Direct Analysis of Glyphosate, Glufosinate and AMPA in Foods Using a Triple Quadrupole LC/MS/MS

Importance of the Topic

Glyphosate and glufosinate are non-selective herbicides extensively applied in agriculture to facilitate pre-harvest weed control. Their degradation product, aminomethylphosphonic acid (AMPA), along with the parent compounds, poses analytical challenges due to their high polarity. Reliable determination of these residues in food commodities is critical for consumer safety and regulatory compliance under varying maximum residue limits (MRLs) imposed by regional authorities.

Objectives and Study Overview

This study aimed to develop a direct, derivatization-free LC-MS/MS method for simultaneous quantification of glyphosate, glufosinate and AMPA in diverse food matrices (flour, whole grain flour, oats, soybeans, and grapes). The method integrates the Quick Polar Pesticides (QuPPe) sample preparation protocol adapted for high-protein and high-lipid samples, followed by triple quadrupole mass spectrometry using MRM detection.

Used Instrumentation

• Liquid Chromatograph: Shimadzu Nexera X2
• Mass Spectrometer: Shimadzu LCMS-8060 with ESI in negative mode
• Column: RESTEK Polar X (2.1 × 30 mm, 2.7 µm)
• Autosampler tubing: PEEK resin to minimize adsorption of polar analytes

Methodology and Sample Preparation

Samples (1–10 g) were weighed into 50 mL tubes and spiked with isotopically labeled internal standards. Water was added and left for 15 minutes, followed by methanol containing 1% formic acid. A 10% aqueous EDTA solution was introduced to chelate metal ions. Samples underwent mechanical shaking, refrigerated centrifugation (13 000 g, –9 °C, 10 min), and filtration (0.22 µm). An aliquot of the extract was diluted with acetonitrile, vortexed, centrifuged (3 000 g, 5 min) and subjected to ultrafiltration (3 kDa) prior to LC-MS/MS analysis.

Analytical Conditions

• Mobile Phase A: 0.5% formic acid in water; B: 0.1% formic acid in acetonitrile
• Gradient: 60% B (0–1 min) → 5% B (2–7 min) → 60% B (7.01–10 min)
• Flow Rate: 0.6 mL/min; Column Temp: 35 °C; Injection: 5 µL
• Ion Source: ESI negative; Probe Voltage: –3.0 kV; Interface Temp: 400 °C
• MRM Transitions: AMPA 110→78.9/110→62.9, Glufosinate 180.1→62.9/180.1→85.0, Glyphosate 168.1→63.0/168.1→78.8

Main Results and Discussion

The method demonstrated linearity from 0.5 to 100 ng/mL with correlation coefficients ≥ 0.999. Limits of quantification (LOQs) were established at 0.5 ng/mL for all analytes, with accuracy between 96 and 104% and repeatability (RSD) < 10%. Recoveries in food matrices ranged from 91.8 to 97.4%, confirming method robustness. In real samples, glyphosate was detected at 0.98 mg/kg and AMPA at 0.08 mg/kg in whole grain flour, while other matrices showed concentrations below LOQs.

Benefits and Practical Applications

• Derivatization-free workflow reduces sample handling time and potential contamination.
• High sensitivity and selectivity enable compliance monitoring at or below regulatory MRLs.
• Universal applicability across varied food matrices simplifies laboratory operations.

Future Trends and Applications

Advancements may include ultrahigh-pressure LC to shorten analysis times, integration with high-resolution mass spectrometry for confirmatory screening, and expansion of the method to other polar pesticides. Automation of QuPPe sample preparation and rapid on-board data processing will further enhance throughput and data quality.

Conclusion

A direct LC-MS/MS approach combined with QuPPe sample preparation provides a rapid, reliable, and sensitive method for quantifying glyphosate, glufosinate, and AMPA in food commodities. The method meets stringent performance criteria and supports regulatory compliance and food safety monitoring.

References

1. European Commission, QuPPe Method. http://quppe.eu (accessed July 10, 2019)
2. Commission Regulation (EU) No 441/2012 of 24 May 2012. EUR-LEX.
3. Codex Pesticide Residues in Food Online Database, Glyphosate (158). FAO/WHO Codex.
4. Ministry of Health, Labour and Welfare, Japan. Public Notice No. 361, 2017.