Analysis of metronidazole and ronidazole in milk using the SCIEX Triple Quad™ 3500 LC-MS/MS System

Significance of the Topic

A reliable, rapid method for detecting nitroimidazole residues in milk is essential due to public health concerns over carcinogenic and mutagenic effects of metronidazole and ronidazole. Regulatory bodies worldwide have banned these compounds in livestock production and established minimum required performance limits (MRPL) to ensure consumer safety.

Objectives and Study Overview

This study aimed to develop and validate a streamlined LC-MS/MS approach using the SCIEX Triple Quad™ 3500 system to quantify metronidazole and ronidazole in milk below established MRPLs. The method was tested on local market samples in India to demonstrate practical applicability.

Methodology and Instrumentation

• Sample Preparation: A five‐step protocol involving acidified acetonitrile extraction, salt-induced phase separation, nitrogen evaporation, and reconstitution in a water–acetonitrile–formic acid mixture, followed by filtration.
• Chromatography: Nexera XR UHPLC with a Phenomenex Luna C18 column (3.0×150 mm, 3 µm), gradient elution with water and acetonitrile (0.1% formic acid), 20 µL injection.
• Mass Spectrometry: SCIEX Triple Quad 3500 operated in positive ESI MRM mode, monitoring two transitions per analyte. Data acquired with Analyst® and processed in MultiQuant™ 3.0.2.

Key Results and Discussion

The method achieved baseline separation with retention times of 1.97 min for metronidazole and 2.25 min for ronidazole. Calibration in milk extract was linear from 0.5 to 20 ppb (r² ≥ 0.99). The limit of detection in aqueous solution was 0.25 ppb (CV < 7%), and the quantification limit in milk was 0.5 ppb. Recoveries at 1 ppb ranged between 90% and 110%, meeting Commission Decision 2002/657/EC criteria. Analysis of market samples revealed no detectable residues.

Benefits and Practical Applications

• Rapid, simple sample preparation suitable for high‐throughput screening.
• High sensitivity and accuracy below regulatory MRPL levels.
• Compatibility with routine QA/QC workflows in food safety laboratories.

Future Trends and Opportunities

Advances may include miniaturized extraction techniques, streamlined on‐line sample cleanup, and integration with high‐resolution MS to extend coverage to additional veterinary drug classes. Automation and multiplexing could further enhance throughput in regulatory monitoring.

Conclusion

The presented LC-MS/MS method offers a robust, validated solution for routine quantification of metronidazole and ronidazole in milk at sub‐ppb levels. Its simplicity and compliance with international guidelines make it readily adoptable in food safety laboratories.

References

1. Cronly M, Behan P, Foley B, Malone E, Martin S, Doyle M, Regan L. Rapid multi-class multi-residue method for the confirmation of chloramphenicol and eleven nitroimidazoles in milk and honey. Food Addit Contam. 2010;27(9):1233–1246.
2. Food Safety and Standards (Contaminants, Toxins and Residues) Regulation. 2011.
3. FSSAI. Method Validation Protocol for Veterinary Drugs Including Antibiotics Chapter 8.
4. Commission Decision 2002/657/EC of 12 August 2002. Official Journal of the European Communities. L221:8–36.