# A Molecularly Imprinted Polymer for Selective Extraction of Phenolic Acids from Human Urine
## 3 Results and Discussion  
### MIP Design, Template, and Analogues Selection  
MIPs are highly reticulated network polymers consisting of a common matrix structure and binding sites formed by a template present during polymer synthesis and monomers are chosen in order to complement functional groups of a template molecule. After remove of the template, binding sites remain that are capable of rebinding the template with high affinity and selectivity. These sites can interact with the template molecules in a non-covalent way, such as via coulombic interactions, hydrogen bonding, ion coordinating, \(\pi\)-\(\pi\) stacking, hydrophobic and van der Waals forces, or by reversible formation of covalent bonds [24]. Non-covalent imprinting is efficient for low-molecular-weight compounds and templates containing acids are often well targeted using basic functional monomers such as vinylpyridine (4-Vpy), or amide monomers such as acrylamide (MA) [18; 24]. Some authors suggested that the best recognition ability depended on the pKa relationship between monomer and template. The basic character of 4-Vpy (pKa = 5.4) makes it a good candidate for phenolic acid templates [10; 18; 22]. [PERSON] et al. also related the rigid structure of templates to the recognition ability of synthesized MIP [19]. Templates with high molecular rigidity (e.g., 4-HBA or 4-HPA) showed a much stronger imprinting effects. Therefore, MIP was synthesized using 4-HPA as a template, 4-Vpy as a functional monomer, ethylene glicol dimethacrylate (EGDMA) as a crosslinker, 2,2'-azobis(2-methyl-propionitrile (AIBN) as polymerization reaction initiator. The polymerization was carried out in a molar ratio of 1:4:20 (template/4-Vpy/EGDMA). Porogen plays an important role in the polymer morphology and also affects the ionic interactions and hydrogen bonds between target and the functional monomer [23]. An aprotic and polar solvent such as \(\Lambda\)CN was chosen as porogen. The optimized volume of porogen was 10 mL.  
Recently, a large number of phenolic acids and other catabolites have been detected in urine after flavonoid intake [2; 5; 25; 26; 27]. Maximum concentration of phenolic metabolites depends of the parent flavonoid and the excretion time [25]. We chose 4-HPA as the target and template analyte because it is determined in higher concentration in urine and presents more flexibility that other phenolic acid molecules. Additionally, maximum peak excretions by 4-HPA were observed between 4-5 h post-consumption [25]. On the other hand, the sorption properties of molecularly imprinted polymers with imprints of phenolic acids and their derivatives were found to depend on the number and location of hydroxyl and carboxyl groups [17]. In order to assess the MIP, we chose other well-known metabolites as analogues present in urine (Figure 1). We chose an isomer of a template, 3-hydroxyphenacetic acid (3-HPA). Furthermore, we chose three compound with structural similarity: 4-hydroxybenzoic acid (4-HBA), 3,4-dihydroxybenzoic acid (protocatechic acid, PCA), and 4-hydroxy-3,5-dimethoxyozoic acid (syringic acid, SyA). Further, 4-HBA also has structural similarity with gallic acid, caffe acid, and vanillic acid, which are well-known metabolites. The last, hippuric acid (HA), proposed as a biomarker for dietary flavonoid intake [3], was included.