Analysis of Veterinary Drug Residues in Animal Muscle Tissue Using a Multi-Residue Screening Method Utilsing LC-MS/MS

Significance of the Topic

The monitoring of veterinary drug residues in animal muscle tissue is critical for ensuring food safety and regulatory compliance. Residues arising from therapeutic treatments or growth promotion practices can pose health risks to consumers and undermine confidence in food products. Implementing a robust, multi-residue analytical method enables laboratories to efficiently screen for a wide range of drug classes using a single workflow.

Objectives and Study Overview

This study aimed to develop and validate a generic multi-residue screening method for over 150 veterinary drugs in animal muscle tissue. Key goals included achieving detection capabilities below established maximum residue limits (MRLs), minimizing sample preparation time, and demonstrating compliance with Commission Decision 2002/657/EC criteria for screening methods.

Methodology and Instrumentation

A generic sample preparation protocol was applied to minced muscle tissues from bovine, porcine, and turkey sources:

• Extraction: Liquid extraction using oxalic acid in acetonitrile
• Clean-up: Dispersive solid-phase extraction (dSPE)
• Storage: Extracts kept at –20 °C and analyzed within 48 hours to preserve analyte stability

The chromatographic separation was performed on a Waters ACQUITY HPLC I-Class Plus system equipped with an ACQUITY UPLC HSS T3 column (2.1 × 100 mm) and an in-line filter. Mobile phases consisted of aqueous 0.1% formic acid with 0.1 mM ammonium formate (A) and 0.1% formic acid in a 50:50 methanol/acetonitrile mixture (B). The gradient elution delivered all analytes between 1.65 and 11 minutes within a total run time of 15 minutes. A 1 µL injection volume and column temperature of 40 °C minimized matrix effects.

Mass spectrometric detection employed a Waters Xevo TQ-XS instrument with electrospray ionization and scheduled multiple reaction monitoring (MRM) with polarity switching. Optimized transitions for each analyte were sourced from Waters’ marketplace.

Main Results and Discussion

Validation followed the Commission Decision 2002/657/EC screening protocol to determine cut-off factors and detection capabilities (CCβ). Three independent batches of spiked and blank tissue samples were evaluated at levels of 0.1, 1.0, and 10 µg/kg. Key findings:

• CCβ values for most compounds were below or equal to 1 µg/kg, surpassing MRL requirements for over 70% of the analytes.
• Ciprofloxacin exemplified reliable differentiation between negative and positive samples at the 0.1 µg/kg and 1.0 µg/kg levels.
• The HSS T3 column delivered sharp peak shapes and resolved isobaric interferences without additional cleanup steps.
• The low injection volume reduced co-extractive load, enhancing long-term system robustness.

Benefits and Practical Applications

This multi-residue method offers several advantages for routine veterinary drug screening:

• High throughput: 15-minute runtime with minimal sample handling.
• Broad scope: Simultaneous monitoring of diverse drug classes in a single assay.
• Regulatory compliance: Validation meets Commission Decision 2002/657/EC screening criteria.
• Enhanced sensitivity: Detection at or below 1 µg/kg for the majority of compounds.

Future Trends and Opportunities

Advancements in analytical chemistry and data processing are expected to further improve multi-residue screening:

• Integration of high-resolution mass spectrometry for expanded screening without preselection.
• Automated sample preparation to reduce manual steps and inter-operator variability.
• Application of machine learning algorithms for rapid peak identification and false-positive reduction.
• Extension to other food matrices, environmental samples, and novel drug metabolites.

Conclusion

The presented LC-MS/MS multi-residue method demonstrates robust performance for screening veterinary drug residues in animal muscle tissue. By combining a streamlined extraction and dSPE cleanup with a sensitive chromatographic and mass spectrometric platform, laboratories can achieve rapid, compliant, and reliable detection of over 150 analytes at or below regulatory limits.

Reference

• Commission Decision 2002/657/EC on the performance of analytical screening methods