# A Molecularly Imprinted Polymer for Selective Extraction of Phenolic Acids from Human Urine
## 3 Results and Discussion
### Selectivity of MIP  
The complete confirmation of the imprinting performance involves the study of the selectivity of the MIP. This was accomplished by determination of binding capacity of the MIP in presence of other structural analogues of the 4-HPA.  
The MISPE procedure was assessed mixing all studies analytes. In the urine sample, the concentration of each phenolic acid was 4 mg/L. Figure 5 shows the comparative recoveries obtained for each. Experiments studying the retention on the NIP in the same conditions than the MIP were done, but the result on NIP were inconsiderable in comparison to MIP.  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Method**} & \multirow{2}{*}{**r\({}^{2}\)**} & \multirow{2}{*}{**LOD**} & \multirow{2}{*}{**LOQ**} & \multicolumn{6}{c}{**Reproducibility (RSD)**} \\ \cline{6-9}  & & & & & \multicolumn{3}{c}{**Inter-Day Precision**} & \multicolumn{3}{c}{**Intra-Day Precision**} \\ \cline{6-9}  & & & & & **2 mg/L** & **40 mg/L** & **2 mg/L** & **20 mg/L** & **40 mg/L** \\ \hline MISPE-LC-DAD-MS & 0.990 & 1.22 & 3.69 & 1.6\% & 2.8\% & 2.8\% & 6.7\% & 3.2\% & 3.1\% \\ \hline \hline \end{tabular}
\end{table}
Table 2: Validation parameter of the MISPE procedure.  
Figure 5: Recoveries percentage of the phenolic acid analogues obtained from the MISPE procedure. In the urine sample, the concentration of each phenolic acid was 4 mg/L. Error bars indicate the standard deviation of replicates (\(n=3\)).  
MIP shows a highly specific molecular recognition for the template (94% of recovery, RSD 1.2%) over similar analogues. Molecular recognition of the compounds was influenced by the presence, number, and position of hydroxyl and carboxyl groups in the aromatic ring [16; 17; 29; 31]. From the compounds tested 3-HPA, isomer of 4-HPA, shows the best structural resemblance, as its only difference is the hydroxyl group position, and recovery value was 88%. All the compounds bear carboxyl and multiple hydroxyl groups being for this reason prone to forming hydrogen bonds with the polymer matrix. Accordingly, strong competition for specific and non-specific binding sites is expected. Moreover, this did not significantly affect the recoveries values of 4-HPA. The retention order of the compounds is associated with their potency to form strong hydrogen bonds and therefore follows the order of the number of hydroxyl and carboxyl groups. Syringic acid (82%) and 4-HBA (80%) are strongly retained, followed by PCA (77%), and HA as the less retained (71%). In addition, the molecular size also conditioned the recoveries values [16; 17; 29; 31].  
These results show that 4-HPA-MIP is an effective sorbent and was successfully applied to the extraction of 4-HPA and other phenolic acids. On the other hand, the advantages of MIP as the selective sorbent was demonstrated because it provides greater specificity compared to other sorbents.  
The selective extraction of these metabolites helps improve the application and results obtained by other less sensitive analysis methods than the validation method. It also facilitates the development of new screening methods as electrochemical sensors to estimate polyphenol excretion (unpublished authors work).