Manal Bitar

Céline Lafarge

Nicolas Sok
email: nicolas.sok@agrosupdijon.fr

Philippe Cayot

Elias Bou-Maroun
email: elias.bou-maroun@agrosupdijon.fr

Molecularly imprinted sol-gel polymers for the analysis of iprodione fungicide in wine: synthesis in green solvent

Keywords: Molecularly imprinted silica, fungicide, wine, experimental design, solid phase extraction, cheap method, green solvent

Iprodione is a fungicide widely used in viticulture in most agricultural countries. It was banned recently in the European community because of its carcinogenic and endocrine disrupting characters.

In this work, a cheap analytical method able to monitor iprodione in a white wine was developed.

Molecularly imprinted sol-gel polymers (MIS) specific to iprodione and using green solvents were synthesized. An experimental design having the following factors (solvent volume and crosslinker quantity) was used to prepare an optimal MIS. In terms of selectivity, the optimal MIS showed the best partition coefficient towards iprodione in a white wine containing four other competing fungicides (procymidone, pyrimethanil, azoxystrobin and iprovalicarb). A solid phase extraction method using the optimal MIS was optimized and applied to analyse iprodione in a white wine. Low detection and quantification limits were reached 11.7 and 39.1 µg/L respectively.

Introduction

Iprodione is a dicarboximide fungicide that is widely used in agriculture around the world. It was authorized to be used on vegetable crops, seed crops, fruit-tree crops, vines, ornamental crops, grasses, fragrance, food, medicinal and culinary plants and field crops (ANSES, 2017).

According to a pesticide-monitoring programme performed by the French Ministry of Agriculture [START_REF] Cugier | Plan de surveillance résidus en viticulture. Campagnes viticoles 1990-2003[END_REF], iprodione was detected in 90% of the grapes analysed samples (47/52) and in 100% of the wines analysed samples (43/43).

In 2008, iprodione was classified as carcinogen category 2 in accordance with (EC) regulation N 1272/2008 of the European Parliament and of the council (Regulation EC 1272[START_REF] Prat | A survey of solvent selection guides[END_REF]. Based on a European Food Safety Authority study, the European Community concluded that there is a high risk that the iprodione uses may expose groundwater to levels exceeding the drinking water limit of 0.1 µg/L (European Food Safety Authority (EFSA), 2016). An evaluation conducted by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) in 2016 showed that iprodione has endocrine disruptor properties. For all these reasons, the (EU) regulation N 2017/2091 decided not to renew the approval of iprodione. Any grace period accorded by the state members expired on 5 June 2018 (Regulation EU 2091, 2017).

In this context, a reliable and cheap method of iprodione analysis should be used in routine in order to check that agriculture products comply with this new regulation. Currently, chromatographic methods coupled to mass spectrometry are used to analysis fungicides in food and environmental samples [START_REF] Castro | Multianalyte, high-throughput liquid chromatography tandem mass spectrometry method for the sensitive determination of fungicides and insecticides in wine[END_REF][START_REF] Elbashir | Application of gas and liquid chromatography coupled to time-of-flight mass spectrometry in pesticides: Multiresidue analysis[END_REF][START_REF] Oliva | Influence of matrix on the bioavailability of nine fungicides in wine grape and red wine[END_REF]. The big advantage of these methods is their low detection limits. However, they are expensive and require high technical experience for the lab staff. As an alternative, we proposed in a previous study a molecularly imprinted solid phase extraction method coupled to high performance liquid chromatography with a UV detection in order to analysis iprodione in wine [START_REF] Bitar | Molecularly imprinted polymer solid phase extraction of fungicides from wine samples[END_REF]. In this previous work, an acrylate base molecularly imprinted polymer (MIP) was used. preconcentration phase. They are shape memory materials. They act as a host for a gest target thanks to a double chemical and steric complementarity. They are able to recognize the target in a complex food (H. [START_REF] Li | Single-hole hollow molecularly imprinted polymer embedded carbon dot for fast detection of tetracycline in honey[END_REF], environmental matrices [START_REF] Bakkour | Molecularly Imprinted Polymers for Compound-Specific Isotope Analysis of Polar Organic Micropollutants in Aquatic Environments[END_REF][START_REF] Ruggieri | Development of molecularly imprinted polymeric nanofibers by electrospinning and applications to pesticide adsorption[END_REF] or biological samples [START_REF] Gao | In Vivo Selective Capture and Rapid Identification of Luteolin and Its Metabolites in Rat Livers by Molecularly Imprinted Solid-Phase Microextraction[END_REF][START_REF] Wei | Imprinted monoliths: Recent significant progress in analysis field[END_REF].

Molecularly imprinted polymers are synthesized in the presence of a template. After solubilization and interaction of a template molecule with a functional monomer in a solvent, a crosslinker is added to form the three-dimensional structure of the polymer in the presence of an initiator. After washing the polymer by an appropriate solvent, the template is removed leaving cavities highly specific to the target.

Most of the imprinted polymers studied in the literature are acrylate based polymers thanks to the high availability of commercial functional monomers and the ease of synthesis in organic solvents. In this work, sol-gel molecularly imprinted polymers (MIS) are developed instead of acrylate based molecularly imprinted polymer (MIP). As they are synthesized in a water/ethanol mixture, MIS respect one of the principle of green chemistry by using safer solvents than those used in MIP synthesis. Prat et al. studied solvents toxicity and elaborated a solvent classification from least toxic to most toxic. Water and ethanol are among the 9 recommended solvents of the 51 studied [START_REF] Prat | A survey of solvent selection guides[END_REF]. Moreover, sol-gel MIS are more stable and specific towards the target species than acrylate based MIP, also allowing faster diffusion of analytes [START_REF] Da Costa Silva | Sol-gel molecular imprinted ormosil for solid-phase extraction of methylxanthines[END_REF].

Molecularly imprinted polymers were already used as solid phase extraction (SPE) material in order to preconcentrate pesticides from real samples. Li et al. used MIS-SPE to extract carbendazim fungicide from vegetables before its determination by UPLC-UV with a detection limit of 3 ng/L (S. [START_REF] Li | Synergetic dual recognition and separation of the fungicide carbendazim by using magnetic nanoparticles carrying a molecularly imprinted polymer and immobilized β-cyclodextrin[END_REF]. They used MIP-SPE to preconcentrate kelthane and pyridaben pesticides from vegetables before they analysis by GC-MS with a detection limit of 1 and 3.2 pg/g respectively (S. [START_REF] Li | Synergetic recognition and separation of kelthane and pyridaben base on magnetic molecularly imprinted polymer nanospheres[END_REF].

The first objective of this study was to obtain a sol-gel molecularly imprinted polymer (MIS) specific to iprodione. In order to get an optimal MIS able to pre-concentrate iprodione, a 2-factors experimental design was developed. In the MIS synthesis, the choice of the relative proportions of the reagents is a crucial point. For this reason, the following 2-factors were studied: the solvent volume and the crosslinker quantity. Selectivity was then checked using the optimal MIS in a white wine containing iprodione and four other competing fungicides. A solid phase extraction (SPE) method was developed using the optimal MIS to monitor iprodione in a white wine. The detection and the quantification limits of this method were determined and compared to the previous method based on MIP [START_REF] Bitar | Molecularly imprinted polymer solid phase extraction of fungicides from wine samples[END_REF] and to a SPE-LC-TOFMS method (Gilbert-López, García-Reyes, Mezcua, [START_REF] Gilbert-López | Determination of Postharvest Fungicides in Fruit Juices by Solid-Phase Extraction Followed by Liquid Chromatography Electrospray Time-of-Flight Mass Spectrometry[END_REF].

Material and methods

Chemicals

Iprodione (97%, CAS number 36734-19-7), Azoxystrobin (Analytical standard, CAS number 131860-33-8), Iprovalicarb (Analytical standard, CAS number 140923-17-7), (3-Aminopropyl)trimethoxysilane (APTMS 97%, CAS number 13822-56-5), tetraethoxysilane (TEOS ≥99%, CAS number 78-10-4), ammonium hydroxide (NH4OH 28-30%, CAS number 1336-21-6), ethanol (≥99.8%, CAS number 64-17-5), methanol (gradient grade, CAS number 67-56-1) and acetonitrile (≥99.9%, CAS number 75-05-8) were purchased from Sigma Aldrich, France. Procymidone (≥98 %, CAS number 32809-16-8) and pyrimethanil (≥98 %, CAS number 53112-28-0) were bought from Chemos GmbH, Regenstauf, Germany. Water used in all experiments was deionised and obtained from an Elga Ionic system PURELAB Option.

Synthesis of the molecularly imprinted and non-imprinted sol-gel polymers

bath under magnetic stirring. The template molecule, iprodione was first solubilized in ethanol (iprodione is sparingly soluble in water, 16 mg⋅L -1 for pH≤8, and slightly soluble in ethanol, 25 g⋅L -1 ; logP = 3.1; pKa = 9.2, logD = 2.25 at pH=10). Then, water was added, followed by the monomer APTMS (highly soluble in water, 1000 g⋅L -1 , and ethanol, 100 g⋅L -1 ; pKa=10.6; from pH1 to 6, logD=-2; logD=0.37 at pH10) and the crosslinker TEOS (TEOS is sparingly soluble in water, 0.4 g⋅L -1 , but reacts with water and slowly hydrolyses; TEOS is miscible with ethanol; logP=3.81). Finally, NH4OH was introduced. The reaction mixture was left under stirring for 20 hours. The polymers were separated from the liquid phase by centrifugation at 10 000 g for 10 minutes at room temperature. In order to eliminate iprodione and free the specific cavities, the polymers were washed several times with ethanol until iprodione was no longer detectable by reversed phase high performance liquid chromatography (HPLC) in washing solvents. After washing, the polymers were dried for 6 h at 60 °C.

In parallel, NIS (Non Imprinted Silica) were synthesized under the same synthesis conditions as those of MIS, but without using the template molecule. NIS served as control polymers.

In a typical synthesis, the following molar ratios were used. For 7.5 or 15 mL of solvent, 0.1 mmol of iprodione, 0.4 mmol of APTMS, 2 or 6 mmol of TEOS and 10 mmol of NH4OH. The proportion ethanol/water was 90/10, v/v. In these conditions, the pH synthesis medium varies between 11.5 and 11.7. In the sol-gel process, the polymer formation pass through hydrolysis of alkoxysilane than condensation. At low pH, the hydrolysis kinetic is fast and the condensation kinetic is slow. The condensation starts when hydrolysis is completed. At high pH, condensation is faster than hydrolysis. pH 11.5-11.7 was chosen to have a good compromise between the rate of hydrolysis and condensation.

In figure 1, the different chemical reactions are presented at the different steps, at the MIS synthesis step (A), washing step of the MIS (B) and and the mixing step with MIS and wine contaminated with iprodione where the interactions between the MIS and iprodione are involved (C).

Binding properties and imprinting factor of MIS/NIS towards iprodione

10 mg of imprinted polymer (MIS) or non-imprinted polymer (NIS) were suspended in 20 mL (50/50, v/v, ethanol/water) solutions containing 1 mmol⋅L -1 of iprodione. The mixture was stirred for 1 h at 25 °C. After removal of the polymer particles by centrifugation at 10 000 g for 10 min at room temperature, the supernatant was diluted 10 times, then filtered using a 2 µm filter and analysed by HPLC for the determination of iprodione.

For HPLC iprodione analysis, a Shimadzu HPLC (LC-20AT) pump equipped with a UV-Visible detector (SPD-20A), a Restek column (Pinacle II, C18 5 μm, 150 x 4.6 mm) and a pre-column (C18, 5 µm, 10 x 4 mm) were used. Separations were conducted at room temperature. Isocratic acetonitrile/water (60/40, v/v) was used as mobile phase and the flow rate was 1mL/min. Iprodione detection was done at 220 nm. A calibration curve was constructed over the range 0.1-500 µmol⋅L -1 of iprodione. Eight standards were prepared in (50/50, v/v, ethanol/water) solutions, the calibration curve equation was y = 47820 x + 254129 (y = peak area; x = concentration) and the correlation coefficient R 2 = 0.9989.

The amount of iprodione retained by the polymer (B, mg of iprodione/g of polymer) was calculated by difference between the initial iprodione concentration (C0, mmol/L) and the free iprodione concentration (F, mmol/L) in the supernatant.

The imprinting factor (IF) was calculated by the equation [START_REF] Spivak | Optimization, evaluation, and characterization of molecularly imprinted polymers[END_REF]:

= = × ×
where K is the partition coefficient K = B/F. The imprinting factor evaluates the specific binding property of each polymer towards iprodione. It takes into account the sorption of iprodione by a MIS and its corresponding control polymer NIS. It allows to get rid of the non-specific interactions that result from the iprodione adsorption on NIS.

Statistical analysis (ANOVA with Tukey's post hoc analysis) was done using (XLSTAT, Addinsoft) software.

Selectivity studies

20 mg of MIS or NIS were suspended in 10 mL of a Burgundy white wine (Chardonnay, Charles Renoir). The same wine was supplemented by the following fungicides: iprodione, procymidone, pyrimethanil, azoxystrobin and iprovalicarb at 0.2 mmol⋅L -1 of each fungicide (cf. figure S1 of the supplementary material section for the fungicide chemical structures). 500 µL of the MIS or NIS suspension was mixed with 500 µL of the supplemented wine in a 2 mL Eppendorf tube. The tubes were then agitated for 3 h using a Stuart rotator SB2. The tubes were finally centrifuged for 10 min at 20 000 g at room temperature. The supernatant were analysed by HPLC using the same system described in the paragraph 2.3 in order to determine the concentration of each of the studied fungicides. Each experiment was repeated three times.

The following gradient was used to separate the 5 fungicides: (solvent A water, solvent B acetonitrile) 50% of B from 0 to 2 min, 70% of B from 2 to 8 min, 90% of B from 8 to 10 min, 50% of B from 10 to 12 min. Finally, a 2 min reconditioning step was applied at the initial conditions. A calibration curve was constructed over the range 0.1-500 µmol⋅L -1 of each fungicide. Eight standards were prepared in (90/10, v/v, water/ethanol) solutions. The calibration curve equations and the correlation coefficients were y = 18648x + 18401 (R 2 = 0.9991) for iprodione; y = 10682x + 8412 (R 2 = 0.9993) for procymidone; y = 7428x -2736 (R 2 = 0.9996) for pyrimethanil; y = 30934x + 7228 (R 2 = 0.9994) for azoxystrobin and y = 7217x -3109 (R 2 = 0.9996) for iprovalicarb.

Statistical analysis (ANOVA with Tukey's post hoc analysis) was done using (XLSTAT, Addinsoft) software.

Molecularly Imprinted Solid Phase Extraction (MISPE)

Empty polypropylene SPE cartridge (1 mL, Supelco, Bellefonte, USA) was packed with 20 mg of MIS or NIS previously suspended in acetonitrile. The polymers (particle size > 20 µm) were packed between two polyethylene frits of 20 µm porosity at each end. The SPE cartridges were firstly preconditioned with 5 mL of methanol or ethanol and then with 5 mL of (water/ethanol, v/v, 87/13). 6 mL white wine sample (Chardonnay, Charles Renoir) spiked with (5 µmol⋅L -1 = 1.65 µg/mL) of iprodione was loaded. The cartridges were subsequently washed with 2 mL of washing solvents, mixture of water/acetone at different proportions: 70/30, 50/50 or 25/75, v/v. The elution step was performed using 1 mL of elution solvents: water/acetone, where the acetone proportion was varied between 75 and 100% (v/v). The effluents from the different SPE steps were analysed by HPLC in order to quantify iprodione using the same conditions described in paragraph 2.3. A calibration curve was constructed over the range 0.1-5 µmol⋅L -1 of iprodione. All experiments were done in triplicate.

Characterisation of the optimal sol-gel polymer

Fourier transform infrared (FTIR) spectra were recorded on a Perkin Elmer spectrum 65 FT-IR spectrometer in the range 4000-500 cm -1 using attenuated total reflectance sampling. 64 scans with a resolution of 4 cm -1 were applied.

For the surface morphological characterization and Energy-dispersive X-ray (EDX) spectrum, samples were suspended in ethanol then a drop was placed on a silicon grid and examined in Hitachi Scanning

Electron Microscope (SEM) SU1510.

The size distribution of the optimal polymer was measured by laser diffraction (Malvern Mastersizer 3000) after suspending 20 mg of polymer powder in 10 mL of water and sonication for 2 min.

Results and discussion

Experimental design for the determination of the optimal polymer

The objective of the experimental design was to develop an optimal polymer for the preconcentration of iprodione fungicide from wine samples. The optimal polymer must have the highest adsorption capacity and the best imprinting factor toward iprodione. The following 2 2 experimental design was applied (table 1). The solvent volume (X1 = V) and the crosslinker quantity (X2 = Q) and were selected as the two factors of the experimental design. Two levels coded (-1) and (+1) were considered for each factor. This experimental design implied the synthesis of 4 MIS (MISa, MISb, MISc and MISd). In parallel to the MIS preparation, 4 NIS were synthesized. They constituted control polymers for the corresponding MIS.

The influence of X1 and X2 factors on the following two variables: the imprinting factor (Y1 = IF) and the iprodione adsorbed amount (Y2 = B) was studied. Y1 and Y2 represent the responses of the experimental design . The determination of IF and B was done three times for each polymer. The results are compiled in table 2.

Table 2. Experimental design factors (X) and responses (Y). Q (crosslinker quantity), S (solvent volume), IF (Imprinting Factor), B (adsorbed iprodione, mg of iprodione/g of polymer).

Experiment N° The significance limit for each response was calculated based on the standard deviation of all experiments "S", it was equal to 1.96S. The significance limit was respectively 0.188 and 1.620 for Y1 and Y2. For both responses, the interactions between the two factors (solvent volume and crosslinker quantity) were the most significant. Results of ANOVA and regression applied to the response of the design of experiment are presented in tables S1 and S2 of the supplementary material section.

Polymer X1 = V X2 = Q Y1 = IF Y2 = B (mg/g) 1 MISa,1 -1 -1 3.
Figure 2 shows that as the solvent volume (V) increases during the synthesis step, the imprinting factor decreases or remain constant (fig. 2A) and the quantity of adsorbed iprodione decreases (fig. 2B). This observation could be explained by a dilution effect. The higher the solvent volume, the lower the reactants meeting probability with monomers, the fewer the cavities specific to iprodione.

Therefore, there is a great interest in decreasing the solvent volume during the MIS synthesis.

Concerning the quantity of crosslinker (Q): When Q increases, the imprinting factor decreases (fig. 2A) but the iprodione binding property remain constant or increases (fig. 2B). This observation could be explained by the increasing of the non-specific interactions between the iprodione and the polymers.

In this case, the non-specific interactions are responsible for the amelioration of iprodione binding properties. Therefore, there is no interest in increasing the crosslinker quantity during the MIP synthesis.

Table 2 shows that MISb (V = 7.5 mL and Q = 6 mmol) had the best iprodione binding property (27.4 mg/g). However, it had the smallest imprinting factor (0.81). MISa (V = 7.5 mL and Q = 2 mmol) had the best imprinting factor (3.2) and the second best iprodione binding property (14.4). Based on the experimental design results, MISa was chosen as the optimal MIS.

The optimal MIS imprinting factor was 3.2. It is higher than the one we determined in a previous study (IF = 2.4) [START_REF] Bitar | Experimental design approach in the synthesis of molecularly imprinted polymers specific for iprodione fungicide[END_REF] that aimed to develop acrylate-based MIP specific to iprodione. On the other hand, the optimal MIS had an adsorption capacity of 14.4 mg/g (mg of iprodione per g of MIS). It is 33 times lower than that of the MIP, which was equal to 473 mg/g. This comparison of the acrylate-based (MIP) and the silica-based (MIS) polymers shows that the MIS are more selective but have a relatively low adsorption capacity which is in agreement with the following studies reports in literature [START_REF] Cummins | A comparative study of the potential of acrylic and sol-gel polymers for molecular imprinting[END_REF][START_REF] Marx | Molecular Imprinting in Thin Films of Organic-Inorganic Hybrid Sol-Gel and Acrylic Polymers[END_REF].

Characterization of the optimal MISa

The optimal MISa was characterized by Scanning Electron Microscopy, laser diffraction particle size analyser and Fourier transform infrared (FTIR) spectroscopy. Results are presented in supplementary materials (figures S2 to S5). SEM micrographs show that the bigger particles size of MISa range from 18 to 61 µm. Particle size distribution of MISa show that the average particle size is 13 ± 3 µm. The EDX spectrum show 3 major peaks corresponding to oxygen (52.6%), silicon (29.9%) and carbon (16.6%). These percentages show that (3-Aminopropyl)trimethoxysilane (APTMS) monomer reacts with tetraethoxysilane (TEOS) to form the sol-gel polymer. FTIR spectrum of MISa show that all the observed peaks are related to the absorption of the TEOS crosslinker and the APTMS monomer after the sol-gel hydrolysis and condensation. The condensed APTMS have several absorption bands: the ones at 2984 cm -1 and 790 cm -1 correspond to the C-H group and the one at 960 cm -1 corresponds to the C-C group. The condensed TEOS has an absorption band at 1060 cm -1 corresponding to the Si-O-Si group.

Selectivity of the optimal polymer MISa

The selectivity of the optimal polymer MISa was studied in a Burgundy white wine supplemented with the five following competing fungicides: iprodione, procymidone, pyrimethanil, iprovalicarb and azoxystrobin. The fungicides choice was based on a study done by the French Ministry of Agriculture in 13 regions representing the main French vineyards. The chosen fungicides were detected in most of the 1 222 wine analysed samples [START_REF] Cugier | Plan de surveillance résidus en viticulture. Campagnes viticoles 1990-2003[END_REF].

The partition coefficient K (mL/g) was calculated for each fungicide. The fungicides partition was determined between MISa or NISa and the white wine. K values were calculated using the equation [START_REF] Duan | A sensitive and selective imprinted solid phase extraction coupled to HPLC for simultaneous detection of trace quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal muscles[END_REF]):

= ×
where B is the fungicide amount retained by the polymer and F the free amount remaining in wine.

V (mL) is the wine volume and m (g) is the polymer mass. The obtained results are presented in figure 3.

Iprodione had the highest K MIS value followed by procymidone. These values are coherent because MISa was synthesized using iprodione as template. Procymidone has a close chemical structure to iprodione compared to the other fungicides. The same order was maintained for K NIS values.

However, K NIS values were much lower than K MIS values. This shows that the molecular imprinting process was successful.

The selectivity of MISa was assessed by the selectivity coefficient α defined as follows:

=

Where the template is iprodione. The calculated α values are: 1.1, 1.2, 1.7 and 2.3. They correspond respectively to procymidone, azoxystrobin, pyrimethanil and iprovalicarb. Procymidone, the fungicide having the closest chemical structure to iprodione, had the lowest α value. Iprovalicarb, with the most distant chemical structure from iprodione, had the highest α value.

The optimal MIS was able to interact with iprodione but to a lesser extent with other competing fungicides in a white wine sample. In order to eliminate the non-specific interactions and to preconcentrate only iprodione, an optimisation of molecularly imprinted solid phase extraction was performed.

Determination of iprodione in white wine by Molecularly Imprinted Solid Phase Extraction (MISPE) using the optimal MISa

Before the application of MISa in the preconcentration of iprodione from a white wine using solid phase extraction (SPE), the optimization of the solvents used during the different steps of the SPE was done. MISa and NISa were packed into two different cartridges. The SPE cartridge filled with MISa was washed with 10 mL of acetonitrile in order to eliminate any residual iprodione. Methanol and ethanol was tested as pre-conditioning solvent. Methanol allowed better sorption of iprodione and gave a better sorption difference between MISa and NISa. The conditioning solvent was water/ethanol, 90/10, v/v. After loading the white wine supplemented with iprodione, three washing solvents were tested: water/acetone, v/v, 70/30, 50/50 and 25/75 respectively. The latter were chosen because it allowed the elution of the total amount of iprodione adsorbed on the NIS. Several elution solvents were used in order to elute all iprodione adsorbed on the MIS. For this reason, the acetone proportion was varied between 75% and 100%. Acetone 100% eliminated the total amount of iprodione adsorbed by MISa. The adsorbed and recovered amount of iprodione during the different SPE steps are compiled in table 3. MISa adsorbed twice as much iprodione as NISa. The washing step removed all of the non-specific interactions that correspond to the amount of iprodione adsorbed on NISa. The iprodione recovered by the elution step corresponds to the specific interactions. It represents 52% of the adsorbed iprodione. Acceptable total recovery percentages (higher than 80 %) were obtained for both MISa and NISa.

Blank white wine was analyzed by HPLC in order to check the specificity of the developed method.

The blank chromatograms showed no interfering peaks at the retention time of iprodione. The limit of detection (LOD) and the limit of quantification (LOQ) of the MISPE method were calculated using the following equations: LOD = (3.3/6) (SD/S) ; LOQ = (10/6) (SD/S) where SD is the standard deviation of the HPLC response and S is the slope of the calibration curve. 6 corresponds to the preconcentration factor of the MIS-SPE method. The calculated LOD and LOQ were respectively 11.7 and 39.1 µg⋅L -1 . The relative standard deviation of the method (n = 3) was 10 %. The quantification limit of this method is lower than that determined by MIP-SPE-HPLC in our previous studies in white wine (LOQ = 422 µg⋅L -1 ) [START_REF] Bitar | Molecularly imprinted polymer solid phase extraction of fungicides from wine samples[END_REF]. It is comparable to that determined by SPE-LC-TOFMS in fruit juices (LOQ = 1.5 µg⋅L -1 ) [START_REF] Gilbert-López | Determination of Postharvest Fungicides in Fruit Juices by Solid-Phase Extraction Followed by Liquid Chromatography Electrospray Time-of-Flight Mass Spectrometry[END_REF] but really less expensive.

Currently, the fungicides detection methods used are mainly based on mass spectrometric detection and are relatively expensive [START_REF] Gilbert-López | Determination of Postharvest Fungicides in Fruit Juices by Solid-Phase Extraction Followed by Liquid Chromatography Electrospray Time-of-Flight Mass Spectrometry[END_REF][START_REF] Hayward | Organohalogen and Organophosphorous Pesticide Method for Ginseng Root. A Comparison of Gas Chromatography-Single Quadrupole Mass Spectrometry with High Resolution Time-of-Flight Mass Spectrometry[END_REF][START_REF] Wong | Multiresidue Pesticide Analysis of Ginseng Powders Using Acetonitrile-or Acetone-Based Extraction, Solid-Phase Extraction Cleanup, and Gas Chromatography-Mass Spectrometry/Selective Ion Monitoring (GC-MS/SIM) or -Tandem Mass Spectrometry (GC-MS/MS)[END_REF].

In parallel, ELISA methods using an immunoadsorbent are used [START_REF] Koch | Modification of a commercially-available ELISA kit to determine chlorothalonil and iprodione concentration on golf course turfgrass[END_REF][START_REF] Watanabe | Immunoassay for iprodione: Key estimation for residue analysis and method validation with chromatographic technique[END_REF]. In addition to their high cost, these methods cannot be used in extreme pH or temperature environments. Preconcentration methods using molecularly imprinted polymers (MIPs) could replace the methods currently used. MIPs are inexpensive materials and resist to extreme temperature and pH conditions [START_REF] Haupt | Molecularly imprinted polymers in analytical chemistry[END_REF][START_REF] Svenson | On the thermal and chemical stability of molecularly imprinted polymers[END_REF].

The developed MIS-SPE-HPLC method has two major limitations: it is time consuming and cannot be applied for fungicides that do not absorb in the UV-visible range.

Conclusion

Molecularly imprinted sol-gel silica (MIS) were developed in this work in order to conduct synthesis in safe condition, and to preconcentrate iprodione fungicide from a Burgundy white wine using solid phase extraction. The analysis was done by a cheap HPLC-UV method. To prepare the optimal MIS for iprodione, an experimental design having the following 2 factors (solvent volume and crosslinker quantity) was developed. The responses of the experimental design were the imprinting factor (IF)

and the binding capacity (BC). The optimal MIS had a higher IF but a lower BC than a previously synthesized acrylate based polymer (MIP) specific to iprodione. The design of experiment showed that the optimal MIS with the higher IF and BC, was obtained with low solvent volume and low crosslinker quantity during its synthesis. The selectivity of the optimal MIS was checked in a white wine containing iprodione and four other competing fungicides. The fungicide with the closest chemical structure to iprodione gave the lowest selectivity factor and the fungicide with the most distant chemical structure to iprodione gave the highest selectivity factor. The optimal MIS was used as packing material in a solid phase extraction (SPE) to pre-concentrate iprodione from a white wine.

After the optimization of the different steps of the SPE, MISPE-HPLC-UV method was able to detect iprodione in a white wine with a detection limit of 11.7 µg⋅L -1 . This method could be used to monitor this forbidden fungicide in wine easily and with a cheap lab equipment such as an UV-VIS-HPLC.

Figure 1 .

 1 Figure captions:Figure 1. Proposed chemical reactions involved in the MIS synthesis. A. Basic hydrolysis and poly-

Figure 2 .

 2 Figure 2. Imprinting factor ± SD (n = 3) (A) and binding capacity ± SD (n = 3) (B) of the MIPs towards

Figure 3 .

 3 Figure 3. Fungicides partition coefficients between MISa or NISa and a Burgundy white wine spiked

  Figure 1.

  Figure 2.

Table 1 . Coded levels and corresponding factors of the 2 2 experimental design.

 1

	Factors	X1	X2
		Solvent	Crosslinker
		volume	quantity
	Levels	(V, mL)	(Q, mmol)
	-1	7.5	2
	1	15	6

Table 3 . Adsorbed and recovered amount of iprodione from white wine using MISPE. Means ± SD (n = 3).

 3

		Adsorbed	Washing recovery	Elution recovery	Total recovery
	Polymer
		(µg)	(µg)	(µg)	(%)
	MISa	8.55 ± 0.85	2.95 ± 0.21	4.43 ± 0.82	86 ± 10
	NISa	3.71 ± 0.73	3.11 ± 0.41	0	84 ± 9

Acknowledgements

This work was supported by the Regional Council of Burgundy and the European Union via FEDER (Fonds Européen de Développement Régional) funding. We are grateful to AgroSup Dijon for cofunding this research program. The authors thank Fleur-Anne RIO and Camille DETANG for their contribution to the experimental work. They thank Anne-Sophie VIAUX for the Laser diffraction analysis and Laurence DUJOURDY for helpful discussion about the design of experiment.