Variable Data-Independent Acquisition (vDIA) Delivers High Selectivity and Sensitivity in Combined Targeted and Untargeted Analyses for Small Molecules

Importance of the Topic

The variable data-independent acquisition (vDIA) method addresses the critical need in small molecule analysis for both high sensitivity in targeted quantitation and comprehensive MS/MS data for untargeted screening. By combining the advantages of narrow-window precursor isolation and full-range fragmentation, vDIA ensures robust identification and quantification while preserving a complete data record for retrospective investigations.

Study Objectives and Overview

This study evaluated a generic vDIA workflow on an Orbitrap-based mass spectrometer using 44 veterinary drug residues in muscle, kidney, milk, and plasma matrices. The aim was to demonstrate that wide-window vDIA can achieve sensitivity and selectivity comparable to data-dependent acquisition (DDA) while generating a full spectrum of MS and MS/MS data suitable for both targeted and non-targeted applications.

Methodology

Sample extracts spiked with calibration standards (0.1 ppt to 500 ppb) were separated by UHPLC using a C18 aQ column under a 6 min gradient from 5 % to 95 % acetonitrile (0.1 % formic acid) at 300 µL/min. MS acquisition comprised a full scan (m/z 100–1000, resolution 70 000 FWHM) followed by five successive fragmentation windows (100–205, 195–305, 295–405, 395–505, 495–1000 m/z) at 17 500 FWHM, yielding a cycle time of approximately 650 ms.

Instrumentation Used

• UHPLC: Thermo Scientific UltiMate 3000 RSLC with Accucore C18 aQ (100 × 2.1 mm, 2.6 µm)
• Mass Spectrometer: Thermo Scientific Q Exactive Focus Orbitrap
• Software: Thermo Scientific TraceFinder 3.2 for targeted quantitation and non-targeted screening

Main Results and Discussion

• Selectivity at Low Levels: All spiked analytes (5 µg/kg antibiotics in muscle/kidney, 1 µg/kg nitroimidazoles in plasma, 1 µg/kg avermectins in milk) were confirmed with multiple high-accuracy fragment ions and matching retention times.
• Linearity: Calibration curves were linear across 0.01–500 µg/kg for all 44 compounds.
• Retrospective Screening: A 1500-component database search identified 30 additional compounds, including cortisol, confirmed by accurate mass, isotope pattern, fragment match, and spectral library search.

Benefits and Practical Applications

• Unified quantitation and screening workflow for food safety, environmental monitoring, and pharmaceutical QA/QC.
• High throughput with a single acquisition method reduces method development time and improves laboratory efficiency.
• Complete data records enable retrospective analysis for emerging contaminants and regulatory follow-up.

Future Trends and Potential Applications

• Integration with advanced data analysis and machine learning to accelerate unknown identification.
• Refinement of isolation window widths and faster scan rates as instrument capabilities evolve.
• Extension to broader small-molecule classes, metabolomics, and biomarker discovery.
• Implementation in clinical and environmental workflows for comprehensive profiling.

Conclusion

vDIA on Orbitrap platforms offers a versatile acquisition strategy that bridges targeted and untargeted analyses. It delivers sensitivity and selectivity on par with DDA while maintaining a full MS/MS record, making it ideal for quantitative workflows and retrospective screening without compromise.

References

• Vogler B. Master’s Thesis, Institute of Organic Chemistry, University of Zürich, Zürich, Switzerland, 2013, p. 76.