## 2 Results and Discussion  
### Optimization of Extraction Parameters  
Even though various approaches have been reported for the determination of CAs in different biological samples, there is a lack of analytical solutions for the simultaneous determination of MHPG, DHPG and VMA using the MEKC-UV method. The goal of our study was to develop and optimize an extraction method for the simultaneous isolation and electrophoretic quantitation of selected norepinephrine pathway metabolites from urine samples.  
In this study, artificial urine was used to evaluate the efficiency of various extraction methods, as its consistent composition facilitates method optimization. However, real urine is essential for final validation due to significant differences in matrix effects. Matrix effects, caused by co-eluting substances, can lead to ion suppression or enhancement in techniques like liquid chromatography-mass spectrometry (LC-MS), affecting analyte detection [36]. Real urine, a complex fluid with diverse endogenous compounds, presents more interfering substances than artificial urine, which typically lacks many metabolites and proteins. While matrix effects are crucial for method development in separation techniques, they are generally less significant during analyte isolation, where selective extraction is prioritized.  
Although CAs have very similar structures, their chemical behaviors, namely interactions with various SPE sorbents, differ significantly. The catecholamines contain a catechol moiety, but differ with substituents and functional groups, which produces significant differences in hydrophobicity and pKa values (Table 1). The literature indicates that CAs are particularly sensitive to minor pH changes, and laboratory guidelines recommend adjusting urine samples to pH 3 to maintain their stability during storage [37]. However, the pH that best promotes the isolation of these analytes from the sample matrix may vary, depending on the pKa and log P values of the individual compounds. This parameter was therefore examined in our study to determine optimal conditions for analyte isolation. Some authors have indicated that the pH of urine samples was crucial for extraction efficiency of analytes and a wide pH variation (i.e., 4.5-8.0) of individual urine samples caused substantial pH differences for the pre-treated urine sample [25]. Therefore, in this study on different extraction approaches, we focused on adjusting the pH value of each sample to a narrow pH range (5.7-6.0).  
In this study, three extraction approaches--LLE, SPE, and a new dispersive solid-phase extraction (DSPE) using a 3D-printed system--were tested for isolating MHPG, DHPG and VMA. Analyte signal intensity was used as a preliminary assessment of extraction efficiency. To identify the most effective conditions for analyte recovery during the development of an extraction method, analyte signal intensity can help compare different extraction procedures such as sorbent choice, solvent choice, pH, or extraction time. It is a useful solution, particularly during method development and optimization. However, we are aware that it is not a substitute for a full validation, which ensures the method's accuracy, precision, selectivity, and robustness [36; 38; 39]. For each of the tested isolation techniques, both the extraction efficiency (R) and the potential for analyte signal enhancement (EF) were evaluated. The extraction efficiency was assessed according to Equation (1) (see Section 3.6) by conducting three independent measurements. If the extraction method yielded an extraction efficiency above 100%, this could result not only from the potential effectiveness of the approach in isolating the selected analytes from the matrix but also from the dissolution of the dry residue, after evaporating the eluent/desorbent, in 50 \(\mu\)L of sample buffer during the final stage of the procedure. Of particular interest were the procedures that allowed for signal enhancement, which was calculated using Equation (2) (see Section 3.6).