Intelligent Reflex Targeted Reinjection Workflow for Pesticide Screening in Food Matrices

Significance of the Topic

Reliable and high-throughput screening of pesticide residues in food matrices is critical for public health and regulatory compliance. As global pesticide use expands and food supply chains become more complex, robust analytical workflows capable of identifying known and emerging contaminants at trace levels are essential. The integration of data-independent acquisition with intelligent reflex automation addresses challenges of matrix interferences and sample throughput, enhancing confidence in result reporting.

Objectives and Study Overview

The primary goal was to develop and demonstrate an automated workflow for routine pesticide screening in broccoli, strawberry, and celery matrices using the Agilent Revident LC/Q-TOF and MassHunter software. Key aims included:

• Implementing an untargeted All Ions acquisition for broad compound coverage.
• Automating targeted MS/MS reinjection for ambiguous or positive hits via an intelligent reflex approach.
• Evaluating method performance across multiple concentration levels and real-world matrices.

Methodology and Instrumentation

Sample Preparation

• QuEChERS extraction: 10 g homogenized sample extracted with 10 mL acetonitrile.
• Dispersive SPE cleanup using Agilent Bond Elut kits tailored for pigmented or general produce.
• Storage of extracts at –20 °C until analysis.

Chromatography and Mass Spectrometry

• Agilent 1290 Infinity II UHPLC with ZORBAX RRHD Eclipse Plus C18 column and guard.
• Gradient elution over 24 min at 0.45 mL/min, 45 °C column temperature.
• Revident LC/Q-TOF MS in positive All Ions mode at collision energies of 0, 20, 40 V.
• Reference masses m/z 121.0509 and 922.0098 for continuous mass calibration.

Data Analysis

• MassHunter Quantitative Analysis 12.1 with embedded LC Screener Tool for untargeted screening.
• SANTE guideline criteria: mass accuracy ≤ 5 ppm, S/N ≥ 3, coelution score ≥ 80, mass match score ≥ 65, retention time window ± 0.4 min.
• Intelligent Reflex Targeted Reinjection (Target Screening Confirmation) to trigger MS/MS for green or orange hits.
• Combined reporting template to merge untargeted and targeted results.

Main Results and Discussion

Mass Accuracy and Isotopic Fidelity

• 98 % of compounds maintained average mass accuracy within ± 2 ppm across eight calibration points and all matrices.
• Over 9,000 data points showed consistent performance at 2.5, 10, 50, 100 ppb levels.

Screening Outcomes

• At 5–100 ppb levels, more than 90 % of the 200 pesticides were confidently identified (green) in all matrices; questionable (orange) designations decreased with increasing concentration.
• No false negatives (red) were observed at tested levels.
• Matrix interferences managed via qualifier/quantifier swap, as demonstrated for methoxyfenozide in strawberry.

Intelligent Reflex Performance

• Automated targeted MS/MS reinjection reduced manual intervention and instrument idle time.
• Example: acephate in celery confirmed by specific fragment ions (library match score 58) whereas broccoli reinjection ruled out false positive fragments.

Benefits and Practical Applications

• Comprehensive screening of known and suspect pesticides with high throughput.
• Automated decision tree minimizes human bias and accelerates sample-to-report timelines.
• Robust performance in challenging matrices supports routine QA/QC and regulatory monitoring.
• Capability for retrospective data mining as new contaminants emerge.

Future Trends and Applications

• Expansion of spectral libraries and retention time databases to cover emerging agrochemicals and transformation products.
• Integration with laboratory information management systems for real-time result tracking.
• Application of machine learning algorithms to refine screening thresholds and reduce false positives.
• Extension of intelligent reflex workflows to other food safety challenges, such as mycotoxins or veterinary drug residues.

Conclusion

The combination of untargeted All Ions acquisition with intelligent reflex targeted reinjection on the Agilent Revident LC/Q-TOF delivers a powerful, automated platform for routine pesticide screening in complex food matrices. The workflow ensures high confidence in compound identification, reduces analyst workload, and supports compliance with stringent regulatory standards.

Reference

1. Carrasco Cabrera L.; Di Piazza G.; Dujardin B.; Medina Pastor P. The 2021 European Union Report on Pesticide Residues in Food. EFSA Journal 2023, 21(4), 7939.
2. 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.
3. Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed, SANTE/11312, European Commission, 2021.
4. USDA Pesticide Data Program website, accessed 2024.