Affiliation:
1. Laboratory for Analytical Chemistry and Residues, Veterinary Department Križevci, Croatian Veterinary Institute, 48260 Križevci, Croatia
2. Laboratory for Residues, Department of Veterinary Public Health, Croatian Veterinary Institute, 10000 Zagreb, Croatia
Abstract
Glyphosate is the most used herbicide in agriculture. Its major metabolite is AMPA (aminomethylphosphonic acid), but N-acetyl-AMPA and N-acetylglyphosate are also metabolites of interest. For risk assessment, a general residue definition was proposed as the sum of glyphosate, AMPA, N-acetyl-glyphosate and N-acetyl-AMPA, expressed as glyphosate. A confirmatory method for glyphosate in fat, liver and kidneys, as well as a confirmatory method for AMPA and N-acetyl-glyphosate in all matrices, are still missing. In this paper, we present a method for the quantitative determination of glyphosate residues and its metabolites AMPA, N-acetyl-AMPA and N-acetyl-glyphosate by liquid chromatography–mass spectrometry (LC-MS/MS) in adipose tissue, liver, eggs, milk and honey without derivatization. Different chromatographic columns were tested, with the Hypercarb column providing the best results. The analytes were eluted with mobile phases of acidified water with 1.2% formic acid and 0.5% formic acid in acetonitrile. Sample purification procedures were also optimized by varying the solvent extraction mixtures (water, methanol and mixture ψ (methanol, water) = 1:1, each with the addition of 1% formic acid (v/v)), using different sorbents in solid phase extraction (SPE) (polymeric cationic (PCX) and anionic (PAX)) and using dispersive solid phase extraction (dSPE) (C18 and PSA) by modifying the extraction procedures. Finally, the analytes were extracted from the samples with 1% formic acid in water (v/v). Milk and adipose tissue were purified by the addition of dichloromethane, while liver and egg samples were purified by SPE with a mixed cation exchange sorbent and ultrafiltration with cut-off filters. The proposed analytical procedures were validated according to SANTE/11312/2021 guidelines: linearity, limits of quantification, precision and accuracy were determined for all matrices. The limits of quantification (LOQs) ranged from 0.025 to 0.2 mg kg−1. Precision, expressed as relative standard deviation, was <20%, while accuracy, expressed as analytical recovery, ranged from 70% to 120%. During method validation, the measurement uncertainty was estimated to be <50% for all analytes. Good validation parameters according to the SANTE document were achieved for all analytes. Therefore, the method can be considered reliable and sensitive enough for routine monitoring of polar pesticides. The application of the accredited method in routine analysis will provide data that are useful for the re-evaluation of risk assessment studies in foods of animal origin.
Reference34 articles.
1. European Food Safety Authority (EFSA) (2017). Peer review of the pesticide risk assessment of the potential endocrine activity of glyphosate. EFSA J., 15, 4979.
2. European Commission (2013). Regulation (EC) No 293/2013 of 20 March 2013 amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for emamectin benzoate, etofenprox, etoxazole, flutriafol, glyphosate, phosmet, pyraclostrobin, spinosad and spirotetramat in or on certain products. Off. J. Eur. Union, L96/1, 252–281.
3. European Food Safety Authority (EFSA) (2019). Review of the existing maximum residue levels for glyphosate according to Article 12 of Regulation (EC) No 396/2005–revised version to take into account omitted data. EFSA J., 17, e05862.
4. European Commission (2021). Commission Implementing Regulation
5. (EU) 2021/601 of 13 April 2021 concerning a coordinated multiannual control programme of the Union for 2022, 2023 and 2024 to ensure compliance with maximum residue levels of pesticides and to assess the consumer exposure to pesticide residues in and on food of plant and animal origin. Off. J. Eur. Union, 64, 29-41.