Determination of Anionic Polar Pesticides in Spinach Using a Novel Application of Torus DEA Column Chemistry by Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

Significance of the Topic

Exposure to anionic polar pesticides in food crops poses significant health and regulatory concerns. Spinach, a leafy vegetable with high water content, can accumulate residues of highly polar compounds such as glyphosate, AMPA, and fosetyl. Reliable monitoring methods are essential for ensuring compliance with maximum residue limits and protecting public health. The development of a robust, sensitive, and cost-effective analytical workflow allows both official control laboratories and food business operators to perform due diligence testing with confidence.

Study Objectives and Overview

This application note presents the validation of a targeted liquid chromatography tandem mass spectrometry method tailored for anionic polar pesticides in spinach. The method combines the Quick Polar Pesticides (QuPPe) extraction protocol with a novel use of the Torus DEA stationary phase in UPLC to achieve improved retention, peak shape, and selectivity. Key aims included demonstrating sufficient sensitivity at parts-per-billion levels, ensuring accurate quantification across multiple analytes, and meeting the performance criteria defined by SANTE guidelines.

Methodology and Instrumentation

The sample preparation employed the QuPPe approach: a 5 gram spinach homogenate was extracted with acidified methanol, vortexed, centrifuged, and filtered without cleanup. Chromatographic separation was achieved on a Torus DEA column (130 Å, 1.7 μm, 2.1×100 mm) using an ACQUITY UPLC I-Class system. A binary gradient of ammonium formate with formic acid and acetonitrile with formic acid at 0.5 mL/min delivered sharp, well-resolved peaks within a 16-minute run time. Mass spectrometric detection was performed on a Xevo TQ-XS instrument in negative electrospray mode. MRM transitions for eleven anionic pesticides and metabolites were optimized by IntelliStart software. Data acquisition and processing utilized MassLynx and TargetLynx XS.

Instrumentation Used

• UPLC system ACQUITY UPLC I-Class with FL Sample Manager
• Torus DEA 130 Å column, 1.7 μm, 2.1×100 mm
• Xevo TQ-XS tandem quadrupole mass spectrometer
• MassLynx MS Software and TargetLynx XS Application Manager

Main Results and Discussion

Validation on spinach according to SANTE criteria demonstrated:

• Limits of quantification of 0.01 mg/kg for nine compounds and 0.05 mg/kg for AMPA and HEPA
• Mean recoveries between 94 and 117 percent for most analytes at 0.01 mg/kg, with RSDr ≤10%
• Excellent linearity (r2 >0.99) across 7.5–200 ppb calibration range
• Clear chromatographic separation of critical isobaric pairs such as AMPA versus fosetyl and phosphonic acid versus fosetyl degradant
• Minimal matrix suppression achieved without cleanup

Retention time variability and ion ratios largely fell within acceptable tolerances, confirming method robustness.

Practical Benefits and Applications

This streamlined approach offers several advantages:

• Single-run analysis of multiple anionic pesticides and metabolites
• No need for derivatization or specialized ion chromatography equipment
• Compatibility with existing UPLC–MS/MS platforms and standard solvents
• High throughput and cost-effectiveness suitable for routine compliance and due diligence testing

Future Trends and Applications

Further development could include expanding the method to other high-water matrices such as lettuce or berries, incorporating isotopically labeled internal standards to enhance quantification accuracy, and reducing run times through faster gradients or smaller particle columns. Advances in high-resolution mass spectrometry may also enable retrospective screening for emerging polar contaminants. Automation of sample preparation and data processing will support high-throughput monitoring in regulatory and industrial laboratories.

Conclusion

The combination of QuPPe extraction with Torus DEA column chemistry and UPLC-MS/MS detection meets stringent validation criteria for anionic polar pesticide analysis in spinach. The method delivers high sensitivity, accuracy, and precision in a cost-effective single-run protocol. Laboratories can adopt this workflow to strengthen surveillance programs and ensure food safety compliance.

References

1. European pesticide database on approved active substances and MRLs in food and feed.
2. Code of Federal Regulations 40 C.F.R. § 180.364 (2018).
3. EURL QuPPe Method for quick polar pesticide extraction.
4. Wuyts B et al. Highly sensitive analysis of polar pesticides in food matrices, Waters Technical Note No. 720005822EN (2016).
5. Wuyts B et al. Improved multi-analyte method for underivatized anionic pesticide analysis, Waters Application Note No. 720006070EN (2017).
6. European Commission SANTE Guidance Document on Analytical Quality Control and Method Validation, document 11813/2017.
7. Waters Torus DEA Column Startup Guide for Polar Pesticide Separations, User Manual No. 720006156EN (2018).