment likelihood, enabling higher-throughput annotation.
• Standardization of CCS measurement protocols to improve inter-laboratory comparability and facilitate regulatory acceptance.
• Application to complex omics workflows (metabolomics, lipidomics), forensic and environmental screening where isomer differentiation is critical.
• Optimization of acquisition strategies (DIA coupled to IM) and faster automated workflows for routine screening and QA/QC use cases.

Conclusion

The travelling-wave cyclic ion mobility–mass spectrometry approach enables high-resolution ion mobility separations and robust CCS measurement for both precursor and product ions. When combined with chromatographic separation, high-resolution MS, in-silico fragmentation and lightweight data-review tools, this multidimensional strategy substantially improves discrimination and annotation of isomeric small molecules across environmental, natural product and pharmaceutical applications. The added dimension of product-ion CCS fingerprints offers a practical pathway to more confident identifications and richer spectral libraries.

Reference

1. Giles, K., et al. A Cyclic Ion Mobility–Mass Spectrometry System. Analytical Chemistry. 2019;91(13):8564–8573.
2. Techniques for sample analysis using product ion collision-cross section information. US Patent US12332210B2.
3. Allen, F., et al. CFM-ID: a web server for annotation, spectrum prediction and metabolite identification from tandem mass spectra. Nucleic Acids Research. 2014;42(Web Server Issue):W94–W99.