## 3 Results and Discussion  
### Optimization of HFM-Protected-MI-MSPE in Soil Samples  
The objective of the present work was to study the performance HFM-protected-MI-MSPE in the analysis of triazines in soil samples by HPLC-UV. These herbicides strongly sorb to soil, thus requiring exhaustive extraction methods [17], which inevitably co-extract high amount of matrix interferens (e.g., humic and fulvic acids, particulated matter, etc.). These facts prevent the possibilityof using simple UV detectors for routine analysis, thus requiring more expensive selective alternatives, such as c or tandem MS-MS for the final quantification. For this purpose, all parameters involved in HFM-protected-MI-MSPE of triazines from soil extracts were optimized. A scheme of the working protocol is shown in Figure 1.  
The first experiments were focused on promoting the migration of analytes from the acetonitrile crude soil sample extracts to the fibre, and on improving selective retention in the MIP microspheres. A set of parallel experiments using MIP and NIP fibres was carried out using 4.5 mL volume standards containing 250 ng of each triazine selected for this study, and the results are shown in Figure 2. It can be seen that the recoveries obtained by HFM-protected-MI-MSPE in acetonitrile solutions were very low (5 to 9%, depending upon the triazine). This fact can be easily understood, taking into account the fact that the interaction of triazines with methacrylic-based MIPs is mainly governed by hydrogen bond interactions, which are always favoured in highly hydrophobic media [18]. For that reason, a good strategy for increasing recoveries was to dilute the acetonitrile extract with toluene (the solvent used as porogen during polymerization) in different proportions. As can be observed, retention on the fibre increased as the content in the toluene increased, being quantitative in all cases for dilutions higher that 1/8. Another successful alternative was to evaporate extracts to dryness, and then redissolve them in 4.5 mL of toluene. Both alternatives gave similar recoveries, and could be selected depending on the concentration range analysed. For high-concentration samples, taking only an aliquot of 500 uL of acetonitrile extract and 1/8 dilution avoids the use of a large volume of toluene. However, for trace analysis where a high concentration factor is needed, evaporation of the complete acetonitrile extract (5 mL) and redissolution in 4.5 mL of toluene should be the chosen protocol, since not only would this increase sensitivity, but it would also avoid the consumption of large amounts of toluene. A washing step was included in order to favour selective recognition of triazines by MIP microspheres, which, at the end, would provide a better sample clean-up. In this sense, and on the basis of previous studies carried out in our laboratory [14], different mixtures of toluene with more polar solvents, such as acetonitrile and methanol in low percentages, were evaluated as washing solutions capable of disrupting non-specific interactions without affecting (if possible) specific interactions. The same conditions described above were used for this set of experiments, but including a washing step of 5 min at 750 rpm with 4.5 mL of toluene mixtures with percentages of ACN or MeOH varying within the 1 to 10% range. According to the obtained results, the mixture consisting of 2% of ACN in toluene was selected as the optimum washing solution, since it permitted almost complete removal of  
Figure 1: Scheme of the hollow-fibre membrane-protected molecularly imprinted microsolid-phase extraction procedure. non-specific interactions with minimal disturbance of specific interactions. Recoveries for all triazines were negligible in NIP fibres, whereas they remained quantitative in MIP fibres, clearly demonstrating an evident imprint effect responsible of the selective molecular recognition.