Improved LC/MS/MS Pesticide Multiresidue Analysis Using Triggered MRM and Online Dilution

Significance of the Topic

Large multiresidue methods capable of analyzing hundreds of pesticide compounds in a single run support efficient monitoring of food, feed and environmental samples to ensure consumer safety and regulatory compliance. Rapid separations on triple quadrupole LC/MS/MS instruments combined with enhanced identification criteria address global trade demands and reduce analysis time and cost.

Goals and Study Overview

This work aimed to develop and validate a rapid LC/MS/MS method for simultaneous analysis of approximately 450 priority pesticides across diverse food matrices. The method uses QuEChERS extraction with direct injection via an online dilution system and leverages triggered multiple reaction monitoring (tMRM) on Agilent 6490 triple quadrupole instruments to enhance identification confidence without extending analysis time.

Methodology and Instrumentation

• Sample Preparation: QuEChERS acetonitrile extraction with acetate buffer partitioning, addition of internal standards, centrifugation; no offline dilution required.
• Online Dilution: Dual high-pressure mixers combine extract with aqueous mobile phase prior to column entry to preserve peak shape of early polar analytes.
• Chromatography: Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1×100 mm, 1.8 µm), 2 μL injection, gradient elution with 10 mM ammonium formate and 0.1 % formic acid, runtime under 10 minutes.
• Mass Spectrometry: Agilent 6490 triple quadrupole MS with Agilent Jet Stream ESI in positive mode, tMRM acquisition of 2 070 transitions (968 primary), 650 ms cycle time, unit resolution.
• MRM Optimization: MassHunter Optimizer selected up to 10 MRMs per analyte; 2–3 primary MRMs collected across peak, additional transitions triggered when threshold exceeded.

Results and Discussion

• Achieved elution of ~450 pesticides in under 10 minutes with uniform peak distribution and optimized separation of critical pairs.
• Online dilution eliminated peak splitting and tailing of polar early eluters without offline dilution steps.
• tMRM provided real-time confirmation by acquiring additional product ions only when analyte signals exceeded defined thresholds, reducing false positives.
• Successful method transfer to three laboratories equipped with identical Agilent systems demonstrated robust reproducibility.
• Validation across high-water, high-acid, high-fat, starch-rich and complex matrices yielded a method LOQ of 0.01 mg/kg for most analyte-matrix combinations, with recoveries and RSDs meeting SANTE guidelines.

Benefits and Practical Applications

• High-throughput screening of multiple pesticide classes in food, feed and environmental monitoring.
• Simplified sample preparation by direct injection of extracts without dilution.
• Improved identification confidence and reduced analyst review time via automated tMRM confirmation.
• Enhanced peak shape and quantitation accuracy for polar analytes.
• Scalable and transferable method suitable for routine laboratory workflows.

Future Trends and Potential Applications

• Expansion of compound libraries to include emerging pesticides and transformation products.
• Integration of automated tMRM scoring into data review and decision-making workflows.
• Extension to other analyte classes such as veterinary drugs, mycotoxins and environmental contaminants.
• Combination with ion mobility or high-resolution MS for further selectivity and confirmation capability.
• Development of advanced online solvent exchange or dilution modules for diverse extraction solvents.

Conclusion

The presented LC/MS/MS multiresidue method combines online dilution and triggered MRM to enable rapid, reliable analysis of ~450 pesticides in under 10 minutes. It meets stringent identification criteria, achieves low-ppb quantitation limits across multiple matrices, and enhances confidence in analyte confirmation. The approach is robust, scalable and readily transferable for routine pesticide residue monitoring.

Reference

1. European Commission Guidance Document SANTE/11945/2015 on Analytical Quality Control and Method Validation for Pesticide Residue Analysis in Food and Feed, 2016
2. Agilent Technologies Technical Overview, Triggered MRM for Simultaneous Quantitation and Confirmation, publication 5990-8461EN, 2013
3. Glauner T, Schuhn B, Kempe G. Application of Triggered MRM Database and Library for Pesticide Analysis. Agilent Application Note 5991-1183EN, 2012
4. Official Methods of Analysis, AOAC Official Method 2007.01 Pesticide Residues by QuEChERS Extraction, 2007
5. Agilent MassHunter Optimizer User Manual, publication G3793-90008, 2014
6. Mastovska K, Zulkoski J, Zweigenbaum J. Triggered MRM LC/MS/MS Method Development, Technical Overview 5991-7195EN, 2017
7. Supplementary Validation Results for Triggered MRM and Online Dilution Method, Agilent 5991-7194EN, 2017