Quick and Sensitive Analysis of Multiclass Veterinary Drug Residues in Meat, Plasma, and Milk on a Q Exactive Focus LC-MS System

Importance of the Topic

The analysis of veterinary drug residues in animal products is critical to ensure food safety and regulatory compliance. Traditional multi-class methods require multiple chromatographic methods and injections to cover diverse compound classes, limiting throughput and retrospective analysis. The variable data-independent acquisition (vDIA) approach addresses these challenges by combining high-resolution full-scan and wide-window MS/MS acquisition in a single run, providing comprehensive, high-quality data for targeted and non-targeted screening.

Objectives and Study Overview

This application note demonstrates a rapid, generic LC-MS/MS method using the Thermo Scientific Q Exactive Focus Orbitrap and vDIA workflow for the quantitation and screening of 44 veterinary drug residues in meat, milk, and plasma. The study aims to achieve high sensitivity, broad dynamic range, and robust retrospective analysis capability with a single chromatographic and mass spectrometric method.

Methodology and Instrumentation

A generic 15-minute LC method was applied on a Thermo Scientific UltiMate 3000 RSLC system with an Accucore C18 aQ column (100 x 2.1 mm, 2.6 μm). The gradient ranged from 5% to 95% acetonitrile with 0.1% formic acid at 300 μL/min. The Q Exactive Focus MS was operated in full-scan (m/z 100–1000, 70,000 FWHM) and vDIA (five isolation windows: 100–205, 195–305, 295–405, 395–505, 495–1000; 17,500 FWHM) modes. Source parameters included 4.4 kV spray voltage, 250°C capillary temperature, 300°C vaporizer temperature, sheath and auxiliary gas flow, and 35 eV HCD energy. Data were processed using TraceFinder 3.2 with a 5 ppm extraction window for quantitation and confirmation.

Used Instrumentation

• Thermo Scientific Q Exactive Focus Orbitrap MS
• Thermo Scientific UltiMate 3000 Rapid Separation LC
• Accucore C18 aQ column (100 x 2.1 mm, 2.6 μm)
• Thermo Scientific TraceFinder software version 3.2

Key Results and Discussion

The vDIA method achieved limits of quantitation between 0.1 and 10 ppb for 44 veterinary drugs with calibration curves spanning 0.5–500 ppb and R2 > 0.99. Fragment ion and isotopic pattern matching provided unambiguous identity confirmation even in complex matrices. For unstable analytes (e.g., abamectin, doramectin), fragment-based quantitation extended sensitivity. Generic full-scan and wide-window MS/MS acquisition enabled retrospective non-targeted screening of a 1500-component database, exemplified by the detection of cortisol.

Benefits and Practical Applications

• Single injection method covering multiple drug classes, reducing analysis time and resources.
• High sensitivity and selectivity with full data records supporting both targeted quantitation and non-targeted screening.
• Compliance with EU regulatory requirements (EC/657/2002) for confirmation via retention time, accurate mass, isotopic ratio, and fragment ions.
• Retrospective data interrogation capability for unknown or newly regulated compounds.

Future Trends and Possibilities

Advancements may include optimized DIA window schemes for enhanced dynamic range, expanded spectral libraries, integration of ion mobility separation, and high-throughput data processing with machine learning. These developments will further streamline multi-residue screening and improve confidence in trace-level detection.

Conclusion

The combination of rapid LC separation, high-resolution Orbitrap detection, and vDIA provides a powerful, single-method solution for comprehensive analysis of multiclass veterinary drug residues in food and biological matrices. This workflow delivers robust quantitation, reliable identity confirmation, and the flexibility needed for retrospective and non-targeted screening.