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Highly Permselective Membrane Surface Modification by Cold Plasma-induced Grafting Polymerization of Molecularly Imprinted Polymer for Recognition of Pyrethroid Insecticides in Fish Rongrong Zhang, Xiaoqing Guo, Xizhi Shi, Aili Sun, Lin Wang, Tingting Xiao, Zigang Tang, Daodong Pan, Dexiang Li, and Jiong Chen Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Nov 2014 Downloaded from http://pubs.acs.org on November 10, 2014

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Analytical Chemistry

Highly Permselective Membrane Surface Modification by Cold Plasma-induced Grafting Polymerization of Molecularly Imprinted Polymer for Recognition of Pyrethroid Insecticides in Fish

Rongrong Zhang†, Xiaoqing Guo†, Xizhi Shi*, Aili Sun, Lin Wang, Tingting Xiao, Zigang Tang, Daodong Pan, Dexiang Li, Jiong Chen

Key Laboratory of Applied Technology of Marine Biology, Ministry of Education, Ningbo University, 818 Fenghua Road, Ningbo 315211, P.R. China

*Corresponding Author: Dr. Xizhi Shi School of marine sciences Ningbo University 818 Fenghua Road, Ningbo 315211 P.R. China E-mail: [email protected] Phone/fax: +86-574-87600551/87608347

† These authors contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT: Specific molecularly imprinted membranes (MIMs) for pyrethroid insecticides were developed and characterized for the first time in this study by cold plasma-induced grafting polymerization using methacrylic acid (MAA) as functional monomer and cypermethrin (CYP) as template. The non-imprinted membranes (NIMs) were also synthesized using the same procedure without the template. Meanwhile, AFM, XPS, ATR-FTIR, contact angle, and permselectivity experiments were conducted to elucidate the imprinting and recognition properties of MIMs. Results demonstrated

that

MIMs

exhibited

excellent

imprinting

effect

and

high

permselectivity. A molecularly-imprinted-membrane assisted solvent extraction (MI–MASE) method based on the MIMs was established. The operating conditions were optimized for group-selective extraction of the five pyrethroid insecticides. Compared with NIMs, higher extraction recoveries (83.8% to 100.6%) of the five pyrethroid insecticides by gas chromatography–electron capture detector(GC-ECD) were obtained using MIMs at three spiked levels in fish samples; the RSD values were lower than 8.3%. The limits of detection (LOD) and quantification (LOQ), are defined as the concentrations at which the signal-to-noise(S/N) ratio is 3:1 and 10:1, respectively, were in the range of 0.26 µg/kg to 0.42 µg/kg and 0.77 µg/kg to 1.27 µg/kg, respectively. No matrix effect of the developed MI–MASE was observed by gas chromatography-tandem mass spectrometry (GC-MS/MS). These results demonstrated a highly selective, efficient, and environment-friendly MI–MASE technique for preconcentration and purification of pyrethroid insecticides from seafood, followed by GC–ECD and GC-MS/MS. The excellent applicability and


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potential of MI–MASE for routine monitoring of pyrethroid pesticides in food samples has also been confirmed.



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Pyrethroid insecticides are extensively and increasingly used for pest control in agriculture, such as control of sea lice (Caligus elongatus) on salmon (Salmo salar) occurring in the UK, Europe, and USA, because of their broad spectrum insecticidal activity and low mammalian toxicity.1,2However, with substantial usage, a considerable amount of their residues is usually released directly into the environment, resulting in contamination of the aquatic ecosystem.3Moreover, given the physicochemical properties of pyrethroid insecticides, such as persistence, solubility, and apt for enrichment, extensive use of pyrethroid insecticides results in accumulation of these substances in marine products. In particular, recent studies have indicated that several pyrethroid insecticides have been found to present endocrine disrupting properties and suspected to affect reproduction and sexual development, as well as the immunesystem.4,5 Fish is one of the main dietary components for people in numerous countries, which results in high exposure to these pollutants accumulating in lipid-rich tissues throughout the food chain.6Therefore, measurement of these substances in fish samples is particularly important for verifying and biomonitoring contamination. Most pyrethroid insecticides analyses have been developed using chromatographic technique, which mainly includes gas chromatography (GC) coupled with electron capture detector (ECD) or mass spectrometry (MS). Although GC–MS could provide structural information of the analytes, this combination is less sensitive compared with GC–ECD.7,8Furthermore, given the effect of complex matrices that may interfere with GC–ECD analysis of the sample extracts, false positive or inaccurate


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quantitation frequently occur.9To reduce or remove matrix interferences during the sample extract, numerous extraction and clean-up methods to improve pretreatment efficiency have been reported in the literature, such as solid-phase extraction (SPE),10 solid-phase microextraction (SPME),11matrix solid-phase dispersion (MSPD),12and Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERs).1Although these methods present relatively high efficiency, low organic solvent consumption, or high automation potential, their relatively strict experimental control, complex operating procedure, or long equilibrium time limit their application to a certain extent. Another alternative technique, namely, membrane-assisted solvent extraction (MASE) based on the diffusion of an organic compound through a non-porous membrane, which presents low solvent consumption, convenience, and potential for automation, has been successfully applied to analysis of organic compounds in samples with complex matrices.13,14The non-porous membrane provides a barrier for interferences in terms of permeation and transport through the membrane, and realizes extraction and enrichment according to the polarity of organic compounds, simultaneously resulting in low permselectivity to specific analytes.15Therefore, surface modification of the membrane by a specific functional base was applied to enhance the selectivity and mass transform capability of the membrane. Molecularly imprinted polymers (MIPs) with high selectivity and excellent physicochemical characteristics could be used as functional base donor, and the molecularly imprinted solid phase extraction (MISPE) has been successfully used for the isolation and clean-up of pyrethroid insecticides in different matrix samples.16,17The potential for



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combining the excellent stability of the membrane with the specific selectivity of MIPs could be used to improve permselectivity for separation of selected molecules from complex mixtures. However, MIPs prepared using traditional methods suffer from several disadvantages, including heterogeneous distribution of the binding sites, poor site accessibility, and low mass transfer.18A new molecular imprinting “grafting from” technique has been applied to overcome these drawbacks.19Such as, the polyethylene membranes were grafted with a thin layer of MIPs using continuous plasma polymerization, which suggested that the good imprinted effect of the modified membranes was obtained.20However, to the best of our knowledge, no study on MIPs modified by cold plasma-induced grafting polymerization and its application to MASE has been reported yet. Therefore, the present study aims to develop a molecularly imprinted membrane (MIMs) by cold plasma-induced grafting polymerization with dense polypropylene (DP) membrane as support membrane and an MIP layer anchored on the out-surface of the DP membrane with CYP as template molecule. Based on MIMs, molecularly-imprinted membrane-assisted solvent extraction (MI–MASE) has been successfully used for extraction and clean-up of target analytes in complex sample matrix. The obtained sensitivity and recovery of the developed method for determination of pyrethroid insecticides are highly satisfactory.

EXPERIMENTAL SECTION Materials and Chemicals. Fenvalerate (FEN), deltamethrin (DEL), cypermethrin

(CYP), cyfluthrin (CYF), and bifenthrin (BIF) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Methacrylic acid (MAA) was obtained from


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Sigma–Aldrich (Steinheim, Germany), and the cross-linker, ethylene glycol dimethacrylate (EGDMA), was sourced from Fluka (Steinheim, USA). HPLC-grade hexane and acetone were purchased from Fisher Scientific Co. (USA). Milli-Q water was obtained from a Milli-Q purification system (Millipore, USA). Dense polypropylene membrane (DP membrane) bags were purchased from Gerstel (Mülheim, Germany). Standard solutions (100 mg/L) of FEN, DEL, CYP, CYF, and BIF were prepared in acetone. A 10 mg/L dilute standard in acetone was prepared weekly. MIM Preparation. The CYP template (1 mmol) and MAA (4 mmol) was dissolved in acetonitrile/acetone (9:1, v/v) and stirred for 2 h. Subsequently, 20 mmol of cross-linker EGDMA was added, and the mixture solution (Solution 1) was purged with nitrogen for 10 min prior to use. Meanwhile, the DP membrane bag was sequentially conditioned with 10 mL ethanol, acetone, and Milli-Q water, and then dried in a vacuum oven at 40 °C. The DP membrane was placed into the reaction chamber of plasma surface treatment apparatus and treated for 60 s under a 30 Pa argon atmosphere delivering 45 W to generate radicals on the surface. Subsequently, the DP membrane was immediately immersed in the “Solution 1” to initiate the graft polymerization reaction, and the functional groups can be grafted on the surface of the DP membrane. The reaction proceeded at 40 °C for 24 h under nitrogen atmosphere. The grafted DP membranes were extracted with acetone through Soxhlet extraction to remove the unreacted monomers, and then sequentially washed with hexane:acetone (9:1, v/v) and acetone until the template molecules were not detectable by GC–ECD.



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Finally, the grafted DP membranes were dried in a vacuum oven at 50 °C. The non-imprinted membranes (NIMs) were similarly prepared without the template. The membrane bags were typically conditioned with sequential preconditioning using hexane and hexane:acetone (9:1, v/v) to remove interfering compounds before further application. Morphological and Chemical Analysis. The chemical structure of the obtained membrane was confirmed by an attenuated total Fourier transform infrared spectroscopy (ATRFT–IR, Nicolet 6700, Thermo, American). The morphological structures of the imprinted and non-imprinted DP membrane before and after grafting were observed by scanning probe microscopes (SPM, Dimension3100V, Veeco, American). X-ray photoelectron spectroscopy (XPS) measurement was conducted on a multifunctional X-ray Photoelectron Spectroscope (AXIS ULTRA DLD, Kratos). The hydrophilicity of membranes was evaluated using water contact angle meter (DIGIDROP, GBX, France). Membrane Permeation Experiment Analysis. To evaluate the permselectivity of MIMs, competitive transport experiments of the five pyrethroid insecticides were carried out in different extraction solvents and homogenization solutions with different ratios of acetonitrile to water. The extraction vial was filled with 15.0 mL solutions having different acetonitrile-to-water ratios of 5:95, 10:90, 20:80 and 30:70 (v/v), respectively, and the membrane bag prepared with MIMs was filled with 0.75 mL n-hexane and extracted at 600 rpm and 40 °C for 30 min.


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Permselectivity was analyzed by comparing the recovery of MIM bags to the analytes and calculating the transporting factor (TF) and permselectivity factor (SF) using the following equations: (1) TF=QMIM/QNIM, where QMIM and QNIM are the extraction amounts of the analytes through MIM and NIM, respectively; (2) SF=Qtemplate/Qanalogue, where Qtemplate and Qanalogue are the extraction amounts of the template and the analogue, respectively.21 Sample Preparation. The fish (Pampus argenteus) samples used in this study were purchased from a local market and confirmed to have no detectable pyrethroid insecticide residues by GC–ECD. The 2.0 g fish muscle samples were precisely weighed and spiked with appropriate levels of the five pyrethroid insecticides. The samples were further homogenized with a blender at 10,000 rpm for 1.0 min and allowed to stand for 60 min. For MI–MASE extraction, 15.0 mL of acetonitrile in water (1:9, v/v) was added to the fish samples, which was then vortexed and ultrasonicated for 2 and 15 min, respectively. Furthermore, the MIM bag was filled with 0.75 mL n-hexane and extracted at 600 rpm and 40 °C for 30 min. After extraction, n-hexane was transferred to the 2.0 mL auto-sampler vial and analyzed by GC–ECD. Each sample treatment was replicated three times. Pyrethroid Insecticide Analysis. Measurement of pyrethroid insecticides was carried out with a GC-2010 gas chromatograph (Shimadzu, Japan) equipped with a 63

Ni electron capture detector (ECD), a split capillary column injector, and a Supel

SPB-5 (30 m × 0.25 mm I.D. × 0.25 µm film thickness) capillary column. Nitrogen (N2) was used as carrier and makeup gas at a flow rate of 1.0 mL/min (constant flow).



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The oven temperature was programmed as follows: 240 °C for 3 min, increased to 290 °C at a heating rate of 5 °C/min, and maintained for 5 min. The injector temperature was 240 °C, and the detector temperature was set at 320 °C. The samples (1.0 µL) were injected using the split mode at a ratio of 30:1.15 Gas Chromatography–Tandem Mass Spectrometry. The quantification of pyrethroid insecticides was performed using an Agilent 7890A GC coupled to an Agilent 7000A triple–quadrupole massspectrometer (Agilent Technologies) operated in multiple reaction monitoring mode (MRM). The GC system was equipped with a7683B autosampler (Agilent Technologies) and an SPB-5 MS capillary column (30 m × 0.25 mm i.d. × 0.25 µm, Supelco). 1.0 µL of samples were injected using the pulsed splitless mode at an injection temperature of 250 °C. The temperature program was as follows: 80 °C for 0.3 min, raised at a rate of 30 °C/min to 180 °C and held for 3 min, raised at a rate of 20 °C/min to 260 °C, and then held for 19 min. The MS transfer line and ion source temperatures were set to 280 and 300 °C, respectively. Nitrogen was applied as the collision gas at a flow of 1.5 mL/min. Helium was applied as the carrier gas at a constant flow of 2.25 mL/min.

RESULTS AND DISCUSSION MIM Preparation and Characterization. In MASE, an environment-friendly

enrichment technique, the properties of the membrane are the most important factor in the specific permeability of analytes and anti-contamination capability during extraction.22Hence,

we

developed MIMs by cold plasma-induced

grafting

polymerization using MAA as functional monomer and CYP as template. The molar


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ratio of template, monomer, and cross-linker at 1:4:20 for molecular imprinting was applied as previously reported.17Observation was first performed on dry membrane samples

under

ambient

atmospheric

conditions

to

better

elucidate

the

physicochemical characteristics of the membrane. Atomic force microscopy (AFM) was used to examine the surface morphology and topography of the investigated DP membrane samples on the order of fractions of a nanometer.23Figure 1 shows that the surface of MIMs is typically nodular (hills and valleys). The nodule size on the membrane surface significantly increased after grafting modification compared with that on the DP membrane, which clearly showed the roughness on the surface of the membrane. In addition, substantial differences can be observed in the topographical structures of the MIMs, NIMs, and DP membranes. The images and surface roughness (Figure 1) of the topographical image for MIMs and NIMs (Figures1A and 1B) show a visually higher roughness surface in comparison with the non-porous DP membranes (Figure 1C). MIMs showed the highest level of roughness. Moreover, the spatial distribution of nodules on the MIMs was more homogeneous compared with the surface of NIMs, which indicates that the surface difference of MIMs and NIMs was caused by imprinting effect. Furthermore, the surface composition of MIMs was analyzed by XPS measurements, which provides quantitative analysis of the atomic composition of a membrane surface by analyzing the binding energies characteristic to each element.24 Figure 2 illustrates the XPS survey scans of the MIMs, NIMs, and DP membranes. The chemical structures of CYP, MAA, and EGDMA are displayed in Figure 3. As shown in Figure 2,



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compared with the untreated DP membrane, significant changes can be observed in the survey spectra of MIMs and NIMs, whereas no obvious difference is observed between the spectra of MIMs and NIMs. This finding suggests that MIMs and NIMs share similar structure. The N 1s peak in Figure 2 shows that the surface of MIMs, NIMs, and DP membranes contained N, which may have originated from N2 used during the preparation of membranes, as described in Section “MIM preparation”. Meanwhile, Table1 shows that the atomic concentration of O 1s and N 1s on MIMs and NIMs evidently increased compared with those of DP membranes; the largest concentrations of O 1s and N 1swere obtained on MIMs. These results indicate that the carboxyl functional groups of MAA were located on the surfaces of the grafted membrane. The lack of CL 2p at about 194.7 eV in MIMs also showed that the template (CYP) was completely removed after being repeatedly washed with hexane:acetone (9:1, v/v) and acetone. To further confirm the type and the relative amounts of different polymeric species, the FT–IR spectra of modified (MIMs and NIMs) and non-modified DP membranes were obtained. As shown in Figure 4, the IR spectra of washed NIMs and MIMs exhibited similar patterns, demonstrating that these membranes have a similar backbone. The grafting degree is most conveniently characterized by the ratio of the heights of the characteristic carbonyl (1700 cm–1 to 1730 cm–1) and (1151 cm–1 and 1294 cm–1) peaks.25,26The IR spectra, which shows that the IR absorbance of the MIMs was higher than that of NIM, illustrate that MIMs have the largest grafting


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content, further indicating that the carbonyl functional groups of the molecular imprinted layer has been cross-linked on the DP membranes. Foulants, which affect the separation and purification of analytes in fish samples, are relatively hydrophobic. In addition, the surface characteristics of hydrophilicity and hydrophobicity are mostly tuned during the manufacture of the membranes mainly to ensure their good adhesive properties, so as to increase permeability and decrease contamination during separations. Hence, an appropriate hydrophilic surface on the membrane is needed. Contact angle measurement is an important tool to evaluate the hydrophilicity extent of the membrane surface. As summarized in Table1 and Figure 5, the contact angle of MIMs and NIMs decreased compared with that of DP membranes, with MIMs exhibiting the smallest water contact angle. The relatively high hydrophilic property of the modified membranes, MIMs and NIMs, suggests that the surface have been modified; thus a specific separation application can be focused on. Presumably, this effect originates from the grafting of carboxyl groups based on the molecular imprint on the DP membrane, consistent with the results of the AFM images and FT–IR, showing that the higher roughness and number of carboxyl groups in comparison with the DP membrane resulted in a lower contact angle. We may conclude that grafting used in the present study is likely to improve the resistance to hydrophobic fouling.27 Evaluation of MIM Selectivity. To evaluate the permselectivity of the synthesized MIMs, MI–MASE based on the MIMs was established, and competitive permeation experiments were performed on the five pyrethroid insecticides. The results are



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summarized in Table 2. We can see that the mean quantitative recoveries of the five pyrethroid insecticides to MASE and NI–MASE at 60 min were evidently improved compared with those obtained at 30 min, and the highest recoveries for the five pyrethroid insecticides were obtained by MASE at 60 min. Moreover, the recoveries of the five pyrethroid insecticides using MI–MASE at 60 min were no obviously different from those at 30 min, and the highest recoveries of 98.5% and 96.9% of CYP were obtained, respectively. The results of the above studies indicated that the transport rates of the pyrethroid insecticides through MIMs were markedly higher than those through NIMs and DP membrane, and the shorter time to reach equilibrium for MI–MASE are required than those needed to MASE and NI–MASE. The reason may be ascribed that the analyte transfer across the NIMs and DP membrane is driven by the difference in concentration with no selectivity. 15 Further, to gain better insight into the permselectivity of MIMs, the TFs for BIF, CYF, CYP, FEN, and DEL were calculated (Table 3), indicating that CYP has higher permeation amount than the other four analogues through MIMs. Meanwhile, DEL exhibited higher recoveries than BIF, CYF, and FEN, which may be due to the similar molecular structure of DEL to that of CYP, except with two bromine atoms instead of the two chlorine atoms, which resulted in higher electron affinity of the oxygen atom of the carbonyl group in CYP ester group compared with that of DEL.28 Therefore, the hydrogen bond between the MAA and the carbonyl group plays an important role in the capture of CYP during the process of MI–MASE from the outer surface of the membrane to its interior. The SF values (Table 3) of MIMs were found to be as high as 1.00 after 30 min or 60 min.


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However, the SF values of NIMs were below or close to 1.00, demonstrating that MIMs could selectively extract CYP, and its extraction yield for CYP was about 4.2 times higher than that of NIMs after 30min. The outer surface of MIMs possessed specific and regular imprinted sites given the imprinting effect of the CYP template, so the MIMs exhibited higher permselectivity to the template than those of other analogues because of the imprinting effect. Therefore, based on the results, we can conclude that CYP selectively permeated through MIMs because of the formed imprinting channels on the surface of membrane, which led to the formation of stronger driving force during the extraction procedure. Moreover, MI–MASE clearly presented higher extraction recoveries for the five pyrethroid insecticides than NI–MASE, indicating that MIMs could preferentially bind the CYP and its analogues to the imprinted sites, followed by exchange and diffusion into the extraction solvent in the membrane bags, which could considerably improve the permeation velocity and amount. Optimization of Extraction Parameters. MIPs are synthetic polymers that can specifically recognize the template or a group of structurally related compounds in an appropriate solvent.29Pyrethroid insecticides typically share a common chemical structure composed of cyclopropane carboxylic acids and different side chain group sizes and shapes (Figure 3). Therefore, we analyzed the group permselective characteristics of MIMs for five pyrethroid insecticides by optimizing the extraction and homogenization solvents. The extraction solvent, as the main parameter affecting the selectivity and extraction efficiency of membrane extraction, was optimized and



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the use of hexane and hexane:acetone (9:1, v/v) for membrane extraction was investigated. Simultaneously, to acquire high extraction efficiency and reduce solvent consumption, different volume ratios of acetonitrile to water were applied and optimized as homogenized solution. The results are shown in Figure 6. By contrast, NI–MASE and MASE exhibited similar extraction efficiencies for the five pyrethroid insecticides; lower recoveries were obtained than those obtained on MI–MASE. Furthermore, when hexane and 10% acetonitrile in water was used as extraction and homogenization solvents, respectively, the highest recoveries of the five pyrethroid insecticides ranging from 86.1% to 96.4% were acquired on MI–MASE compared with NI–MASE and MASE. These results clearly show that group-selective permeability for the five pyrethroid insecticides was obtained through MI–MASE under optimized extraction conditions because of the common core structure of pyrethroid insecticides. Therefore, hexane and 10% acetonitrile in water were subsequently used as extraction and homogenized solvents, respectively. MI–MASE Application on Fish Samples. Compared with the conventional membrane-modified technique, the developed MIM “grafting from” technique on DP membrane

by

cold

plasma-induced

grafting

polymerization

showed

high

permselectivity and excellent application capability. To further evaluate the reliability and anti-fouling properties of MIMs, fish (Pampus argenteus) samples from the local aquaculture were further acquired and confirmed to lack detectable pyrethroid insecticides. Validation of the MI–MASE method was performed following standard procedure. Fish sample analysis by MI–MASE was performed under the following


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optimal conditions: 40 °C extraction temperature, 30 min extraction time, 0.75 mL extraction volume, hexane as extraction solvent, and 10% acetonitrile in water as homogenized solvent. Meanwhile, validation of the developed MI–MASE method was processed based on linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) to evaluate the feasibility and potential of the proposed MI–MASE for analyzing pyrethroid insecticides. The comparison of accuracy (recovery) and precision (RSD) of the method applied for the fish samples are presented in Table 4. Each sample was extracted in triplicate. Compared with NI–MASE and MASE methods, the mean quantitative recoveries of the five pyrethroid insecticides after MI–MASE using different MIMs significantly improved, ranging between 83.8% and 100.6%. Meanwhile, RSD is generally used to characterize the precision of an analytical method. The RSD values were below 8.3% for the five pyrethroid insecticides, which simultaneously demonstrated the reproducibility of the proposed cold plasma-induced grafting polymerization approach to obtain the reproducible MIMs; the typical chromatograms of fish samples analyzed by MI–MASE, NI–MASE, and MASE are shown in Figure 7. Cleaner chromatograms were obtained using GC–ECD. These results indicate the satisfactory accuracy and precision of MI–MASE coupled to GC–ECD for detecting pyrethroid insecticides in fish samples. The LOD and LOQ values for the five pyrethroid insecticides under optimum extraction conditions are listed in Table 5. The LOD and LOQ values obtained by MI–MASE ranged between 0.26 and 0.42 µg/kg and between 0.77 and 1.27 µg/kg, respectively. To evaluate the matrix interference on trace analytes,



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matrix-matched calibration curves were obtained by extracting the five different spiked concentrations of pyrethroid insecticides at 5.0, 10.0, 25.0, 50.0 and 100.0 µg/L. As shown in Table 5, excellent linearity with correlation coefficients of R2>0.9959 was obtained for each of five pyrethroid insecticides, indicating that no chemical interferences induced by matrix effect were found and matrix interference was minimal. In addition, MI–MASE exhibited high extraction efficiency and capability offouling. These experimental results clearly showed that MI–MASE has better permeation selectivity for eliminating matrix interference compared with NI–MASE and MASE. The MI–MASE method coupled with GC–ECD was successfully applied in the detection of pyrethroid insecticides in fish samples. Furthermore, in view of interferences may occur when samples containing non-target electron-adsorbing components were analyzed by GC-ECD, the applicability of MI-MASE was confirmed by GC-MS/MS. As shown in Table 6, the values of mean quantitative recoveries of 81.9-101.5% and RSD of 3.2-8.3% for the five pyrethroid insecticides after MI–MASE coupled with GC-MS/MS were acquired, respectively, which are mostly in accordance with those obtained by GC-ECD. Meanwhile, no significant interferences from matrix effects or other compounds were observed (Figure 8). These results demonstrated that the MI-MASE method has good practicability for quantitatively determining pyrethroid insecticides in fish samples.

CONCLUSIONS

MIMs for CYP were prepared by cold plasma-induced grafting polymerization and characterized for the first time in this study. MI–MASE method based on MIMs was


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established under optimized conditions. Subsequently, a novel membrane extraction method for specific group selective recognition of five pyrethroid insecticides through GC–ECD was validated and successfully applied to the extraction of pyrethroid insecticides in fish samples. These data showed that good grafting of a molecularly imprinted layer on the outer surface of DP membranes by cold plasma-induced grafting polymerization can be processed, and MI–MASE based on MIMs could be a valuable tool for the enrichment and clean-up of pyrethroid insecticides from fish samples. Compared to conventional MASE, the method exhibited excellent permselectivity, extraction efficiency and lower time consumption. Furthermore, in contrast to the conventional LLE and SPE, the developed MI–MASE has a lower organic solvent and time consumption, moreover, in view of its simplicity, less labour-consuming and reusability after a simple cleaning procedure, the high throughout and low cost for sample preparation can be processed. The ease of performance of the developed MI–MASE method shows high potential for routine analysis of pyrethroid insecticides in monitoring programs.

ACKNOWLEDGEMENTS

This work was supported by the public science and technology research funds projects of ocean（201405035）, the National Natural Science Foundation of China (31001139), National Key Technology Research and Development Program of the Ministry of Science and Technology of China(2012BAK08B01-2), Natural Science Foundation of Ningbo (2013A610167), Scientific Research Fund of Zhejiang Provincial Education Department (pd2013101) and the K.C. Wong Magna Fund of Ningbo University.



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Figure Captions:

Figure 1. Atomic force microscopy (AFM) images of (A) MIMs, (B) NIMs, and (C) DP membranes. Figure 2. XPS survey of (A) MIMs, (B) NIMs, and (C) DP membranes. Figure 3. Chemical structures of pyrethroid insecticides. Figure 4. FT–IR spectra of (A) MIMs, (B) NIMs, and (C) DP membranes. Figure 5. Contact angle images of (A) MIMs, (B) NIMs, and (C) DP membranes. Figure 6. Optimization of extraction and homogenization solvents. Hexane: (A) MI–MASE, (B) NI–MASE, (C) MASE; hexane :acetone (9:1, v/v): (D) MI–MASE, (E) NI–MASE, (F) MASE. Analyte concentration: 5 µg/kg. Extraction conditions: 600 rpm, 40 °C; n=3. Figure 7. Chromatographic profiles of 2.0 g fish samples spiked with 10 µg/kg each of BIF, CYF, CYP, FEN, and DEL after MI–MASE, NI–MASE, and MASE. (A) reference standard; (B) blank sample; (C) after MI–MASE; (D) after NI–MASE; (E) MASE. Figure 8. MRM chromatograms of GC-MS/MS analysis of 2.0 g fish samples spiked with 10 µg/kg each of BIF, CYF, CYP, FEN, and DEL. (A) Blank fish sample; (B) Reference standard; (C) Spiked fish sample.


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Tables:

Table 1. Atomic concentration and contact angle Atomic Concentration(%) Type

Contact angle O 1s

N 1s

C 1s

MIMs

18.2

12.8

68.7

73.2 ± 4.8

NIMs

16.7

10.8

72.4

90.0 ± 4.9

DP membranes

8.7

2.8

88.3

104.5 ± 1.8



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Table 2. Recoveries of pyrethroid insecticides in 15.0 mL distilled water on MI–MASE, NI–MASE, and MASE using hexane as extraction solvent at 40 °C and 600 rpm (analyte concentration, 5.0 µg/L). 60 min

MI–MASE

NI–MASE

MASE

30 min

Recovery(%)

RSD(%)

Recovery(%) RSD(%)

BIF

45.7

6.5

43.8

6.2

CYF

68.1

5

65.8

5.3

CYP

98.5

4.4

96.9

5.6

FEN

49.6

7.6

45.1

7.2

DEL

82.5

6.7

79.9

5.9

BIF

41.6

7.0

33.9

7.5

CYF

50.9

8.1

32.1

8.4

CYP

47.4

5.1

22.8

6.9

FEN

46.4

8.0

33.9

7.5

DEL

49.9

7.1

25.4

6.9

BIF

98.5

7.3

63.5

5.6

CYF

101.4

5.4

56.8

5.1

CYP

95.8

6.7

63.4

6.8

FEN

90.6

5.4

54.7

6.4

DEL

92.4

6.3

55.9

4.9


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Table 3. Permselectivity capability of MIMs and NIMs MIMs

NIMs

Type TF30 min

TF60 min

SF30 min

SF60 min

SF30 min

SF60 min

BIF

1.29

1.10

2.21

2.16

0.67

1.14

CYF

2.05

1.34

1.47

1.45

0.71

0.93

CYP

4.26

2.08

1.00

1.00

1.00

1.00

FEN

1.33

1.07

2.15

1.99

0.67

1.02

DEL

3.14

1.65

1.21

1.19

0.90

0.95



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Table 4. Analysis of pyrethroid insecticides in spiked fish samples using MI–MASE, NI–MASE, and MASE; n=3. Spiked concentrations Pyrethroid 5.0 µg/kg

10.0 µg/kg

15.0 µg/kg

insecticides

MI–MASE

NI–MASE

MASE

Recovery (%)

RSD(%)

Recovery(%)

RSD(%)

Recovery(%)

RSD(%)

BIF

89.6

6.5

85.3

7.3

84.6

4.9

CYF

88.5

5.5

84.3

5.2

83.8

6.5

CYP

100.6

6.2

95.8

5.8

95.1

8.3

FEN

89.3

7.3

85.1

8.2

84.7

4.1

DEL

96.6

3.7

92.0

4.7

91.3

5.8

BIF

52.3

7.8

49.8

5.3

49.5

4.7

CYF

45.9

9.2

43.7

5.0

44.0

6.9

CYP

45.6

5.3

43.4

7.1

43.2

6.5

FEN

52.1

8.6

49.6

6.8

49.7

4.8

DEL

48.0

7.4

45.7

6.7

45.5

7.5

BIF

72.3

6.9

68.9

4.6

68.6

6.2

CYF

75.6

5.8

72.0

5.4

71.4

6.4

CYP

76.3

4.2

72.6

5.2

72.2

7.1

FEN

70.9

5.7

67.5

7.1

67.0

5.2

DEL

82.1

7.1

78.2

5.5

78.0

6.5


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Table 5. MI–MASE validation data. Pyrethroid

Linearity R2

insecticides

LOD(µg/kg)a

LOQ(µg/kg)b

range(µg/L)

BIF

5.0–100.0

0.9983

0.42

1.27

CYF

5.0–100.0

0.9962

0.30

0.89

CYP

5.0–100.0

0.9977

0.26

0.77

FEN

5.0–100.0

0.9959

0.31

0.93

DEL

5.0–100.0

0.9982

0.27

0.80

a

S/N ratio=3;b S/N ratio=10.



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Table 6. Precursor and product ions for MS/MS determination of five pyrethroid insecticides and Analysis of pyrethroid insecticides in spiked fish samples using MI–MASE by GC-MS/MS, n=3 Spiked concentrations Pyrethroid

Precursor

Product ion CE

insecticides ion (m/z)

(m/z)

5.0 µg/kg Recovery (%)

RSD (%)

10.0 µg/kg Recovery RSD (%) (%)

15.0 µg/kg Recovery (%)

RSD (%)

BIF

181

166,165

15，25

82.2

4.4

84.0

5.2

82.6

4.7

CYF

163

127,91

5，15

88.0

3.2

83.7

5.6

81.9

7.3

CYP

181.1

152.1,127.1

25，30

101.5

5.6

91.7

4.3

92.0

4.1

FEN

167.1

125,89.1

10，40

84.7

4.1

86.7

3.6

85.1

8.3

DEL

253

172,93

10，25

95.2

3.4

88.0

6.1

90.3

7.0

*


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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Figure 7



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Figure 8


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