# A Molecularly Imprinted Polymer for Selective Extraction of Phenolic Acids from Human Urine
## 3 Results and Discussion
### Optimization of MISE Procedure  
In this study, volume and concentration of sample, loading, washing, and elution solvents and volumes were optimized. Before the sample loading, the cartridge was conditioning with 2 mL of water and then 2 mL of ACN. The loading solvent is chosen in order to allow rebinding of the analyte to specific sites, whereas the elution solvent has to be optimized according to its ability to disrupt the analyte-polymer interaction. In accordance with the literature, the solvent used as porogen during polymerization was chosen as the loading solvent. Then, different concentrations of 4-HPA (4 to 100 mg/L) in water were prepared to assess loading concentration. In this concentration range, results obtained did not show significant difference in the recovery. Therefore, lower concentration of 4 mg/L was chosen as the working concentration. Loading was carried out maintaining the sample onto the cartridge during 5 min and then it was passed through the column under vacuum conditions. Subsequently, different sample volumes (0.5-2 mL) were tested to evaluate the loading. The retained 4-HPA was evaluated and calculated as the difference between 4-HPA total amounts loaded onto the cartridge initially, and collected 4-HPA in the fractions from the loading and washing steps. In each process, the obtained extracts were injected into the HPLC-DAD-MS system to evaluate the best procedure. Before the elution, the washing step was carried out in order to remove the interfering compounds non-specifically retained. The presence of non-specific binding sites is attributed to the high amount of monomer used during MIP synthesis by the non-covalent approach, and it was well known that polar and protic solvents weakened the non-covalent specific interactions between template and MIP. In this step, different washing solvents and volumes were investigated in this study: water, acetonitrile, methanol, and mixtures of water:ACN (80:20 and 90:10). The results indicated that best MIP recoveries and purification effect were achieved when water was used as the washing solvent (see Figure 5). Washing volume was also optimized and a volume of 1 mL of water was used as the optimum washing condition. On the other hand, the different recoveries obtained for NIP and MIP indicated that the interactions between the template and MIP were specific. In the wash step, analyte is lost when NIP is used as an adsorbent. To ensure complete elution of the adsorbed analyte from the MISEPC procedure, it is necessary to find out the appropriate solvent. Consequently, the efficacy of various solvents for the elution of 4-HPA was evaluated. The solvents tested were MeOH and ACN, with or without 1% formic, mixtures of both in different proportions (20:80; 50:50; 80:20). MeOH with acetic acid in proportion (8:2) was also tested. The best recovery was obtained using acetonitrile with 1% formic acid. Different volumes of elution have also been tested. These studies revealed that the optimum volume of elution was 1.5 mL. In urine samples, the recovery obtained from 4-HPA under the MISEP optimized conditions were of 94% (RSD 1.2).