## 2 Materials and Methods
### DVB Calibration Levels  
Before initiating the validation phase for the UHPLC-MS/MS approach, the HLMs matrix was made inactive by adding DMSO (2%) and incubating it at 50 \({}^{\circ}\)C for 5 min. The incorporation of this precautionary estimate targeted alleviating the probable metabolic special effects of HLMs [35; 36; 37] on the analytes under investigation, specifically DVB and EFB. A validation matrix was specifically created for HLMs studies to evaluate the  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**UPLC**} & \multicolumn{2}{c}{**TQD MS**} \\ \hline \multirow{4}{*}{Eclipse plus-C8 reversed column} & Particle size & 3.5 μm & Positive ESI source \\ \cline{2-5}  & Internal diameter & 2.1 mm & The extractor voltage & 3.0 (V) \\ \cline{2-5}  & Length & 50.0 mm & Cone gas rate & 100 L/hr \\ \cline{2-5}  & Column T & 22.0 \(\pm\) 2.0 \({}^{\circ}\)C & \multicolumn{2}{c}{The RF lens voltage} & 0.1 (V) \\ \hline \multirow{4}{*}{Isocratic mobile phase system} & \multirow{4}{*}{Aqueous part} & 0.1\% Formic acid in H\({}_{2}\)O & \multirow{2}{*}{ESI} & \multirow{2}{*}{Nitrogen gas} \\ \cline{3-3} \cline{5-5}  & & 55\% & & 100 L/hr \\ \cline{3-3} \cline{5-5}  & & pH: 3.2 & & 350 \({}^{\circ}\)C \\ \cline{2-5}  & Organic part & 45\% ACN & & Capillary voltage: 4 KV \\ \cline{2-5}  & Injection volume: & 5.0 μm & Argon gas & 0.14 mL/min \\ \cline{2-5}  & Flow rate & 0.5 mL/min. & Mode & MRM \\ \hline \hline \end{tabular}
\end{table}
Table 1: UPLC-MS/MS tuned parameters.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Time Segment** & **Rt** & **Mass Transitions (_m/z_)** \\ \hline \multirow{3}{*}{MRM detection segments} & 0.0 to 0.65 min & DVB (0.44 min) & 393\(\rightarrow\)336 (CE \({}^{\text{a}}\):32 and CV \({}^{\text{b}}\): 28) \\ \cline{1-1} \cline{3-3} \cline{5-5}  & & 393\(\rightarrow\)58 (CE:36 and CV: 46) \\ \cline{1-1} \cline{3-3} \cline{5-5}  & 0.65 to 1.0 min & EFB (IS; 0.77 min) & 540\(\rightarrow\)359 (CE: 56 and CV: 36) \\ \cline{1-1} \cline{3-3} \cline{5-5}  & & 540\(\rightarrow\)116 (CE: 56 and CV: 32) \\ \hline \hline \end{tabular}  
* Dissociation energy, \({}^{\text{b}}\) the voltage of the cone.  
\end{table}
Table 2: MRM features for the quantitative estimation of DVB and EFB.  
metabolic stability of DVB. The experimental steps included adding 30 \(\mu\)L of inactivated HLMs, which had a protein at 1 mg/mL, to 1 mL of metabolic matrix. The metabolic buffer solution was formulated by combining 0.1 M sodium phosphate at a pH of 7.4. In addition, the buffer solution contained 3.3 mM of MgCl\({}_{2}\) and 1 mM of NADPH. The objective of utilizing this technology was to simulate the features of genuine in vitro incubation for practical experiments. The DVB (WK2 and WK3) underwent a series of dilutions using the inactive HLMs matrix in order to create DVB calibration standards, also known as CSs. Therefore, a grand total of eight CSs were generated at 1 to 3000 ng/mL. Additionally, four quality controls (QCs) were formulated, as follows: 2400 ng/mL (the high QC, HQC), 900 ng/mL (the medium QC, MQC), 3 ng/mL (the lower QC, LQC), and 1 ng/mL (LLOQ). During the multi-step dilution process, strict methods were followed to maintain the HLMs matrix at a level exceeding 90%. The target of this activity was to decrease the possible influences of matrix dilution, thereby reproducing the conditions of in vitro incubation. The QCs were utilized as unknowns, and their values were determined by applying the regression equation generated from the parallel injection of DVB CSs. An internal standard solution of EFB WK at 20,000 ng/mL was added into 100 \(\mu\)L to 1 mL of all DVB CSs and QCs.