Validated Method for the Determination of Phenylbutazone in Horse Meat with LC-MS/MS

Significance of the Topic

The presence of phenylbutazone, a nonsteroidal anti-inflammatory drug, in horse meat poses a potential risk to human health and violates food safety regulations. Routine monitoring is essential to ensure compliance with European Commission limits and to prevent illegal residues entering the food chain.

Aims and Overview of the Study

This work describes the development and in-house validation of a sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantifying phenylbutazone in horse meat. The method achieves a limit of quantification below the 5 µg/kg action limit set by the European Commission and is suitable for routine food safety testing.

Methodology and Instrumentation

Sample preparation combines enzymatic deconjugation with a two-step cleanup to remove fats and matrix interferences, followed by direct LC-MS/MS analysis:

• Enzymatic hydrolysis of phenylbutazone conjugates using β-glucuronidase in acetate buffer
• Extraction with acetonitrile and defatting on a silica SPE cartridge
• C18 SPE cleanup, evaporation and reconstitution in mobile phase
• UHPLC separation on a C18 column with a water–formic acid/acetonitrile gradient
• Triple quadrupole MS in negative heated electrospray SRM mode for quantification and confirmation

Main Results and Discussion

The method exhibits the following performance characteristics:

• Linearity from 0 to 30 µg/kg (R2 = 0.994)
• Repeatability (%RSD) 2–6% at 2.5–7.5 µg/kg; intermediate precision ~10% at 5 µg/kg
• Accuracy (recovery) between 95.6% and 103.9%
• Limit of detection 0.8 µg/kg; limit of quantification 2.0 µg/kg
• Decision limit (CCα) 1.0 µg/kg; detection capability (CCβ) 1.29 µg/kg
• No significant matrix effects observed between solvent and meat extracts

Benefits and Practical Applications

This validated protocol delivers high sensitivity and specificity for phenylbutazone in complex meat matrices. The robust cleanup prevents matrix suppression, and the streamlined sample work-up supports high throughput in food safety laboratories. It meets regulatory requirements and can be applied to both horse and beef meat screening.

Future Trends and Potential Applications

Advances in automated sample prep and ultra-high-resolution MS may further reduce analysis time and improve sensitivity. Expanding the method to other veterinary drug residues and alternative matrices (e.g., feed, plasma) will enhance surveillance capabilities. Integration with data-driven quality control systems can support real-time compliance monitoring.

Conclusion

The described LC-MS/MS method is fit for routine detection of phenylbutazone in horse meat, offering reliable quantification, regulatory compliance, and operational efficiency in food safety testing.

Used Instrumentation

• Thermo Scientific UltiMate 3000 UHPLC system
• Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer
• Silica and C18 SPE cartridges on a 16-port vacuum manifold
• Standard lab equipment (centrifuge, vortex, evaporator, pH meter)

References

1. Commission Decision 2002/657/EC on analytical method validation criteria
2. Jedziniak P. et al. Anal. Chim. Acta 2010;672:85–92
3. ISO 11843: Capability of detection (1997)