## 3 Results and Discussion
### Optimization of QuEChERS Extraction Conditions
#### 3.2.2 Multivariable Method  
After having concluded in the extraction solvent (acetonitrile) and the suitable salts combination for the extraction and the cleanup steps of the QuEChERS method, more specific parameters should be checked: the sample amount, the sorbents amount, the solvent volume and the percent (%) content of acetic acid in the extraction solvent. For that purpose and since the parameters were satisfactory (7), a screening experimental design was employed in order to evaluate their effect on the analytes' recoveries. The parameters were: (x\({}_{1}\)) the sample amount (mL), (x\({}_{2}\)) the solvent volume (ACN) (mL), (x\({}_{3}\)) the quantity of MgSO4 (g), (x\({}_{4}\)) the quantity of NaCl (g), (x\({}_{5}\)) the quantity of PSA (mg), (x\({}_{6}\)) the quantity of C18 (mg) and (x\({}_{7}\)) the percent content of CH\({}_{3}\)COOH (%). Thus, a 27\({}^{-4}\) Plackett-Burman design was employed, and each of the seven parameters were investigated at two levels, a high (\(-1\)) and a low (\(+1\)). Eight runs in total were generated, plus three central points (0) (Table S4), and the measured response of each run was the mean recovery of the target EDCs. QuEChERS extraction was performed in milk fortified at the same concentration level, 100 ng g\({}^{-1}\). An effect was considered significant when it was above the standard error at the 95% confidence level (\(p<0.05\)), which is denoted by the vertical line on the Pareto chart. Positive signs indicate that increasing the value of this variable there is also an enhancement in the measured response, being in this case the mean recovery of the target EDCs. Negative signs indicate, respectively, a negative correlation with the measured response. In the studied experimental domain (see Pareto chart in Figure S5), all factors  
Figure 1: The effect of the extraction salts from a milk sample fortified at 100 ng g\({}^{-1}\) on the analytes’ recovery. Figure 2: Influence of the different salts’ combination of the dSPE cleanup step on the analytes recovery from a milk sample spiked at 100 ng g\({}^{-1}\): (**a**) MgSO4/PSA; (**b**) MgSO4/PSA/C18; (**c**) MgSO4/PSA/GCB and (**d**) MgSO4/PSA/C18/GCB. proved to be statistically significant at a 95% confidence level except the amount of MgSO\({}_{4}\) which was excluded from the study (Table S5). The effect of the acetonitrile volume and the sample amount were found to be significant; however, these variables were not taken into consideration for further investigation since it was found from previous experiments that the optimum solvent-to-sample amount ratio would be 1:1. Moreover, using a small volume of solvent (e.g., 5 mL) resulted in various practical problems (e.g., difficulty in the organic phase separation) during the experimental setup. Three parameters remained as the most crucial ones and were subjected to further study via a central composite design (CCD): the amounts of NaCl and C18 and the percent (%) content of acetic acid in the extraction solvent. CCD was a fractional factorial 2\({}^{3}\) design which included six-star points in the distance \(\alpha\) (\(\alpha\) = 1.682) from the central point. Seventeen experiments were run in total in random order and three variables were checked, each one at five levels (\(-\alpha\), \(-1\), \(0\), \(1\), \(+\alpha\)) and three central points (0) (Table S6). The main effects, interaction effects, as well as quadratic effects were evaluated through analysis of variance (ANOVA) at spiking concentration level of 100 ng g \({}^{-1}\) (Table S7). Lack of fit was also checked and was shown to be not significant relative to the pure error, indicating a good response to the model (Table S7). The normality assumption was satisfied as the residual plot approximated along a straight line (Figure S6). Pareto chart (Figure S7) indicated the linear (L), quadratic (Q) as well as the interaction effects on the method performance.