## 3 Results
### UHPLC-MS/MS Approach  
Some stationary phases were evaluated, including HILIC columns. Neither the DVB nor the EFB were resolved or retained. Utilizing a C8 column has demonstrated advantageous outcomes as the reversed stationary phase. Conversely, when using the UHPLC-MS/MS approach to resolve DVB and EFB, the use of a C18 column causes the analytes to be retained in the chromatographic system. However, the observed targets, specifically DVB and EFB, with longer tails of the peak, exhibited inadequate distinctions of the main peak, and a prolonged elution period. The use of an Agilent C8 column (Eclipse plus), characterized by specific parameters such as a 50 mm length, a 3.5 mm particle size, and a 2.1 mm inner diameter, produced favorable results in terms of elution time and the shapes of analytical peaks. Table 4 shows a thorough compilation of several experiments performed to improve and determine the best characteristics for extracting, separating, and evaluating DVB and EFB peaks. The main goal of these experiments was to achieve a desirable feature, such as an acceptable peak shape and a reduced running duration. So as to enhance the specificity and sensitivity features of the UHPLC-MS/MS instrument, the MRM analyzer mode was put in place to accurately detect and measure the levels of DVB and EFB. The target of this research was to investigate any possible interference produced by the HLMs matrix ingredients (Figure 4). \begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{2}{c}{**Physicochemical Properties**} & \multicolumn{2}{c}{**Water Solubility**} \\ \hline Formula & C\({}_{21}\)H\({}_{21}\)FN\({}_{6}\)O & Solubility & \(8.60\times 10^{-2}\) mg/mL; \(2.19\times 10^{-4}\) mol/L \\ Molecular weight & 392.43 g/mol & Log S (ESOL) & \(-3.66\) \\ Heavy atoms num. & 29 & Class & Soluble \\ Arom. heavy atoms num. & 19 & Solubility & \(2.32\times 10^{-1}\) mg/mL; \(5.92\times 10^{-4}\) mol/L \\ Rotatable bonds num. & 2 & Log S (Ali) & \(-3.64\) \\ Fraction Csp3 & 0.24 & Class & Soluble \\ Num. H-bond donors & 3 & Solubility & \(8.66\times 10^{-5}\) mg/mL; \(2.21\times 10^{-7}\) mol/L \\ TPSA & 94.04 Å\({}^{2}\) & Log S (SILICOS-IT) & \(-6.66\) \\ Num. H-bond acceptors & 4 & Class & Poorly soluble \\ \cline{2-3} \multicolumn{2}{c}{Molar refractivity} & 120.28 & Brenk & 0 alert \\ \hline \multicolumn{2}{c}{**Lipophilicity features**} & PAINS & 0 alert \\ \hline Log Po/w (XLOGP3) & 1.64 & Leadlikeness & No; 1 violations: MW \(>350\) \\ Log Po/w (iLOGP) & 2.26 & Synthetic accessibility & 3.20 \\ \cline{2-3} \multicolumn{2}{c}{Log Po/w (MLOGP)} & 2.31 & \multicolumn{2}{c}{**Pharmacokinetics**} \\ \cline{2-3} Log Po/w (SILICOS-IT) & 3.27 & GI absorption & High \\ Log Po/w (WLOGP) & 2.21 & P-gp substrate & Yes \\ Consensus Log Po/w & 2.34 & Permeant to BBB & No \\ \hline \multicolumn{2}{c}{**Druglikeness features**} & Inhibitor of CYP1A2 & Yes \\ \hline [PERSON] & Yes & Inhibitor of CYP2D6 & Yes \\ [PERSON] & Yes; 0 violation & Inhibitor of CYP3A4 & No \\ [PERSON] & Yes & Inhibitor of CYP2C9 & No \\ [PERSON] & Yes & Inhibitor of CYP2C19 & No \\ [PERSON] & Yes & Skin permeation (Log Kp) & \(-7.53\) cm/s \\ The score of bioavailability & 0.55 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: ADME characteristics of DVB derived by the freely available online SwissADME software. The MRM chromatograms of the negative control (Figure 5A) and positive control (Figure 5B) for DVB in the HLMs did not show any carry-over impact. Figure 5C shows the mass chromatograms obtained from the MRM analysis of DVB CSs and EFB. The range of concentrations covered is from 1 to 3000 ng/mL for DVB CSs and 2000 ng/mL for EFB. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
\begin{tabular}{c} Optimum \\ shape \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Tuned UHPLC-MS/MS parameters. Figure 4: MRM (two mass transitions) mass spectrum of DVB [M + H]\({}^{+}\) (**A**) and Encorafenib [M + H]\({}^{+}\) (**B**) represented as two mass transitions.