Completely Automated Extraction and Determination of Antibiotics in Eggs using a Robotic Autosampler and LC-MS/MS Platform

Importance of the topic

Eggs may contain antibiotic residues that pose health risks and promote bacterial resistance. Reliable, high-throughput screening methods are essential to ensure consumer safety and regulatory compliance.

Objectives and Study Overview

This work describes the full automation of liquid–liquid extraction and phospholipid cleanup for antibiotic analysis in eggs. The goal was to streamline sample preparation, improve reproducibility, and achieve sensitive LC-MS/MS detection with minimal matrix interference.

Methodology

Homogenized egg samples were spiked with a deuterated internal standard and extracted automatically using an 80:20 acetonitrile:water solvent. Mixing was performed with the GERSTEL QuickMix at 2000 rpm for 2.5 minutes, followed by centrifugation. Supernatants were passed through Oasis PRiME HLB cartridges for phospholipid removal, then diluted and transferred for LC-MS/MS analysis. Calibration standards and QC levels were prepared in eggs to cover relevant concentration ranges.

Applied Instrumentation

• GERSTEL MPS robotic PRO sampler with QuickMix option, CF-200 centrifuge, and universal filtration
• Agilent 1260 HPLC system
• Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm)
• Agilent Ultivo Triple Quadrupole Mass Spectrometer with jet-stream ESI

Main Results and Discussion

The automated mixing using QuickMix produced the most uniform extraction compared to traditional agitator methods, reducing mixing time by over 90%. The SPE cleanup effectively removed co-extracted phospholipids, as confirmed by phospholipid monitoring. Calibration curves for six antibiotics exhibited excellent linearity (R2 ≥ 0.99). Limits of quantitation were five times lower than industry minimum reporting limits. Accuracy ranged from 94.6% to 115% and precision (RSD) from 1.23% to 11.2% across QC levels.

Benefits and Practical Applications

Automating the entire extraction and cleanup workflow minimizes manual errors, enhances reproducibility, and increases throughput. The method meets regulatory requirements for antibiotic residue monitoring in eggs and can be applied in food safety laboratories for routine screening.

Future Trends and Potential Applications

Further development may include extension to other food matrices, integration with online data processing platforms, miniaturization of extraction volumes, and the use of greener solvents. Advancements in robotics and MS technologies will continue to drive high-throughput, cost-effective monitoring of veterinary drug residues.

Conclusion

The study demonstrates a robust, fully automated LC-MS/MS workflow for antibiotic analysis in eggs, combining fast, efficient extraction, reliable phospholipid removal, and precise quantitation. This platform supports high-throughput operations and stringent food safety standards.

References

• Waters Application Note: Simple and Effective Cleanup for UPLC-MS/MS Determination of Veterinary Drug Residues in Eggs.