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Molecular imprinting technique, regarded as one of the current state-of-the-art researches, was incorporated with the simple dispersive solid-phase extraction (MI-DSPE) in this work for the extraction of triazine herbicides in grape seeds. The atrazine molecularly imprinted polymers (MIPs) were successfully prepared and characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The imprinting particles were used as the adsorbent in DSPE. Thus, a simple, rapid and selective method based on MIPs coupled with DSPE was established for the simultaneous cleaning-up and quantitative extraction of four triazine herbicides in grape seeds. The experiment parameters, including type of washing solvents, washing time and type of eluting solvents, were investigated and optimized. The performance of the present method was validated by high-performance liquid chromatography. Good linear responses were obtained in concentration range of 0.010–5.0 µg g−1 with correlation coefficients (r2) higher than 0.9993. The recoveries at two spiked levels (1.0 and 2.0 µg g−1) were between 81.2 and 113.0% with relative deviations ranging from 1.2 to 10.7%. The limits of detection were ranged between 0.006 and 0.013 µg g−1, which were lower than the values required by European regulations.
Procyanidins (PAs) are a type of polymeric flavan-3-ol compounds. Studies show that the antioxidant activities of PAs are 20 times and 50 times more powerful than vitamin C and vitamin E, respectively (1). Nowadays, PAs are used in human nutriments and preventive medicines for the purpose of scavenging reactive oxygen and nitrogen species (2–4), modulating immune functions and platelet activation (5–8) and producing vasorelaxation (9). PAs can be found in large quantities in the extract of grape seeds (10). In order to control the growth of grass and broadleaf weeds in farmland, triazine herbicides are widely used in agriculture around the world (11). However, many studies have shown that triazine herbicides represent potentials for toxic effects on human health (12–14). Therefore, many countries have formulated the maximum residue limits (MRLs) of triazine herbicides (15–17).
To detect pesticides residual, effective and sensitive analytical methods play an important part. In recent years, many research groups have made extraordinary efforts on developing various methods of sample pretreatment for the determination of triazine herbicides in foods and agricultural products (18–22). Most of the developed methods, such as liquid–liquid extraction (23), solid-phase extraction (24), solid-phase microextraction (25), pressurized microwave-assisted extraction (26), dynamic microwave-assisted extraction-solidification of ﬂoating organic drop (15), cloud point extraction (27), matrix solid-phase dispersion (28), dispersive solid-phase extraction (DSPE) (29) and pressurized liquid extraction (30), are generally combined with gas chromatography, liquid chromatography (31, 32), gas chromatography–mass spectrometry and high-performance liquid chromatography (HPLC)–mass spectrometry (33, 34) for the separation and quantification of triazine herbicides. DSPE, belonging to the “QuEChERS” (quick, easy, cheap, effective, rugged and safe) method, is a relatively new and effective approach to extract trace triazine herbicides from complex matrix. DSPE procedure can be carried out by dispersing the adsorbents in a sample solution. However, the dispersive adsorbents frequently used, including primary secondary amine, octadecylsilane (C18) and graphitized carbon black, are lack of necessary selectivity for triazine herbicides.
Molecularly imprinted polymers (MIPs), referring to a type of synthesized materials for molecular recognition, have drawn great attention owing to their importance in wide areas including separation, sensors and catalysis (35–37). Comparing with natural recognition materials like antibodies, MIPs are more stable and easy to handle. During sample pretreatment, both matrix compounds and the target analytes are bound onto the atrazine-MIP sorbent. Thus, the ideal washing solvent should disrupt the binding forces between the MIP surface and interfering substances while it should not disrupt the forces binding the analyte to the MIP. That is, for the distribution, coefficient of the interferents toward the MIP should be low, while that of the analyte should be high (38). However, the eluting solvent should disrupt the forces binding the analyte to the MIP. Thus, by using the molecularly imprinting technique, the effect of selection, enrichment and extraction can be obtained.
In this article, atrazine MIPs, synthesized by simple precipitation polymerization, were employed as the dispersive adsorbent of DSPE for the selective extraction of triazine herbicides from grape seeds, followed by the determination of triazine herbicides using HPLC-ultraviolet spectrometry.
HPLC-grade solvents used including methanol and acetonitrile were produced by Fisher Scientific Company (USA). Methacrylic acid (MAA) was purchased from Fuchen Chemical Reagent Company (Tianjin, China). Ethylene glycol dimethacrylate (EGDMA) was supplied by Acros (New Jersey, USA). 2,2′-Azobisisobutyronitrile (AIBN) was produced by Shanghai Haoshen Chemical Reagent Corp. (Shanghai, China). Four triazine herbicides, including atrazine, atraton, simetryn, prometryn and terbutryn, were obtained from Aladdin Industrial Corporation (Shanghai, China). All other reagents used were of analytical grade and were purchased from Beijing Chemical Works (Beijing, China).
The mixed stock solution of the four triazine herbicides was prepared in acetonitrile at the concentration of each analyte of 50 µg mL−1 and stored at 4°C. And the working solutions of the triazine herbicides employed in the following experiments were freshly prepared by diluting the stock solution with acetonitrile.
The HPLC system (LC-20ADXR, Shimadzu, Japan), equipped with a SIL-20A automated sample injector, two LC-20AD pumps, a CTO-20A thermostatted column compartment and a SPD-20A UV–Vis detector, was used for triazine herbicides analysis. An eclipse XDB-C18 column (150 mm × 4.6 mm, 3.5 µm particle size, Agilent, USA) was applied to HPLC. An ultrasonic generator (KQ2200E, Kunshan Ultrasonic Instruments, China) and a high-speed centrifuge (HC-2066, Anhui USTC Zonkia Scientific Instruments, China) were employed for the sample pretreatments. A scanning electron microscope (SEM, SU-8020, Hitachi, Japan) was used to obtain the morphology of the MIPs synthesized and the non-imprinted polymer particles (NIPs). A Fourier transform infrared spectrometer (FT-IR, Nicolet Avatar 360, Thermo Fisher Scientific Inc., USA) was employed to verify the synthesis of MIPs.
Three kinds of powder of grape seeds cultivated in different regions were purchased from local stores and named as Sample 1, Sample 2 and Sample 3, respectively. Except for the experiments mentioned in “Analysis of real samples,” which were performed with all three samples, all other experiments were carried out with Sample 1. Recovery experiments were performed by spiking Sample 1 with a proper volume of triazine herbicides working solution.
The MIPs were synthesized by polymerization reaction according to literature (39). Template atrazine (87.5 mg) and MAA (140 µL) were dissolved in acetonitrile (40 mL) in a round-bottom flask (100 mL). The mixture was shaken with an ultrasonic water bath at room temperature until a clear solution was obtained. The solution was kept for 4 h at room temperature. Then EGDMA (1,780 µL) and AIBN (80 mg) were added into the above solution under sonication. Then the flask was purged with a gentle flow of nitrogen for 10 min and sealed under the nitrogen atmosphere followed by water bath, of which the temperature was gradually increased from room temperature to 60°C in 30 min and kept for 24 h for polymerization reaction. The MIP particles produced were centrifuged at 4,000 r.p.m. and washed by the mixture of methanol and acetic acid (80 : 20, v/v) repeatedly until the template atrazine in the supernatant could not be detected by HPLC-UV. Finally, the obtained MIP particles were dried in the air. The NIPs were synthesized with the same procedure described above, except the addition of template atrazine.
The MIPs and NIPs with the weight ranging from 20 to 130 mg were placed into individual 3 mL polypropylene microcentrifuge tubes followed by the addition of 1 µg mL−1 solutions of atrazine. These mixtures were uniformly dispersed by sonication and then incubated overnight at room temperature. After that, the supernatants were filtered by 0.22 µm filter membranes and analyzed by HPLC-UV.
The morphology of the MIPs was observed using a SEM after coating the samples with a thin layer of gold. The IR spectra of MIPs before and after washing off the template atrazine were recorded on a FT-IR spectrometer using KBr pellet in the wavenumber range of 400–4,000 cm−1.
About 0.5 g of grape seed powder sample and 3 mL of acetonitrile were added into a 10 mL centrifuge tube followed by shaking for 30 min (40). The mixture was centrifuged at 4,000 r.p.m. for 4 min and the supernatant was transferred to a 4 mL centrifuge tube containing 80 mg of MIPs. The MIPs were dispersed by sonication for 1 min and the mixture was incubated at room temperature for 10 min. Then, the mixture was centrifuged at 8,000 r.p.m. for 6 min and the supernatant was discarded. The MIPs obtained was washed with 2 mL acetonitrile for 10 min under sonication. The analytes were eluted from the MIPs with 2.5 mL of the mixture of methanol and acetic acid (80 : 20, v/v) by sonication for 10 min twice. The eluents obtained were combined and transferred to a 10 mL glass tube, followed by evaporating to dryness under mild nitrogen stream at 50°C. The residue was redissolved in 150 µL methanol. Finally, the solution was filtered by 0.22 µm filter membrane and analyzed by HPLC-UV. All experiments were performed in triplicate. After the MI-DSPE procedure, the MIPs were dried and recycled.
HPLC-grade acetonitrile (A) and deionized water (B) were employed as the mobile phase constituents for gradient elution. A gradient program was carried out as follows: 0–8 min, 80–70% B; 8–18 min, 70–60% B; 18–25 min, 60–55% B; 25–30 min, 55–45% B; 30–40 min, 45–10% B. The temperature of column oven, the flow rate of mobile phase and the volume of sample injection were set at room temperature, 0.8 mL min−1 and 20 µL, respectively.
The molecularly imprinting polymers prepared were successfully incorporated with the simple dispersive solid-phase extraction (MI-DSPE) in this work for the simultaneous cleaning-up and quantitative extraction of triazine herbicides in grape seeds. Major extraction conditions were optimized. Satisfactory extraction recoveries were obtained.
The atrazine MIPs, synthesized by using precipitation polymerization, were in the form of spherical particles. Compared with bulk polymerization, precipitation polymerization avoided grinding and sieving the synthetic polymers. The characteristics and morphologies of the MIPs synthesized were studied by FT-IR and SEM. Figure 1 shows the SEM images of the MIPs synthesized in this work and the reported literature (41). These particles are in the form of near-perfect spheres with the average diameter of 300 nm. Amplification image of MIP synthesized in this work (Figure 1a) exhibits the specific open-framework structure, which was favor of adsorption. The IR spectra of MIPs, NIPs and atrazine are presented in Figure 2. The bands at 3,562 and 3,554 cm−1 of NIPs and MIPs after washing off the template atrazine, respectively, which are assigned to –OH stretching vibration of MAA, are observed red shift (3,430 cm−1) in the spectrum of MIPs before washing off the template atrazine due to the hydrogen-bond interaction between atrazine and MAA. The characteristic band of atrazine at 1,550 cm−1, existed in the spectrum of MIPs before washing off the template atrazine, disappears in the spectrum of MIPs after washing off the template atrazine, indicating that the template atrazine is washed off completely.
Figure 3 shows the results obtained by direct binding experiments of atrazine to the MIPs and NIPs synthesized. As expected, the MIPs bound more atrazine than the NIPs. The binding efficiency increases obviously with the amount of MIPs increasing in the range of 20–100 mg.
In this study, the spiked grape seed samples at a concentration level of 3 µg g−1 were employed for the optimization of the MI-DSPE conditions. The HPLC peak areas of analytes were used to evaluate the extraction efficiency.
Various washing solvents including acetonitrile, dichloromethane, acetone and ethanol were tested. The volume of each washing solvent is set to 4 mL. The experimental results (Figure 4a) indicate that the recoveries of analyte obtained by acetonitrile and dichloromethane are better than that obtained by acetone and ethanol. Considering the environmental fate of dichloromethane (42), acetonitrile was selected as the washing solvent. The extraction efficiencies of analytes for various washing time ranged from 5 to 60 min were examined. The results show that the HPLC peak areas reach the maximum value at the washing time of 10 min (Figure 4b). Thus, 10 min was chosen as the optimum washing time.
Four milliliters of the mixture of methanol and acetic acid were employed for the elution of analytes. The influences of the proportion of methanol and acetic acid were investigated. As shown in Figure 5, the elution efficiency increases with the increasing of acetic acid proportion of eluents. However, the time of evaporation of eluting solvents increases obviously if the ratio of methanol and acetic acid is <4 : 1. Therefore, the mixture of methanol and acetic acid with the ratio of 4 : 1 was used in following experiments.
Linearity, precision, recovery, limits of detection (LODs) and limits of quantification (LOQs) of the present method were investigated by performing a series of experiments under the optimal experimental conditions.
Working curves were constructed by plotting the areas of the HPLC peaks versus the concentrations of spiked triazine herbicides in the range of 0.010–5.0 µg g−1. The correlation coefficients (r2) of the working curves are in the range of 0.9993–0.9999, and the LODs and LOQs of the present method, which were considered as the lowest concentration of a certain analyte detected and quantified at the signal-to-noise ratio of 3 and 10, are in the range of 0.006–0.013 and 0.02–0.04 µg g−1, respectively (Table I).
Recovery and precision of the present method were assessed at two spiking levels (1 and 2 µg g−1) in terms of intraday repeatability (n = 3) and interday reproducibility (three consecutive days). The detailed results are listed in Table II. The recoveries of analytes were in the range of 81.2–113.0%. The relative standard deviations (RSDs) of the intra- and interday analyses were ranged from 1.2–7.8 to 4.9–10.7%, respectively.
Three grape seed samples cultivated in different regions were analyzed by the present method under the optimized MI-DSPE conditions and the results are listed in Table III. The HPLC chromatogram of Sample 1 is shown in Figure 6. In comparison with the sample without pretreatment by MI-DSPE, a considerable decrease in the amount of the matrix interference peaks is observed. The recoveries of four triazine herbicides are in the range of 81.2–113.0% with RSDs between 0.7 and 11.4% for two spiking concentrations (1 and 2 µg g−1), indicating that the present method was suitable for the quantification of triazine herbicides in the complex samples. In order to examine the significance levels of the analytical results, the Student's t-test was performed (43). The results, which are listed in Table III, indicate that the differences between most of the analytical values and the spiked concentrations are not significant.
In this work, MIPs were prepared by simple precipitation polymerization and performed as the dispersive sorbents for MI-DSPE procedure. According to the obtained results, the facility for selective extraction and sample clean-up of the proposed MI-DSPE method were demonstrated. This LODs and LOQs obtained by this work can meet the European Union standards for MRLs of triazine herbicides in grape seed and have a promising application future.
This work was supported by National Natural Science Foundation of China (Nos 21405057, 21207047, 21075049 and 21105037) and Scientific Forefront and Interdisciplinary Innovation Project of Jilin University (No. 450060481018).