## 2 Results and Discussion
### Optimization of Extraction Parameters
#### 2.1.2 Solid-Phase Extraction
According to the manufacturer, ABN sorbents are resistant to deconditioning, meaning they do not exhibit poor analyte-solvent interactions due to phase collapse. One of the key advantages is the elimination of the need for conditioning and equilibration steps. Additionally, these sorbents offer protein exclusion, and their wash and elution protocols reduce or eliminate matrix interferences [40]. In our study, we followed the manufacturer's recommended procedure, which did not require an activation step. After applying the urine sample, the sorbent was washed with a water:MeOH mixture (95:5, \(v/v\)), and pure MeOH was used as the eluent. However, under these conditions, unsatisfactory extraction efficiency was observed for DHPG and MHPG. For VMA, which has the most acidic properties among the analyzed compounds, the R value was only 21.2 \(\pm\) 2.6%. Anion exchange (AX) columns are filled with a sorbent modified with a quaternary amine group and a chloride counterion. This positively charged sorbent retains acidic (negatively charged) analytes through nonpolar and strong anion exchange interactions. According to the manufacturer's recommended procedure, the sorbent was not activated, and the analytes were eluted using a mixture of MeOH and FA (98:2, \(v/v\)). After applying the sample, potential contaminants were removed with 1 mL of a 0.05 M ammonium acetate pH 6:MeOH mixture (95:5, \(v/v\)), followed by 1 mL of MeOH. However, this procedure was not effective for DHPG and MHPG, with a low R value for DHPG (9.1 \(\pm\) 2.3%), and no effective for MHPG. The best results were obtained for VMA (53.4 \(\pm\) 3.7%). This difference is likely due to the pKa values of the analytes. VMA, which has a carboxyl group, is retained on the AX sorbent. While DHPG also has a carboxyl group, its higher polarity compared to VMA may explain its weaker retention. MHPG, likely due to the absence of acidic groups, is not retained on the AX sorbent. Cation exchange (CX) columns contain a sorbent modified with a sulfonic acid group, which retains basic (positively charged) analytes through nonpolar interactions and strong cation exchange functions. This sorbent is designed to extract positively charged basic/cationic compounds. The dual \"mixed-mode\" retention mechanism of these sorbents enables the use of 100% organic solvents during the wash step, effectively removing interferences without compromising analyte recovery. Analyte elution is achieved at high pH, neutralizing the positive charge on the analytes. The recommended eluent, a mixture of MeOH:NH4 OH (95:5, \(v/v\)), was applied. Similar to AX sorbent, no preliminary activation of the CX sorbent was necessary, and after the sample was applied, the sorbent was washed with 1 mL of 0.05 M ammonium acetate (pH 6), followed by 1 mL of methanol. The highest extraction efficiency was observed for MHPG, with an R of 30.5 \(\pm\) 3.9%, while for VMA, it was only 12.8 \(\pm\) 3.5%. DHPG, being the most polar compound, was not effectively extracted under these conditions. SPE columns with polystyrene-divinylbenzene (PS-DVB) sorbent are commonly used for retaining neutral and aromatic compounds, making them suitable for screening applications involving a broad range of analytes. In the case of SPE with PS-DVB cartridges, three types of eluents were tested: pure acetone, an ACN:MeOH mixture (50:50, \(v/v\)), and a CH\({}_{2}\)Cl\({}_{2}\):IPA:NH4 OH mixture (78:20:2, \(v/v/v\)). As shown in Table 4, the highest extraction efficiency for DHPG (140.4 \(\pm\) 3.5%) was achieved with acetone as the eluent, leading to a signal enhancement of up to 1.4 times. In contrast, the extraction efficiencies for MHPG and VMA were 50.5 \(\pm\) 2.9% and 67.5 \(\pm\) 4.1%, respectively, with no potential for signal enhancement. The other eluents tested were ineffective for tested analytes.