From data-independent acquisition (DIA) to targeted MS/MS: Automatic reinjection for additional confirmation in suspect screening

Importance of the Topic

The monitoring of multiple pesticide residues in food matrices is critical for ensuring public health and regulatory compliance. High-resolution mass spectrometry (HRMS) methods, such as data-independent acquisition (DIA), offer comprehensive coverage but often require manual follow-up to resolve ambiguous identifications. Introducing an automated reflex workflow from DIA to targeted MS/MS enhances throughput and confidence in suspect screening.

Objectives and Study Overview

This study demonstrates an intelligent reinjection approach that seamlessly transitions from an all-ions DIA screening of complex food samples to a targeted MS/MS confirmation sequence. Using a broccoli extract spiked with over 200 pesticides at concentrations ranging from 0.625 to 100 ppb, the workflow aims to comply with SANTE guidelines for identification and quantification, while minimizing manual intervention and downtime.

Methodology and Instrumentation

Sample preparation involved a QuEChERS protocol optimized for pigmented fruits and vegetables, followed by spiking with a pesticide mix and heavy labeled standards. Initial screening was performed using all-ions non-targeted acquisition in both positive and negative ion modes with collision energies of 20 V and 40 V. Data processing in MassHunter Quantitative Analysis 12.1 employs the SureMass algorithm for profile-mode feature detection, enabling high sensitivity and accuracy. Upon flagging of suspects based on mass accuracy, coelution score, and signal-to-noise criteria, the system automatically appends targeted MS/MS experiments to the worklist using retention time and m/z information.

Instrumentation Used

• Agilent 1290 Infinity II LC
• Agilent Revident LC/Q-TOF with thermally controlled flight tube and new detector
• Agilent MassHunter Quantitative Analysis 12.1 with LC Screener Tool and SureMass algorithm

Main Results and Discussion

A total of 214 pesticides were detected, including five isomeric pairs, with mass accuracy predominantly within ±2 ppm. Over 99% of abundance measurements at 5 and 10 ppb exhibited relative standard deviations below 20%. Calibration curves showed linearity (R2 ≥ 0.99) across the tested range. The intelligent reflex workflow successfully converted 18 of 22 questionable suspects into confirmed identifications via targeted MS/MS, illustrating enhanced specificity and reduced manual processing. The SureMass algorithm provided improved trace detection by analyzing full-profile peaks rather than centroids.

Benefits and Practical Applications

This automated DIA-to-targeted MS/MS strategy significantly reduces instrument idle time and analyst workload by eliminating manual method setup and reinjection steps. The combined approach delivers rapid, reliable screening and confirmation of hundreds of residues in complex matrices, supporting routine food safety and quality control laboratories.

Future Trends and Possibilities

Advancements may include integration of machine learning models for real-time suspect prioritization and further expansion of spectral libraries to cover emerging contaminants. Miniaturized HRMS platforms and cloud-based data analytics could enable on-site screening with automated confirmation, accelerating decision making in regulatory and industrial environments.

Conclusion

The presented intelligent reflex workflow leverages the strengths of DIA and targeted MS/MS on an Agilent Revident LC/Q-TOF platform to achieve high-confidence, high-throughput pesticide screening in food matrices. Automated reinjection streamlines suspect confirmation, improving laboratory productivity and data reliability.

References

1. Yannell, K.; et al. Enhanced Food Safety Testing: A Pesticide Screening Methodology Using the Agilent 6546 LC/Q-TOF and MassHunter Quantitative Analysis Software 10.0 LC/Q-TOF Screener Tool. Agilent Technologies Application Note, 5994-0738EN, 2019.
2. European Commission. Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed. SANTE/11312/2021.