Automated Online Multi-Residue LC-MS/MS Method for the Determination of Antibiotics in Chicken Meat

Significance of the Topic

Antibiotics are routinely administered to poultry for treatment, prevention and growth promotion. Improper or excessive use leads to residues in meat that can pose health risks and undermine food safety standards. European regulations set maximum residue limits (MRLs) to protect consumers, creating a need for reliable, high-throughput analytical methods.

Objectives and Overview

This study presents a confirmatory multi-residue method for quantifying 36 antibiotics from seven classes in chicken meat. The approach integrates online sample cleanup with liquid chromatography–tandem mass spectrometry (LC-MS/MS) to meet EU Commission Decision 2002/657/EC requirements while maximizing throughput and cost-effectiveness.

Methodology and Instrumentation

The workflow combines a simple extraction and an automated on-line cleanup:

• Sample preparation: Homogenize ~150 g chicken, weigh 500 mg aliquot.
• Extraction: Add internal standard and acetonitrile/2 % TCA, vortex, centrifuge and filter.
• Online cleanup: Load extract onto TurboFlow Cyclone P column in the Transcend TLX-1 system, where macromolecules are flushed away and analytes retained.
• Analytical separation: Transfer analytes to a BetaSil Phenyl/Hexyl column for gradient LC separation (19 min run time).
• Detection: Use heated-ESI SRM on TSQ Quantum Access MAX triple quadrupole, quantifying via matrix-matched calibration and sulfaphenazole as internal standard.

Used Instrumentation

• Transcend TLX-1 online sample preparation system with TurboFlow Cyclone P column
• BetaSil Phenyl/Hexyl analytical column (50 × 2.1 mm, 3 µm)
• TSQ Quantum Access MAX triple quadrupole mass spectrometer
• Aria operating software and Xcalibur data system
• PAL autosampler with 100 µL syringe

Main Results and Discussion

Validation followed EU 2002/657/EC guidelines:

• Specificity: SRM transitions and ion ratios within permitted tolerance for all analytes.
• Linearity: Correlation coefficients > 0.99 over 0–400 µg/kg.
• Accuracy: Recoveries ranged 71 – 120 % across three spike levels.
• Precision: Repeatability RSD ≤ 27 %, intermediate precision ≤ 34 %.
• Detection limits: LOQs between 1 and 25 µg/kg, all below respective MRLs; LODs from 0.3 to 40 µg/kg.
• Decision limit (CCα) and detection capability (CCβ) values complied with regulatory thresholds.
• Throughput: Approximately 40 samples per day including preparation and analysis.

Benefits and Practical Applications

The method offers:

• High sample throughput from streamlined online cleanup and a single extraction.
• Reduced solvent consumption and maintenance demands due to clean extracts.
• Robust quantification of multiple antibiotic classes in compliance with EU law.
• Applicability in food safety laboratories for routine monitoring and regulatory enforcement.

Future Trends and Applications

Potential developments include:

• Expansion to additional veterinary drugs and emerging contaminants.
• Integration with high-resolution MS for broader screening capabilities.
• Application to other food matrices (milk, eggs, seafood).
• Advances in miniaturized and automated workflows to further increase throughput.
• Coupling with rapid screening techniques for tiered analysis strategies.

Conclusion

An automated TLX-MS/MS approach effectively quantifies 36 antibiotic residues in chicken meat with sensitivity, specificity and throughput suitable for regulatory laboratories. The method meets EU performance criteria and substantially reduces sample cleanup time and maintenance requirements.

References

1. European Commission. Council Regulation (EEC) No. 2377/90: Community procedure for establishing MRLs. Off. J. Eur. Comm. L156:23 (1994).
2. Martos PA, Jayasundara F, Dolbeer F, et al. Multiclass, multiresidue LC-MS/MS screening of 39 drugs in veal muscle. J. Agric. Food Chem. 58:5932-5944 (2010).
3. Bousova K, Mittendorf K. Multi-residue automated turbulent flow on-line LC-MS/MS determination of antibiotics in milk. Thermo Fisher Scientific Method 63551 (2012).
4. EU Commission Decision 2002/657/EC. Off. J. Eur. Commun. L221/8 (2002).