## 3 Results and Discussion
### ELISA Method Validation  
A protocol for using the ELISA kit has been successfully developed and optimized for its use in the analysis of corticosteroids in cosmetic formulations. Calibration curves were generated by spiking a known amount of target analytes in the cosmetic matrices (facial cream corresponding to cream A used for the HPLC-MS method and serum) suitably dissolved in THF and diluted 1:5 (_v_/_v_) in PBS. The matrices were selected based on the fact that generally the illegal addition of corticosteroids involves preparations for facial application [35]. These calibration curves were compared with those obtained by spiking target analytes in a mixture of PBS/THF. In the case of FN, BD, and DP, it was not possible to generate calibration curves since there were no significant changes in the signal even in the presence of high concentrations of these targets with respect to the blank. The primary antibody immobilized on the microwell plate was probably not able to recognize these \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Steeroid**} & \multicolumn{2}{c}{**Spiked**} & \multicolumn{2}{c}{**Matrix Effect \%**} & \\ \cline{3-5}  & **Concentration** & **(ng/mL)** & **Cream A** & **Cream B** & **Serum** \\ \hline \multirow{4}{*}{BM 17-V} & 0.3 & 0.47 & \(-\)0.48 & \(-\)0.47 \\  & 0.5 & 0.26 & \(-\)0.25 & 0.21 \\  & 10.0 & 0.04 & \(-\)0.32 & 0.26 \\  & 20.0 & 0.01 & \(-\)0.01 & \(-\)0.29 \\ \hline \multirow{4}{*}{BC} & 0.3 & 0.46 & 0.81 & 0.71 \\  & 0.5 & 0.21 & 0.06 & \(-\)0.02 \\  & 10.0 & 0.12 & \(-\)0.03 & 0.08 \\  & 20.0 & \(-\)0.49 & 0.82 & \(-\)0.12 \\ \hline \multirow{4}{*}{BCDP} & 0.3 & 0.91 & \(-\)0.66 & \(-\)0.97 \\  & 0.5 & 0.11 & \(-\)0.44 & \(-\)0.14 \\  & 10.0 & 0.11 & \(-\)0.28 & \(-\)0.12 \\  & 20.0 & 0.06 & 0.21 & \(-\)0.18 \\ \hline \multirow{4}{*}{MP} & 0.3 & 0.87 & \(-\)0.33 & \(-\)0.33 \\  & 0.5 & 0.19 & \(-\)0.07 & \(-\)0.41 \\  & 10.0 & 0.18 & \(-\)0.06 & \(-\)0.36 \\  & 20.0 & 0.01 & \(-\)0.48 & \(-\)0.38 \\ \hline \multirow{4}{*}{BD} & 0.3 & 0.15 & 0.07 & 1.01 \\  & 0.5 & 0.09 & 0.12 & \(-\)0.14 \\  & 10.0 & 0.09 & \(-\)0.38 & \(-\)0.51 \\  & 20.0 & \(-\)0.11 & \(-\)0.36 & 0.71 \\ \hline \multirow{4}{*}{FN} & 0.3 & 0.33 & \(-\)0.40 & \(-\)0.40 \\  & 0.5 & 0.05 & \(-\)0.53 & \(-\)0.52 \\  & 10.0 & 0.15 & \(-\)0.48 & \(-\)0.47 \\  & 20.0 & \(-\)0.04 & \(-\)0.23 & \(-\)0.45 \\ \hline \multirow{4}{*}{FM} & 0.3 & 0.72 & \(-\)0.27 & \(-\)0.53 \\  & 0.5 & 0.16 & \(-\)0.07 & 0.08 \\  & 10.0 & 0.07 & \(-\)0.12 & \(-\)0.07 \\  & 20.0 & \(-\)0.08 & \(-\)0.15 & \(-\)0.06 \\ \hline \multirow{4}{*}{DM} & 0.3 & 0.64 & \(-\)0.05 & \(-\)0.13 \\  & 0.5 & 0.10 & \(-\)0.15 & \(-\)0.02 \\ \cline{1-1}  & 10.0 & 0.26 & \(-\)0.15 & \(-\)0.03 \\ \cline{1-1}  & 20.0 & 0.05 & \(-\)0.11 & \(-\)0.05 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Matrix effect on spiked samples in different matrices. corticosteroids. Indeed, the specifications of the used commercial kit did not report these target analytes among those that can be quantified. The calibration curves (Figure 2) showed a high matrix effect; indeed, the LODs and dynamic ranges proved that the matrix significantly influences the performance of the method. Table 3 shows the LOD values and upper limits obtained for all the target analytes in the different matrices. As expected, the best performances were obtained by spiking corticosteroids in the solvent PBS/THF without the matrices. The dynamic ranges for some analytes were wide (up to 3 orders of magnitude), thus showing that to obtain a significant change in the signal a large difference in concentration of the target analyte is required. For FM and DM, the lowest LODs were obtained; and for the other target analytes the minimum detectable concentrations were also compatible with the concentrations of corticosteroids that are commonly added to cosmetic preparations.