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

Analysis of veterinary drug residues is essential for ensuring food safety, protecting public health, and meeting regulatory requirements. Rapid, sensitive, and comprehensive detection of multiple drug classes in animal products helps prevent unauthorized or harmful residues reaching consumers.

Study Objectives and Overview

This application note introduces a streamlined workflow for multiclass veterinary drug analysis using ultrafast chromatography coupled with variable data-independent acquisition (vDIA) on a Q Exactive Focus Orbitrap mass spectrometer. The primary objectives were:

• Develop a single chromatographic and mass spectrometric method covering 44 veterinary drugs across meat, milk, and plasma matrices.
• Demonstrate quantitative performance over eight calibration points (0.1–500 ppb) with full-scan and MS/MS confirmation.
• Enable non-targeted and retrospective screening within the same dataset.

Methodology and Instrumentation

The method combines a rapid generic LC gradient with a novel vDIA scheme to acquire full-scan and MS/MS data across wide m/z windows.

• Liquid Chromatography: Thermo Scientific UltiMate 3000 RSLC with Accucore C18 aQ column (100 x 2.1 mm, 2.6 μm); 6 min gradient from 5% to 95% acetonitrile with 0.1% formic acid; total cycle time 15 min; flow rate 300 µL/min.
• Mass Spectrometry: Q Exactive Focus Orbitrap; full-scan resolution 70 000 @ m/z 200 (100–1000 m/z); vDIA resolution 17 500 with five overlapping MS/MS windows (100–205, 195–305, 295–405, 395–505, 495–1000 m/z); HCD energy 35 eV; source conditions: spray voltage 4.4 kV, capillary temp. 250 °C, heater temp. 300 °C.
• Data Processing: Thermo Scientific TraceFinder v3.2; chromatogram extraction ±5 ppm; isotopic pattern, accurate mass, retention time, and fragment ion confirmation; non-targeted screening via a 450-component database.

Key Results and Discussion

The method achieved limits of quantitation (LOQs) between 0.1 and 10 ppb for 44 analytes with linear calibration (R2 > 0.99) from 0.5 to 500 ppb. Variable DIA provided:

• High sensitivity and selectivity comparable to targeted MS/MS methods.
• Robust confirmation using precursor ion ratios, isotopic patterns, and 1–5 fragment ions free of interferences in complex matrices.
• Full data recording enabling retrospective confirmation of unexpected compounds such as cortisol in muscle extracts.

Benefits and Practical Applications

This workflow reduces analysis time and resource use by replacing multiple injections and methods with a single run capable of:

• Simultaneous quantitation of diverse drug classes in meat, milk, and plasma.
• Retrospective and non-targeted screening for unknown or emerging contaminants.
• Compliance with EU regulation EC/657/2002 through comprehensive identity confirmation.

Future Trends and Opportunities

Potential developments include expanding compound libraries, integrating machine-learning tools for non-targeted identifications, and transferring vDIA strategies to other high-resolution platforms. Automation and miniaturized workflows could further increase throughput for routine surveillance.

Conclusion

The Q Exactive Focus LC-MS with vDIA offers a robust, sensitive, and versatile solution for multiclass veterinary drug residue analysis. It delivers comprehensive quantitative and qualitative data in a single, high-throughput method, meeting regulatory demands and supporting retrospective screening capabilities.

Reference

• Scheibner O., Bromirski M. Application Note 614: Quick and Sensitive Analysis of Multiclass Veterinary Drug Residues in Meat, Plasma, and Milk on a Q Exactive Focus LC-MS System. Thermo Fisher Scientific, 2016.