Determination of Glyphosate, Glufosinate and their Relevant Metabolites in Soybean Extracts Using UPLC-MS/MS with the Torus DEA Column

Significance of the Topic

The widespread use of glyphosate and glufosinate as non-selective and selective herbicides has raised concerns over residue levels in food commodities such as soybean. These compounds and their metabolites are highly polar and ionic, presenting analytical challenges for conventional reversed-phase LC methods. Sensitive, reliable detection at regulatory limits is essential for food safety monitoring and compliance with maximum residue limits (MRLs).

Objectives and Overview of the Study

This study aimed to develop and demonstrate a targeted UPLC-MS/MS method for simultaneous quantification of glyphosate, glufosinate and their key metabolites in soybean extracts. By employing a Torus DEA column, the method avoids derivatization steps while providing adequate retention and separation of polar anionic pesticides. The application note evaluates method performance against U.S. tolerances for residues in soybean.

Methodology and Instrumentation

Sample preparation involved extraction of ground soybean with aqueous acetic acid and EDTA to precipitate proteins and solubilize analytes. A pass-through cleanup using an Oasis HLB SPE cartridge removed particulates and non-polar co-extractives. Extracts were diluted, filtered and injected onto a Torus DEA column under a rapid gradient of ammonium formate/formic acid and acetonitrile/formic acid. Detection employed MRM transitions optimized on a tandem quadrupole mass spectrometer.

Used Instrumentation

• Oasis HLB SPE Cartridge (60 mg)
• Torus DEA Column, 1.7 µm, 2.1 × 100 mm
• ACQUITY UPLC H-Class Bio System with FTN Sample Manager
• Xevo TQ-XS Mass Spectrometer (ESI-)
• MassLynx v4.2 Software
• TargetLynx XS Application Manager

Main Results and Discussion

The method delivered excellent retention time stability, peak shape and sensitivity for all analytes. Calibration over 2.5–40 mg/kg for glyphosate and 0.25–4 mg/kg for glufosinate yielded r2 values >0.99 and residuals within 20%. For matrix-matched spikes at 0.5 and 5 mg/kg, intra-day precision (n=9) showed RSDs ≤6%. Ion ratios and retention times met stringent tolerances, confirming selectivity and reproducibility without derivatization.

Benefits and Practical Applications of the Method

By circumventing derivatization, this workflow reduces sample preparation time and complexity. The Torus DEA column provides robust retention of polar analytes in a single run, while the Xevo TQ-XS ensures low detection limits compatible with regulatory MRLs. Laboratories can adopt this approach for routine monitoring of glyphosate and glufosinate residues in soybean and potentially other high-fat/protein matrices.

Future Trends and Applications

Advances may include integration of isotopically labeled internal standards to improve quantitation accuracy, expansion to high-resolution MS platforms for untargeted screening, and automation of SPE cleanup for higher sample throughput. Development of multipesticide methods on anionic polar columns could broaden the scope of routine residue analysis in various crops.

Conclusion

This application demonstrates that UPLC-MS/MS with a Torus DEA column and pass-through SPE cleanup provides a sensitive, precise and efficient solution for determining glyphosate, glufosinate and their metabolites in soybean. The method meets regulatory performance criteria and can be implemented by analytical laboratories after in-house validation.

Reference

1. U.S. EPA (2017) 40 C.F.R. § 180.473 Glufosinate Ammonium; Tolerances for Residues.
2. U.S. EPA (2017) 40 C.F.R. § 180.364 Glyphosate; Tolerances for Residues.
3. Wuyts B. et al. (2018) Determination of Anionic Polar Pesticides in Spinach Using Torus DEA Column. Waters Application Note.
4. Shah D. et al. (2018) Determination of Anionic Polar Pesticides in Wheat Flour Extracts Using UPLC-MS/MS with Torus DEA Column. Waters Application Note.
5. Chamkasem N., Harmon T. (2016) Direct Determination of Glyphosate, Glufosinate, and AMPA in Soybean and Corn by Liquid Chromatography–Tandem Mass Spectrometry. Anal Bioanal Chem, 408(18), 4995–5004.
6. European Commission (2017) SANTE 11813/2017 Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residues.
7. Waters Corporation (2017) Torus DEA Column Startup Guide for Polar Pesticide Separations.