## 2 Results and Discussion

Table 1: Signal intensities of the analytes obtained using different sample preparation protocols (each experiment was repeated six times \(n=6\)). Figure 1: Comparison of two media for the homogenization step: perchloric (PCA) and formic acid (FA) solutions in methanol (PCA/MeOH vs. FA/MeOH). Electrophrogram of brain samples spiked with nine neurotransmitters (NTs) (each analyte at the concentration of 10 \(\upmu\)g/mL) obtained after homogenization with either FA/MeOH (**a**) or PCA/MeOH (**b**) solutions without further purification. The separation was carried out using the optimized micellar electrokinetic chromatography with spectrophotometric detection (MEKC–UV/VIS) method. Capillary electrophoresis (CE) conditions: uncoated fused silica capillary (75 \(\upmu\)m i.d. (internal diameter) 60.2 cm length); \(\lambda\) = 200 nm; injection time 8 s; applied voltage 25 kV; temperature 25 (\(\pm\)0.1) \({}^{\circ}\)C. The background electrolyte (BGE) consisted of 10 mM sodium tetraborate decahydrate, 30 mM SDS, 15% (_v/v_) MeOH, and 25 mM alpha cyclodextrin (\(\upalpha\)-CD), adjusted to pH 9.36 using 1 M sodium hydroxide (NaOH). Legend: 5-HT—serotonin; VMA—vanillylandnelic acid; HVA—homovanillic acid. Screen DAU (CSDAU) columns, dispersive liquid-liquid microextraction (DLLME), and solid-phase microextraction (SPME). The SPE coatings and eluents were chosen on the basis of previously published data related to the preparation of biological samples for the isolation of some NTs or their metabolites [15; 16]. Comparing the already published data with our studies, the most appropriate SPE column conditioning, equilibration, and sample eluent media were chosen (Table 2). Indeed, our experiments proved that for acidic metabolites (VMA and HVA), isolation with the use of the SPE SCX-based method and 0.1% FA in acetonitrile (ACN)/MeOH as the elution solvent worked efficiently (Figure 2b, Table 1). SPE allowed to obtain purified brain samples that were enriched with VMA and HVA, since the signal intensities (the height of the analyte peaks) were 15 times higher than those obtained after the analysis of brain homogenates without the SPE procedure. Moreover, the baseline was free of interfering signals from the sample matrix (Figure 2a). On the other hand, a lower extraction efficiency was observed for 5-HT due to the basic physicochemical properties of this biogenic amine. The signal intensities of other basic compounds, such as A, NA, DA, L-Trp, L-Tyr, were below the LOD (Figure 2b). \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Type of SPE Resin**} & **Column Conditioning and** & **Elution With 2 \(\times\) 200 \(\mu\)L of** \\  & **Equilibration with a Mixture of** & \\ \hline \multirow{2}{*}{Strong Cation Exchange (SCX)} & 1 mL 0.1\% FA in ACN (apparent pH 3), & MeOH or \\  & 1 mL MeOH, & 0.1\% FA in ACN/MeOH (1:1, \%/) or \\  & 1 mL H\({}_{2}\)O & MeOH + 0.2 M NH\({}_{4}\)OH \\ \hline \multirow{2}{*}{Clean Screen DAU (CSDAU)} & 1 mL MeOH, & \\  & 1 mL H\({}_{2}\)O (pH 7) & MeOH + 0.2 M NH\({}_{4}\)OH \\ \hline \hline \end{tabular}
\end{table} 
Table 2: SPE settings for the clean-up and preconcentration of NTs from brain sample homogenates (200 \(\mu\)L of sample). The SPE columns were washed each time with 1 mL of H\({}_{2}\)O, and the eluents were evaporated to dryness (45 \({}^{\circ}\)C/1 h) (each experiment was repeated six times \(n=6\)). Figure 2: Electrophogram of (**a**) the blank brain control sample after the SPE SCX sample clean-up with 0.1% FA in ACN as the eluent, (**b**) the brain sample spiked with nine NTs (each analyte at the concentration of 10 \(\mu\)g/g) obtained after the SPE SCX sample clean-up with 0.1% FA in ACN/MeOH (1:1, \(\
u\)/\(\
u\)), (**c**) the brain sample spiked with nine NTs (each analyte at the concentration of 10 \(\mu\)g/mL) obtained after the SPE SCX sample clean-up with MeOH as the eluent.