Multiclass Residue Analysis of Veterinary Drugs in Animal Tissues and Eggs Using Liquid Chromatography Coupled With Tandem Mass Spectrometry (LC/MS/MS)

Significance of the Topic

The widespread use of veterinary drugs in food-producing animals raises concerns over residues entering the human food chain. Regulatory authorities worldwide enforce maximum residue limits (MRLs) to protect public health and limit antimicrobial resistance. A robust, high-throughput analytical workflow capable of simultaneously screening multiclass veterinary drug residues in diverse animal-derived matrices is essential for effective monitoring and compliance.

Objectives and Study Overview

This study presents an integrated LC-MS/MS method for the simultaneous screening and quantification of over 180 veterinary drug residues spanning 32 chemical classes (β-agonists, tetracyclines, macrolides, β-lactams, sulfonamides, quinolones, avermectins, glucocorticosteroids, NSAIDs, peptides, anticoccidiosis agents, and more) in animal tissues (muscle and liver from swine, bovine, chicken, eel) and hen eggs. The goal was to develop a rapid, reliable workflow—from sample preparation through data analysis—that meets sensitivity, accuracy, and precision requirements for regulatory testing in a single analytical run.

Methodology and Instrumentation

• Sample Preparation:
  • Homogenization of tissues or whole eggs followed by protein precipitation with acidified acetonitrile (5% formic acid)
  • Cleanup using Agilent EMR-Lipid dispersive SPE to remove lipids selectively
  • Final polish step with Agilent dSPE kit for further matrix removal
• Instrumental Analysis:
  • Agilent 1290 Infinity II LC system with Binary Pump, Multisampler, and Multicolumn Thermostat
  • Agilent Jet Stream Ionization Source
  • Triple quadrupole mass spectrometers tested: 6460, 6470, and 6495 models
• Calibration and Validation:
  • Matrix-matched calibration standards (0.1 – 40 ng/g) in pork, swine liver, eel, chicken, and egg matrices
  • Linearity assessed over nine concentration levels, R² ≥ 0.99 for 98% of compounds
  • Limits of detection (LOD) and quantification (LOQ) determined by replicate injections at 1, 5, and 20 ng/g
  • Recovery and precision evaluated at three spiking levels (1, 5, and 20 ng/g)

Main Results and Discussion

The method delivered excellent analytical performance across all matrices:

• Linearity: R² ≥ 0.99 for most analytes over the 0.1–40 ng/g range.
• Recovery: At the 5 ng/g level, 92% of compounds yielded recoveries within 80–120%. A small subset showed recoveries outside this range due to instability or strong matrix effects.
• Precision: Over 85% of compounds exhibited repeatability (%RSD) ≤ 15% at 5 ng/g; only a limited number of analytes exceeded 20% RSD, often those prone to degradation or low response.
• Sensitivity: LOQs ranged from 0.2 to 8.6 ng/g depending on compound and instrument, with the 6495 system generally offering the lowest LOQs.
• Instrument Comparison: The 6495 Triple Quadrupole LC/MS achieved comparable linearity and precision to the 6460 and 6470 models but offered improved sensitivity for particularly low-level analytes.

Benefits and Practical Applications

This multiclass workflow delivers:

• High throughput: Single-run analysis of 180+ veterinary drugs reduces total testing time and labor.
• Regulatory compliance: Meets MRL requirements for a broad spectrum of drug classes in Chinese and international standards.
• Versatility: Applicable to multiple animal-derived foods including muscle, liver, fish, and eggs.
• Robustness: Effective cleanup of proteins and lipids ensures reliable quantification in complex matrices.

Future Trends and Opportunities

Advancements may include:

• Extension to emerging veterinary drugs and metabolites to maintain regulatory relevance.
• Integration with high-resolution mass spectrometry for comprehensive non-targeted screening.
• Automation of sample preparation steps to further enhance throughput and reproducibility.
• Application of machine learning algorithms for rapid data processing and anomaly detection.

Conclusion

The described LC-MS/MS workflow provides a comprehensive, sensitive, and efficient solution for multiclass veterinary drug residue analysis in animal-derived food products. Its validated performance on Agilent 6460, 6470, and 6495 instruments supports reliable routine monitoring and regulatory compliance while significantly improving laboratory throughput.

Reference

1. Ministry of Agriculture (MOA) China, Announcement No. 235: Maximum Residue Limits in Animal-Derived Food Samples (2002).
2. L. Zhao, D. Lucas. Multiresidue Analysis of Veterinary Drugs in Bovine Liver by LC/MS/MS. Agilent Technologies Application Note, 5991-6096 (2015).
3. T. Anumol, J. Stevens, J. Zweigenbaum. Analysis of Veterinary Drugs in Meat with the Agilent 6495 Triple Quadrupole LC/MS. Agilent Technologies Application Note, 5991-7895EN (2017).