Utility of the ACQUITY UPLC I-Class System and Ion Mobility in a Routine Workflow to Understand the Challenge of Analyzing Fluoroquinolone Antibiotic Residues

Significance of the topic

Fluoroquinolone antibiotics are widely used in veterinary medicine but pose a risk of residue in food products. Reliable detection and identification of these compounds and their gas phase protonated forms, known as protomers, are critical to ensure food safety and regulatory compliance. Incorporating ion mobility spectrometry into an ultra high performance liquid chromatography workflow adds an orthogonal separation dimension, improving specificity and confidence in residue analysis.

Objectives and Study Overview

This study aimed to evaluate the ACQUITY UPLC I-Class System coupled with ion mobility mass spectrometry for routine screening of fluoroquinolone residues in crude porcine tissue extracts. Key goals included the identification of multiple protomer forms, determination of collision cross section values for each species, and assessment of the impact of complex matrices on protomer ratios.

Methodology and Instrumentation

Porcine muscle samples were fortified with 25 veterinary drugs and extracted using a mixed aqueous organic solvent followed by centrifugation. Chromatographic separation employed an ACQUITY UPLC I-Class System with a BEH C18 column (1.7 μm, 100 x 2.1 mm) at 40 C and a water-acetonitrile gradient (both containing 0.1 percent formic acid). Mass analysis used a SYNAPT G2-S HDMS with electrospray ionization in positive mode. Ion mobility measurements were performed with N2 drift gas, T Wave velocity of 900 msec-1, and a pulse height of 40 V. Data were acquired with MassLynx software and processed in Waters UNIFI for protomer characterization and CCS library building.

Main Results and Discussion

Ion mobility enabled clear separation of nine fluoroquinolone compounds into two protomeric species each, yielding 18 distinct features. Collision cross section differences between protomers ranged from 6 to 12 A2, exemplified by ciprofloxacin values of 108.7 A2 and 119.1 A2. Fragmentation spectra acquired simultaneously for each protomer allowed assignment of protonation sites on acidic or basic moieties. Matrix effects were observed as fluctuations in protomer abundance across replicate porcine extracts. Spectral cleanup via ion mobility filtering removed background interferences and delivered mass accuracy below 1 ppm and CCS error within 2 percent.

Practical Benefits and Applications

Integrating ion mobility with UPLC-MS enhances routine residue screening by providing:

• Additional identification parameter in the form of CCS values
• Orthogonal separation to resolve protomer and matrix interferences
• Simultaneous acquisition of precursor and fragment ion data for all components
• Retrospective data analysis capabilities for method optimization

Future Trends and Opportunities

Expansion of CCS reference libraries and further software advancements will streamline high throughput screening. Emerging workflows may couple ion mobility with data independent acquisition modes for comprehensive non-targeted analysis. Standardization of CCS measurements across labs will improve inter-laboratory reproducibility and support regulatory testing.

Conclusion

This work demonstrates the utility of the ACQUITY UPLC I-Class system combined with ion mobility spectrometry for robust identification of fluoroquinolone protomers in complex tissue matrices. The approach adds confidence through CCS measurements and spectral cleanup, supporting reliable residue analysis in food safety applications.

Reference

• McCullagh M Stead S Williams J de Keizer W Bergwerff A Identification of multiple sites of intra molecular protonation and fragmentation patterns for fluoroquinolone antibiotics in porcine extracts using travelling wave ion mobility MS Waters Appl Note 720004720EN 2013
• Verdon E Couedor P Roudaut B Sanders PJ Multiresidue method for ten quinolone antibacterial residues in animal tissues by LC fluorescence detection AOAC Int 2005 88 1179 1192
• Kaufmann A Butcher P Maden K Widmer K Giles K Uria D Are LC ESI Tandem quadrupole fragmentation ratios unequivocal confirmation criteria Rapid Commun Mass Spectrom 2009 23 985 998
• Mol HG Zomer P de Koning M Validation of a screening method for pesticides in fruits and vegetables based on LC Orbitrap MS Anal Bioanal Chem 2012 403 2891 2908
• Croley TR White KD Callahan J Musser SM The chromatographic role in high resolution MS for non targeted analysis J Am Soc Mass Spectrom 2012 23 1569
• Commission Decision 2002 657 EEC Official Journal European Communities 2002
• FDA Final Decision Docket 2000N 157 Withdrawal of enrofloxacin approval in poultry 2000
• FDA CVM June 2 1997 Order prohibits extra label use of fluoroquinolones and glycopeptides
• EU Regulation 1831 2003 on additives for use in animal nutrition Official Journal EU L268 29 43 2003
• EU Regulation 2377 90 MRLs for veterinary medicinal products Official Journal EU L24 1 8 1990
• Lalli PM Iglesias BA Toma HE de Sa GF Daroda RJ Silva Filho JC Szulejko JE Araki K Eberlin MN Protomers formation separation and characterization via travelling wave ion mobility MS J Mass Spectrom 2012 47 712 719