## 2 Basis of the QuEChERS Method  
The QuEChERS was designed as a multiresidue approach for the determination of multiple analytes (more than 200 pesticides) at the same time, involving simultaneous extraction and clean-up of samples (particularly, fruit and vegetables) [17]. This method is based on the dispersion of salts (salting-out effect) to extract and isolate a wide variety of analytes from complex matrices in addition to the subsequent clean-up of the sample extract obtained. In this sense, there are two clear steps in this approach:  
1. Extraction step based on partitioning via salting-out extraction, achieving an equilibrium between an aqueous and an organic phase. 2. Clean-up step carried out by dispersive solid-phase extraction (dSPE) using different sorbent materials and salts to remove matrix interferences. The original QuEChERS procedure involves using 10 g of sample, 10 mL of organic solvent (acetonitrile, ACN), 5 g of partitioning salts (4 g MgSO\({}_{4}\) and 1 g NaCl) and 175 mg of clean-up sorbents (25 mg primary secondary amine (PSA) and 150 mg MgSO\({}_{4}\)) [17]. Accordingly, the sample is first subjected to a solid-liquid extraction (SLE) with ACN carried out by manual shaking and followed by the salt partitioning step with MgSO\({}_{4}\) and NaCl to promote water partition from the organic layer and its dehydration. The addition of these salts enables a decrease in the solubility of polar compounds in the aqueous phase and removal of water from the organic phase. To achieve efficient and homogeneous interaction among the salts, the organic solvent and the sample, a stirring process followed by centrifugation is carried out, which allows for the separation of both phases. Afterward, an aliquot of the supernatant corresponding to the organic phase is recovered for the subsequent dSPE clean-up step. PSA is a weak anion exchange sorbent; thus, it can interact strongly with the acid matrix interferents that may have been co-extracted during the process, such as sugars, fatty acids and organic acids, promoting their elimination from the ACN phase. Conversely, the addition of MgSO\({}_{4}\) in the clean-up step removes the residual water content in the extract and improves the interaction of the above-mentioned matrix interferences on the PSA sorbent, leading to a final extract with less polarity due  
Figure 1: (**a**) Overview of the main extraction methods used for pyrrolidine alkaloids from food and feed, and (**b**) evolution of the number of published articles using QuEChERS for the determination of pyrrolidine alkaloids in food and feed samples over the last 11 years (2011–2022). Data obtained from Scopus, Web of Science and Google search engines up to March 2022. LLE: liquid–liquid extraction; SLE: solid–liquid extraction; SPE: solid–phase extraction. to the precipitation of the polar interferets which improves their retention in the PSA sorbent. After a brief shaking and centrifugation, the supernatant is recovered and can be directly analyzed by GC or LC [15; 16; 17; 18]. Figure 2 shows the schematic layout of the original QuEChERS procedure. Nevertheless, since its origin, despite its high efficiency, the QuEChERS concept has evolved to be adapted to other analytes and matrices. In this sense, due to its flexibility, several modifications have been successfully introduced in its key parameters in order to improve its extraction efficiency, to spread its potential application to a wide range of matrices and to achieve simultaneous extraction of multiple compounds belonging to different chemical families [15; 16]. Some of the most notable modifications in the QuEChERS procedure are depicted in Figure 3.  
Evolution of the Original QuEChERS Method and Its Application to the Determination of Pyrrolizidine Alkaloids in Food and Feed Samples  
Table 1 summarizes the different QuEChERS strategies carried out within the last 11 years for the extraction and analysis of PAs and PANOs in different food and feed products.