Analytical Performances of prototype assays suitable for validation
Figure 1: Structures of glyphosate and its metabolites (AMPA, N-acetyl-glyphosate and N-acetyl-AMPA)
Glyphosate (N-(phosphonomethyl)glycine), is an organophosphorus broad spectrum, non-selective, post-emergence herbicide. It is a chemical used worldwide in plant protection and its global consumption has increased over the years. The global glyphosate market worth $8 billion in 2022 is estimated to reach $10.6 billion by 2030, recording a compound annual growth rate (CAGR) of 3.6% for the period 2022-2030.
Glyphosate has been thoroughly assessed by Member States, the European Chemicals Agency (ECHA) and the European Food Safety Authority (EFSA) in recent years due to its controversial toxicity. The last update in the renewal process refers to the adoption of Implementing Regulation (EC) 2023/2660 by the Commission in November 2023 to renew the approval of glyphosate for 10 years.
Maximum Residue Levels (MRLs) are set with a view to protect vulnerable groups, by taking into account toxicological safety thresholds as calculated in the toxicological assessment (Regulation (EC) No 396/2005). In the case of glyphosate, a revision of MRLs has been suggested, considering inclusion of metabolites. For enforcement purposes, the “main residue definition” included the sum of glyphosate, aminomethylphosphonic acid (AMPA) and N-acetyl-glyphosate for plants with glyphosate-tolerant genetically modified varieties currently available on the market, whereas glyphosate only can be considered a sufficient marker for enforcement in conventional crops.
For risk assessment, a general residue definition covering both conventional and genetically modified crops was proposed as the sum of glyphosate, AMPA, N-acetyl-glyphosate and N-acetyl-AMPA, expressed as glyphosate. In the case of maize, the MRL was set to 1 mg/kg in Regulation (EC) No 396/2005. The structures of glyphosate and its metabolites (AMPA, N-acetyl-glyphosate and N-acetyl-AMPA) are reported in figure 1.
A rapid and fully automated lateral flow immunoassay for the detection of glyphosate in maize is under development in HOLiFOOD (WP2). The aim of the assay is to generate glyphosate contamination data allowing both the analysis of on-site commodities and facilitating, through automation, the management and collection of data in quality control laboratories.
The present report describes the achievement of Milestone M7, i.e. to obtain a prototype assay showing analytical performances suitable for validation. Different strip tests and reagent batches were produced and tested for these purposes.
The optimized protocol for maize analysis is shown in figure 2. The prototype reagents were proven to be stable for 60 days either at room temperature (+20°C) and at refrigerate conditions (+4°C). A preliminary estimation of the test sensitivity and precision showed that the optimized assay is able to discriminate maize samples contaminated at 100 %MRL (1 mg/kg), 10% MRL (0.1 mg/kg) – as candidate screening detection limit – and 0% MRL, to be confirmed in full validation experiments. Finally, assay cross reactivity was investigated. The antibody showed cross reactivity of 92% for N-Acetyl glyphosate and no cross reactivity for AMPA. A LC-MS confirmatory method to be used in the validation study was implemented.
In conclusion, the obtained results showed the suitability of the developed assay for full validation in maize and analysis automation (extraction, lateral flow analysis and data collection/processing).
Figure 2 : Optimized lateral flow immunoassay protocol
