A Modified Orbitrap Exploris MS used for Multi-class Veterinary Drug Screening and Quantitation by High Resolution Mass Spectrometry (HRMS)

Significance of the Topic

The analysis of veterinary drug residues in animal-derived foods is critical for ensuring food safety and regulatory compliance.

By employing high-resolution mass spectrometry (HRMS), laboratories can simultaneously screen and quantify a broad spectrum of compound classes within a single analysis.

Such comprehensive monitoring supports efficient control of maximum residue limits (MRLs) and protects public health.

Objectives and Study Overview

This study aimed to develop and validate an HRMS-based workflow for multi-class veterinary drug screening in bovine muscle, adhering to AOAC SMPR 2018.010 performance requirements.

The research evaluated both data-independent acquisition (DIA) and data-dependent acquisition (DDA) strategies for their screening confidence and quantitation accuracy across 170 target analytes.

Methodology

Sample preparation followed a QuEChERS protocol on 5 g of homogenized bovine muscle, using acetonitrile extraction, salt partitioning, and cleanup with C18 sorbent.

Extracts were dried, reconstituted in mobile phase, and injected (5 µL) into an ultrahigh-performance liquid chromatography (UHPLC) system under a 17-minute gradient.

DIA and DDA HRMS acquisitions covered an m/z range of 140–1100 Da with full-scan resolution at 60,000 FWHM and MS² resolution at 15,000 FWHM, applying stepped normalized collision energies.

Used Instrumentation

• Thermo Scientific™ Vanquish™ Flex UHPLC System
• Thermo Scientific™ Accucore™ VDX C18 column (100 × 2.1 mm, 2.6 µm)
• Modified Thermo Scientific™ Orbitrap Exploris™ mass spectrometer
• TraceFinder™ and mzVault™ software for spectral matching and data processing

Main Results and Discussion

Instrument detection limits (IDLs) for 170 compounds ranged from 0.02 to 8 ppb, with calibration coefficients (r²) above 0.995 for the majority of analytes.

Probability of detection (POD) testing at half and full MRLs demonstrated POD values ≥0.80, requiring both precursor and fragment masses within ±5 ppm and signal-to-noise ≥3.

Analysis of a certified reference material (BOTS-1) containing incurred residues showed quantitation within 10% of true values across multiple drug classes and excellent reproducibility.

Both DIA and DDA workflows delivered comparable performance for confident screening and accurate quantitation.

Benefits and Practical Applications

• Simultaneous multi-class screening and quantitation in a single analytical run
• Compliance with international guidelines (AOAC SMPR and SANTE) for residue monitoring
• High confidence in identification via library matching and stringent mass accuracy criteria
• Applicability in routine quality assurance, diagnostic, and regulatory laboratory settings

Future Trends and Opportunities

Expansion of HRMS spectral libraries to include emerging veterinary drugs will enhance monitoring coverage.

Advances in instrument speed and sensitivity are expected to further lower detection limits and increase throughput.

Integration of automated workflows and machine learning approaches could streamline data processing and improve identification confidence.

Conclusion

The modified Orbitrap Exploris platform demonstrates robust capability for comprehensive veterinary drug screening and quantitation in bovine muscle.

Both DIA and DDA acquisition modes achieved high sensitivity, accuracy, and reproducibility in line with AOAC performance criteria.

This workflow provides a reliable solution for regulatory compliance and food safety monitoring.

References

• AOAC SMPR 2018.010. Standard Method Performance Requirements for Veterinary Drug Residue Analysis.
• European Commission SANTE/12682/2019. Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residues.