Abstract
Carbamates (CMs) and organophosphates (OPs) are widely used pesticides with known neurotoxicity arising from the inhibition of acetylcholinesterase (AChE). When AChE is active, in vitro, it can hydrolyze certain substrates to colored products while in the presence of an inhibitor this color development is decreased. Based on this principle, an AChE assay for CM and OP compounds was optimized and validated for carbofuran, carbofuran-3-hydroxy and dichlorvos in lettuce and strawberry extracts. The analytical performance of the assay was confirmed by an accredited liquid chromatography tandem mass spectrometry (LC–MS/MS) method. The developed AChE assay achieved low limits of detection (LODs) at the part per billion (ppb) level, depending the analyte inhibitory strength, recovery rates higher than 70% and good repeatability. Moreover, the toxic unit (TU) approach was applied, for extracts containing the validated analytes, and antagonism was noticed in all cases. Overall, the developed method is rapid, simple, cost-effective and may find application as a low-cost pre-screening tool of AChE inhibitors presence. Last but not least, this study can be considered a guide on development, validation and benchmarking of bioassays in food safety, a topic, which is commonly mispresented in the available literature.
Highlights
The ever-increasing demand for food production requires a widespread use of pesticides to prevent pests and boost agricultural productivity
Phosphate buffer saline (PBS) tablets, AChE from Electrophorus electricus, acetylthiocholine iodide (AThI, purity >99%), 5,50 -dithio bis-2-nitrobenzoic acid (DTNB, purity >99%) indoxyl acetate (IDA, purity >95%), a-naphthyl acetate (α-NAc, purity >98%), fast blue B salt (FBBS, purity 95%), z-sep sorbent, 0.22 μm syringe filters, 5 mL syringes were from Sigma-Aldrich (Prague, Czech Republic). 96-microwell plates were supplied by Gama Group (Ceske Budejovice, Czech Republic)
The toxic unit (TU) approach was applied to investigate the effect of mixtures containing two pesticide residues, as a simplified analogue towards real life conditions, since samples may be contaminated with multiple residues
Summary
The ever-increasing demand for food production requires a widespread use of pesticides to prevent pests and boost agricultural productivity. CMs and OPs show relatively high acute toxicity and their residues in food may result in chronic toxicity incidents [2]. The toxicity of CMs and OPs arises from the inhibition of acetylcholinesterase (AChE), a vital enzyme in the neural system of insects and mammals including humans. AChE activity is reduced in the presence of CMs or OPs, due to carbamylation or phosphorylation of serine hydroxyl group in the enzyme active cite [3], respectively. This results in acetylcholine accumulation, which can
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