## 3 Results and Discussion
### Optimized AChE Assay Application in Lettuce and Strawberry
#### 3.2.1 Carbofuran Validation and Benchmarking  
Carbofuran analysis was successfully validated using the optimized AChE assay in lettuce and strawberry (Table 3). The obtained recoveries (\(n=6\)) at two spiking levels, 0.020 mg Kg\({}^{-1}\) and 0.100 mg Kg\({}^{-1}\), were higher than 70% in both matrices. Better repeatability was noticed in lettuce compared to strawberry and a LOD equal to 0.013 mg Kg\({}^{-1}\) was achieved for lettuce and 0.012 mg Kg\({}^{-1}\) for strawberry. Worthy to notice is that LODs do not allow us to determine the measurement of uncertainty and this is a reason why screening methods results need to be verified by instrumental methods. Other spectrophotometric AChE assays have also achieved LODs at the ppb level [20; 21], however, insufficient validation was reported (solely focused on LODs). Comparing to EC methods, an AChE based EC sensor was utilized for carbofuran detection in cucumber achieving worse LODs than our study (0.22 uM meaning 0.048 mg Kg\({}^{-1}\)) [37]. In another paper, a non-enzymatic EC sensor was used and lower LOD was reported for carbofuran residues in grape, orange, tomato and cabbage matrices [14]. However, such a sensor is not available at the market probably due to a complicated manufacture. Although it was not feasible to achieve LODs lower than EU MRLs (0.002 mg Kg\({}^{-1}\) and 0.005 mg Kg\({}^{-1}\) for lettuce and strawberry, respectively), the developed assay was able to detect carbofuran about two times lower than the Chinese MRL (0.020 mg Kg\({}^{-1}\) for fruits and vegetables) [38] and ten times lower than the Indian MRL (0.100 mg Kg\({}^{-1}\) for fruits and vegetables) [39]. Our method can enhance food testing in these countries, as carbofuran is commonly detected in foodstuffs originating from China [40] or India [41]. Additionally, the AChE assay can be used, in the EU, as a cost-effective pre-screening tool for imported food. Moreover, the developed method can be beneficial also for checking the EU acute reference dose (ARfD) exceedance. In detail, carbofuran ARfD is equal to 0.00015 mg Kg\({}^{-1}\) Bow\({}^{-1}\) and the AChE assay could enable the concentration control that might, under certain consumption conditions, approach or even exceed it. Considering, e.g., an individual with a 70 kg body weight, the ARfD is 0.0105 mg, and then this value would be achieved when eating 150 g of a contaminated foodstuff containing 0.070 mg Kg\({}^{-1}\) of carbofuran residues. In fact, such cases have been recently reported in the EU Rapid Alert System for Food and Feed (RASFF) portal (shorturl.at/quavKO,last accessed 10/01/2020). For example, carbofuran was detected at concentrations higher than 0.070 mg Kg\({}^{-1}\) both in eggplants and goji berries in Germany, Czech Republic and Switzerland the last two years. To benchmark AChE assay analytical performance, the same strawberry and lettuce samples were analyzed by an accredited LC-MS/MS reference method (Figure 2). Recoveries (\(n=6\)) at two spiking levels, 0.020 mg Kg\({}^{-1}\) and 0.100 mg Kg\({}^{-1}\), were higher than 80% in both matrices by using the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction. Although the QuEChERS extraction has been addressed as the golden standard for multiple residues extraction, an ethyl acetate extraction was modified for the AChE assay. In contrast to QuEChERS extraction, which includes several analytical steps, the developed ethyl acetate extraction, did not include any centrifugation step resulting in a faster extraction protocol. Another advantage was that ethyl acetate can be evaporated considerably faster compared to acetonitrile (QuEChERS solvent) due to a lower boiling point and thus a reduced extraction time was achieved. Furthermore, acetonitrile is related to difficulties associated with the removal of water molecules, therefore, ethyl acetate (a less polar solvent) fits better to the sample preparation simplification concept.