Identification of Multiple Sites of Intra-Molecular Protonation and Different Fragmentation Patterns within the Fluoroquinolone Class of Antibiotics in Porcine Muscle Extracts Using Travelling Wave Ion Mobility Mass Spectrometry

Importance of the Topic

The monitoring of fluoroquinolone antibiotic residues in food of animal origin is critical for public health and regulatory compliance. These compounds are widely used in livestock for infection control and growth promotion, but their persistence in tissues can contribute to antimicrobial resistance. Advanced analytical techniques that improve selectivity, sensitivity, and confidence in residue identification are essential for ensuring food safety and meeting international maximum residue limits.

Study Objectives and Overview

This study explored the use of ultra-performance liquid chromatography coupled with travelling wave ion mobility high-definition mass spectrometry (UPLC-IMS HDMS) to:

• Resolve multiple intra-molecular protonation sites (protomers) of fluoroquinolones in porcine muscle extracts.
• Obtain single-component precursor and fragmentation spectra for each protomer.
• Evaluate the potential of drift time as an additional identification point.

Methodology and Instrumentation

Extracts from fortified porcine muscle were prepared by homogenization, centrifugation, and dilute-and-shoot. UPLC separations were performed on an ACQUITY UPLC BEH C18 column using a water/acetonitrile gradient with 0.1% formic acid. Ionization was by positive ESI (2.0 kV) on a SYNAPT G2-S mass spectrometer. Travelling wave ion mobility used N₂ or CO₂ as drift gas. Data were acquired in HDMSE mode, capturing precursor and fragmentation information in a single injection.
Used instrumentation:

• ACQUITY UPLC System (Waters Corporation)
• ACQUITY UPLC BEH C18 Column, 1.7 µm, 50 × 2.1 mm
• SYNAPT G2-S Mass Spectrometer with ESI source
• MassLynx and DriftScope Software

Key Results and Discussion

• Ciprofloxacin eluted at 2.19 min, with accurate mass [M+H]+ at m/z 332.1410 (0 ppm error).
• Ion mobility separated two protomers with drift times of 4.34 ms and 5.48 ms, corresponding to protonation on acidic and basic sites.
• Single-component MSE fragmentation showed protomer-specific fragments: acidic-site protomer yielded m/z 314 and 231; basic-site protomer yielded m/z 288 and 245.
• Doubly charged species (m/z 157, 166) were confirmed by ion mobility and shown to depend on ion source conditions.
• CO₂ drift gas increased ion mobility resolution (Rs up to 4.13) compared to N₂, enabling baseline separation of fluoroquinolone protomers.
• Orthogonal gas-phase separation improved spectral cleanliness and reduced matrix interferences, minimizing sample preparation.

Benefits and Practical Applications

• Additional identification point via protomer drift time enhances method specificity.
• Simultaneous acquisition of accurate precursor and fragmentation spectra in one run increases throughput.
• Improved resolution of coeluting compounds and matrix components reduces false positives.
• Supports robust method development and inter-laboratory reproducibility by visualizing protonation variability.

Future Trends and Potential Applications

• Extension of protomer separation strategies to other antibiotic classes and small-molecule residues.
• Optimization of drift gas composition and mobility conditions for even higher resolution.
• Integration of drift time data into regulatory confirmatory workflows as a standard identification parameter.
• Application of IMS-based cleanup approaches to reduce reliance on solid-phase extraction.
• Use of protomer profiling in pharmacokinetic and bioavailability studies.

Conclusion

Travelling wave ion mobility mass spectrometry coupled to UPLC provides powerful orthogonal separation and single-component fragmentation analysis for fluoroquinolone antibiotics in complex tissue matrices. The ability to resolve and characterize multiple protonation sites enhances identification confidence, improves spectral purity, and offers new identification points for regulatory compliance. Continued development of IMS-based approaches will further advance residue monitoring and analytical robustness.

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