Measurement of Underivatized Glyphosate and Other Polar Pesticides in Multiple Matrices Using Reversed-Phase Liquid Chromatography and Tandem Mass Spectrometry

Significance of the topic

Reliable quantification of glyphosate, glufosinate and related polar degradation products is critical for environmental monitoring, food safety and regulatory compliance. Their high polarity and strong interaction with metal surfaces in liquid chromatography systems create challenges for retention, peak shape and detection at sub-µg/L levels.

Objectives and study overview

This work demonstrates a streamlined workflow for the determination of eight underivatized polar pesticides (glyphosate, glufosinate, AMPA, MPPA, HEPA, NAG, ethephon and fosetyl) in water, wine and honey matrices. The study aims to achieve low detection limits, robust chromatography and reproducible quantitation through optimized sample preparation, reversed-phase separation and tandem mass spectrometry.

Used instrumentation

• Agilent Infinity II 1290 LC system (binary pump, multisampler, column thermostat)
• PEEK-lined stainless steel capillaries and Deactivator Additive for metal passivation
• Prototype superficially porous reversed-phase column (600 bar max)
• Agilent 6470A Triple Quadrupole LC/MS with Jet Stream ESI source

Methodology and sample preparation

Water samples were centrifuged, filtered (0.2 µm PES) and acidified to 0.1 % formic acid. Red wine was diluted ten-fold with 0.1 % formic acid. Honey extracts were prepared by vortexing 0.5 g honey with 5 mL 0.5 % formic acid, filtering and further diluting before injection. Chromatography employed a large injection volume (25 µL) of near-100 % aqueous extract on a superficially porous reversed-phase column at 40 °C, with a 0.1 % formic acid mobile phase containing deactivator additive and a short 8 min gradient. Tandem MS used dynamic multiple reaction monitoring in positive and negative polarity to maximize sensitivity for each analyte.

Main results and discussion

Chromatograms showed sharp, non-tailing peaks for all eight compounds with retention times spanning 0.97 to 4.49 min. Matrix-matched calibrations delivered excellent linearity (R2 > 0.999) over broad concentration ranges (0.025–100 µg/L for water, 2.5–1000 µg/L for honey, 5–1000 µg/L for wine). Method detection limits ranged from 0.01 to 0.05 µg/L for various analytes, with signal-to-noise ratios above 10 in sub-µg/L spiked samples. Ten replicate injections of glyphosate in honey yielded area RSD of 5.6 % and retention time RSD of 0.6 %.

Benefits and practical applications

• Minimal sample preparation aligned with chromatographic conditions enables high throughput.
• PEEK-lined flow path and deactivator additive prevent metal artefacts and tailing.
• Large aqueous injections improve sensitivity without compromising peak shape.
• Dual-polarity MRM acquisition increases versatility and signal optimization by compound and matrix.
• Applicable to environmental water testing, food safety screening and industrial QA/QC.

Future trends and possibilities

Ongoing developments in column chemistries may further enhance retention of highly polar analytes under reversed-phase conditions. Integration of high-resolution mass spectrometry could expand target lists to include emerging polar contaminants. Automation of sample preparation and online coupling with solid-phase extraction may improve throughput for routine monitoring. Data-driven analytics and machine learning approaches offer potential for advanced matrix-matched calibration and predictive maintenance of metal-sensitive systems.

Conclusion

The presented workflow provides a robust solution for analyzing underivatized glyphosate and seven polar pesticides in diverse matrices. By harmonizing simple sample preparation, a novel reversed-phase column and sensitive triple quadrupole detection, the method achieves low detection limits, high reproducibility and linearity suitable for environmental and food safety applications.

Reference

• J. Hsiao et al., “Surface Passivation Strategies for Polar Pesticide Analysis,” Analytical Chemistry, 2018, 90, 9457–9464.
• US EPA Method Detection Limit (MDL) Procedure, Revision 2, December 2016.