Automated QuEChERS Extraction for Pesticides Analysis in Dry and Wet Matrix

Significance of the topic

Automated, standardized sample preparation for multi‑residue pesticide analysis addresses critical needs in food safety testing: consistent recovery at low µg/kg levels, high throughput to meet laboratory demand, and traceable compliance with regulatory guidelines (SANTE 11312/2021). QuEChERS is the preferred extraction approach for diverse food matrices, but manual workflows limit reproducibility and increase labor. Integrating QuEChERS with autosampler automation and tandem MS detection enables robust, repeatable quantitation of hundreds of analytes from single extractions while reducing operator variability.

Objectives and study overview

This application note demonstrates a fully automated QuEChERS workflow implemented on a CTC PAL3 Series 2 RTC autosampler and validated for combined LC/MS/MS and GC/MS/MS pesticide analysis. Goals were to: automate extraction, salting‑out, dilution and injection; integrate sample workup scripting into a single acquisition platform (Agilent MassHunter); and evaluate method performance in wet (tomato) and dry (wheat flour) matrices for ~350 pesticide targets according to SANTE acceptance criteria.

Methodology and instrumentation

The workflow begins with manual weighing and optional fortification; all subsequent liquid handling and phase partitioning are automated by the PAL3. Key automated steps: solvent addition (ACN), vortex mixing, centrifugation, transfer of acetonitrile supernatant to salt tubes for partitioning, second centrifugation, and either direct LC injection or dilution prior to GC injection. Fast‑wash routines minimize carryover.

Calibration employed matrix‑matched standards (multiple levels spanning the working range) with 1/x weighting for linear regression. Performance was assessed using five technical replicates at 10 or 20 µg/kg (QC level), reporting recovery, repeatability (RSD), linearity (R²) and method sensitivity.

Instrumentation

• CTC PAL3 Series 2 RTC autosampler with Method Composer scripting and modules: liquid syringe, dilutor, vortex, centrifuge combi, dilutor multi, solvent and fast wash modules, LC injection valve, and tray racks.
• Agilent 6475 triple quadrupole LC/MS with Agilent Jet Stream ESI operating in dynamic MRM with polarity switching; Agilent 1290 Infinity III LC (ZORBAX RRHD Eclipse Plus C18, 2.1 × 150 mm, 1.8 µm).
• Agilent 8890 GC coupled to Agilent 7010D triple quadrupole GC/MS (HES 2.0 source) configured with multimode inlet (MMI), two 15 m HP‑5ms UI columns with mid‑column backflush via PUU and PSD.
• MassHunter Acquisition and Quantitative Analysis software with the Pesticide & Environmental Pollutants MRM database for dMRM transitions and method management.
• Consumables: Agilent QuEChERS extraction kit, appropriate vials and inlet liners, GC columns and MS tuning per vendor recommendations.

Main results and discussion

• Analyte scope: approximately 350 pesticide residues across LC‑amenable and GC‑amenable compound classes were covered by the combined workflow.
• Linearity: 98% of analytes achieved R² ≥ 0.99 using matrix‑matched calibration with 1/x weighting, indicating excellent linear response across the chosen concentration range.
• Recovery and precision: Meeting SANTE 11312/2021 acceptance (mean recoveries 40–120% and RSD ≤ 20%) was achieved for the majority of targets. Using LC/MS/MS, ~96% of analytes in tomato and ~93% in flour met criteria at 10 µg/kg. Using GC/MS/MS, ~97% of targets met acceptance in both matrices at the same level.
• Throughput and runtime: The LC or GC method runtime was ~20 minutes per injection (20–20.75 min listed), enabling routine throughput compatible with typical laboratory schedules. Integration of sample preparation with acquisition allowed single‑click end‑to‑end runs.
• Automation benefits: The PAL3 script reconstructed the full QuEChERS sequence (extraction, salting‑out, dilution, injection) and was embedded into MassHunter methods, enabling reproducible, standardized runs and optional standalone autosampler operation.

Benefits and practical applications of the method

1. Improved reproducibility and reduced operator variability by automating all liquid‑handling and phase‑separation steps after weighing.
2. High analytical scope—one extraction supports both LC‑ and GC‑MS/MS analysis of several hundred pesticides—reducing sample consumption and lab workload.
3. Regulatory compliance—validated performance meets SANTE 11312/2021 criteria for linearity, recovery and precision at low µg/kg levels, supporting routine monitoring programs and MRL enforcement.
4. Operational efficiency—single software platform for sample prep scripting and data acquisition simplifies method deployment and lowers training burden.

Future trends and applications

• Expanded automation: incorporating on‑deck automated weighing and onboard standard addition (fortification) to further reduce manual steps and increase traceability.
• Broader analyte panels: extending target lists including metabolites and polar pesticides, with simultaneous use of high‑resolution MS for non‑target screening and retrospective analysis.
• Informatics integration: automated flagging, QA/QC workflows and LIMS connectivity, augmented by machine‑learning tools for anomaly detection and faster data review.
• Miniaturization and green chemistry: reduced solvent volumes and micro‑QuEChERS formats to lower consumable use and waste.

Conclusion

The automated PAL3 QuEChERS workflow coupled to Agilent triple quadrupole LC/MS and GC/MS platforms delivers robust, high‑coverage pesticide residue analysis in wet and dry food matrices. The method achieves excellent linearity, acceptable recoveries and repeatability for the vast majority of ~350 targets using matrix‑matched calibration, and satisfies SANTE/11312/2021 validation requirements. Integration of sample preparation scripting into the acquisition environment streamlines routine testing and increases laboratory throughput and reproducibility.

Reference

SANTE 11312/2021: Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed. European Commission, 2026.