FURTHER INVESTIGATIONS INTO CHARGED ISOMER SPECIES OF THE FLUOROQUINOLONE CLASS OF ANTIBIOTICS USING LINEAR TWIM AND CYCLIC ION MOBILITY

Significance of the Topic

Fluoroquinolone antibiotics are widely used in veterinary and human medicine. Their zwitterionic nature and structural diversity demand high-resolution analytical techniques to distinguish charge-site isomers (protomers). Ion mobility spectrometry (IMS) offers rapid gas-phase separation based on collisional cross section (CCS), improving specificity in residue analysis, contamination control, and regulatory compliance.

Objectives and Study Overview

This study aimed to:

• Compare linear travelling-wave IMS (TWIM) and cyclic IMS (cIM) in resolving fluoroquinolone protomers.
• Generate an expanded CCS library for eighteen fluoroquinolones.
• Evaluate a multi-pass calibration strategy to estimate CCS values at increasing IMS resolution.

Methodology

Reverse-phase liquid chromatography was performed on a C18 column (1.7 µm, 2.1×100 mm) using a formic acid/acetonitrile gradient. Positive-ion electrospray ionization (ESI) generated [M+H]+ precursor ions. Both LC-MS and direct infusion experiments were conducted.

Instrumentation Used

Experiments employed a quadrupole–ion mobility–time-of-flight platform with:

• Linear TWIM module (resolution R≈40 Ω/ΔΩ).
• Cyclic IMS device capable of 1–5 passes (resolution R≈65 to ≈145 Ω/ΔΩ).
• IMS/ToF calibration kit and multi-pass CCS calibration according to established protocols.

Results and Discussion

Cyclic IMS achieved baseline separation of fluoroquinolone protomer pairs at R≈65–145, outperforming linear TWIM. Cross-platform comparison showed ΔCCS <1.1 % (RMS 0.54 %) between single-pass cIM and linear TWIM reference values. Multi-pass cIM CCS estimates yielded ΔCCS <2.1 % (RMS 0.97 %) relative to linear TWIM. Eighteen charged-isomer CCS values were characterized; notably, danofloxacin exhibited three distinct protomers resolved at five passes (R≈145).

Benefits and Practical Applications

Enhanced IMS resolution increases peak capacity and enables detection of multiple protonation sites within a single molecule. The expanded CCS library supports robust identification of antibiotic residues in complex matrices, improving QA/QC workflows and environmental monitoring.

Future Trends and Applications

Ongoing advances may include higher-resolution cyclic devices, integration with machine learning for CCS prediction, and application to broader classes of small molecules and biotherapeutics. The methodology supports rapid screening in food safety, pharmaceutical analysis, and environmental testing.

Conclusion

Cyclic IMS significantly enhances separation of fluoroquinolone charge-site isomers compared to linear TWIM. The multi-pass calibration strategy delivers reliable CCS estimates across increasing resolutions. The expanded CCS database for eighteen fluoroquinolones underpins improved analytical specificity and facilitates future method development.

Reference

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