Screening Method for Fluoroquinolones Using an Advanced UHPLC Column and System Combination

Importance of the Topic

Fluoroquinolones represent a class of broad-spectrum antibiotics critically monitored in pharmaceutical and environmental testing. Their chemical structure, featuring polyaromatic rings, fluorine atoms, and a carboxylic acid moiety, can interact with silanol groups on conventional stationary phases, leading to peak tailing, poor resolution, and compromised quantitative results. Developing a robust, high-throughput screening method for fluoroquinolones is essential for compliance with regulatory limits, ensuring food safety, and supporting quality control in drug development.

Objectives and Study Overview

This application aimed to demonstrate a rapid UHPLC screening approach for 25 fluoroquinolone compounds using Thermo Scientific Hypersil GOLD VANQUISH 1.9 µm column in combination with a Vanquish UHPLC system. The study focused on achieving improved peak shape, high resolution, and exceptional retention time precision without relying on mass spectrometric detection, thereby simplifying instrument requirements and reducing operational costs.

Methodology and Instrumentation

A non-linear gradient method was employed over a 24-minute run time, optimizing separation of complex fluoroquinolone mixtures. Key UHPLC conditions included:

• Column: Hypersil GOLD VANQUISH, 1.9 µm, 200 × 2.1 mm
• Mobile phases: A—0.1% formic acid in water; B—0.1% formic acid in acetonitrile
• Gradient profile: Initial 10% B held for 1 min, ramped to 60% B at 17 min (non-linear curve value 8), then returned to starting composition by 20 min
• Flow rate: 0.4 mL/min; Column temperature: 40 °C; Injection volume: 1 µL; UV detection at 254 nm

The Vanquish UHPLC system modules comprised:

• Binary Pump H with low pulsation and non-linear gradient capability
• Split Sampler HT with intelligent sample pre-compression
• Active pre-heater; Column Compartment H; Diode Array Detector HL; LightPipe flow cell
• Data acquisition: Chromeleon 7.2 SR2 chromatography data system

Sample preparation involved dissolving each analyte at 1 mg/mL in water/methanol (1:1, v/v), aided by formic acid to ensure complete solubility, followed by dilution to 100 µg/mL.

Main Results and Discussion

The non-linear gradient provided adequate separation for all 25 target compounds, with some closely eluting peaks still resolved sufficiently for consistent identification. Retention time reproducibility was outstanding, with relative standard deviations below 0.1% across six replicate injections. Typical USP resolution values exceeded 1.0 for challenging pairs, and large resolution factors (up to 35) were observed for early and late eluting analytes. Chromatogram overlays confirmed retention time RSDs below 0.05% for most peaks, underlining the benefit of the system’s low extra-column volume and stable flow delivery.

Benefits and Practical Applications

• Simple UV-based screening replaces more complex MS approaches while maintaining required resolution and reproducibility
• High sensitivity and peak capacity for trace-level analysis
• Robust, high-throughput method suitable for routine QC laboratories and regulatory monitoring
• Reduced solvent usage and run time compared to conventional HPLC methods

Future Trends and Potential Applications

Advances in UHPLC column chemistries and pump technologies will continue to push analysis speed and resolution boundaries. Integration of intelligent software wizards and automated data processing will streamline method development and reporting. Expanding similar non-linear gradient workflows to other challenging analyte classes (e.g., polar pesticides, biologically active peptides) can further enhance laboratory productivity and compliance.

Conclusion

This work illustrates that the Hypersil GOLD VANQUISH UHPLC column combined with the Vanquish UHPLC system and Chromeleon software enables a reliable, non-linear gradient screening method for 25 fluoroquinolones using only UV detection. The approach delivers exceptional peak shapes, high resolution, and outstanding retention time precision, offering a streamlined alternative to MS-based methods for routine screening.

Reference

• Derek Hillbeck, "Screening Method for Fluoroquinolones Using an Advanced UHPLC Column and System Combination," Thermo Fisher Scientific Application Note AN21500-EN, 2016.