Rapid Screening Method for Veterinary Antibiotics Using an Advanced Solid Core UHPLC Column and UHPLC System with MS/MS Detection

Significance of the Topic

The rapid and reliable screening of veterinary antibiotics in food and feed matrices is essential for ensuring consumer safety, enforcing regulatory limits, and maintaining public confidence in animal-derived products. Efficient multi-analyte methods reduce laboratory turnaround times, lower per-sample costs, and support high-throughput testing demands in quality control and food safety laboratories.

Objectives and Study Overview

This study aimed to develop and demonstrate a fast UHPLC-MS/MS screening method capable of detecting 36 commonly used veterinary antibiotics—covering sulfonamides, penicillins, macrolides, fluoroquinolones, and tetracyclines—within a single, sub-five-minute analysis cycle. Key goals included achieving adequate separation of isobaric or co-eluting compounds and leveraging high-pressure UHPLC for throughput enhancement.

Methodology and Instrumentation

Sample Preparation:

• Primary standards (1 mg/mL) prepared in water/methanol (1:1, v/v), diluted to 100 ng/mL in 90:10 water/methanol.
• Virtuoso Vial Identification System used for sample tracking.

UHPLC Conditions:

• Column: Accucore Vanquish C18, 1.5 µm solid core, 100 × 2.1 mm.
• Mobile phases: 0.1% formic acid in water (A) and methanol (B).
• Gradient: 10%–90% B over 4.375 min; total cycle <9 min.
• Flow rate: 400 µL/min; column temperature: 40 °C.

MS/MS Detection:

• Instrument: Thermo Scientific Vanquish UHPLC with TSQ Vantage triple quadrupole.
• Ionization: Positive-mode HESI; SRM transitions optimized for each analyte.
• Source parameters: spray voltage 5000 V; vaporizer 500 °C; sheath gas 75 arb; aux gas 20 arb.

Main Results and Discussion

The method resolved all 36 antibiotics within a 5 min detection window, achieving baseline separation of critical isobaric pairs such as sulfadoxine and sulfadimethoxine. System pressures ranged from 840 to 1210 bar, demonstrating robust operation at high flow rates. Representative SRM chromatograms confirmed sharp, symmetric peaks and adequate sensitivity across compound classes.

Benefits and Practical Applications

The developed approach delivers:

• Rapid screening cycles (<5 min detection, <9 min total).
• High sample throughput and cost efficiency.
• Effective separation of isobaric and structurally similar antibiotics.
• Compatibility with standard triple quadrupole MS systems.

It is well suited for routine screening in food safety, veterinary residue monitoring, and regulatory compliance laboratories.

Future Trends and Applications

Emerging directions include:

• Integration with automated sample preparation and data processing workflows.
• Expansion of analyte panels to novel antimicrobial compounds and metabolites.
• Adoption of even smaller particle UHPLC columns and ultra-high-pressure platforms for further time reduction.
• Coupling with high-resolution MS for non-targeted screening and confirmatory analyses.

Conclusion

This work demonstrates a robust, high-throughput UHPLC-MS/MS screening method for 36 veterinary antibiotics using a 1.5 µm solid core C18 column and a high-pressure UHPLC system. The approach offers rapid analysis, reliable resolution of isobaric compounds, and practical applicability for routine food and feed safety testing.