## 2 Basis of the QuEChERS Method
### Modifications in the Extraction Solvent  
In the original QuEChERS method, the extraction solvent proposed is ACN, as it extracts a broad range of organic compounds without leading to co-extraction of lipophilic materials [17]. Moreover, this organic solvent is less polluting than others, such as methanol, and it promotes protein precipitation, which can be considered a first clean-up step in \begin{table}
\begin{tabular}{c c c} \hline \hline
**Sample Type** & **Sample Amount (g)** & **Water Addition (g)** \\ \hline Fruit, vegetables and other & & \\ products with \textgreater{}80\% water & 10 g & - \\ content & & \\ Fruit, vegetables and other & & \\ products with 25–80\% water & 10 g & \(\textgreater{}\) kg * \\ content & & \\ Cereals & 5 g & 10 g \\ Dried fruits & 5 g & 7.5 g \\ Honey & 5 g & 10 g \\ Spices & 2 g & 10 g \\ \hline \hline \end{tabular}  
* *xg: 10 g—water amount in 10 g of sample.  
\end{table} 
Table 2: Proposal of the amount of sample and water addition to perform QuEChERS depending on the type of sample (data obtained and adapted from reference [39]). complex matrices, such as food and feed. Nevertheless, over time, many solvent modifications have been proposed in this procedure to improve the extraction efficiency of different analytes from diverse matrices. The most common is to use acidified ACN with some type of acid, such as formic acid (the most extended), acetic acid or citric acid. Another common practice is to use mixtures of ACN with other solvents, such as water, methanol, ethyl acetate, dichloromethane or hexane. In other cases, ACN can be substitute by other organic solvents, such as methanol, or even used combinations of different solvents without ACN [15; 16]. Accordingly, in the case of PAs, several authors have chosen to perform the first extraction step of the QuECHERS procedure with acidified ACN, mainly using different percentages of formic acid ranging from 0.1% to 2% (Table 1). For instance, [PERSON] et al. (2020) compared the extraction of PAs with non-acidified ACN and with ACN acidified with 1% formic acid [26]. The results revealed that the recoveries of the targeted PAs evaluated improved 6-18% when ACN was acidified. Likewise, [PERSON] et al. (2011) also used acidified ACN to extract 14 PAs and PANOs from different products (food supplements, honey and feed), but using in this case 1% acetic acid [30]. Conversely, some authors have also used mixtures of ACN with other solvents for the extraction of PAs in the QuECHERS procedure (Table 1). In this sense, [PERSON] et al. (2022) combined ACN with an acidified aqueous solution with 0.5% formic acid (75:25, \(v/v\)) for the extraction of 28 PAs and PANOs from dried teas and herbs [25]. One of the main reasons why authors selected acidified polar solvents or acidified aqueous solutions is due to the great polarity of PANOs, as is essential to simultaneously extract both PAs and PANOs from the samples [40]. [PERSON] et al. (2014) observed that the pH of the matrices influenced the extraction efficiency of basic analytes such as PAs and PANOs [29]. In this sense, matrices with pH values >5 did not provide acceptable recovery values, whereas in food matrices with pH values <4.5, good recoveries were achieved. For this reason, they decided to add 2% formic acid to the extraction solvent, observing an improvement in the recovery of those problematic analytes in the less acidic food matrices [29]. As the authors stated, the extraction at lower pH probably prevented or at least minimized the interaction between the analytes and the matrix through their charged functional groups. However, in contrast, [PERSON] et al. (2021) achieved the best extraction of PAs from forage grass under basic conditions, by using a mixture of ACN with an aqueous solution of ammonium carbonate (84:16, \(v/v\)) at pH 8.5 [35]. Nonetheless, in this case, only five PAs were used as target analytes (retrosine, senkirkine, seneciphylline, monocottaline and senecionine), and no PANOs were included in the determination.