## 3 Results and Discussion
### Optimization of MISPE Conditions  
The major purpose of the present paper is the application of MISPE for the determination of polymyrsins in water samples. Accordingly, 1 mL of water (fortified concentration: 10 mg L\({}^{-1}\) of CS) was loaded both on MIP and NIP cartridges and different washing and elution conditions were tested. Initially, it was observed that most of CS-loaded samples were neither retained by MIP nor by NIP likely due to the high initial flow-rate used (-10 mL min\({}^{-1}\)). By reducing the flow-rate, an increase on the retention of CS was observed although recoveries were still very low (<50%). Thus, it was decided to stop the flow during the loading step, allowing loading solution to be in contact with the polymers during different times (1-5 min). Increasing the loading time from 1 to 3 min, the recovery increased from 68.8% to 97.8%, but, no evident improvement on recovery was observed for times longer than 3 min. Hence, a loading time of 3 min was selected for further experiments. These results suggested that both the big size of CS and the low porosity of the polymers have a negative impact on mass transfer kinetics and thus CS need longer times to reach binding sites.  
Figure 2: Scanning electron micrographs of MIP and NIP obtained at 10,000\(\times\) magnification.  
Washing is a crucial step in MISPE since it has to be able to reduce non-specific interactions, as much as possible, keeping unaltered interactions inside the binding sites. Accordingly, after loading, washing solutions (1 mL) containing different amounts of MeOH in water (100%, 75%, 50%, 25% and 0%) were assayed. As presented in Figure 3A, when MeOH content is higher than 75%, large amount of CS was washed off, suggesting that interactions based on hydrogen bonding are taking place between binding sites and CS, and thus they are disrupted in the presence of a polar protein solvent (MeOH). However, for MeOH content lower than 50%, quantitative recoveries of CS in MIP cartridges were achieved. In parallel, imprinting factor (IF) (expressed as IF = RMIP/RNIP, where RMIP and RNIP are the recoveries of CS from MIP and NIP cartridges, respectively) reached a maximum value of 1.5 when a mixture of MeOH:water (50:50, _v/v_) was added. Therefore, 1 mL of MeOH:water (50:50, _v/v_) was selected as optimum washing solution since, under these conditions, non-specific interactions were minimized and satisfactory recoveries (93.7% in average) of CS on MIP was achieved.  
Finally, MeOH and mixtures of AM:MeOH and FA:MeOH were tested as elution solutions. The obtained results, shown in Figure 3B, show that elution efficiencies of MeOH and AM:MeOH (1:99, _v/v_) were extremely achieving recoveries of CS lower than 20%. In contrast, the presence of FA improved the elution ability of MeOH, confirming, as mentioned above, that CS interactions with MIP were mainly governed by hydrogen bonding. It was clear that the highest recovery (95.1%) was achieved when the percentage of FA in MeOH was up to 2% and thus it was chosen as optimum. Then, the effect of elution volume was studied and it was observed that the recovery increased with the rising of elution volume, however, there was no significant improvement on recovery for volumes higher than 2 mL. Accordingly, 2 mL of 2% FA in MeOH was sufficient to elute CS from MIP cartridge.