Chemicals

Acrylamide (AA), methacrylate (MAA), 4-vinylpyridine (4-VP), trimethylolpropane trimethacrylate (TRIM), ethylene glycol dimethacrylate (EGDMA), and azodiisobutyronitrile (AIBN) were purchased from the Aladdin Reagent Corporation (Shanghai). Ethyl chrysanthemate and phenyl ether-biphenyl eutectic mixture were acquired from Beijing Honghu United Chemical Products Co., Ltd. (Beijing, China). The above reagents were all of analytical grade. HPLC-grade acetic acid, methanol, and acetonitrile were obtained from Thermo Fisher Technologies (Shanghai). Pyrethroid pesticide standard solutions (≥ 98% purity), including cypermethrin (CYP), cyfluthrin (CYF), deltamethrin (DEL), cyhalothrin (CYH), and fenvalerate (FEN), were obtained from Anpu Experimental Technology Co., Ltd. The cucumber, pear, and apple samples were purchased from local supermarkets.

Instruments and Conditions

Scanning electron microscope (SEM) images were captured using a SU8010 microscope (Hitachi, Japan). The experiments were carried out on a GC-2010 Plus GC (Shimadzu, Japan) furnished with an electron-capture detector (ECD). A DB-5MS capillary column (30 m×0.32 mm×0.25 μm) was utilized for the separation. High-purity nitrogen was used as a carrier gas, and the flow rate was 1.30 mL/min. The column temperature program was from 60ºC to 180ºC at 20ºC·min⁻¹, maintained for 1 min, rose 10ºC·min⁻¹ to 220ºC, and then increased 2ºC·min⁻¹ to 250ºC, held for 3 min, and finally rose 30ºC min⁻¹ to a final temperature of 280ºC, which was maintained for 30 min. The temperatures of the injector and detector were established at 280ºC and 300ºC, respectively. The carrier gas injection mode was a splitless inlet with an injection volume of 1 μL.

Preparation of the MIPs

The pyrethroid pesticide molecularly imprinted polymer was prepared using precipitation polymerization. A total of 0.5 mmol of ethyl chrysanthemate (Eth) and 0.5 mmol of phenyl ether-biphenyl eutectic mixture (Diphyl) were dissolved in 40 mL of acetonitrile in a glass flask. Subsequently, different amounts of AA and MAA were added to the solution, and the resultant solution was further shaken for 30 min to enable the template molecule and functional monomer to fully act. After adding different amounts of EGDMA and TRIM and 20 mg of AIBN, the mixture was deoxygenated with nitrogen for 10 min and sealed. The glass flask was then placed in a water bath at 60ºC and shaken at 160 rpm for 24 h. At the end of the reaction, the resulting polymer was eluted repeatedly with an acetic acid-methanol (2:8, v/v) solution until the template molecules were no longer detected on an ultraviolet (UV) spectrophotometer. Finally, the acquired MIP powder was washed three times with methanol to eliminate excess acetic acid and then dried under a vacuum oven at 40ºC. The preparation diagram of the MIP is presented in Fig. 1a).

Fig. 1.

a) The preparation diagram of the MIP and b) the application of the MIP-SPE.

As a comparison, the non-imprinted polymer (NIP) was synthesized following the same procedure without the template.

Adsorption Properties of the MIPs

Five pyrethroid pesticides that are detected more frequently and pose greater safety risks were selected for the kinetic and isothermal adsorption tests. First, 50 mg of the MIP and 50 mg of the NIP were measured and placed in a 2 mL centrifuge tube. Second, 2 mL of pyrethroid methanol solutions with different mass concentrations were added sequentially, and the supernatant was centrifuged by shaking for a certain time at room temperature. Finally, the contents of the target substances in the supernatant were identified through the use of gas chromatography equipped with an electron capture detector (GC-ECD). Equation (1) was adopted to calculate the adsorption capacity of the polymers, and the adsorption kinetics and isothermal adsorption curves of the MIP and NIP were drawn to ascertain the optimal adsorption saturation time and mass concentration:

where Q represents the adsorption of the polymer to the target after adsorption equilibrium (mg/g); C₀ indicates the initial content of the target substance methanol solution (mg/g); C_e stands for the content of the target in the supernatant after adsorption equilibrium; V refers to the volume of the target substance methanol solution; and m is the weighing capillary for the MIP or NIP.

Characterization of the MIPs

The surface morphology and structure of the synthesized polymers were characterized by scanning electron microscopy (SEM).

Preparation of the Molecularly Imprinted Solid-Phase Extraction (MI-SPE) and Optimization of the Solid-Phase Extraction Conditions

The synthesized molecularly imprinted polymer powder was filled into the solid-phase extraction (SPE) columns by using the dry packing method. As illustrated in Fig. 1b), first, a sieve plate with a pore size of 20 μm was fitted into the bottom of a cylindrical tube, and then 50 mg of the MIP was filled in the column. Finally, the packing material was compacted from the top with the sieve plate. A total of 2 mL of a 10 mg/L pyrethroid standard solution was added to the prepared MI-SPE, and the loading solution was then optimized according to the recovery rate of the target compound.

Actual Sample Processing

A total of 2.0 g each of pear, apple, and cucumber samples were added to a mixed standard solution containing five types of pyrethroid pesticides. The samples were weighed and subsequently placed in a 50 mL centrifuge tube. Totals of 10 mL of acetonitrile, 4 g of anhydrous magnesium sulfate, and 1 g of sodium chloride were then added to the samples. This was then homogenized at high speed for 5 min, left to stand for 30 min, centrifuged at 4000 r/min for 5 min, and extracted repeatedly three times. The supernatants were then combined, and 1 mL of the supernatants was used for the purification. The purification process followed the optimized extraction conditions of the molecularly imprinted solid-phase extraction (MI-SPE) column, and the target analyte was detected using GC-ECD.

Selection of Double Template Molecules

In the selection of template molecules, pyrethroid insecticides are composed of chrysanthemum acid esters and halogenated derivatives of their acids and alcohols, while ethyl chrysanthemate is an important intermediate of pyrethroid insecticides, and phenyl ether-biphenyl eutectic mixture is also a benzene ring structure that most pyrethroid insecticides have. These two template molecules are the primary structural frameworks of pyrethroid insecticides. In theory, it is feasible to prepare polymers using ethyl chrysanthemate and phenyl ether-biphenyl eutectic mixture as double template molecules. Fig. 2 indicates that in the actual adsorption experiment, the adsorption capacity of the molecularly imprinted polymers prepared by employing double template molecules (ethyl chrysanthemate: phenyl ether-biphenyl eutectic mixture = 1:1, n/n) was superior to that of molecularly imprinted polymers prepared by using a single template. This may be due to the double template molecules that can make the polymer produce a large number of imprinted cavities that are more matched with the structure of the target analytes and simultaneously increase the adsorption capacity of the target substances [34, 35]. Therefore, ethyl chrysanthemate and phenyl ether-biphenyl eutectic mixture were chosen as double template molecules for the preparation of the pyrethroid pesticide molecularly imprinted polymer.

Fig. 2.

Adsorption capacities of the different template molecules (Eth: ethyl chrysanthemate, Diphyl: phenyl ether-biphenyl eutectic mixture).

The Screening and Optimization of the Preparation System for the Molecularly Imprinted Polymers

A precipitation polymerization approach was employed to produce the pyrethroid pesticide molecularly imprinted polymer [36]. To obtain a pyrethroid pesticide molecularly imprinted polymer with a high adsorption capacity and strong specificity, the polymer preparation system was screened and optimized. MAA, AA, and 4-VP were selected as functional monomers in this experiment. It was found that the prepared polymer was a cloudy, milky white liquid when 4-VP was used as the monomer through the preparation experiment of the polymer. Although the white solid was precipitated, it easily agglomerated after template elution and vacuum drying. Therefore, 4-VP was not suitable as the monomer for preparing the polymer, and MAA and AA were selected as monomers for the subsequent experiment. The binary crosslinking agents, EGDMA and TRIM, were selected as crosslinking agents. Table 1 shows the types of monomers and crosslinking agents, and the ratios of monomers, templates, and crosslinking agents in the polymer preparation process were optimized. The results showed that when ethyl chrysanthemate and phenyl ether-biphenyl eutectic mixture were mixed in a ratio of 1:1 as template molecules with AA as the functional monomer, EGDMA as the crosslinking agent (the molar ratio of the three substances was 1:4:20), and acetonitrile as the pore-forming agent, the prepared polymer had the highest adsorption capacity. At this point, MIPs with good adsorption capacities for pyrethroid pesticides were obtained. Therefore, this system was chosen as the final preparation system for the polymer.

Morphological and Structural Characterization of the MIPs

The imprinted polymer (MIP) and non-imprinted polymer (NIP) prepared in this experiment were characterized using a scanning electron microscope. As shown in Fig. 3, the left image shows the appearance and morphology of the MIP, which shows that there are abundant reticular pore structures similar to honeycomb shapes, and there are many irregularities on the polymer surface. This was because after the template molecules in the MIP were eluted, imprinting cavities that matched the target molecule were generated on the surface, increasing the binding sites of the polymer to the target molecule [37]. The right image shows the NIP without template molecules, and the particles in the NIP aggregates exhibit irregular states with varying particle sizes, relatively flat surfaces, and no imprinted pores. Compared with the NIP, the MIP had a higher adsorption performance for pyrethroid pesticides.

Adsorption Properties of the MIPs

Five typical pyrethroid pesticides with high detection frequency and great potential safety hazards were selected, and their dynamic and static adsorption experiments were conducted. This allowed for the determination of the adsorption capacity of the molecularly imprinted polymer synthesized under the optimal polymerization system. The dynamic and static adsorption curves were drawn, and their performance was evaluated. It is evident from the dynamic adsorption curve in Fig. 4 (a-e) that the dynamic adsorption patterns of the MIP and NIP for the five pyrethroid pesticides were basically the same. With an increase in time, the adsorption curve gradually showed an upward trend until it reached equilibrium after 120 min. Thus, 120 min was regarded as the saturation adsorption time.

Fig. 3.

SEM images of the MIP (left) and the NIP (right).

The static adsorption curve in Fig. 4 (f-j) indicates that the MIP and NIP exhibited good adsorption performances for the five pyrethroid pesticides in target solutions with different concentration gradients. The adsorption capacities of the MIP and NIP for cypermethrin presented unsaturated states; that is, the adsorption capacities displayed upward trends with the growth of the pesticide concentration, while the adsorption capacities for beta-cypermethrin, cypermethrin, fenvalerate, and deltamethrin gradually increased with the increase in the pesticide concentration and then achieved a balance. At a low concentration of pyrethroid pesticides, the adsorption capacity of the MIP was similar to that of the NIP, which may result from the nonspecific property of the polymer. At high concentrations, the adsorption capacity of the MIP for the target substance was greater than that of the NIP. When the concentration exceeded 40 mg/L, the increase in concentration caused the specific binding site of the polymer to play a role. Hence, the MIP has a specific adsorption capacity for the target substance.

Study of the Specific Adsorption of the MIPs

Organophosphorus pesticide (parathion-methyl) was selected to investigate the class-specific adsorption performance of the pyrethroid molecularly imprinted polymer. Fig. 5a) shows that the adsorption results of the MIP and NIP for parathion-methyl, cyhalothrin, cyfluthrin, cypermethrin, deltamethrin, and fenvalerate were 248, 440, 420, 420, 438, 431 μg/g and 159, 205, 194, 183, 187, 158 μg/g, respectively. The adsorption capacity of the MIP for pyrethroid pesticides was significantly greater than that of organophosphorus pesticides, which indicated that the synthesized MIP exhibited class-specific adsorption for pyrethroid pesticides.

Establishment of the Pyrethroid Pesticide Molecularly Imprinted Solid-Phase Extraction Method

In order to obtain the MIP-SPE with a specific adsorption and enrichment effect, the extraction conditions of the MIP-SPE prepared through the method described in the Materials and Methods Section were optimized. We selected different loading solutions (acetonitrile, 80% acetonitrile-water, methanol, 80% methanol-water, and 50% methanol-water) to analyze the retention effect of the target substances in the MIP-SPE (Fig. 5b)). The results showed that when 80% acetonitrile-water was selected as the loading solution, the retention effect of the target analyte in the MIP-SPE was the best.

To reduce the interference of impurities in the sample matrix, methanol, acetonitrile, and water were selected as the leachates. It can be concluded from Fig. 5c) that when water and methanol were selected as the leachates, the target substance was eluted and lost, which reduced the recovery rate. When acetonitrile was used as the eluent, the recovery rate of the target substance was enhanced, which may have been due to an increase in the specific force between the target substance and the binding site.

Fig. 5c) shows that methanol eluted the target substance from the solid-phase extraction column to varying degrees. Therefore, when investigating the influence of the eluent on the target recovery rate, not only were different volumes of methanol selected, but acidified methanol was also chosen as the eluent to compare the target recovery rate. Fig. 5d) shows that the use of 3 mL of 20% acetic acid-methanol as the eluent improved the target recovery rate. Therefore, 3 mL of 20% acetic acid-methanol was selected as the eluent.

Fig. 4.

The dynamic adsorption curves (a-e) and the static adsorption curves (f-j) of the MIP and NIP. (a, f) cyfluthrin; (b, g) cyhalothrin; (c, h) cypermethrin; (d, i) fenvalerate; (e, j) deltamethrin.

Fig. 5.

The adsorption capacity of both the MIP and NIP for the various types of pesticides a) and a comparison of the recovery rates of the five pyrethroid pesticides among the different loading solutions b), Leachate c) and eluent d) (PAM: parathion-methyl; CYH: cyhalothrin; CYF: cyfluthrin; CYP: cypermethrin; DEL: deltamethrin; FEN: fenvalerate).

To sum up, 3 mL of methanol and 3 mL of water were employed as the activation solution, 1 mL of 80% acetonitrile-water target solution was used as the loading solution, 1 mL of acetonitrile was taken as the leachate, and 3 mL of 20% acetic acid-methanol was regarded as the eluent. The flow rate was controlled at 1 mL/min. Under these conditions, the recovery rate of the pyrethroid target substances was the maximum.

Methodology Evaluation

Linear Range and Detection Limit

In this experiment, a pear was chosen as the sample matrix, and the blank sample extracting solution purified using the MIP-SPE was used to prepare the five pyrethroid pesticide mixed standard solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg/L. The standard curve was plotted with the concentrations of the five pyrethroid pesticides as the x-axis and the peak area as the y-axis. The signal-to-noise ratios (S/N) that equaled 3 and 10 were the limits of detection and quantification for each component, respectively. Table 2 and Fig. 6 reveal that the five pyrethroid pesticides had excellent linear relationships within the range of 0.01-0.5 mg/L, the correlation coefficients were all above 0.9930, and there were favorable chromatographic separation effects. When the S/N equaled three, the minimum limit of detection (LOD) of the pyrethroid pesticides in the pear samples ranged between 15.5 and 25.8 ng/L, and when the S/N equaled 10, the minimum limit of quantification (LOQ) ranged from 54.3 to 88.1 ng/L. Therefore, this experiment met the quantitative determination requirements of pyrethroid pesticide residues in agricultural products. The detection limit, linear range, and recovery rate of the method were 0.011-0.018 ng/g, 0.1-200 ng/g, and 94.3-102.7%, respectively.

Table 2.

The linear equation, correlation coefficient (R²), linear range, LOD, and LOQ of the five pyrethroid pesticides.

Pyrethroid pesticides	Linear equation	Correlation coefficient (R2)	Linear range mg/L	LOD ng/L	LOQ ng/L
CYP	Y = 372460X + 7135.9	0.9949	0.01-0.5	25.6	87.8
CYF	Y = 381850X + 1893.6	0.9932	0.01-0.5	23.4	79.8
DEL	Y = 383015X + 4716.7	0.9940	0.01-0.5	25.8	88.1
CYH	Y = 371140X + 8560.1	0.9935	0.01-0.5	15.7	54.3
FEN	Y = 260529X + 3951.4	0.9932	0.01-0.5	18.9	65.1

Fig. 6.

Standard chromatogram for the five pyrethroid pesticides according to the GC-ECD (0.02 mg/L).

Table 3.

Recovery rates and RSDs (n = 3) of the five pyrethroid pesticides in the different agricultural products.

Substrate	Addition levels
	0.01 mg/kg		0.02 mg/kg		0.05 mg/kg
	Average recovery rates (%)	Relative standard deviations (%)	Average recovery rates (%)	Relative standard deviations (%)	Average recovery rates (%)	Relative standard deviations (%)
Pear	81.6-94.8	0.6-4.8	72.8-98.5	1.3-4.2	87.6-10.2.8	1.2-3.6
Cucumber	74.8-94.6	0.3-5.5	81.8-104.6	1.7-3.1	77.8-110.2	1.7-2.3
Apple	77.4-89.4	0.6-2.3	75.4-102.4	2.2-5.1	79.4-106.4	0.6-2.6