## 3 Results and Discussion
### Binding Optimization
The imprinted polymers prepared via thermal heating show _Bmax_ values in the range of 82.9-115.4 \(\upmu\)mol\(\cdot\)mg\({}^{-1}\) and _Kd_ values ranging from 4.9 to 25.6 \(\upmu\)M (Table 2). The exhibition of specific and non-specific binding behavior and equivalent _Bmax_ and _Kd_ in MW- and TH-assisted polymerization can be an indication that apparently radiation did not affect the formation of cavities with complementary features. Considering the variation in template-functional monomer molar ratio, there is, in general, an increase in _Bmax_ from a molar ratio of 1:2 to 1:4 in both preparation systems. MIP 3 and 6 present the higher values (115.6 \(\pm\) 5.5) \(\upmu\)mol\(\cdot\)mg\({}^{-1}\) and (115.4 \(\pm\) 4.5) \(\upmu\)mol\(\cdot\)mg\({}^{-1}\), respectively. To examine the impact of the non-selective interactions of the analyte and polymer on the _Kd_ and _Bmax_ descriptor values, the binding differential was examined through _IF_. For the ease of polymer comparison, _IF_ values were determined using the line of ligand conservation method (see Section 2). Data revealed that the six prepared polymers follow the same _IF_ decaying values, in the range of 1.0-2.0 mM (Figure 8). In general, MIP  
Figure 7: Equilibrium binding isotherms for 2C-B incubated in MIP 1–6 (\(\bullet\)) and NIP 1–6 (\(\blacktriangle\)) at 25 \({}^{\circ}\)C. Non-linear fitting using Langmuir (—) and Freundlich (\(\cdot\cdot\)) models are pictured. Data represents mean \(\pm\) SD, \(n=3\). distribution factor \(D\) falls (Equation (5)), while in the NIP isotherm curve the curvature is less pronounced, which could be explained by the lack of specific binding, promoting less variation in the middle range of studied \(F\) concentrations. This data provides information on at which concentrations the MIP can be more efficient than its non-imprinted pair. The selectivity of MIPs depends on their ability to maintain the fidelity of the binding sites formed by template imprinting. Molecular selectivity was established from investigations conducted under competitive batch binding conditions with 2C-B analogues and other common NPS, using MIP 3 (Figure 9). Selectivity was higher for ketamine and lower for compounds chemically similar to 2C-B, such as 2C-I and 2C-C. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & \multicolumn{4}{c}{**Langmuir**} & \multicolumn{4}{c}{**Freundlich**} \\ \cline{2-10}  & \multicolumn{2}{c}{**Isotherm Parameters**} & \multicolumn{2}{c}{**Fitting Parameters**} & \multicolumn{2}{c}{**Isotherm Parameters**} & \multicolumn{2}{c}{**Fitting Parameters**} \\ \cline{2-10}  & \(B_{max}\) & \(Kd\) & \(R^{2}\) & \(SS\) & \(P\)**(AIC)** & \(A\) & \(m\) & \(R^{2}\) & \(SS\) & \(P\)**(AIC)** \\ \hline MIP 1 & (75.9 \(\pm\) 1.6) & (8.6 \(\pm\) 0.8) & 0.9797 & 266 & \(>\)99.9 & (23.8 \(\pm\) 3.6) & (0.17 \(\pm\) 0.03) & 0.8146 & 2426 & \(<\)0.01 \\ MIP 2 & (108.3 \(\pm\) 1.6) & (32.8 \(\pm\) 2.0) & 0.9932 & 138 & \(>\)99.9 & (16.9 \(\pm\) 0.3) & (0.02 \(\pm\) 0.02) & 0.9443 & 1421 & \(<\)0.01 \\ MIP 3 & (115.6 \(\pm\) 5.5) & (26.7 \(\pm\) 6.2) & 0.9695 & 2031 & 8.8 & (24.3 \(\pm\) 2.9) & (0.24 \(\pm\) 0.02) & 0.9829 & 1566 & 91.2 \\ MIP 4 & (82.9 \(\pm\) 2.2) & (4.9 \(\pm\) 0.6) & 0.9658 & 562 & \(>\)99.9 & (29.2 \(\pm\) 3.4) & (0.16 \(\pm\) 0.02) & 0.9005 & 1632 & \(<\)0.01 \\ MIP 5 & (96.6 \(\pm\) 1.4) & (6.8 \(\pm\) 0.4) & 0.9921 & 167 & \(>\)99.9 & (32.2 \(\pm\) 4.2) & (0.16 \(\pm\) 0.02) & 0.8594 & 2966 & \(<\)0.01 \\ MIP 6 & (115.4 \(\pm\) 4.5) & (25.6 \(\pm\) 4.4) & 0.9556 & 1367 & 99.1 & (23.1 \(\pm\) 3.4) & (0.24 \(\pm\) 0.02) & 0.9255 & 2294 & 0.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Non-linear fitting parameters (mean \(\pm\) SD, \(n\) = 2) through [PERSON] and [PERSON] isotherm models. Figure 8: Imprinting factor of three MW-assisted MIP/NIP preparations. Figure 9: Selectivity factor of MIP 3 towards different similar structures.