Trace Level Determination of Eleven Antiprotozoal Agents in Eggs after Simple Matrix Clean-Up

Importance of the Topic

The presence of coccidiostat residues in chicken eggs poses a significant food safety concern due to strict EU maximum residue limits (MRLs). Reliable trace-level detection of eleven approved antiprotozoal agents is essential for regulatory compliance and consumer protection.

Goals and Study Overview

This work aimed to develop an automated on-line solid phase extraction coupled with UHPLC–MS/MS for simultaneous quantification of eleven coccidiostats in eggs after a streamlined acidified acetonitrile extraction and simple matrix clean-up.

Methods and Instrumentation

Sample Preparation:
• Homogenization of egg white and yolk from six eggs
• Extraction with acidified acetonitrile, salt-induced phase separation, centrifugation
• Evaporation to dryness and reconstitution in 50% water/acetonitrile

On-Line SPE–UHPLC–MS/MS Workflow:
• Automated solid phase extraction using Thermo Scientific SolEx HRP cartridge
• Dual-gradient Dionex UltiMate 3000 system with dedicated loading, analytical and dilution pumps for controlled dilution before/after SPE
• Separation on Thermo Scientific Acclaim PolarAdvantage II (2.1×150 mm, 3 µm) column
• Detection by triple quadrupole tandem mass spectrometer in ESI positive/negative SRM mode

Instrumentation:

• Dionex UltiMate 3000 UHPLC with dual ternary and binary pumps, autosampler and column compartment
• SolEx HRP SPE cartridge and optional TurboFlow Cyclone-P comparison cartridge
• Viper fingertight fittings for low-dead-volume switching
• Triple quadrupole mass spectrometer with SRM transitions for each analyte

Results and Discussion

Comparison of SolEx HRP with Cyclone-P showed superior balanced retention of polar and non-polar coccidiostats on SolEx HRP. The three-pump configuration enabled on-line dilution to improve retention and peak focusing. Two complementary methods covered all eleven analytes in separate runs (polar vs non-polar transfer solvents). UHPLC achieved baseline resolution, and on-line SPE reduced baseline noise by over four-fold compared to off-line SPE, effectively doubling sample throughput (3 h vs 6 h for ten samples). Calibration curves were linear (r>0.987), limits of detection ranged from 0.02 to 0.5 µg/kg, and limits of quantitation from 0.06 to 1.5 µg/kg, all below EU MRLs. Recoveries at 30 µg/kg spiking level were 90–118%, with measurement uncertainties of 10–37%.

Benefits and Practical Application

This fully automated approach minimizes manual labor and solvent exposure while enhancing reproducibility, sensitivity and data quality. It is well suited for routine screening and confirmatory analysis in official food control laboratories.

Future Trends and Opportunities

Further integration of high-throughput on-line SPE–LC–MS platforms may enable multiplexed multi-class residue screening. Advances in microflow LC and faster gradient cycles could further boost throughput. Expansion to other veterinary drug classes and continuous on-line monitoring are promising developments.

Conclusion

The developed on-line SPE–UHPLC–MS/MS method meets EU regulatory criteria, delivering robust, sensitive and high-throughput determination of eleven coccidiostats in chicken eggs.

Reference

• Kämpfer P. et al., Thermo Fisher Custom Application Note 116
• Regulation (EC) No 1831/2003, No 37/2010, No 124/2009
• Commission Decision 2002/657/EC