Application of a Triggered MRM Database and Library for the Quantitation and Identification of Pesticides in Food Extracts

Significance of the Topic

Accurate detection and identification of pesticide residues in food are essential for consumer safety and compliance with regulatory maximum residue limits (MRLs). Traditional LC–MS/MS methods based on two MRM transitions can suffer from false positives in complex matrices. The use of triggered multiple reaction monitoring (MRM) combined with a spectral library enhances selectivity and confirmation capability without sacrificing throughput.

Objectives and Overview of the Study

This study aimed to develop a comprehensive triggered MRM database and spectral library covering over 300 pesticide analytes compatible with LC–MS/MS. A targeted method for 120 pesticides was demonstrated on diverse food commodities (lemon, tomato, green tea, chamomile, ginger). The work included method development, in-house validation for key matrices, and examples illustrating the elimination of false positives through library matching.

Methodology and Instrumentation

Sample Preparation

• QuEChERS extraction with citrate buffering for fruits, vegetables, dried chamomile, and green tea.
• Dispersive SPE cleanup using primary–secondary amine and graphitized carbon black.
• Matrix-matched calibration prepared in spiked QuEChERS extracts at 1–100 µg/kg.

Method Development

• LC separation on ZORBAX RRHD Eclipse Plus C18 column (2.1 × 150 mm, 1.8 µm) with a water/methanol gradient containing 0.1% formic acid and 5 mM ammonium formate.
• Agilent Jet Stream positive electrospray ionization optimized for sensitivity.
• Dynamic MRM for routine quantitation (two transitions per compound).
• Triggered MRM mode enabling acquisition of up to eight additional fragment transitions upon exceeding compound-specific thresholds.
• Optimization of precursor/product ions, fragmentor voltages, and collision energies via MassHunter Optimizer.
• Construction of a triggered MRM spectral library using MassHunter Quantitative Analysis (over 2 000 transitions for >300 pesticides).

Used Instrumentation

• Agilent 1290 Infinity UHPLC system: Binary Pump, High-Performance Autosampler, Thermostatted Column Compartment.
• Agilent 6460 Triple Quadrupole LC/MS with Agilent Jet Stream ESI source.
• MassHunter Workstation software for acquisition, optimization, and library management.

Results and Discussion

The triggered MRM database provided 2–10 transitions per pesticide, enabling full product ion spectra at low concentrations. Constant cycle times preserved quantifier peak quality even when triggering confirmatory ions. Calibration curves for representative analytes (e.g., oxamyl) showed near-identical slopes and linearity (R² > 0.997) between dynamic and triggered MRM. Limits of quantitation (LOQs) were <5 µg/kg for all 120 pesticides, with over 80% achieving <1 µg/kg in complex matrices. Repeatability was excellent (RSD <5% for most compounds at 1 µg/kg spike). Matrix effects (suppression/enhancement) affected up to 90% of analytes but were compensated by matrix-matched calibration. Triggered MRM spectra library matching required a score >75/100 to confirm identity. Examples of potential false positives (e.g., tebuthiuron in chamomile) were successfully flagged by poor match scores, preventing misidentification.

Benefits and Practical Applications

• Enhanced confidence in pesticide identification through spectral library matching.
• Reduction of false positives in complex food matrices.
• High throughput quantitation of hundreds of residues in a single run.
• Compliance with SANCO/12495/2011 validation criteria for LOQ, linearity, and precision.
• Scalable method scope by using only one primary transition as trigger.

Future Trends and Potential Applications

• Expansion of triggered MRM libraries to include additional emerging contaminants.
• Automation of real-time library matching and flagging in routine QC workflows.
• Integration with data-independent acquisition (DIA) strategies for broader screening.
• Application to other regulated domains such as veterinary drug residues and environmental monitoring.
• Advances in MS hardware to further improve sensitivity and speed.

Conclusion

Triggered MRM combined with a comprehensive spectral library significantly improves the selectivity, sensitivity, and reliability of pesticide residue analysis in food extracts. The approach meets stringent regulatory requirements, maintains high throughput, and minimizes false positives by leveraging optimized fragment transitions and automated library matching.

Reference

1. Regulation (EC) No 396/2005 of the European Parliament and of the Council on maximum residue limits of pesticides in or on food and feed of plant and animal origin.
2. European Guideline SANCO/12495/2011: Method validation and quality control procedures for pesticides residue analysis in food and feed.
3. Identification and Quantification of Pesticides in Chamomile and Ginger Extracts Using an Agilent 6460 Triple Quadrupole LC/MS System with Triggered MRM, Application Note 5990-8460EN.
4. Triggered MRM: Simultaneous Quantification and Confirmation Using Agilent Triple Quadrupole LC/MS Systems, Technical Overview 5990-8461EN.
5. EN 15662:2008, Foods of plant origin—Determination of pesticide residues using GC–MS and/or LC–MS/MS following QuEChERS sample preparation.
6. Pesticide Dynamic MRM Compound Database for Screening and Identification Using the Agilent Triple Quadrupole LC/MS Systems, Technical Note 5990-4255EN.