## 3 Results
### HPLC-MS/MS Method Validation
#### 3.1.5 Analytic Stability  
In the storage conditions tested, Fingolimod and Fingolimod-P stock and working solutions were stable for, respectively, 7 and 2 months at \(-\)20 \({}^{\circ}\)C, since the absolute peak areas of the working solutions confirmed the stability of both analytes in these storage conditions, with differences always \(\leq\)15% compared to the mean peak area measured a t = 0 (Table 5). \begin{table}
\begin{tabular}{c c c c c c c c c c c c} \hline \hline  & \multicolumn{8}{c}{**Fingolimod**} \\ \cline{2-13}
**QC Conc.** & \multicolumn{3}{c}{**Fingolimod Recovery**} & \multicolumn{3}{c}{**Fingolimod-D4 (IS) Recovery**} & \multicolumn{3}{c}{**Fingolimod MF**} & \multicolumn{3}{c}{**IS-Normalized MF**} \\ \cline{2-13}
**(ng/mL)** & **Mean** & \(\pm\)**SD** & **CV** & **Mean** & \(\pm\)**SD** & **CV** & **Mean** & \(\pm\)**SD** & **CV** & **Mean** & \(\pm\)**SD** & **CV** \\ \hline
**4.0 (6)** & \(88.0\%\) & 2.3\% & 2.7\% & 10.7\% & 87.0\% & 10.7\% & 12.3\% & 1.281 & 0.178 & 13.9\% & 1.123 & 0.033 & 2.9\% \\
**100.0 (6)** & \(90.6\%\) & \(9.7\%\) & 10.7\% & 10.7\% & 10.7\% & 1.037 & 0.077 & 7.4\% & 0.956 & 0.040 & 4.2\% \\ \hline \multicolumn{13}{c}{**Fingolimod-P**} \\ \hline
**QC Conc.** & \multicolumn{3}{c}{**Fingolimod-P Recovery**} & \multicolumn{3}{c}{**Fingolimod-P-D4 (IS) Recovery**} & \multicolumn{3}{c}{**Fingolimod-P MF**} & \multicolumn{3}{c}{**IS-Normalized MF**} \\ \cline{2-13}
**(ng/mL)** & **Mean** & \(\pm\)**SD** & **CV** & **Mean** & \(\pm\)**SD** & **CV** & **Mean** & \(\pm\)**SD** & **CV** & **Mean** & \(\pm\)**SD** & **CV** \\ \hline
**4.0 (6)** & \(73.4\%\) & 4.3\% & 5.9\% & 71.7\% & 9.3\% & 13.0\% & 1.331 & 0.195 & 14.7\% & 1.109 & 0.054 & 4.9\% \\
**100.0 (6)** & \(75.5\%\) & 9.4\% & 12.5\% & & & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Fingolimod and Fingolimod-P recovery and matrix effect in human plasma calculated on six different matrices. \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline  & \multicolumn{8}{c}{**QCs Calculated Concentrations (ng/mL) in Six Replicates**} \\ \cline{2-10}
**QCs Nominal** & \multicolumn{3}{c}{**Intra-Day**} & \multicolumn{3}{c}{**Inter-Day**} \\ \cline{2-10}
**Concentrations (ng/mL)** & \begin{tabular}{c} **Mean** \\ **(ng/mL)** \\ \end{tabular}  & \(\pm\)**SD** & **Accuracy** & \begin{tabular}{c} **Precision** \\ **(CV)** \\ \end{tabular}  & \begin{tabular}{c} **Mean** \\ **(ng/mL)** \\ \end{tabular}  & \(\pm\)**SD** & **Accuracy** & \ \begin{tabular}{c} **Precision** \\ **(ng/mL)** \\ \end{tabular}  & \(\pm\)**SD** & **Accuracy** & \begin{tabular}{c} **Precision** \\ **(CV)** \\ \end{tabular}  \\ \hline
**Fingolimod** & \(0.3\) (6) & \(0.30\) & \(0.02\) & \(99.5\%\) & \(8.3\%\) & \(0.30\) & \(0.01\) & \(101.6\%\) & \(3.4\%\) \\
**4.0 (6)** & \(4.24\) & \(0.20\) & \(105.9\%\) & \(4.7\%\) & \(4.16\) & \(0.23\) & \(103.9\%\) & \(5.4\%\) \\
40.0 (6) & \(39.52\) & \(1.45\) & \(98.8\%\) & \(3.7\%\) & \(36.93\) & \(1.50\) & \(92.3\%\) & \(4.1\%\) \\
100.0 (6) & \(94.52\) & \(4.64\) & \(94.5\%\) & \(4.9\%\) & \(10.28\)Stability of Singolimod and Fingolimod-P in plasma was evaluated at two concentrations (4.0 and 100 ng/mL) under four conditions (Table 6): (i) bench-top stability (4 h at room temperature); (ii) stability after two freeze-thaw cycles; (iii) autosampler stability (48 h at 6 \({}^{\circ}\)C); (iv) long-term storage stability (1, 3, 6 and 12 months at \(-20\)\({}^{\circ}\)C and at \(-80\)\({}^{\circ}\)C).