## 3 Results and Discussion
The mean R\({}^{2}\) obtained in the experimental measure was 0.9998, demonstrating that linearity proved to be acceptable for the quantification of tRES. The obtained linear regression equation was \(y=400\),628\(x-144\),146, where \(y\) stands for the peak area (AU), and \(x\) stands for the tRES concentration (\(\mu\)g/mL). Method specificity was analyzed with respect to the risk of interference between the lipids and surfactants composing SLN and their effect in the tRES retention time [46]. The three distinct samples were filtered before analysis by the proposed HPLC method. When comparing the results with a chromatogram of tRES standard solution of 50 \(\mu\)g/mL (Figure 3a,b), no interferences between the components of SLN could be recorded. As shown in Figure 3a, for all the tested formulations, the filtrate of blank SLN (F1) did not exhibit any peak at the tRES retention time. These results demonstrate the specificity of the proposed HPLC-PDA to identify and to quantify the _trans_ isomeric form (tRES) released from SLN. Three different tRES standard solutions, each concentration prepared in triplicate (n = 3) (1.0, 50.0, and 100.0 \(\mu\)g/mL), were analyzed in three consecutive days. The results obtained for the assessment of precision are shown in Table 2. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & \multicolumn{4}{c}{Precision} & \multicolumn{2}{c}{Accuracy} \\ \hline \multirow{2}{*}{**Day**} & \multirow{2}{*}{\begin{tabular}{c} tRES \\ **Concentration/(ug/mL)** \\ \end{tabular} } & \multicolumn{2}{c}{**Intraday**} & \multirow{2}{*}{\begin{tabular}{c} **Interday** \\ **SD 7/(AU)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Resp** \\ **\(\star\)/(\%)** \\ \end{tabular} } & \multirow{2}{*}{\
\begin{tabular}{c} **Resp** \\ **\(\star\)/(\%)** \\ \end{tabular} } & \multirow{2}{*}{
\begin{tabular}{c} **Experimental** \\ **Concentration/(ug/mL)** \\ \end{tabular} } & \multirow{2}{*}{**Recovery/(\%)**} \\ \hline
1 & & 10,916 & 2.87 & & & \\
2 & 1.0 & 9867 & 2.35 & 19150 & 4.83 & 4.626 \(\pm\) 0.009 & 92.53 \(\pm\) 0.19 \\
3 & & 632 & 0.16 & & & \\ \hline
1 & & 9623 & 0.05 & & & \\
2 & 50.0 & 91,044 & 0.45 & 662346 & 3.17 & 50.57 \(\pm\) 0.228 & 101.15 \(\pm\) 0.46 \\
3 & & 205,253 & 0.95 & & & \\ \hline
1 & & 66,372 & 0.16 & & & \\
2 & 100.0 & 68,212 & 0.17 & 1439947 & 3.47 & 99.64 \(\pm\) 0.170 & 99.64 \(\pm\) 0.170 \\
3 & & 84,338 & 0.19 & & & \\ \hline \hline \end{tabular}  
\end{table} 
Table 2: Results achieved for the intraday and interday precision of the method and results found for method accuracy. Figure 3: Retention time recorded in the chromatogram of the (**a**) filtrated drug-free SLN formulation (F1) (**b**) and in the chromatogram of _trans_-resveratrol (tRES) standard solution (50.0 \(\mu\)g/mL). The obtained results document the acceptable intraday and interday precisions of our method. All the data were within the acceptance criteria of RSD lower than 5.0%, which attributes a high degree of agreement between the results when analyzed with the same experimental conditions. The method proved to be accurate, not exceeding \(\pm\) 10.0% [44], demonstrating the proximity of experimental results with the real value. Robustness can be measured by assessing the effect of small fluctuations in the chromatographic experimental conditions on the retention time and obtained RSD value [46]. Robustness was evaluated by analyzing the 50.0 \(\upmu\)g/mL tRES standard solution. The results are shown in Table 3. The standard solution was prepared in triplicate (n = 3). No significant changes in the retention time were observed when flow was changed, and a similar result was shown when varying the temperature. The tested variations were among the values allowed by ANVISA [44], for which the RSD is \(\pm\) 5.0%. The method was shown to be robust for the flow but not for the temperature. The LOD and LOQ obtained in this work were 1.29 \(\upmu\)g/mL and 4.29 \(\upmu\)g/mL, respectively.