Enhanced Sensitivity in Multi-Residue Pesticide Analysis by LC-MS/MSUsing High-Pressure Feed Injection

Enhanced Sensitivity in Multi-Residue Pesticide Analysis by LC-MS/MS Using High-Pressure Feed Injection — Poster Summary

Importance of the Topic

The routine monitoring of pesticide residues in food commodities requires sensitive, robust, and high-throughput multi-residue methods. Typical QuEChERS extracts are prepared in acetonitrile, which creates a strong solvent matrix that can compromise reversed-phase LC separations, especially for early-eluting analytes. Improving injection strategies to mitigate solvent effects while preserving method simplicity and throughput is therefore crucial for lowering limits of detection and ensuring reliable quantification across hundreds of pesticides in a single LC-MS/MS run.

Objectives and Study Overview

This poster evaluated the impact of high-pressure feed injection versus classical flow injection on chromatographic peak shape and analytical sensitivity in a comprehensive pesticide residue LC-MS/MS workflow. The goals were to: demonstrate reduction of solvent mismatch effects from acetonitrile-based QuEChERS extracts, quantify the improvement in peak shape and signal linearity with increased injection volumes, and show practical implications for limits of detection and data review efficiency in routine multi-residue analysis.

Methodology

• Sample matrix: Spinach prepared by QuEChERS extraction using 10 mL acetonitrile from 10 g sample; extract analyzed directly without solvent exchange or concentration.
• Chromatography: Reversed-phase C18 column (2.1 x 150 mm, 1.8 µm) at 40 °C; mobile phases: water + 5 mM ammonium formate + 0.1% formic acid (A) and methanol + 5 mM ammonium formate + 0.1% formic acid (B); flow 0.4 mL/min with gradient from 5% to 100% B and 3 min hold.
• Injection modes compared: classical flow injection versus feed injection using a high-pressure hybrid multisampler configured for a controlled on-line dilution (feed speed 10 µL/min producing ~1:40 dilution during injection).
• MS detection: Triple quadrupole MS in dynamic MRM (dMRM) mode; polarity switching (positive/negative); 1590 MRMs monitored; optimized source gases and voltages as reported.
• Standards: Commercial and custom multiresidue pesticide mixes spiked into spinach QuEChERS extracts; injection volumes tested from 0.2 µL to 2 µL.

Used Instrumentation

• Agilent 1290 Infinity III High Speed Pump (G7120A)
• Agilent 1290 Infinity III Hybrid Multisampler (G7137B) capable of up to 1300 bar and offering feed injection
• Agilent 1290 Infinity III Multicolumn Thermostat (G7116B)
• Agilent 6495D Triple Quadrupole LC/MS (G6495D)
• Agilent ZORBAX RRHD Eclipse Plus C18 column, 2.1 x 150 mm, 1.8 µm (PN 959759-902)
• QuEChERS EN extraction kit and standard laboratory equipment (shaker, centrifuge)

Main Results and Discussion

• Solvent mismatch effects: Classical flow injection of acetonitrile QuEChERS extracts produced peak broadening and splitting for early-eluting pesticides as injection volume increased, with clear deterioration at 2 µL for several compounds.
• Feed injection performance: With on-line dilution provided by feed injection (effective ~1:40 dilution at 10 µL/min feed speed), chromatograms showed no solvent-related artifacts across the tested injection volumes, even at higher volumes that caused problems in flow injection mode.
• Signal linearity and saturation: For classical flow injection, peak area generally increased linearly with injection volume, but peak height reached a saturation point as peak broadening increased—indicating limited peak capacity and reduced sensitivity gains from larger injections. In feed injection mode both peak area and peak height increased linearly across the tested range (0.2–2 µL) for representative analytes (e.g., aminocarb, pymetrozine), demonstrating absence of saturation and preservation of peak shape.
• Data quality and workflow: Feed injection produced symmetrical, well-defined peaks that enabled reliable automated integration and reduced need for manual reintegration, streamlining data review in high-throughput pesticide residue analyses.

Benefits and Practical Applications

• Improved sensitivity: Ability to use larger injection volumes without peak distortion increases signal-to-noise and lowers practical limits of detection for trace-level pesticides.
• Method simplicity: Direct injection of QuEChERS acetonitrile extracts is preserved, avoiding additional evaporation/reconstitution or solvent-exchange steps that add labor and potential analyte loss.
• Robust high-throughput operation: Compatibility with long, sub-2 µm columns and the capability to operate at pressures up to 1300 bar support demanding multi-residue methods that monitor hundreds of compounds per run.
• Better automation and data quality: Symmetrical peaks reduce manual post-processing and improve reproducibility in routine labs (QA/QC, regulatory testing, contract labs).

Future Trends and Potential Applications

• Broader adoption of high-pressure feed injection is likely as laboratories seek to combine simplified sample prep with increased sensitivity for trace contaminants in food, environmental, and biological matrices.
• Integration with larger multi-analyte panels and expanding dMRM methods will benefit from the reduced solvent effects and improved peak capacity provided by feed injection.
• Potential to couple feed injection with advanced data processing workflows and targeted screening libraries to accelerate routine surveillance and incident response.
• Further optimization could investigate feed-dilution ratios, injection kinetics, and applicability to other challenging solvent/matrix combinations beyond acetonitrile QuEChERS extracts.

Conclusion

High-pressure feed injection using a hybrid multisampler mitigates solvent mismatch effects inherent to acetonitrile-based QuEChERS extracts and enables larger injection volumes without compromising chromatography. This yields improved peak shape, linear increases in peak height and area, enhanced sensitivity, and more reliable automated integration for comprehensive multi-residue pesticide LC-MS/MS assays. The approach preserves simple sample preparation while extending method performance for trace-level detection in routine food safety testing.

References

1. Naegele E., et al. Feed Injection at Elevated Pressure Using the Agilent 1290 Infinity III Hybrid Multisampler. Agilent Technologies Application Note, Publication Number 5994-8478EN, 2025.
2. Naegele E., et al. Improved Peak Shape and Lower LOQs in Pesticide Analysis. Agilent Technologies Application Note, Publication Number 5994-6125EN, 2024.
3. Kornas P., et al. Analysis of 764 Pesticides in Tomato Using an Agilent 6495D Triple Quadrupole LC/MS System. Agilent Technologies Application Note, Publication Number 5994-8004EN, 2025.
4. Quantitative Analysis of Multiresidue Pesticides in Food Matrices Using Agilent 6470 Triple Quadrupole LC/MS system – Method Protocol, 2022.