Determination of a Single Methodology for the Analysis and Quantitation of Multi- class Veterinary Drugs in Different Animal Matrices Used for Consumption

Significance of the topic

The presence of veterinary drug residues in meat poses health risks for consumers and necessitates rigorous monitoring to comply with strict maximum residue limits (MRLs). A streamlined analytical approach is essential for food safety laboratories to efficiently detect and quantify a broad spectrum of drugs at levels ranging from sub-µg/kg to tens of thousands µg/kg.

Objectives and overview

The study aimed to establish a single LC-MS/MS method capable of quantifying over 200 multi-class veterinary drugs in beef, pork and chicken tissues with one chromatographic run. This unified workflow is intended to replace multiple specialized extraction and detection protocols, enabling both targeted quantitation and screening across diverse meat matrices.

Methodology and instrumentation

The method integrates a modified QuEChERS extraction with reversed-phase LC separation and triple-quadrupole MS:

• Sample preparation: 2.0 g of homogenized meat extracted with 10 mL acetonitrile/water (80:20), shaken 10 min, centrifuged and filtered through 0.45 µm PVDF.
• Chromatography: 3 µL injection onto a 100×2.1 mm, 2.6 µm C18 Polar Endcapped column; gradient elution with water/0.1% FA and methanol/0.1% FA at 300 µL/min; column at 30 °C.
• Mass spectrometry: Thermo Scientific TSQ Endura triple-quadrupole with heated electrospray ionization (HESI) in positive/negative modes; spray voltage 3.5 kV (positive), 2.5 kV (negative); vaporizer 300 °C; capillary 250 °C; sheath gas 40 arb; auxiliary gas 5 arb; collision gas 1.5 mTorr; 3–5 SRM transitions per analyte with optimized collision energies.
• Calibration: Matrix-matched curves from 0.05 to 300 µg/kg (12 levels) plus blank; internal standards at 20 ppb.

Results and discussion

The single-run method demonstrated strong performance:

• Linearity: r²>0.990 for over 95% of compounds across matrices.
• Precision: %RSD<20% at the lowest levels; <15% at LOQ.
• LODs ranged from 0.05–100 µg/kg and LOQs from 0.1–200 µg/kg, meeting or exceeding regulatory MRLs.
• Challenges: Exclusion of highly polar or poorly ionized analytes; broad peaks for certain acidic drugs; careful handling of mixed standards for stability.
• Incurred analytes: Nicotinamide at 100–500 µg/kg and riboflavin (vitamin B2) at 100–150 µg/kg detected in blank matrices, highlighting sensitivity and matrix interferences.

Benefits and practical applications

• Consolidates multiple protocols into one LC-MS/MS run, reducing analysis time and solvent use.
• Supports simultaneous quantitative and targeted screening of multi-class drugs.
• Features an integrated compound database for rapid method setup in data processing software.
• Meets regulatory requirements for routine QA/QC and high-throughput food safety testing.

Future trends and opportunities

Potential enhancements include:

• Adoption of high-resolution MS for non-targeted screening and retrospective analysis.
• Automated and miniaturized sample preparation to increase throughput and reduce solvent consumption.
• Integration with informatics and AI for real-time monitoring and rapid decision support.
• Development of green analytical approaches to cover emerging contaminants and improve sustainability.

Conclusion

A robust, single-run LC-MS/MS method was developed for quantifying over 200 veterinary drugs in multiple meat matrices. The workflow meets stringent regulatory MRLs with high sensitivity, precision and throughput, streamlining food safety testing for both targeted and screening applications.

Reference

1. (EU) Regulation 1831/2003/EC; Directive 2009/8/EC.
2. U.S. Department of Agriculture. QuEChERS method for pesticide and veterinary drug residue analysis.