Analysis of Polyether Antibiotics in Animal Feeds Using Post-Column Derivatization

Significance of the Topic

Polyether antibiotics such as salinomycin, monensin and narasin are widely used as feed additives and regulated by international standards. Reliable quantification in animal feeds is critical for ensuring food safety and compliance. Traditional microbiological assays are time consuming, whereas HPLC with post-column derivatization offers rapid, sensitive and automated analysis.

Objectives and Overview of the Study

• Develop and validate a post-column visible photometric derivatization HPLC method for three polyether antibiotics in feed samples.
• Demonstrate method performance in terms of linearity, repeatability, recovery and separation efficiency.

Methodology

• Sample preparation: Extraction of feed according to official feed analysis standards.
• Chromatographic separation: Reversed-phase C18 column with isocratic elution using water, methanol and acetic acid in a 60:940:1 volumetric ratio at 0.6 mL per minute and 40 °C.
• Post-column derivatization: Reaction with vanillin in acidic methanol at 95 °C in a thermostatted coil.
• Detection: Photometric measurement at 520 nm.

Used Instrumentation

• Nexera lite HPLC system
• CRB-40 chemical reaction box with uniform air circulation
• DGU-405 degassing unit
• SPD-40V UV-VIS detector with inert cell
• SIL-40 autosampler

Main Results and Discussion

• All calibration curves showed excellent linearity with determination coefficients above 0.999 over relevant concentration ranges.
• Repeatability at 0.25 µg/mL yielded retention time and peak area RSD values below 1.0 percent.
• Spike recovery in feed extracts at 0.5 µg/mL ranged from 100.6 to 104.6 percent with chromatographic resolution above 1.9 between all analyte peaks.
• The post-column derivatization provided stable, sensitive detection and complete separation from potential coeluting components.

Benefits and Practical Applications

• Significant reduction in analysis time with total pretreatment around 20 minutes compared to 16 to 24 hours for microbiological assays.
• Fully automated workflow from instrument startup to shutdown improves efficiency and reduces manual labor.
• High precision and accuracy meet regulatory requirements for feed additive monitoring.
• Suitable for routine quality control in feed production and regulatory laboratories.

Future Trends and Potential Applications

• Coupling with mass spectrometry for enhanced specificity and lower detection limits.
• Expansion to multi-residue screening of emerging veterinary drugs in feeds and environmental matrices.
• Development of portable HPLC systems for on-site rapid testing.
• Integration with automated sample preparation for high-throughput analysis.

Conclusion

The presented post-column derivatization HPLC method enables fast, reliable and fully automated quantification of polyether antibiotics in animal feeds. It meets regulatory standards, delivers excellent linearity, precision and recovery, and significantly enhances laboratory productivity.

References

1. Public Notice No 750 on Feed Additives, Ministry of Agriculture, Forestry and Fisheries, Japan, July 24, 1976.
2. Feed Analysis Standards, Food Safety and Consumer Affairs Bureau, MAFF Notice No 19/14729, April 1, 2008.
3. Ministerial Ordinance No 35 on Specifications and Standards of Feeds and Feed Additives, MAFF, July 24, 1976.