## 2 Basis of the QuEChERS Method

Table 1: Application of modified QuaCELERs to the determination of pyrrolidine alkaloids in food and food samples. Due to the multiple advantages of the QuECHERS method (simplicity, cost-effectiveness and ability to perform multiple extraction of different analytes), many of the works reviewed carried out the simultaneous co-analysis of PAs with other compounds, such as other plant toxins (mainly tropane alkaloids, among others such as glycoalkaloids, isoquinoline alkaloids, ergot alkaloids, opium alkaloids, etc. ), pesticides, myocarditis, drugs, phytoestrogens, etc. [19; 23; 29; 30; 31; 32; 33; 34; 35]. However, despite its advantageous properties, the QuECHERS procedure has been scarcely employed for the analysis of PAs compared to SPE, as previously mentioned (Figure 1). Nonetheless, its application to the analysis of PAs is expected to increase significantly in the coming years, as it is quicker and more environmentally friendly than conventional SPE. In this context, [PERSON] et al. (2017) compared both types of extraction techniques for the analysis of nine PAs in honey samples, concluding that SPE, besides being more tedious and time-consuming than QuECHERS, also provided worse recovery values for the analytes [19]. Conversely, some authors carried out a first extraction with H\({}_{2}\)SO\({}_{4}\) followed by the addition of zinc dust prior to the QuECHERS method. This first step is performed with the aim of reducing the PANOs to their corresponding PAs form (which is their tertiary base form) [19; 20; 34]. For this reason, in these works, only PAs are determined in the honey and pollen samples, while PANOs are not determined nor quantified. This reduction procedure is more indicated for the analysis of PAs by GC, as PANOs cannot be analyzed with this technique because they are unstable at the temperatures needed for volatilization [2; 37; 38]. Therefore, as PAs and PANOs are both toxicologically important and both forms need to be included in the analytical determination of these alkaloids [1; 14], this reduction procedure can be carried out to ensure the extraction and determination of both types. However, in all the works reviewed for the determination of PAs by QuECHERS extraction, none of them used GC for the instrumental analysis of the sample extracts obtained after the QuECHERS procedure (Table 1). In contrast, all of them used LC coupled to tandem mass spectrometry (LC-MS/MS), which it is generally preferred over GC, as no derivatization of PAs is required; thus, sample preparation is easier and quicker [2]. Moreover, LC-MS/MS achieves the individual detection and quantification of both PAs and PANOs. Therefore, one of the reasons to use the zinc reduction strategy with LC analysis may be the acquisition of less commercial standards to perform the total quantification of PAs, as it avoids acquiring commercial standards for PANOs [2]. However, one drawback of this reduction procedure is that it does not perform individual analysis of PAs, and consequently, it is not possible to determine the contamination profile of PAs and PANOs in the samples or the origin of the contamination source. Moreover, the reduction step increases the time of the analytical method, as it requires 1 h and 30 min [20], which makes the extraction process more tedious and time-consuming. Regarding the LC-MS/MS instrumental analysis of the works reviewed, it is observed that all of them used electrospray ionization (ESI) in positive ion mode as ionization source (Table 1). Nonetheless, some works also indicated the simultaneous determination in both positive and negative ion mode [30; 31; 32]. However, the reason is that in these works, in addition to PAs, other compounds are also included in the chromatographic analysis, as previously indicated (e.g., plant toxins, mycotoxins, pesticides, phytoestrogens, etc.). Thus, the ionization in negative ion mode is employed for the identification of these other analytes.