High-throughput Screening Method for Multiple Classes of Antibiotics in Milk Using Automated Sample Preparation and LC-MS/MS

Significance of the Topic

Veterinary antibiotics are routinely used in livestock to prevent and treat diseases, but residues in milk can pose health risks and must be monitored under regulatory maximum residue limits. A rapid, multi-class screening approach enhances food safety control and laboratory efficiency.

Objectives and Study Overview

This study sought to develop a high throughput, sensitive, and reproducible LC-MS/MS screening method for multiple classes of antibiotic residues in milk, minimizing manual sample handling through automated online extraction.

Methodology and Instrumentation

Sample preparation involved protein precipitation of 100 µL milk with 900 µL 50 mM ammonium acetate in 50 % acetonitrile/water containing 7.5 mM EDTA, followed by centrifugation and direct injection. Online cleanup employed two TurboFlow Cyclone columns in tandem for extract filtration and matrix removal in under 15 minutes per sample. Chromatographic separation used a Phenyl/Hexyl HPLC column at 50 °C with a gradient of aqueous formic/trifluoroacetic acid, methanol, and isopropanol/acetone. Detection was by heated electrospray positive ion SRM on a triple quadrupole mass spectrometer. Key method parameters—ion transitions, collision energies, resolution, and gas settings—were optimized by direct infusion of standards.

Instrumentation Used

• Thermo Scientific Aria TLX-2 system with TurboFlow Cyclone 0.5×50 mm and Cyclone P 0.5×50 mm columns
• Thermo Scientific TSQ Quantum Ultra triple stage quadrupole with heated ESI source
• BETASIL Phenyl/Hexyl HPLC column (3.0×50 mm, 3 µm) and HOT POCKET heater

Results and Discussion

The method showed linear response (R²>0.99) and precision (RSD<15 %) over 50–500 µg/L. Limits of detection ranged from 0.4 to 5.2 µg/L, below most regulatory limits. Matrix effects varied by analyte and fat content, with suppression up to 85 % for albendazole and enhancement for difloxacin, but quantitation remained accurate using matrix-matched calibration. Blank samples exhibited no interfering peaks; real milk samples screened negative for residues above method LODs.

Benefits and Practical Applications

• Automated online extraction reduces manual steps and sample prep time to ~15 minutes per sample.
• Sensitive SRM detection enables multi-class antibiotic screening at low ppb levels.
• High throughput supports large sample loads in QA/QC and regulatory laboratories.

Future Trends and Opportunities

Future developments may include incorporation of isotopically labeled internal standards for improved compensation of matrix effects, extension to other dairy matrices, use of high-resolution MS for non-targeted screening, and integration into fully automated laboratory workflows.

Conclusion

This automated TurboFlow LC-MS/MS method provides a rapid, sensitive, and reproducible platform for multi-class antibiotic residue screening in milk. It offers significant gains in throughput and data quality while meeting regulatory requirements.

References

• Blasco C, Torres CM, Pico Y. Progress in analysis of residual antibacterials in food. Trends Anal Chem. 2007;26(9):895–913.
• Koesukwiwat U, Jayanta S, Leepipatpiboon N. Solid-phase extraction for multiresidue determination of sulfonamides, tetracyclines, and pyrimethamine in bovine milk. J Chromatogr A. 2007;1149(1):102–111.
• Kantiani L, Farré M, Sibum M, Postigo C, Alda MD, Barceló D. Fully automated analysis of beta-lactams in bovine milk by online SPE-LC-ESI-MS/MS. Anal Chem. 2009;81(11):4285–4295.