## 3 Results and Discussion
### Comparison Between HPLC-MS Method and Immunoassay  
Several observations can be made based on the results obtained using the two different methods for detecting corticosteroids within cosmetic matrices. As regards performance in terms of detection limits, these appear to be comparable for the two approaches. Indeed, depending on the analyte, the immunological assay guarantees the detection of the target even at low concentrations, showing the possibility of competing with the reference HPLC-MS technique. However, from the point of view of the matrix effect, the bioassay is greatly affected by the different type of cosmetic, thus making calibration necessary for each different type of matrix. Given the possibility of testing several samples in a single analysis and given the reproducibility of the data, the need to perform a specific calibration for each matrix is not a problem. Furthermore, despite having a significant matrix effect, the LODs and LOQs are suitable for the detection of these target analytes in the different matrices; therefore, the ELISA method is sufficiently sensitive for its application in this field. Recovery and precision are also higher in the case of the HPLC-MS method. The selectivity of the reference HPLC-MS method is high, but the ELISA test is not able to identify and discriminate different corticosteroids, instead only providing their generic presence. As regards quantification, the immunoassay for some analytes (BC, BM 17-V, MP) presents very wide dynamic ranges that correspond to low sensitivities and to a more imprecise quantification compared to that obtainable by HPLC-MS. Despite this, however, considerations must be made regarding ease of use and rapidity of analysis, which are very different for the two approaches. In fact, the immunoassay requires less complex instrumentation, high-throughput screening, and an easy-to-use procedure, which therefore considerably simplifies the analysis process. In particular, these analyses can also be conducted by non-specialized personnel and outside of centralized laboratories, unlike HPLC-MS techniques, which instead require specific training for qualified personnel [51]. These two techniques therefore offer different advantages, which makes them complementary and suitable for different contexts. Furthermore, the expansion of the use of the commercial ELISA kit that allows for the broadening of the spectrum of targets and matrices to be detected and quantified represents a novelty of this study.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Steenoid**} & \multicolumn{2}{c}{**Spiked Concentration (ng/mL)**} & \multicolumn{2}{c}{**Recovery (\%)**} \\ \cline{2-5}  & **Cream** & **serum** & **Cream** & **Serum** \\ \hline \multirow{3}{*}{FM} & 0.7 & 2 & 75 & 71 \\  & 7 & 4 & 82 & 79 \\  & 12 & 7 & 90 & 89 \\ \hline \multirow{3}{*}{DM} & 2 & 1 & 72 & 70 \\  & 30 & 60 & 85 & 87 \\  & 50 & 100 & 92 & 90 \\ \hline \multirow{3}{*}{BC} & 2 & 15 & 73 & 79 \\  & 30 & 3000 & 79 & 80 \\  & 50 & 5000 & 88 & 85 \\ \hline \multirow{3}{*}{BM 17-V} & 20 & 80 & 72 & 71 \\  & 250 & 700 & 75 & 78 \\ \cline{1-1}  & 400 & 50,000 & 83 & 81 \\ \hline \multirow{3}{*}{MP} & 60 & 10 & 71 & 73 \\  & 20,000 & 15,000 & 79 & 78 \\ \cline{1-1}  & 30,000 & 25,000 & 81 & 85 \\ \hline \hline \end{tabular}
\end{table}
Table 4: Recovery for spiked concentrations in different matrices.  
Regarding the applicability of the two methods to different types of formulations, we analyzed spiked samples at medium concentration. The results were in line with the cosmetic products involved in the method validation. In particular, we tested four more creams--two oil/water and two water/oil emulsions--and three more serums.