Analysis of Nitroimidazoles in Egg Using Agilent Captiva EMR—Lipid and LC/MS/MS

Significance of the Topic

Effective monitoring of veterinary drug residues in animal-derived foods is critical for food safety and regulatory compliance. Nitroimidazoles, a class of antibiotics once used in poultry production, pose health risks and are banned in many regions. Robust analytical workflows are needed to detect trace levels of these compounds and their metabolites in complex matrices such as egg.

Objectives and Study Overview

This study aimed to develop and validate a streamlined method for the simultaneous determination of four nitroimidazoles (metronidazole, dimetridazole, ronidazole, ipronidazole) and their hydroxy metabolites in chicken eggs. The focus was on achieving high accuracy, precision, and efficient removal of lipids and pigments prior to LC/MS/MS analysis.

Methodology and Instrumentation Used

A QuEChERS-based extraction in acetonitrile with 5% formic acid was employed to precipitate proteins and extract analytes from 5 g egg samples. Cleanup utilized a 3 mL Agilent Captiva EMR—Lipid cartridge operated in pass-through mode to selectively remove lipid components. Chromatographic separation was performed on an Agilent 1290 Infinity LC with a Poroshell 120 EC-C18 column (3.0×100 mm, 2.7 µm) using a gradient of 0.1% formic acid in water and acetonitrile. Detection employed an Agilent 6495B triple quadrupole MS in positive electrospray mode with optimized MRM transitions. A complementary GC/MS full-scan evaluation on an Agilent 9000/7000 system assessed residual matrix interferences.

Main Results and Discussion

Calibration was linear (R² 0.993–0.995) over 0.1–50 ng/g for most analytes (0.5–100 ng/g for HMMNI). Recoveries across low (1 ng/g), mid (5 ng/g), and high (10 ng/g) QC levels ranged from 86.5% to 118.3%, with RSDs below 6%. GC/MS full scans demonstrated 76% reduction in matrix signals after EMR—Lipid cleanup. Visual inspection of dried extracts confirmed substantial removal of pigments and lipids, ensuring clean LC/MS/MS analysis.

Benefits and Practical Application of the Method

• The pass-through EMR—Lipid protocol simplifies sample cleanup by eliminating multiple pH adjustments and SPE steps.
• High recoveries and low variability meet regulatory validation criteria for residue analysis.
• Efficient lipid removal reduces matrix effects, extending instrument uptime and data quality.
• The workflow is adaptable to other fatty matrices and multi-residue panels in food safety laboratories.

Future Trends and Potential Applications

Integration of EMR cleanup with automated QuEChERS platforms could boost throughput in routine testing. Expansion to high-resolution mass spectrometry may enable broader screening of emerging veterinary drugs. Coupling with miniaturized sample preparation and direct-infusion MS could further streamline residue monitoring in various food matrices.

Conclusion

The combined QuEChERS extraction and Agilent Captiva EMR—Lipid cleanup provides a rapid, robust, and selective method for trace analysis of nitroimidazoles and their metabolites in egg. Validation data demonstrate excellent accuracy, precision, and matrix removal efficiency, supporting its implementation in food safety and regulatory laboratories.

References

1. Council Regulation (EEC) No. 2377/90 on maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Official Journal of the European Communities L224, 1990.
2. Cronly M., et al. Rapid confirmatory method for the determination of 11 nitroimidazoles in egg using liquid chromatography tandem mass spectrometry. J. Chromatogr. A 2009, 1216, 8101–8109.
3. Granja R., et al. Determination and confirmation of metronidazole, dimetridazole, ronidazole, and their metabolites in bovine muscle by LC-MS/MS. Food Addit. Contam. A 2013, 30, 970–976.
4. Mitrowska K., Posyniak A., Zmudzki J. Multiresidue method for the determination of nitroimidazoles and their hydroxy-metabolites in poultry muscle, plasma, and egg by isotope dilution LC-MS. Talanta 2010, 81, 1273–1280.