Alternative Proteins Testing Application Compendium

Significance of the Topic

Ensuring the safety and quality of foods consumed globally requires rapid, accurate methods to detect trace levels of hundreds of potential contaminants in complex matrices. Regulatory agencies set maximum residue limits (MRLs) for pesticides at parts-per-billion levels that vary over a wide dynamic range. Traditional sample preparation and single-technique analyses often fall short of delivering both the sensitivity and broad concentration coverage needed to enforce these limits in a timely, cost-effective manner.

Study Goals and Overview

This work demonstrates a single-workflow approach to quantify over 1,000 pesticide residues in a high-pigment, high-oil commodity (tomato) according to the EU SANTE 11312/2021 guideline. Using a single QuEChERS extraction combined with both LC/MS/MS (764 analytes) and GC/MS/MS (341 analytes, 84 overlapping) platforms, the method was validated for linearity, sensitivity, recovery, and precision over four orders of magnitude without extensive instrument maintenance.

Methodology and Instrumentation

Sample preparation:

• Weigh 10 g of homogenized tomato.
• Spike QC samples at 10 µg/kg and equilibrate 15 min.
• Perform QuEChERS EN extraction (10 mL water + 10 mL ACN/1% acetic acid).
• Clean up crude extract directly by dilution for LC or by Captiva EMR pass-through for GC.

LC/MS/MS analysis (Agilent 1290 Infinity II + 6470B TQ):

• Reversed-phase C18 column, 20 min gradient.
• Dynamic MRM with 1,590 transitions, dual polarity.
• Matrix-matched calibration 0.5–100 µg/L, 1/x weighting.

GC/MS/MS analysis (Agilent 8890 + 7010C TQ or 7000E TQ):

• Dual 15 m HP-5ms UI columns with midcolumn backflush.
• MMI splitless injection and temperature-programmed liner.
• Dynamic MRM with 2,093 transitions.

Key Findings and Discussion

Linearity and Calibration:

• LC/MS/MS: R² ≥ 0.99 for > 98 % of 764 analytes over 0.5–100 µg/L.
• GC/MS/MS (7000E): R² ≥ 0.99 over 0.1–5,000 µg/L for > 98 % of 341 analytes.
• GC/MS/MS (7010C with HES): R² ≥ 0.99 over 0.1–1,000 µg/L with superior sensitivity.

Sensitivity (Instrument LOD):

• LC/MS/MS: > 98 % analytes S/N ≥ 10 at ≤ 10 µg/L.
• GC/MS/MS (7000E & 7010C): > 88 % analytes S/N ≥ 10 at 1 µg/L in tomato extract.

Recovery and Precision:

• Mean recoveries at 10 µg/kg within 40 – 120 % for > 97 % LC and GC targets.
• RSDr ≤ 20 % for > 98 % of all technical replicates.

Benefits and Practical Applications

• Single QuEChERS extraction saves time and cost versus multiple prep methods.
• Combined use of LC/MS/MS and GC/MS/MS provides full coverage of > 1,000 pesticides.
• Midcolumn backflush and Captiva EMR cleanup extend instrument uptime and reduce maintenance.
• Wide dynamic range calibration minimizes re-injection or dilution steps for commodities with varied MRLs.

Future Trends and Potential Uses

As food safety regulations expand to more compounds and emerging contaminants, workflows that integrate UHPLC/QqQ, GC/QqQ, and high-resolution MS will become increasingly vital. Automation in sample cleanup (e.g., pass-through EMR), broader spectral libraries, and enhanced cell-based interference control (He KED and reaction gases) will help laboratories scale up throughput while maintaining the highest levels of data quality and compliance.

Conclusion

This application note illustrates an efficient, robust workflow for the simultaneous quantitation of over 1,000 pesticide residues in tomato across challenging matrices. By combining QuEChERS extraction with optimized cleanup, midcolumn backflush, and dual LC and GC triple quadrupole MS platforms, the method meets SANTE guidelines for sensitivity, linearity, recovery, and precision with minimal instrument downtime.

Reference

• SANTE 11312/2021: Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed.
• Kornas P.; Agilent application note 5994-5303EN, 2023.