Accepted Manuscript Title: A Molecularly Imprinted Polymer for the Selective Solid-Phase Extraction of Dimethomorph from Ginseng Samples Author: Xuanwei Xu Shuang Liang Xinxin Meng Min Zhang Ying Chen Dan Zhao Yueru Li PII: DOI: Reference:
S1570-0232(15)00133-6 http://dx.doi.org/doi:10.1016/j.jchromb.2015.02.033 CHROMB 19344
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
15-11-2014 21-2-2015 25-2-2015
Please cite this article as: X. Xu, S. Liang, X. Meng, M. Zhang, Y. Chen, D. Zhao, Y. Li, A Molecularly Imprinted Polymer for the Selective Solid-Phase Extraction of Dimethomorph from Ginseng Samples, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.02.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript
A Molecularly Imprinted Polymer for the Selective Solid-Phase Extraction of Dimethomorph from Ginseng Samples Xuanwei Xua,*, Shuang Liangb, Xinxin Menga, Min Zhanga, Ying Chena, Dan Zhaoa , Yueru Lia a Ginseng and Antler Products Testing Center of the Ministry of Agricultural PRC, Jilin Agricultural University, Changchun,
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Jilin, China
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b College of Resources and Environment Science, Jilin Agricultural University, Changchun, Jilin, China
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ABSTRACT
A molecularly imprinted polymer (MIP) was synthesized and evaluated to selectively extract dimethomorph from ginseng samples. Dimethomorph molecularly imprinted polymers with template to monomer molar ratios were contrived and
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developed via precipitation polymerization employing methacrylic acid as functional monomer, ethylene dimethacrylate as cross-linker and butanone:N-heptane (7:3, v:v)as porogen. The LOD (limit of detection) of this method was 0.002mg·kg-1,
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and the LOQ (limit of quantification) was 0.005mg·kg-1. The different spiked level of ginseng was 0.1mg·kg-1, 1.0mg·kg-1, 5.0mg·kg-1, and the average recovery of dimethomrph was 89.2-91.6%. Under the optimized condition, good linearity was obtained from 0.01 to 5 mg·kg-1 (r2≥0.9997) with the relative standard deviations of less than 3.20%. This proposed
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MISPE-GC procedure eliminated the effect of template leakage on quantitative analysis and could be applied to direct
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determination of dimethomrph in ginseng samples.
Keywords: Molecularly imprinted polymers; Dimethomorph; Precipitation polymerization; Solid-phase extraction; Ginseng samples
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1. Introduction
Dimethomrph (4-[3-(4-chlorophenyl)-3-(3, 4-dimethyloxyphenyl) propenyl] morpholine) is a kind of
morpholine fungicide developed by BASF. [1] It is cinnamic acid analogs, concentrated fungicide. It is mainly used for preventing and curing plant disease like Plasmopara viticola, Phytophthora infestans and Pseudoperonospora cubensis. Dimethomrph is mainly applied on grapevines, apples, ginsengs, tomatoes, potatoes, cucumbers, Chinese cabbage and other crops [2]. Molecular imprinting is a versatile and facile method for preparing synthetic polymers with predetermined molecular recognition properties and is presently attracting widespread interest, especially as the technological potential of molecularly imprinted polymers (MIPs) in chromatographic separations
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[3],solid-phase extraction [4]and catalysis[5]has now been established.Among these methods, precipitation polymerization is the simplest and the most efficient because it does not require any surfactant or interfering additives to be used. Molecular imprinting is considered as an elegant and convenient technology that can introduce special recognition and binding sites in imprinted materials, which are
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chemically and geometrically complementary to the template [6]. Compared to biological counterparts, molecularly imprinted polymers (MIPs) are more stable, less costly, and easier to produce [7]. Their use as
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sorbent material for SPE is one of the most exciting applications of MIPs because it would provide a
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simple and effective pretreatment method for complex samples. The traditional MIPs for compound analysis are prepared by using one kind of compound as template[8-11], which may be influenced by
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template leaking when using MIPs as SPE sorbents. As the filler of solid phase extraction，the MIP was get into solid phase extraction column[12,13].Its application was evaluated.
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The aim of the present work was to demonstrate the feasibility of using a molecular imprinting solid phase extraction (MISPE) cartridge for the selective clean-up and quantification of trace amounts of dimethomrph in ginseng samples. Dimethomorph molecularly imprinted polymers with template to
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monomer molar ratios were contrived and developed via precipitation polymerization employing
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methacrylic acid as functional monomer, ethylene dimethacrylate as cross-linker and butanone and N-heptane as porogen. The synthesized MIP enabled direct determination of the target compound. Combination of gas chromatography with MIP-SPE could be successfully used for quality control of
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pesticide residues. The experimental results indicated that: the extracts of ginseng could be effectively separated by MISPE，a high degree of cleaning up and acceptable recoveries were obtained. The application of MISPE in pre-treatment of dimethomrph was further developed in the thesis and provided a new way in samples analysis and detection. Following a conventional non-covalent imprinting protocol, several binding rebinding parameters have been evaluated in an attempt to extract selectively dimethomrph from ginseng samples. 2. Experimental 2.1 Materials and Chemicals The active compound of dimethomorph (97.6%) was purchased from BASF SE (Fig.1). Chromatographic grade acetonitrile (ACN), Azobiisobutyronitrile (AIBN), Methacrylic acid (MAA) and
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Ethylene dimethacrylate (EDMA) were purchased from Beijing Chemical Factory (Beijing, China). Inhibitor in MAA was removed by cleanup on activated alumina columns. EDMA was extracted with 25% aqueous sodium hydroxide three times to remove the inhibitor prior to use. AIBN was recrystallized from methanol before use. Methanol, Acetic acid, Butanone, N-heptane and Tetrahydrofuran (THF) were all
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analytical grades and obtained from Beijing Chemical Factory (Beijing, China). Water was doubly distilled.
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2.2 Apparatus and chromatographic conditions
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Gas chromatographic analysis was performed on an Aglient GC system, model Aglient 6890N, equipped with micro-electron capture detector (µ-ECD) and HP-1 capillary column (30m×0.25mm i.d.
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×0.25µm). A 1µL aliquot of the standard/sample was injected in the splitless mode and analyzed under the following conditions. The initial temperature of the column was maintained at 120℃ for 1 min, raised
to
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260℃ at 20℃·min−1 and maintained at 260℃ for 22 min. The injector, detector temperatures were 280℃ and 280℃. The pre-polymerizations were detected by Ultraviolet spectrometry (UV-2450, SHIMADZU,
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Japan) and Fourier-transform infrared spectrometry (FTIR-IRAffinity-1, SHIMADZU, Japan). The
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morphology of MIPs was characterized by scanning electron microscopy (SSX-550, SHIMADZU, Japan). 2.3 Preparation of Pre-polymerisation
0.017 mmol·L-1 of dimethomorph and a series of various concentrations of MAA (0.034 mmol·L-1,
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0.068 mmol·L-1, 0.102 mmol·L-1, 0.136 mmol·L-1) were prepared in butanone:N-heptane（7:3, v:v）solution, then dimethomorph solution mixed in identical volumes respectively. The pre-polymerisations were kept in water bath at 30℃ for 5 h and then placed in the refrigerator（0℃）overnight. The pre-polymerisations were scanned by UV spectroscopy. Dimethomorph were prepared separately in THF. Then dimethomorph solution mixed with equivalent volumes of various concentration of MAA for preparation of different pre-polymerisations. The pre-polymerisations were treated in the same manner as described above. The IR spectra of pre-polymerisations in THF were detected by a KBr crystal demountable assembly. 2.4 Preparation of Polymers The pre-polymerisations were incubated in a bath at 30℃ for 5 h to prearrange dimethomorph and MAA. EDMA and AIBN were added sequentially to the solutions, the solutions were oscillated to make them uniform. The pre-polymerisations then purged with a gentle flow of nitrogen for 10 min. The 3
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polymerization was initiated at 60℃ for 24 h. The generated polymeric particles were gathered by centrifugation at 4000 rpm for 10 min. To remove the template bound within the polymer matrix, the resulting powder was washed for 30 min with a mixture of methanol and acetic acid (9:1, V: V).The washing procedure was repeated until no more dimethomorph could be detected in the washing solution.
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These MIPs were then washed with methanol to eliminate residual acetic followed by acetonitrile three times. And corresponding nonimprinted polymers were synthesized at the same experimental conditions by
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omitting the template and treated in an identical manner as described above. Finally, the MIPs and NIPs
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were dried under vacuum at 40℃ overnight. Before use,each cartridge was activated by treatment with the same solvent used in the loading step.
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2.5 Characterization of Polymers
The morphology of the produced polymer particles were analyzed by scanning electron microscopy. The samples were prepared in methanol and sputtered a thin gold film prior to measurement in SEM.The
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structure of the polymers was characterization by FTIR spectroscopy in the range of 4000-500 cm-1 by KBr pellet method.
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2.6 Equilibrium Adsorption Experiment
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For adsorption isotherm studies, 20 mg of polymer was placed in an Erlenmeyer flask containing 50 mL of dimethomorph (20-200 mg·L-1) prepared in acetonitrile/water (7:3, V: V). The solution was shaken for 24 hours at room temperature at a speed of 150 rpm. Upon equilibration, all samples were filtered
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through a 0.22µm filter to minimize the interference of particles during analysis. The residual concentration of dimethomorph was analyzed by GC. The amount of dimethomorph was determined from the difference in concentrations at the beginning and at the end of each batch test. Before analysis, 10 mL of rinse solution was loaded onto the conditioned MIP cartridge. After a washing step with 1 mL acetonitrile, dimethomorph was recovered with 1 mL methanol. 2.7 Solid Phase Extraction (SPE) Studies In order to establish the optimum conditions under which the template can be recognized by the corresponding MIP, a standard solution of dimethomorph was initially prepared in various proportions (10-100%) of ACN/H2O and DMF/H2O mixture. To a 3 mL empty polypropylene solid phase extraction cartridges, 200 mg corresponding control polymer NIP was packed between two polypropylene frits. Before analyte loading, the polymer was conditioned with 1 mL methanol, 1mL acetonitrile and 1 mL H2O. 4
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In the loading step, 2 mL of dimethomorph solution (20 mg·L-1) prepared in a mixture of ACN/H2O (7: 3, V: V) was passed through the cartridge. Finally, dimethomorph was eluted with 1 mL of methanol. Ginseng powder samples were diluted with acetonitrile and were then filtered through a 0.22 µm syringe filter and were kept in the freezer until their use. Before analysis, 10 mL of ginseng samples were loaded
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onto the conditioned MIP cartridge. After a washing step, dimethomorph was recovered with methanol. 3. Results and discussion
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3.1 Synthesis and characteristics of the MIP
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The application of MIPs as sorbents in SPE is limited because the possibility of leaking of residual template molecules that remains trapped in the polymers after they have been washed extensively. [14].Thus, the best way to overcome this problem is the use of an analogue of the target molecule during
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MIP design and production, as mimic molecularly imprinted polymer [15–17].The literatures show that the ratio of template to functional monomer would affect adsorption capacity of target molecular
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[18].Therefore, four kinds of polymers were produced using different template to monomer molar ratios via precipitation polymerization to investigate the impacts of MAA on the adsorption. Four molar ratios
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between dimethomorph and MAA with1:2, 1:4, 1:6 and 1:8 were arranged. They were devised to survey
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the effects of template to monomer molar ratio on absorbability of polymers. The best molar ratio is 1:4.The objective of this paper is to select appropriate ratio of template-monomer to prepare a high adsorbing MIP.The molar ratio of dimethomorph to EDMA was selected at 1:20 to ensure the formation of
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defined imprinted cavities because with small molecule as template, high quantity of cross-linker was essential to yield proper special cavities into the polymer matrix. Solvent acts as an important role in the synthesis of the self-assembly type molecularly imprinted polymers. An appropriate porogen could not only dissolve template, monomer and cross-linker, but with poorly polar to reduce the interference between template and monomer. The porogen, butanone:N-heptane （7:3, v:v）is a moderately polar solvent that can be micron grade microspheres. 3.2 Morphology and Structure Characterization of the Polymers Traditional interactions between template and monomer include hydrogen bond, ion pair, hydrophobic interactions, π-π interactions. To illustrate the interaction sites of template and monomer, IR spectra of pre-polymerisations were recorded in the range of 4000-500 cm-1 by a KBr crystal demountable assembly. In this study, using infrared spectroscopy technique for the preparation of imprinted polymer MIP and NIP 5
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blank polymer micro structure analysis and whether the formation of hydrogen bonds that can be seen from Figure 2 NIP (a) and MIP (b) infrared spectra, infrared spectra NIP (a) in the absorption 3541cm-1 redshifted 18cm-1 with respect to MIP (b) of 3523cm-1.It demonstrated that the dimethomorph interacted with MAA might via hydrogen bonds.
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Scanning electron microscopy and FTIR were utilized to reflect the shape and structure of the synthesized polymers. As shown in Figure 3, spherical MIPS were prepared with precipitation
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polymerization and a serious agglomeration was appeared in NIPs. The images of MIP (Figure A) showed
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small microspheres, whereas that of NIPs (Figure B) showed highly agglomerated and irregular particles. We observed that only MIP (Figure A) was isolated in the form of monodispersed microsphere. It was gained that with increasing amounts of MAA, the MIPs were built in large size beads.
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3.3 Binding capacity of the MIP
After synthesis, equilibrium binding experiments of the MIP and NIP were performed at 25℃. Fig. 4
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shows the experimental binding isotherms of MIP (and NIP) amount as a function of dimethomorph concentration. The binding assay was repeated three times and the average values were obtained. The
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results showed that the binding capacity of MIP or NIP increased with the increasing of dimethomorph
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concentrations. Although NIP showed a similar trend in terms of target adsorption, its adsorption capacities were significantly changed. MIP exhibited higher adsorption capacity for the target molecule than NIP. Numerous and precise imprinting sites were found in MIP, resulting in specific adsorption and
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higher adsorption capacity. By contrast, the weak adsorption of the template on NIP was likely due to non-specific interactions with the polymer matrix, the absence of imprinting molecules, and the lack of suitable recognition sites and imprinting cavities in NIP. As it was shown in Fig.4, the adsorptive maximum capacities of MIP and NIP were 3.48 mg·g−1 and 2.41 mg·g−1, respectively. It was concluded that the prepared MIP could selectively recognize of the template molecule. Fig. 4 also showed that MIP has high adsorption capacity toward the dimethomorph, this might because that dimethomorph have similar chemical structures and functional groups. The presence of imprinting sites in MIP that complement the template molecules was vital for specific adsorption [19].The consequences from Scatchard analysis were attributed to the fact that the binding affinity of the imprinted polymer was originated from the specific sites created by the imprinting process and mainly driven by hydrogen binding[20]. 6
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3.4 Optimization of the MISPE In order to obtain the optimum selectivity and recovery, the selection and volume of washing and elution solvents were optimized [21-23]. To interrupt the non-selective interactions with the interferences present in the sample matrix, different washing solvents including methanol, acetonitrile, water,
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dichloromethane, acetone and acetonitrile-water (9:1, 7:3 and 1:1 v:v) were evaluated. The results in Fig.5 showed that acetonitrile and dichloromethane had higher elution strength, which resulted in a lower
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recovery. Although methanol as the washing solvent provided the best recoveries nearly 100%, the
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impurities elimination efficiency was not sufficient, and the main cause probably lied in the interference of polar solvent to the hydrogen-bonding based recognition of MIP coating. Moreover, the recoveries of dimethomorph were not obviously decreased with the increasing volume of acetonitrile–water (7:3, v: v)
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fom 0.1 mL to 5.0 mL.Considering the recoveries, impurities elimination efficiency and economic factors, 5.0 mL of acetonitrile–water (7:3, v: v) was used as the washing solution for further work.
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The elution solvent was also a key factor that affected the recovery. Different elution solvents were investigated to identify its influence on desorption of dimethomorph from the MIM cartridges and the
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results of Fig.6 indicated that acetonitrile–dichloromethane–acetic acid (65:30:5, v: v: v) as elution solvent
dichloromethane-acetic,
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had the best elution ability. Different elution solvents including methanol-acetic, methanol-acetic, acetone-acetic,
chloroform-acetic
and
acetonitrile-dichloromethane-acetic
(85:10:5, 65:30:5 and 45:50:5, v: v: v) were evaluated. The results in Fig.6 showed that chloroform-acetic,
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acetonitrile-acetic, dichloromethane-acetic, acetone-acetic, acetonitrile-dichloromethane-acetic (85:10:5, v: v: v) had higher elution strength, which provided the satisfied recoveries.Howerver, it was found that these elution solvents were not only able to wash out the interfering compounds presented in samples, but also able to wash out most of dimethomorph bound to both MIP and NIP. So it was suggested that acetonitrile-dichloromethane-acetic (65:30:5, 45:50:5, v: v: v) and chloroform-acetic were employed. Furthermore, the recoveries of chloroform-acetic and acetonitrile-dichloromethane-acetic (45:50:5, v: v: v) obtained from MIP were more than 60%, while those obtained from NIP were less than 30%.Moreover, different volumes of acetonitrile–dichloromethane–acetic acid (65:30:5, v: v: v) (1.0–10.0mL) were tested. The recoveries of dimethomorph were not obviously decreased with the increasing volume of acetonitrile-dichloromethane-acetic (65:30:5, v: v: v) from 0.1 mL to 5.0 mL. Therefore, considering the recoveries,
impurities
elimination
efficiency
and
economic
factors,
5.0
mL 7
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acetonitrile–dichloromethane–acetic acid (65:30:5, v: v: v) was chosen as the elution solvent for further work. 3.5 Validation of the MISPE-GC method The validation of specificity, linearity, limit of detection, recovery and precision for the proposed
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method were determined.Specificity was checked by analyzing twenty blank ginseng samples. No interfering peaks could be detected at the retention time of dimethomorph. The linearity and regression
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study were performed for dimethomorph standard graph to generate calibration curve. The high correlation
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coefficient (r=0.9997)indicated good linearity over the concentrations ranged from 0.01 to 5.0 mg·kg-1 for dimethomorph in ginseng samples.For the recovery study,each ginseng sample spiked at 0.1,1.0 and 5.0 mg· kg-1 dimethomorph and they were used for validation of the extraction procedure and MISPE clean up.
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The peak areas for spiked samples were compared with those of standards to determine the recovery. The recovery and repeatability of the method at spiked levels.Recoveries of dimethomorph on MIP cartridge
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from ginseng samples were between 89.2% and 91.6% with relative standard deviations of 3.20%. The limit of detection (LOD) was calculated in blank extracts as the lowest analyte concentration that yielded a
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signal-to-noise (S/N) ratio of 3. The LOD (limit of detection) of this method was 0.002mg·kg-1, and the
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LOQ (limit of quantification) was 0.005mg·kg-1. The data reported indicate that the above method for the analysis of dimethomorph in ginseng samples can achieve good recovery and repeatability. The developed MISPE-GC method was evaluated by the linearity, precision, repeatability, recovery,
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detection limits, Accuracy and precision of the MISPE-GC method were assessed by performing replicate analyses of the spiked samples in five replicates in the same day and consecutive three days.The intra-day precision and accuracy of the method evaluated as RSD was 2.12% and the inter-day reproducibility was 2.31%. Additionally, chromatograms (Fig.7) of spiked sample indicated that MISPE exhibited cleaner eluents (A: before MISPE; B: after MISPE). The retention time of dimethomorph ((Z)-isomer) was about 22.05 min and dimethomorph ((E)-isomer) was about 23.50 min. 4. Conclusions In this paper, the
developed
chromatographic
solid-phase extraction and GC was
method
involving
molecularly imprinted
appropriate for the analysis of dimethomorph in ginseng
samples. A MIP of dimethomorph was synthesized developed via precipitation polymerization for the separation and preconcentration of dimethomorph. Through evaluated in a series of adsorption experiments, 8
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the polymer exhibited good recognition and selective ability, suggesting that it could be a useful tool for analytical purposes. Furthermore, a method was successfully developed to detect dimethomorph at low concentration levels in ginseng samples using this MIP as enrichment sorbent of SPE coupled with GC. Compared to the
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methods used for determining dimethomorph in ginseng samples, the method developed in this article exhibited better characteristics such as sensitivity and facility. The presented MISPE-GC method combined
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the advantages of MIP and SPE, which could be potentially applied for the determination of dimethomorph
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in ginseng samples. Good figures of merit were attained, such as low LOQ, wide linear range，good precision and accuracy and its high selectivity. This paper also offered a new method to determine other
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analyte in different samples.
REFERENCES
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[1] J. H. Matt, S. Takayukim, J.Agric Food Chem. 48 (2000) 5825.
[2] H.B. Christensen, K.Granby, Food Addit Contam A. 18 (2001) 867-870.
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[3] B.Sellergren, J. Chromatogr.A 906 (2001) 227. [4] L. I.Andersson, J. Chromatogr. B 739 (2000) 163.
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[5] G. Wulff, Chem. Rev. 102 (2002) 1.
[6] L.I. Andersson, J. Chromatogr. B 745 (2000) 3.
[7] G. Vlatakis, L.I. Andersson, R. Miller, K. Mosbach, Nature 361 (1993) 645.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
[8] C. Zhao, T. Zhao, X. Liu, H. Zhang, J. Chromatogr. A 1217 (2010) 6995. [9] F. Puoci, C. Garreffa, F. Iemma, R. Muzzalupo, U.G. Spizzirri, N. Picci, Food Chem.93 (2005) 349. [10] C. Baggiani, L. Anfossi, P. Baravalle, C. Giovannoli, G. Giraudi, C. Barolo, G. Viscardi, J. Sep. Sci. 32 (2009) 3292. [11] Z. Zhang, H. Zhang, Y. Hu, S. Yao, Anal. Chim. Acta 661 (2010) 173. [12] S. Pardeshi, R. Dhodapkar, A. Kumar, Food Chem. 146 (2014) 385-393. [13] T. Yasuyama, H. Matsunaga, S. Ando, T. Ishizuka, Chem. Pharm. Bull. 61 (2013) 546-549. [14] A. Ellwanger, C. Berggren, S. Bayoudh, C. Crecenzi, L. Karlsson, P.K. Owens, K.Ensing, P. Cormack, D. Sherrington, B. Sellergren, Analyst 126 (2001) 784. [15] T. Dandekar, P. Argos, Biochem. Mol. Biol. 4 (1993) 75. [16] X. Feas, J.A. Seijas, M.P. Vazquez-Tato, P. Regal, A. Cepeda, Anal. Chim. Acta 638(2009) 209. [17] L.M. He, Y.J. Su, Y.Q. Zheng, X.H. Huang, L. Wu, Y.H. Liu, Z.L. Zeng, Z.L. Chen, J.Chromatogr. A 1216 (2009) 6196. [18] Y. Wang, E. L. Wang, Z. M. Wu, H. Li, Z. Zhu, X. S. Zhu, Y. Dong, Carbohydr. Polym. 101 (2014) 517. 9
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[19] O.S. Amuda, A.A. Giwa, I.A. Bello, Biochem. Eng. J. 36 (2007) 174. [20] M. Radhika, K. Palanivelu, J. Hazard. Mater. 138 (2006) 116. [21] F.X. Qiao, Y.R.Geng, C, Q.He, Y.P.Wu, P.Y.Pan, J. Chromatogr. B 745 (2000) 3. [22] Z.R.Lian, X.L.He, J.T.Wang, J. Chromatogr. B 957 (2014) 53-59.
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[23] H.B. Guo, K.Y.Liu, Y.H.Liu, B.H.Fang, M.L, L.M.He, Z.L.Zeng, J. Chromatogr. B 879 (2011) 181-185.
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*Highlights (for review)
1. This method employing butanone and N-heptane as porogen. 2. The porogen is a moderately polar solvent that can be micron grade microspheres. 3. This method combined the advantages of MIP and SPE and applied it to ginseng.
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ed
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4. This paper offered a new method to determine other analyte in different samples.
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Fig.1. Chemical structure of dimethomorph
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Figure1
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Fig.2. IR spectrum scanning curve
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Figure 2
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Fig.3. SEM micrographs of (A) MIP, (B) NIP
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Figure3
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Figure 4
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Fig.4. The binding isotherm of dimethomorph on MIP and NIP (n = 3)
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Figure 5
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1: Methanol; 2: Acetonitrile; 3: Dichloromethane; 4: Water; 5: Acetone; 6: Acetonitrile/water (1:1, v/v); 7:
Acetonitrile/water (7:3, v/v); 8: Acetonitrile/water (9:1, v/v)
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Fig.5. Effect of washing solvents on the loss of dimethomorph
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Figure 6
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1: Methanol-acetic (95:5, v/v); 2: Acetonitrile-acetic (95:5, v/v); 3: Dichloromethane-acetic (95:5, v/v); 4:
Chloroform-acetic (95:5, v/v); 5: Acetone-acetic (95:5, v/v); 6: Acetonitrile-dichloromethane-acetic (85:10:5,
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v/v/v); 7: Acetonitrile-dichloromethane-acetic (65:30:5, v/v/v); 8: Acetonitrile-dichloromethane-acetic (45:50:5,
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v/v/v)
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Fig.6. Effect of elution solvents on the recovery of dimethomorph
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Figure 7
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Fig.7.Chromatograms of the spiked ginseng samples (A: before MISPE; B: after MISPE)
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S1570-0232(15)00133-6 http://dx.doi.org/doi:10.1016/j.jchromb.2015.02.033 CHROMB 19344
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
15-11-2014 21-2-2015 25-2-2015
Please cite this article as: X. Xu, S. Liang, X. Meng, M. Zhang, Y. Chen, D. Zhao, Y. Li, A Molecularly Imprinted Polymer for the Selective Solid-Phase Extraction of Dimethomorph from Ginseng Samples, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.02.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript
A Molecularly Imprinted Polymer for the Selective Solid-Phase Extraction of Dimethomorph from Ginseng Samples Xuanwei Xua,*, Shuang Liangb, Xinxin Menga, Min Zhanga, Ying Chena, Dan Zhaoa , Yueru Lia a Ginseng and Antler Products Testing Center of the Ministry of Agricultural PRC, Jilin Agricultural University, Changchun,
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Jilin, China
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b College of Resources and Environment Science, Jilin Agricultural University, Changchun, Jilin, China
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ABSTRACT
A molecularly imprinted polymer (MIP) was synthesized and evaluated to selectively extract dimethomorph from ginseng samples. Dimethomorph molecularly imprinted polymers with template to monomer molar ratios were contrived and
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developed via precipitation polymerization employing methacrylic acid as functional monomer, ethylene dimethacrylate as cross-linker and butanone:N-heptane (7:3, v:v)as porogen. The LOD (limit of detection) of this method was 0.002mg·kg-1,
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and the LOQ (limit of quantification) was 0.005mg·kg-1. The different spiked level of ginseng was 0.1mg·kg-1, 1.0mg·kg-1, 5.0mg·kg-1, and the average recovery of dimethomrph was 89.2-91.6%. Under the optimized condition, good linearity was obtained from 0.01 to 5 mg·kg-1 (r2≥0.9997) with the relative standard deviations of less than 3.20%. This proposed
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MISPE-GC procedure eliminated the effect of template leakage on quantitative analysis and could be applied to direct
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determination of dimethomrph in ginseng samples.
Keywords: Molecularly imprinted polymers; Dimethomorph; Precipitation polymerization; Solid-phase extraction; Ginseng samples
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1. Introduction
Dimethomrph (4-[3-(4-chlorophenyl)-3-(3, 4-dimethyloxyphenyl) propenyl] morpholine) is a kind of
morpholine fungicide developed by BASF. [1] It is cinnamic acid analogs, concentrated fungicide. It is mainly used for preventing and curing plant disease like Plasmopara viticola, Phytophthora infestans and Pseudoperonospora cubensis. Dimethomrph is mainly applied on grapevines, apples, ginsengs, tomatoes, potatoes, cucumbers, Chinese cabbage and other crops [2]. Molecular imprinting is a versatile and facile method for preparing synthetic polymers with predetermined molecular recognition properties and is presently attracting widespread interest, especially as the technological potential of molecularly imprinted polymers (MIPs) in chromatographic separations
1
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[3],solid-phase extraction [4]and catalysis[5]has now been established.Among these methods, precipitation polymerization is the simplest and the most efficient because it does not require any surfactant or interfering additives to be used. Molecular imprinting is considered as an elegant and convenient technology that can introduce special recognition and binding sites in imprinted materials, which are
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chemically and geometrically complementary to the template [6]. Compared to biological counterparts, molecularly imprinted polymers (MIPs) are more stable, less costly, and easier to produce [7]. Their use as
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sorbent material for SPE is one of the most exciting applications of MIPs because it would provide a
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simple and effective pretreatment method for complex samples. The traditional MIPs for compound analysis are prepared by using one kind of compound as template[8-11], which may be influenced by
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template leaking when using MIPs as SPE sorbents. As the filler of solid phase extraction，the MIP was get into solid phase extraction column[12,13].Its application was evaluated.
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The aim of the present work was to demonstrate the feasibility of using a molecular imprinting solid phase extraction (MISPE) cartridge for the selective clean-up and quantification of trace amounts of dimethomrph in ginseng samples. Dimethomorph molecularly imprinted polymers with template to
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monomer molar ratios were contrived and developed via precipitation polymerization employing
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methacrylic acid as functional monomer, ethylene dimethacrylate as cross-linker and butanone and N-heptane as porogen. The synthesized MIP enabled direct determination of the target compound. Combination of gas chromatography with MIP-SPE could be successfully used for quality control of
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pesticide residues. The experimental results indicated that: the extracts of ginseng could be effectively separated by MISPE，a high degree of cleaning up and acceptable recoveries were obtained. The application of MISPE in pre-treatment of dimethomrph was further developed in the thesis and provided a new way in samples analysis and detection. Following a conventional non-covalent imprinting protocol, several binding rebinding parameters have been evaluated in an attempt to extract selectively dimethomrph from ginseng samples. 2. Experimental 2.1 Materials and Chemicals The active compound of dimethomorph (97.6%) was purchased from BASF SE (Fig.1). Chromatographic grade acetonitrile (ACN), Azobiisobutyronitrile (AIBN), Methacrylic acid (MAA) and
2
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Ethylene dimethacrylate (EDMA) were purchased from Beijing Chemical Factory (Beijing, China). Inhibitor in MAA was removed by cleanup on activated alumina columns. EDMA was extracted with 25% aqueous sodium hydroxide three times to remove the inhibitor prior to use. AIBN was recrystallized from methanol before use. Methanol, Acetic acid, Butanone, N-heptane and Tetrahydrofuran (THF) were all
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analytical grades and obtained from Beijing Chemical Factory (Beijing, China). Water was doubly distilled.
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2.2 Apparatus and chromatographic conditions
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Gas chromatographic analysis was performed on an Aglient GC system, model Aglient 6890N, equipped with micro-electron capture detector (µ-ECD) and HP-1 capillary column (30m×0.25mm i.d.
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×0.25µm). A 1µL aliquot of the standard/sample was injected in the splitless mode and analyzed under the following conditions. The initial temperature of the column was maintained at 120℃ for 1 min, raised
to
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260℃ at 20℃·min−1 and maintained at 260℃ for 22 min. The injector, detector temperatures were 280℃ and 280℃. The pre-polymerizations were detected by Ultraviolet spectrometry (UV-2450, SHIMADZU,
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Japan) and Fourier-transform infrared spectrometry (FTIR-IRAffinity-1, SHIMADZU, Japan). The
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morphology of MIPs was characterized by scanning electron microscopy (SSX-550, SHIMADZU, Japan). 2.3 Preparation of Pre-polymerisation
0.017 mmol·L-1 of dimethomorph and a series of various concentrations of MAA (0.034 mmol·L-1,
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0.068 mmol·L-1, 0.102 mmol·L-1, 0.136 mmol·L-1) were prepared in butanone:N-heptane（7:3, v:v）solution, then dimethomorph solution mixed in identical volumes respectively. The pre-polymerisations were kept in water bath at 30℃ for 5 h and then placed in the refrigerator（0℃）overnight. The pre-polymerisations were scanned by UV spectroscopy. Dimethomorph were prepared separately in THF. Then dimethomorph solution mixed with equivalent volumes of various concentration of MAA for preparation of different pre-polymerisations. The pre-polymerisations were treated in the same manner as described above. The IR spectra of pre-polymerisations in THF were detected by a KBr crystal demountable assembly. 2.4 Preparation of Polymers The pre-polymerisations were incubated in a bath at 30℃ for 5 h to prearrange dimethomorph and MAA. EDMA and AIBN were added sequentially to the solutions, the solutions were oscillated to make them uniform. The pre-polymerisations then purged with a gentle flow of nitrogen for 10 min. The 3
Page 3 of 18
polymerization was initiated at 60℃ for 24 h. The generated polymeric particles were gathered by centrifugation at 4000 rpm for 10 min. To remove the template bound within the polymer matrix, the resulting powder was washed for 30 min with a mixture of methanol and acetic acid (9:1, V: V).The washing procedure was repeated until no more dimethomorph could be detected in the washing solution.
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These MIPs were then washed with methanol to eliminate residual acetic followed by acetonitrile three times. And corresponding nonimprinted polymers were synthesized at the same experimental conditions by
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omitting the template and treated in an identical manner as described above. Finally, the MIPs and NIPs
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were dried under vacuum at 40℃ overnight. Before use,each cartridge was activated by treatment with the same solvent used in the loading step.
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2.5 Characterization of Polymers
The morphology of the produced polymer particles were analyzed by scanning electron microscopy. The samples were prepared in methanol and sputtered a thin gold film prior to measurement in SEM.The
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structure of the polymers was characterization by FTIR spectroscopy in the range of 4000-500 cm-1 by KBr pellet method.
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2.6 Equilibrium Adsorption Experiment
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For adsorption isotherm studies, 20 mg of polymer was placed in an Erlenmeyer flask containing 50 mL of dimethomorph (20-200 mg·L-1) prepared in acetonitrile/water (7:3, V: V). The solution was shaken for 24 hours at room temperature at a speed of 150 rpm. Upon equilibration, all samples were filtered
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through a 0.22µm filter to minimize the interference of particles during analysis. The residual concentration of dimethomorph was analyzed by GC. The amount of dimethomorph was determined from the difference in concentrations at the beginning and at the end of each batch test. Before analysis, 10 mL of rinse solution was loaded onto the conditioned MIP cartridge. After a washing step with 1 mL acetonitrile, dimethomorph was recovered with 1 mL methanol. 2.7 Solid Phase Extraction (SPE) Studies In order to establish the optimum conditions under which the template can be recognized by the corresponding MIP, a standard solution of dimethomorph was initially prepared in various proportions (10-100%) of ACN/H2O and DMF/H2O mixture. To a 3 mL empty polypropylene solid phase extraction cartridges, 200 mg corresponding control polymer NIP was packed between two polypropylene frits. Before analyte loading, the polymer was conditioned with 1 mL methanol, 1mL acetonitrile and 1 mL H2O. 4
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In the loading step, 2 mL of dimethomorph solution (20 mg·L-1) prepared in a mixture of ACN/H2O (7: 3, V: V) was passed through the cartridge. Finally, dimethomorph was eluted with 1 mL of methanol. Ginseng powder samples were diluted with acetonitrile and were then filtered through a 0.22 µm syringe filter and were kept in the freezer until their use. Before analysis, 10 mL of ginseng samples were loaded
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onto the conditioned MIP cartridge. After a washing step, dimethomorph was recovered with methanol. 3. Results and discussion
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3.1 Synthesis and characteristics of the MIP
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The application of MIPs as sorbents in SPE is limited because the possibility of leaking of residual template molecules that remains trapped in the polymers after they have been washed extensively. [14].Thus, the best way to overcome this problem is the use of an analogue of the target molecule during
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MIP design and production, as mimic molecularly imprinted polymer [15–17].The literatures show that the ratio of template to functional monomer would affect adsorption capacity of target molecular
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[18].Therefore, four kinds of polymers were produced using different template to monomer molar ratios via precipitation polymerization to investigate the impacts of MAA on the adsorption. Four molar ratios
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between dimethomorph and MAA with1:2, 1:4, 1:6 and 1:8 were arranged. They were devised to survey
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the effects of template to monomer molar ratio on absorbability of polymers. The best molar ratio is 1:4.The objective of this paper is to select appropriate ratio of template-monomer to prepare a high adsorbing MIP.The molar ratio of dimethomorph to EDMA was selected at 1:20 to ensure the formation of
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defined imprinted cavities because with small molecule as template, high quantity of cross-linker was essential to yield proper special cavities into the polymer matrix. Solvent acts as an important role in the synthesis of the self-assembly type molecularly imprinted polymers. An appropriate porogen could not only dissolve template, monomer and cross-linker, but with poorly polar to reduce the interference between template and monomer. The porogen, butanone:N-heptane （7:3, v:v）is a moderately polar solvent that can be micron grade microspheres. 3.2 Morphology and Structure Characterization of the Polymers Traditional interactions between template and monomer include hydrogen bond, ion pair, hydrophobic interactions, π-π interactions. To illustrate the interaction sites of template and monomer, IR spectra of pre-polymerisations were recorded in the range of 4000-500 cm-1 by a KBr crystal demountable assembly. In this study, using infrared spectroscopy technique for the preparation of imprinted polymer MIP and NIP 5
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blank polymer micro structure analysis and whether the formation of hydrogen bonds that can be seen from Figure 2 NIP (a) and MIP (b) infrared spectra, infrared spectra NIP (a) in the absorption 3541cm-1 redshifted 18cm-1 with respect to MIP (b) of 3523cm-1.It demonstrated that the dimethomorph interacted with MAA might via hydrogen bonds.
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Scanning electron microscopy and FTIR were utilized to reflect the shape and structure of the synthesized polymers. As shown in Figure 3, spherical MIPS were prepared with precipitation
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polymerization and a serious agglomeration was appeared in NIPs. The images of MIP (Figure A) showed
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small microspheres, whereas that of NIPs (Figure B) showed highly agglomerated and irregular particles. We observed that only MIP (Figure A) was isolated in the form of monodispersed microsphere. It was gained that with increasing amounts of MAA, the MIPs were built in large size beads.
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3.3 Binding capacity of the MIP
After synthesis, equilibrium binding experiments of the MIP and NIP were performed at 25℃. Fig. 4
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shows the experimental binding isotherms of MIP (and NIP) amount as a function of dimethomorph concentration. The binding assay was repeated three times and the average values were obtained. The
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results showed that the binding capacity of MIP or NIP increased with the increasing of dimethomorph
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concentrations. Although NIP showed a similar trend in terms of target adsorption, its adsorption capacities were significantly changed. MIP exhibited higher adsorption capacity for the target molecule than NIP. Numerous and precise imprinting sites were found in MIP, resulting in specific adsorption and
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higher adsorption capacity. By contrast, the weak adsorption of the template on NIP was likely due to non-specific interactions with the polymer matrix, the absence of imprinting molecules, and the lack of suitable recognition sites and imprinting cavities in NIP. As it was shown in Fig.4, the adsorptive maximum capacities of MIP and NIP were 3.48 mg·g−1 and 2.41 mg·g−1, respectively. It was concluded that the prepared MIP could selectively recognize of the template molecule. Fig. 4 also showed that MIP has high adsorption capacity toward the dimethomorph, this might because that dimethomorph have similar chemical structures and functional groups. The presence of imprinting sites in MIP that complement the template molecules was vital for specific adsorption [19].The consequences from Scatchard analysis were attributed to the fact that the binding affinity of the imprinted polymer was originated from the specific sites created by the imprinting process and mainly driven by hydrogen binding[20]. 6
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3.4 Optimization of the MISPE In order to obtain the optimum selectivity and recovery, the selection and volume of washing and elution solvents were optimized [21-23]. To interrupt the non-selective interactions with the interferences present in the sample matrix, different washing solvents including methanol, acetonitrile, water,
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dichloromethane, acetone and acetonitrile-water (9:1, 7:3 and 1:1 v:v) were evaluated. The results in Fig.5 showed that acetonitrile and dichloromethane had higher elution strength, which resulted in a lower
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recovery. Although methanol as the washing solvent provided the best recoveries nearly 100%, the
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impurities elimination efficiency was not sufficient, and the main cause probably lied in the interference of polar solvent to the hydrogen-bonding based recognition of MIP coating. Moreover, the recoveries of dimethomorph were not obviously decreased with the increasing volume of acetonitrile–water (7:3, v: v)
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fom 0.1 mL to 5.0 mL.Considering the recoveries, impurities elimination efficiency and economic factors, 5.0 mL of acetonitrile–water (7:3, v: v) was used as the washing solution for further work.
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The elution solvent was also a key factor that affected the recovery. Different elution solvents were investigated to identify its influence on desorption of dimethomorph from the MIM cartridges and the
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results of Fig.6 indicated that acetonitrile–dichloromethane–acetic acid (65:30:5, v: v: v) as elution solvent
dichloromethane-acetic,
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had the best elution ability. Different elution solvents including methanol-acetic, methanol-acetic, acetone-acetic,
chloroform-acetic
and
acetonitrile-dichloromethane-acetic
(85:10:5, 65:30:5 and 45:50:5, v: v: v) were evaluated. The results in Fig.6 showed that chloroform-acetic,
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acetonitrile-acetic, dichloromethane-acetic, acetone-acetic, acetonitrile-dichloromethane-acetic (85:10:5, v: v: v) had higher elution strength, which provided the satisfied recoveries.Howerver, it was found that these elution solvents were not only able to wash out the interfering compounds presented in samples, but also able to wash out most of dimethomorph bound to both MIP and NIP. So it was suggested that acetonitrile-dichloromethane-acetic (65:30:5, 45:50:5, v: v: v) and chloroform-acetic were employed. Furthermore, the recoveries of chloroform-acetic and acetonitrile-dichloromethane-acetic (45:50:5, v: v: v) obtained from MIP were more than 60%, while those obtained from NIP were less than 30%.Moreover, different volumes of acetonitrile–dichloromethane–acetic acid (65:30:5, v: v: v) (1.0–10.0mL) were tested. The recoveries of dimethomorph were not obviously decreased with the increasing volume of acetonitrile-dichloromethane-acetic (65:30:5, v: v: v) from 0.1 mL to 5.0 mL. Therefore, considering the recoveries,
impurities
elimination
efficiency
and
economic
factors,
5.0
mL 7
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acetonitrile–dichloromethane–acetic acid (65:30:5, v: v: v) was chosen as the elution solvent for further work. 3.5 Validation of the MISPE-GC method The validation of specificity, linearity, limit of detection, recovery and precision for the proposed
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method were determined.Specificity was checked by analyzing twenty blank ginseng samples. No interfering peaks could be detected at the retention time of dimethomorph. The linearity and regression
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study were performed for dimethomorph standard graph to generate calibration curve. The high correlation
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coefficient (r=0.9997)indicated good linearity over the concentrations ranged from 0.01 to 5.0 mg·kg-1 for dimethomorph in ginseng samples.For the recovery study,each ginseng sample spiked at 0.1,1.0 and 5.0 mg· kg-1 dimethomorph and they were used for validation of the extraction procedure and MISPE clean up.
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The peak areas for spiked samples were compared with those of standards to determine the recovery. The recovery and repeatability of the method at spiked levels.Recoveries of dimethomorph on MIP cartridge
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from ginseng samples were between 89.2% and 91.6% with relative standard deviations of 3.20%. The limit of detection (LOD) was calculated in blank extracts as the lowest analyte concentration that yielded a
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signal-to-noise (S/N) ratio of 3. The LOD (limit of detection) of this method was 0.002mg·kg-1, and the
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LOQ (limit of quantification) was 0.005mg·kg-1. The data reported indicate that the above method for the analysis of dimethomorph in ginseng samples can achieve good recovery and repeatability. The developed MISPE-GC method was evaluated by the linearity, precision, repeatability, recovery,
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detection limits, Accuracy and precision of the MISPE-GC method were assessed by performing replicate analyses of the spiked samples in five replicates in the same day and consecutive three days.The intra-day precision and accuracy of the method evaluated as RSD was 2.12% and the inter-day reproducibility was 2.31%. Additionally, chromatograms (Fig.7) of spiked sample indicated that MISPE exhibited cleaner eluents (A: before MISPE; B: after MISPE). The retention time of dimethomorph ((Z)-isomer) was about 22.05 min and dimethomorph ((E)-isomer) was about 23.50 min. 4. Conclusions In this paper, the
developed
chromatographic
solid-phase extraction and GC was
method
involving
molecularly imprinted
appropriate for the analysis of dimethomorph in ginseng
samples. A MIP of dimethomorph was synthesized developed via precipitation polymerization for the separation and preconcentration of dimethomorph. Through evaluated in a series of adsorption experiments, 8
Page 8 of 18
the polymer exhibited good recognition and selective ability, suggesting that it could be a useful tool for analytical purposes. Furthermore, a method was successfully developed to detect dimethomorph at low concentration levels in ginseng samples using this MIP as enrichment sorbent of SPE coupled with GC. Compared to the
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methods used for determining dimethomorph in ginseng samples, the method developed in this article exhibited better characteristics such as sensitivity and facility. The presented MISPE-GC method combined
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the advantages of MIP and SPE, which could be potentially applied for the determination of dimethomorph
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in ginseng samples. Good figures of merit were attained, such as low LOQ, wide linear range，good precision and accuracy and its high selectivity. This paper also offered a new method to determine other
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analyte in different samples.
REFERENCES
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[1] J. H. Matt, S. Takayukim, J.Agric Food Chem. 48 (2000) 5825.
[2] H.B. Christensen, K.Granby, Food Addit Contam A. 18 (2001) 867-870.
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[3] B.Sellergren, J. Chromatogr.A 906 (2001) 227. [4] L. I.Andersson, J. Chromatogr. B 739 (2000) 163.
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[5] G. Wulff, Chem. Rev. 102 (2002) 1.
[6] L.I. Andersson, J. Chromatogr. B 745 (2000) 3.
[7] G. Vlatakis, L.I. Andersson, R. Miller, K. Mosbach, Nature 361 (1993) 645.
Ac ce p
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
[8] C. Zhao, T. Zhao, X. Liu, H. Zhang, J. Chromatogr. A 1217 (2010) 6995. [9] F. Puoci, C. Garreffa, F. Iemma, R. Muzzalupo, U.G. Spizzirri, N. Picci, Food Chem.93 (2005) 349. [10] C. Baggiani, L. Anfossi, P. Baravalle, C. Giovannoli, G. Giraudi, C. Barolo, G. Viscardi, J. Sep. Sci. 32 (2009) 3292. [11] Z. Zhang, H. Zhang, Y. Hu, S. Yao, Anal. Chim. Acta 661 (2010) 173. [12] S. Pardeshi, R. Dhodapkar, A. Kumar, Food Chem. 146 (2014) 385-393. [13] T. Yasuyama, H. Matsunaga, S. Ando, T. Ishizuka, Chem. Pharm. Bull. 61 (2013) 546-549. [14] A. Ellwanger, C. Berggren, S. Bayoudh, C. Crecenzi, L. Karlsson, P.K. Owens, K.Ensing, P. Cormack, D. Sherrington, B. Sellergren, Analyst 126 (2001) 784. [15] T. Dandekar, P. Argos, Biochem. Mol. Biol. 4 (1993) 75. [16] X. Feas, J.A. Seijas, M.P. Vazquez-Tato, P. Regal, A. Cepeda, Anal. Chim. Acta 638(2009) 209. [17] L.M. He, Y.J. Su, Y.Q. Zheng, X.H. Huang, L. Wu, Y.H. Liu, Z.L. Zeng, Z.L. Chen, J.Chromatogr. A 1216 (2009) 6196. [18] Y. Wang, E. L. Wang, Z. M. Wu, H. Li, Z. Zhu, X. S. Zhu, Y. Dong, Carbohydr. Polym. 101 (2014) 517. 9
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[19] O.S. Amuda, A.A. Giwa, I.A. Bello, Biochem. Eng. J. 36 (2007) 174. [20] M. Radhika, K. Palanivelu, J. Hazard. Mater. 138 (2006) 116. [21] F.X. Qiao, Y.R.Geng, C, Q.He, Y.P.Wu, P.Y.Pan, J. Chromatogr. B 745 (2000) 3. [22] Z.R.Lian, X.L.He, J.T.Wang, J. Chromatogr. B 957 (2014) 53-59.
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[23] H.B. Guo, K.Y.Liu, Y.H.Liu, B.H.Fang, M.L, L.M.He, Z.L.Zeng, J. Chromatogr. B 879 (2011) 181-185.
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10
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*Highlights (for review)
1. This method employing butanone and N-heptane as porogen. 2. The porogen is a moderately polar solvent that can be micron grade microspheres. 3. This method combined the advantages of MIP and SPE and applied it to ginseng.
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ed
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cr
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4. This paper offered a new method to determine other analyte in different samples.
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Fig.1. Chemical structure of dimethomorph
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Figure1
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Fig.2. IR spectrum scanning curve
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Figure 2
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Fig.3. SEM micrographs of (A) MIP, (B) NIP
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Figure3
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Figure 4
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Fig.4. The binding isotherm of dimethomorph on MIP and NIP (n = 3)
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Figure 5
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1: Methanol; 2: Acetonitrile; 3: Dichloromethane; 4: Water; 5: Acetone; 6: Acetonitrile/water (1:1, v/v); 7:
Acetonitrile/water (7:3, v/v); 8: Acetonitrile/water (9:1, v/v)
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Fig.5. Effect of washing solvents on the loss of dimethomorph
Page 16 of 18
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Figure 6
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1: Methanol-acetic (95:5, v/v); 2: Acetonitrile-acetic (95:5, v/v); 3: Dichloromethane-acetic (95:5, v/v); 4:
Chloroform-acetic (95:5, v/v); 5: Acetone-acetic (95:5, v/v); 6: Acetonitrile-dichloromethane-acetic (85:10:5,
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v/v/v); 7: Acetonitrile-dichloromethane-acetic (65:30:5, v/v/v); 8: Acetonitrile-dichloromethane-acetic (45:50:5,
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v/v/v)
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Fig.6. Effect of elution solvents on the recovery of dimethomorph
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Figure 7
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Fig.7.Chromatograms of the spiked ginseng samples (A: before MISPE; B: after MISPE)
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