Automated QuEChERS Extraction for Pesticides Analysis in Dry and Wet Matrix

Significance of the topic

The accurate, reproducible quantitation of pesticide residues in food matrices is critical for regulatory compliance, consumer safety, and environmental monitoring. QuEChERS extraction is a broadly used approach for multi-residue pesticide analysis, but manual sample preparation is labor‑intensive and can introduce variability. Automating QuEChERS workflows reduces hands‑on time, improves standardization and traceability, and enables high-throughput routine testing that meets strict method validation criteria such as SANTE 11312/2021.

Objectives and overview of the study

This application note presents an automated end‑to‑end QuEChERS workflow implemented on the CTC PAL3 Series 2 RTC autosampler and integrated into Agilent MassHunter software. The goal was to combine automated extraction, salting‑out, dilution and injection with LC/MS/MS and GC/MS/MS detection (Agilent 6475 LC/TQ and 7010D GC/TQ) to quantify several hundred pesticide residues in wet (tomato) and dry (flour) matrices within ~20 minute chromatographic runs, while conforming to SANTE 11312/2021 acceptance criteria.

Methodology

The workflow starts with manual weighing and fortification of samples (typical aliquots: 8 g tomato and 4 g flour), followed by a largely automated QuEChERS extraction sequence executed by the PAL3 autosampler. Key automated steps include solvent addition (e.g., 8 mL ACN), vortex mixing, centrifugation, transfer of ACN supernatant to vials preloaded with QuEChERS salt (0.5 g), phase partitioning, final centrifugation and either dilution for GC analysis or direct injection for LC analysis. Fast‑wash needle cleaning and thorough post‑wash routines were used to minimize carryover.

Calibration and QC: matrix‑matched calibration curves were prepared across multiple concentration levels with 1/x weighting; calibration linearity acceptance was R² ≥ 0.99. Method precision was evaluated by preparing five independent technical replicates of prespiked QC samples at 10 or 20 µg/kg and assessing recovery repeatability (RSD ≤ 20% per SANTE guidance).

Used instrumentation

• Automated sample preparation: CTC PAL3 Series 2 RTC autosampler configured with vortex, centrifuge combi, dilutor, solvent and fast wash modules, LC injection valve and PAL Method Composer scripting.
• LC/MS/MS: Agilent 1290 Infinity III LC coupled to Agilent 6475 triple quadrupole with Agilent Jet Stream ESI operated in dynamic MRM (dMRM). Column: Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 × 150 mm, 1.8 µm. Run time ~20 min; mobile phases 5 mM ammonium formate in water and MeOH (0.1% formic acid) at 0.4 mL/min.
• GC/MS/MS: Agilent 8890 GC with Agilent 7010D triple quadrupole; multimode inlet (MMI) and mid‑column backflush configuration using two Agilent J&W HP‑5ms columns (15 m each). Run time ≈20.75 min; helium carrier, solvent vent injection.
• Software and libraries: Agilent MassHunter acquisition and quantitative analysis with Pesticide & Environmental Pollutants (P&EP) MRM database; PAL3 scripts created with CTC Method Composer and integrated into MassHunter.

Main results and discussion

- Scope: The combined LC and GC workflow covered approximately 350 target pesticide analytes from a single QuEChERS extract (Venn distribution in tomato indicated complementary coverage between LC and GC enabling broad multispecies analysis).
- Linearity: Using matrix‑matched calibration and 1/x weighting, 98% of target compounds achieved R² ≥ 0.99, indicating excellent linear response across the tested range.
- Recoveries and precision: Following SANTE 11312/2021 criteria (mean recoveries 40–120% and RSD ≤ 20%), LC/MS/MS results showed acceptance for ~96% of analytes in tomato and ~93% in flour at 10 µg/kg. GC/MS/MS met the criteria for ~97% of analytes in both matrices at the same level. These outcomes demonstrate that the automated extraction and handling produce reliable quantitative results at low µg/kg levels.
- Throughput and robustness: The integrated PAL3 automation shortened hands‑on time, reduced operator variability, and provided reproducible sample preparation that is directly linked to the LC/GC acquisition methods in a single software environment, enabling one‑click start of full analysis sequences.

Benefits and practical applications of the method

• High throughput multi‑residue pesticide screening in routine food testing laboratories—one extraction supports both LC and GC detection to maximize analyte coverage.
• Improved reproducibility and traceability versus manual QuEChERS handling, reducing inter‑operator variability and potential sample contamination.
• Regulatory compliance: method performance (linearity, recovery, precision) aligns with SANTE 11312/2021 acceptance criteria, making the workflow suitable for regulatory monitoring and QA/QC programs.
• Operational efficiency: PAL3 scripting and MassHunter integration permit standardized procedures, automated QC sample handling, and reduced manual labor.

Future trends and applications

• Extended automation: further integration of automated weighing and on‑autosampler fortification can fully eliminate manual sample handling and increase throughput in high‑volume labs.
• Expanded analyte panels and AI‑assisted data review: larger MRM libraries and machine‑learning tools for automated flagging of outliers and review of chromatographic quality will streamline result validation.
• Miniaturization and green analytical chemistry: reduction of solvent volumes and waste via optimized micro‑extraction variants compatible with autosampler workflows.
• Cross‑platform digitalization: tighter laboratory information management system (LIMS) connectivity and method metadata capture for audit trails and regulatory reporting.

Conclusion

The automated QuEChERS workflow implemented on the CTC PAL3 autosampler and integrated with Agilent 6475 LC/TQ and 7010D GC/TQ instruments delivers robust, high‑coverage pesticide residue analysis for both wet and dry food matrices. The approach meets SANTE 11312/2021 validation criteria for linearity, recovery and precision for the vast majority of the investigated compounds, while significantly reducing manual effort and improving reproducibility—making it practical for routine regulatory and quality control laboratories.

Reference

1. European Commission. SANTE 11312/2021: Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed. 2021 (as cited in the application note).