Detection of Six Zeranol Residues in Animal- derived Food by HPLC-MS/MS

Significance of the Topic

The non-steroidal estrogenic agent zeranol is used to stimulate growth in livestock but poses endocrine disruption risks in humans. Its residues in animal-derived foods are regulated or banned in many countries, requiring sensitive assays for food safety monitoring.

Objectives and Study Overview

The study aimed to establish a robust LC-MS/MS method for simultaneous detection of six zeranol residues (α-zearalanol, β-zearalanol, α-zearalenol, β-zearalenol, zearalanone, zearalenone) in various matrices including muscle, liver, milk, and eggs.

Methodology

Sample preparation involved extraction of tissues, eggs, and milk with acetonitrile, followed by pH adjustment and cleanup on mixed-mode cation exchange SPE cartridges. Extracts were dried, reconstituted in aqueous acetonitrile, and centrifuged prior to analysis.

Instrumentation

HPLC was performed on a Surveyor system with a Hypersil GOLD column (150×2.1 mm, 5 µm) using a gradient of water with 0.1% formic acid (A) and acetonitrile (B) at 250 µL/min. MS/MS analysis employed a Thermo TSQ Quantum triple quadrupole with electrospray ionization in negative mode. SRM transitions were optimized for each analyte with specific collision energies.

Main Results and Discussion

Limits of detection for all six residues were 0.1 µg/kg, and limits of quantitation were 1.0 µg/kg, meeting EU and Chinese MRLs (2 µg/kg in meat, 10 µg/kg in liver). Extraction recoveries ranged from 65% to 115% with acceptable precision. SRM chromatograms demonstrated clear resolution and quantification capability.

Benefits and Practical Applications

The method delivers high sensitivity, selectivity, and reproducibility for routine monitoring of zeranol residues in food safety laboratories. Its compliance with international regulations supports quality assurance in the meat and dairy industries.

Future Trends and Potential Applications

Advancements may include automation of sample preparation, expansion to additional food matrices, integration with high-resolution MS for non-target screening, and development of faster chromatographic protocols for high-throughput analysis.

Conclusion

The developed LC-MS/MS assay provides a reliable tool for detecting low-level zeranol residues in diverse food samples, ensuring regulatory compliance and consumer safety.

Reference

• Leffers H, Næsby M, Vandelbo B, Skakkebæk N, Jørgensen M. Oestrogenic potencies of zeranol, oestradiol, diethylstilboestrol, bisphenol-A and genistein: implications for exposure assessment of potential endocrine disrupters. Human Reproduction. 2001;16(5):1037-1045.
• Liu S, Lin YC. Transformation of MCF-10A human breast epithelial cells by zeranol and estradiol-17beta. Breast J. 2004;10(6):514-521.
• Council Directive 96/23/EC on measures to monitor certain substances and residues in live animals and animal products. Official Journal of the European Communities L125. 23 May 1996:10-32.
• Ministry of Agriculture, P.R. China. Maximum Limit Standard of the Veterinary Drugs in Animals. Regulation 235. 2002.
• European Food Safety Authority. Opinion on hormone residues in bovine meat and meat products. EFSA Journal. 2007;510:1-62.