Multiclass veterinary drug screening and quantitation by high-resolution mass spectrometry (HRMS) using the Orbitrap Exploris 120 mass spectrometer

Significance of the Topic

Veterinary drugs are widely administered to livestock to maintain animal health and productivity. Monitoring their residues in edible tissues is critical to ensure consumer safety and regulatory compliance. High-resolution mass spectrometry (HRMS) offers the sensitivity and selectivity needed to detect multiple drug classes at or below maximum residue limits (MRLs).

Objectives and Study Overview

This study demonstrates a single HRMS-based method for screening and quantitating 109 veterinary drug residues in bovine muscle according to AOAC SMPR 2018.010 performance requirements. The method employs both data-independent acquisition (DIA) and data-dependent acquisition (DDA) to achieve reliable detection and confirmation at half the lowest global MRLs.

Methodology and Instrumentation

Sample preparation used a modified QuEChERS extraction followed by cleanup. Dispersive SPE with CEC18 sorbent was selected after comparing freezing and SPE cartridges due to its simplicity and reproducibility.

Used Instrumentation

• UHPLC: Thermo Scientific Vanquish Flex system with Accucore VDX column (100 × 2.1 mm, 2.6 µm) at 40 °C, gradient elution (water/0.05% formic acid and methanol–acetonitrile/0.05% formic acid).
• Mass Spectrometer: Thermo Scientific Orbitrap Exploris 120; full scan at 60,000 FWHM, MS2 at 15,000 FWHM.
• Acquisition Modes: DIA with variable isolation windows (4 vs 17) and DDA with inclusion list.
• Data Processing: Thermo Scientific TraceFinder with mzVault and offline mzCloud libraries, 5 ppm mass tolerance.

Results and Discussion

Cleanup comparison demonstrated that CEC18 dispersive SPE provided recoveries of 70–120% for most analytes with RSDs below 10%. A freezing step was found impractical for high-throughput work.

DIA and DDA both achieved a probability of detection (POD) ≥ 0.8 at half MRL for the majority of compounds. Increasing the number of DIA isolation windows from 4 to 17 improved fragment detection and confirmation at low ng/g levels (e.g., monensin at 1 ng/g).

Quantitation using a certified reference material (BOTS-1) yielded results within 15% of certified values for target analytes, demonstrating accuracy and precision.

Benefits and Practical Applications

The approach allows a single HRMS run to screen and confirm over 100 heterogeneous veterinary drugs in a complex food matrix, reducing method count and labor. It supports regulatory surveillance and quality-assurance workflows in food safety laboratories.

Future Trends and Applications

• Optimization of DIA isolation schemes to balance scan speed and specificity.
• Expansion of high-quality spectral libraries for emerging compounds and metabolites.
• Automation of sample preparation and data processing using advanced software pipelines.
• Application to other food matrices and integration with targeted MS/MS quantitation for confirmatory analysis.

Conclusion

The Orbitrap Exploris 120 HRMS platform, combined with optimized QuEChERS extraction and CEC18 dispersive SPE cleanup, delivers robust multi-class veterinary drug screening and quantitation in bovine muscle. Both DIA and DDA workflows meet AOAC SMPR criteria, enabling high-confidence detection at stringent MRLs.

References

• European Commission Decision 2002/657/EC on method performance and result interpretation for veterinary residues.
• Electronic Code of Federal Regulations Title 21, Subchapter E – Animal Drugs, Feeds, and Related Products.
• AOAC SMPR 2018.010: Multi-class veterinary drug residue methods.
• Guidance Document SANTE/12682/2019: Confirmation criteria for qualitative methods.