Determination of Lasalocid and Tylosin at Therapeutically Relevant Levels in Animal Feed Using the ACQUITY UPLC H-Class System and ACQUITY QDa Mass Detector

Significance of the topic

Regulatory laboratories require reliable methods to verify labeled levels of veterinary drugs in animal feed. Matrix complexity and evolving formulations often hinder conventional HPLC detectors and complicate quantification. The integration of a compact mass detector addresses these challenges by enhancing selectivity and reducing sample preparation demands.

Objectives and overview of the study

This study compared the performance of traditional HPLC with photodiode array or fluorescence detection against a UPLC system coupled with a compact mass detector for the quantification of lasalocid and tylosin at therapeutically relevant levels in diverse feed matrices.

Methodology and instrumentation

• Standards and samples: Lasalocid sodium and tylosin standards prepared in acidified methanol or phosphate buffer/methanol; feeds included soy, corn and non-grain bases in pellet and meal forms.
• Lasalocid extraction: 10 g feed with acidified methanol, sonication and orbital shaking, followed by filtration.
• Tylosin extraction: 10 g feed with phosphate buffer and methanol, analogous procedure.
• HPLC-FLD and HPLC-PDA: Waters Alliance 2695, Waters 474 fluorescence detector, Waters 996 PDA detector; isocratic or gradient methods, injection volumes of 20 μL.
• UPLC-MS: Waters ACQUITY UPLC H-Class System, ACQUITY QDa Mass Detector, Empower 3 CDS software; CORTECS UPLC C18+ and BEH C18 columns; gradient elution, SIR and full scan at unit mass resolution.

Main results and discussion

• Calibration curves for both analytes exhibited correlation coefficients above 0.9989 across methods.
• Deviations between HPLC and UPLC-MS results for lasalocid ranged from 2.3% to 27.5% and for tylosin from 0.1% to 11.8%, generally within acceptable regulatory limits.
• The compact mass detector achieved baseline separation and unambiguous identification with simplified sample preparation.
• Ten replicate injections demonstrated excellent repeatability (retention time RSD 0.07%, area RSD 1.2%).
• Tylosin urea adduct (TUA) was detected selectively by UPLC-MS at m/z 958.6 and 1018.7, including residual levels undetectable by conventional HPLC.

Benefits and practical applications of the method

• Superior selectivity and reduced interference improve confidence in feed supplement quantification.
• Streamlined sample preparation protocols and rapid system readiness lower operational complexity.
• Cost effective adoption of mass detection in routine feed regulatory testing.
• Capability to detect unexpected feed-related adducts such as TUA enhances quality control.

Future trends and potential applications

• Expanded multi-analyte workflows for simultaneous screening of additional veterinary drugs.
• Refined extraction protocols to further simplify laboratory operations.
• Integration of compact mass detectors for real-time feed monitoring in industrial settings.
• Development of certified reference materials for comprehensive method validation.

Conclusion

The study demonstrated that a UPLC system coupled with a compact mass detector provides a sensitive, selective and user-friendly solution for the determination of lasalocid and tylosin in complex feed matrices. The approach meets regulatory requirements, offers excellent reproducibility and reveals additional analyte forms such as TUA, highlighting its potential for broad implementation in feed testing laboratories.

References

1. AOAC INTERNATIONAL. Official Methods of Analysis, 19th edition, 2012.
2. AOAC INTERNATIONAL. Official Method 2008.01, in Official Methods of Analysis, 19th edition, 2012.
3. Association of American Feed Control Officials. Proficiency Testing Program Method Performance Reports, 2015.
4. Houglum JE, Tasler MK. Liquid chromatographic assay of tylosin in animal feeds. Journal of AOAC International. 1996;79(3):369–374.