Fast separation of coccidiostats

Importance of the Topic

Coccidiostats are widely employed in poultry and other livestock feeds to suppress protozoan infections and maintain animal health. Regulatory bodies and food safety authorities are increasingly focused on monitoring these compounds to prevent residue accumulation and potential resistance development. Fast, reliable analytical methods are therefore essential for quality control in feed production, veterinary pharmacology, and compliance with evolving legislation.

Objectives and Overview

This application note presents an optimized UHPLC method using a solid-core Phenyl-Hexyl column to separate four key coccidiostats. The goal is to significantly accelerate analysis time and reduce per-sample costs compared to conventional HPLC protocols, while preserving chromatographic resolution and reproducibility.

Methodology and Instrumentation

Sample preparation involved dissolving reference standards of 4-amino-3,5-dinitrobenzamide, nitromid, zoalene, and ethopabate at 1 mg/mL in water/acetonitrile (20:80 v/v), with working solutions at 0.1 mg/mL in water/acetonitrile (80:20 v/v). Vials were labeled via an automated Virtuoso system.

UHPLC conditions for the final method:

• Column: Accucore Phenyl-Hexyl, 2.6 µm, 100 × 2.1 mm
• Mobile phases: A = water/methanol 95:5 v/v; B = water/methanol 5:95 v/v
• Gradient: 0–0.07 min 15% B; 0.07–1.47 min to 85% B; 1.48 min return to 15% B; hold to 4.7 min
• Flow rate: 0.6 mL/min; column temperature: 50 °C with pre-heater; injection volume: 1 µL
• Detection: Diode array at 268 nm

Used Instrumentation

• Thermo Scientific Vanquish Flex Quaternary UHPLC System
• Quaternary Pump F and System Base modules
• Split Sampler FT with SmartInject technology
• Column Compartment H with Active Pre-heater
• Diode Array Detector HL with LightPipe flow cell
• Thermo Scientific Chromeleon 7.2 SR4 software

Main Results and Discussion

By raising the column temperature from 40 °C to 50 °C and increasing flow from 0.4 to 0.6 mL/min, all four analytes eluted within 2.5 minutes, with full equilibration under 5 minutes. Twenty-four replicate injections showed retention time RSDs below 0.1%, far surpassing typical legacy HPLC precision. Peak resolution between closely eluting pairs exceeded 3.5, and peak shapes remained sharp across runs.

Comparison with a conventional 150 × 4.6 mm column (15 min run, 1.0 mL/min) demonstrated a three-fold throughput gain and a five-fold reduction in solvent use and waste generation.

Benefits and Practical Applications

• Rapid turnaround of results supports high-throughput screening in feed safety laboratories.
• Lower solvent consumption reduces operational costs and environmental impact.
• High retention time stability enhances data confidence for regulatory compliance.

Future Trends and Prospects

Continued development of UHPLC stationary phases and system hardware will drive further reductions in analysis time. Integration with mass spectrometry could expand selectivity for multi-residue screening. Automation and digital tracking systems will streamline sample handling and data management, meeting stricter regulatory demands and enabling real-time monitoring.

Conclusion

The described UHPLC method achieves sub-5-minute separation of four coccidiostats with exceptional precision and reduced solvent usage. It offers a robust, high-throughput alternative to legacy HPLC, aligning with modern demands for rapid, cost-effective feed safety analysis.

References

1. FVE. Position paper on coccidiostats. Federation of Veterinarians of Europe; 2004.
2. D. Hillbeck. Improved analysis of veterinary drug coccidiostats using a Hypersil GOLD Phenyl HPLC column. Thermo Fisher Scientific Application Note; 2017.
3. Thermo Fisher Scientific. Accucore HPLC Columns Technical Guide; 2017.