## 2 Results and Discussion
### Optimization of Micellar-Electrokinetic Chromatography as Separation Method  
The separation parameters of the MEKC-UV (DAD) method were developed, optimized, and discussed in our previous paper [29]. In that publication, we provided a detailed analysis of the optimization of the separation buffer composition. Due to the diverse pKa values of the selected panel of CAs, their simultaneous determination is an analytical challenge. The chemical structures of these compounds contain multiple functional groups that can ionize over a wide pH range, making the selection of optimal buffer ingredients a complex task. In our study, a borate buffer was chosen, as catechol compounds and some substituted catechol (like CAs) can become charged in a weakly alkaline electrolyte. Once ionized, the hydroxyl groups are capable of forming complexes with borate ions, allowing for adequate electrophoretic mobility. However, due to insufficient separation of all analytes, the addition of surfactants and an organic modifier was explored. It was shown that the presence of 50 mM SDS and 15% methanol in the separation buffer significantly improved separation efficiency. A borate buffer without SDS or with SDS concentrations below 50 mM did not provide satisfactory separation of the compounds. Additionally, the 15% (\(v/v\)) methanol as an organic modifier improved separation efficiency by influencing buffer viscosity.  
The optimized separation buffer, composed of 5 mM sodium tetraborate decahydrate, 50 mM SDS, 15% MeOH (\(v/v\)), and 150 mM boric acid, was further enhanced with the addition of an imidazolium-based ionic liquid (1 mM 1-hexyl-3-methylimidazolium chloride). The application of this ionic liquid in the background electrolyte (BGE) created a dynamic double-coating effect, resulting in greater repeatability of analyte migration times and satisfactory separation of the analytes [42]. A representative electropherogram showing the separation of DHPG, VMA, and MHPG is presented in Figure 2.  
It should be pointed out that electromigration-based procedures require capillary rinsing after each run to ensure proper repeatability of migration times in individual analyses. Regarding the potential carry-over effect, baseline control was also performed by analyzing the separation buffer alone. To check for any residual analytes carried over from one sample to the next, a pure separation buffer was performed after the analysis of a sample containing high concentrations of the analytes. No analyte signals were detected, indicating that there was no carry-over from the previous sample.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**3D-DSPE-Based Extraction Procedures**} & \multicolumn{2}{c}{**DHPG**} & \multicolumn{2}{c}{**MHPG**} & \multicolumn{2}{c}{**VMA**} \\ \cline{2-7}  & **R \(\pm\) SD (\%)** & **EF** & **R \(\pm\) SD (\%)** & **EF** & **R \(\pm\) SD (\%)** & **EF** \\ \hline Sorbent & Eluent & & & & & \\ \hline \multirow{2}{*}{DVB 30 mg} & Aceton & 350.6 \(\pm\) 16.5 & 3.5 & 324.4 \(\pm\) 7.1 & 3.2 & 228.9 \(\pm\) 11.0 & 2.3 \\ \cline{2-7}  & ACN:MeOH 50:50 & 292.2 \(\pm\) 15.4 & 2.9 & 249.3 \(\pm\) 11.2 & 2.5 & 214.9 \(\pm\) 10.5 & 2.1 \\ \hline \hline \end{tabular}
\end{table}
Table 5: The results of extraction efficiency (R) with SD value and enhancement factor (EF) for 3D-DSPE-based extraction procedures depending on eluent (n = 3).