INVESTIGATIONS INTO CROSS-PLATFORM AND LONG-TERM ROBUSTNESS OF A CCS METRIC

Significance of the Topic

Ion mobility coupled with mass spectrometry delivers collision cross section (CCS) values that add an orthogonal separation dimension to traditional liquid chromatography and mass‐to‐charge measurements. Reliable CCS metrics are critical for confident compound identification in forensic toxicology, food safety, environmental monitoring and pharmaceutical analysis. Ensuring long‐term and cross‐platform consistency underpins the integration of CCS into routine screening workflows and searchable libraries.

Objectives and Overview

This study evaluates the robustness of CCS measurements over extended periods (up to four years), across multiple instrument platforms and laboratories. It encompasses hundreds of small molecules relevant to forensic toxicology, pesticides, veterinary drugs, flavonoids and steviol glycosides. Key aims include assessing inter‐ and intra‐laboratory reproducibility, cross‐platform comparability between Waters Vion IMS Q‐ToF, SYNAPT G2‐Si and cyclic ion mobility (cIM) devices, and establishing routine calibration strategies.

Methodology and Instrumentation

Chromatography was performed using UPLC systems with ACQUITY HSS C18 and BEH C18 columns, as well as micro‐flow ionKey iKey PCA BEH C18 devices. Ion mobility separations employed travelling wave IM (TWIM) on Q‐ToF platforms and multi‐pass cyclic IM for increased resolution. Mass analysis used Waters Vion IMS Q‐ToF and SYNAPT G2‐Si instruments, with ESI in positive and negative modes under HDMSE acquisition. Calibration relied on leucine enkephalin lockmass and an IMS/ToF CCS calibration kit. Instrument resolutions were ~30 000 (Vion), ~20 000 (SYNAPT) and up to 65 Ω/ΔΩ (cIM).

Main Results and Discussion

• Cross‐platform comparison of 435 forensic toxicology analytes yielded CCS deviations within 2% (R² > 0.997).
• Inter‐site and intra‐site reproducibility confirmed CCS delta < 2% across laboratories.
• Continuous monitoring of QC mixes over 10 weeks produced 18 974 detections with < 1% CCS deviation (RMS error 0.26%).
• Steviol glycosides in complex food matrices were detected over three orders of magnitude dynamic range with < 1% CCS deviation.
• Fluoroquinolone protomer CCS values measured by linear IM and cIM remained within 2% over three years.
• Pesticide library measurements (200 analytes, 2 000 observations) across four years showed CCS deltas consistently < 2% for m/z 123–859.

Benefits and Practical Applications

• Enhanced analytical specificity by combining CCS with m/z and retention time, reducing false positives/negatives in non‐targeted screening.
• Robust searchable CCS libraries support forensic, food safety and environmental testing.
• Single‐point CCS calibration simplifies routine workflows and quality control.
• Applicable to QA/QC in pharmaceutical development and regulatory compliance.

Future Trends and Applications

• Expansion of curated CCS libraries covering broader chemical space.
• Integration of predictive modeling and machine learning for CCS estimation and annotation.
• Standardization of CCS protocols across instrument vendors and laboratories.
• Advances in cyclic ion mobility for higher resolution separations and isomer discrimination.
• Real‐time CCS‐driven decision support in automated screening workflows.

Conclusion

The study demonstrates that CCS measurements can be obtained reproducibly (< 2% deviation) across multiple platforms, laboratories and over extended timeframes. A single TWIM CCS calibration strategy supports long‐term stability and cross‐platform comparability, validating CCS as a reliable metric for enhancing compound identification confidence in diverse analytical applications.

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