An efficient, rapid, and selective method for sample pretreatment, namely, molecularly imprinted matrix solid-phase dispersion (MI-MSPD) coupled with gas chromatography (GC), was developed for the rapid isolation of four phosphorothioate organophosphorus pesticides (tolclofos-methyl, phoxim, chlorpyrifos, and parathion-methyl) from carrot and yacon samples. New molecularly imprinted polymer nanomicrospheres were synthesized by using typical structural analogue tolclofos-methyl as a dummy template via surface grafting polymerization on nanosilica. Then, these four pesticides in carrot and yacon were extracted and adsorbed using the imprinted nanomicrospheres and further determined by gas chromatography. Under the optimized conditions, a good linearity of four pesticides was obtained in a range of 0.05–17.0 ng·g−1 with
Carrot is among the top ten most economically important vegetable crops in the world, in terms of both area of production and market value [
Sample extraction, purification, and enrichment of pretreatment procedure are very crucial steps in an analytical process when the target compounds are detected. Currently, there have been a great number of the traditional pretreatment methods in pesticide residue analysis, such as Soxhlet extraction, liquid-liquid extraction (LLE), solid-phase extraction (SPE), and ultrasonic extraction. These methods not only require lots of organic solvents, but also need plenty of time; moreover, they might cause environmental pollutions. Compared with the traditional pretreatment methods, a promising technique named matrix solid-phase dispersion (MSPD) has been developed and extensively applied in recent years, which enables extraction and cleanup to be performed in one single step [
Molecularly imprinted polymers (MIPs) are artificially synthesized macromolecular materials, which are highly cross-linked polymers and are able to recognize the target molecules by imprinting the molecules during polymer synthesis through covalent or noncovalent interactions [
The aim of this study is to employ tolclofos-methyl molecularly imprinted nanomicrospheres as a dispersant of MSPD based on supporting materials of nanosilica microspheres, which was used for the rapid screening and adsorbing of four phosphorothioate pesticides from the extracted solution of carrot and yacon. The nanomicrospheric imprinted polymers of tolclofos-methyl were synthesized via a method of grafting technique on the surface of nanosilica. The obtained MIPs were used as the adsorbent of matrix solid-phase dispersion to extract and preconcentrate the four phosphorothioate pesticides from the extracted solution of the carrot and yacon samples. It especially was very significant and novel in process of separation of the sample that magnesium chloride was used for improving adsorbance of MIP nanomicrospheres towards pesticides in a process of pretreatment of the carrot and yacon samples. Then the factors affecting the preconcentration and separation of the analytes were discussed in detail and the applicability of this method was evaluated.
The pure product of tolclofos-methyl with purity of
Analysis of four phosphorothioate pesticides was performed using an Agilent 7890 gas chromatograph (Agilent, CA, USA) equipped with a nitrogen phosphorus detector (NPD). The separation was performed on an Agilent DB-35ms column (30 m × 320
The morphological features of the imprinted polymer grain were examined under a JEOL JSM-5610LV scanning electron microscope (JEOL, Tokyo, Japan). Infrared analyses between 400 and 4000 cm−1 of the MIP- and NIP-imprinted polymer grains were performed in a VERTEX 70-IR spectrometer (Bruke, Germany) where KBr was used to prepare the samples.
Nanosilica was pretreated in order to eliminate any surface contaminants and to activate the surface silanol groups for silanization. In a typical experiment, 15 g silica gel was pretreated by soaking in 10% HCl solution for 8 h and was rinsed thrice by deionized water.
Active nanosilica was prepared as follows: 10 g of pretreated nanosilica, 300 mL of acetic acid, and 35 mL of ammonia water were added to 140 mL of pure water; then the mixture was carried out under nitrogen atmosphere at 45°C for 1 h with magnetic stirring. After centrifugation at 8000 rpm for 10 min at 4°C, the sediment of active nanosilica was obtained and washed thrice with absolute ethyl alcohol, rinsed by deionized water, and then dried in vacuum at 60°C before use.
Dried active nanosilica (5 g), gamma-(methacryloyloxy) propyl trimethoxy silane (20 mmol), and 10 mL absolutely dry toluene were introduced into a conical flask under the atmosphere of nitrogen. At a constant temperature of 90°C and with continuous stirring, the reaction was allowed to proceed for 20 h. The particles were then separated from the mixture via centrifugation. The product was washed with toluene for five times and then washed with methanol for five times in order to remove the excessive gamma-(methacryloyloxy) propyl trimethoxy silane. At the end, the obtained gamma-(methacryloyloxy) propyl trimethoxy silane nanosilica was dried under vacuum at room temperature.
1.0 mmol of tolclofos-methyl was dissolved into 40 mL acetonitrile, followed by the addition of 15 mmol of ethylene glycol dimethacrylate (EGDMA) and 3 mmol g of
Molecularly imprinted polymer nanomicrospheres were prepared by synthesis of tolclofos-methyl imprinted polymers on the surface of gamma-(methacryloyloxy) propyl trimethoxy silane nanosilica. 5.0 g of activated gamma-(methacryloyloxy) propyl trimethoxy silane nanosilica was mixed fully with prepolymerization mixture and then 0.5 mL initiator solution was added into this prepolymerization mixture. After the system was degassed for 20 min using nitrogen, the sealed system was heated in a water bath shaker at 60°C for 18 h under nitrogen protection to produce polymerized mixture. Then the resulting nanomicrospheres were cooled to room temperature.
The obtained polymer nanomicrospheres were washed with methanol-acetic acid (v/v, 9 : 1), methanol, and acetonitrile solution till no template could be detected. And then the solid was dried in vacuum at 40°C, resulting in a complex of tolclofos-methyl imprinted polymers with nanosilica. Synthesis of nonimprinted polymer microspheres on the surface of nanosilica was carried out in a similar manner but without the addition of template. Finally, a small part of the particles of sediment was scanned by JEOL JSM-5610LV electron microscope.
This test was performed according to Section
Then clear solution was discarded, and the sediment was taken out. 10 mL acetonitrile- trifluoroacetic acid (99 : 1, v/v) was added to extract sediments by vibrating for 30 min and then the acetonitrile phase was separately filtered and the filtered acetonitrile solution was evaporated to dryness and dissolved in 1.0 mL dichloromethane of chromatographic grade again. The dichloromethane solution was filtered through a 0.22
50.0 mg of the MIPs and NIPs was placed in vial containing 5 mL of 5.0
50 mg microsphere particles of MIP were, respectively, weighed in two-group 5 mL test tube (group A and group B), and 1 mL methanol was added to each tube. Then, 400
Then clear solution was discarded, and the sediment was taken out. 10 mL acetonitrile- trifluoroacetic acid (99 : 1, v/v) was added to extract the three groups of sediments by vibrating for 30 min; then the acetonitrile phase was separately filtered and the filtered acetonitrile solution was evaporated to dryness and dissolved in 1.0 mL dichloromethane of chromatographic grade again. The dichloromethane solution was filtered through a 0.22-
The recognition studies of adsorption capacity were performed with iprobenfos which is the structurally similar compound of the four pesticides.
The mixed standard solution of the four pesticides (phoxim, tolclofos-methyl, chlorpyrifos, and parathion-methyl) and iprobenfos, each of 5 mg·L−1, were prepared in 10 mL of the methanol. And then 20 mg MIPs was added to this liquid phase. The mixture was shaken for 3 hours at room temperature. After the solutions had been centrifuged, the concentrations of the four pesticides and iprobenfos in the supernatants were determined by GC.
The same procedure was performed for the NIPs.
In this experiment, after fresh carrot and yacon samples were homogenated through tissue homogenizer, which were collected from supermarket of Huazhong Agricultural University in Wuhan city. An aliquot of 0.2 g of samples was placed in an agate mortar and grounded firmly with the pestle. Then, 0.3 g of MIPs and 0.05 mL 10% magnesium chloride (MgCl2) prepared with pure water were added to the agate mortar. Intimate contact between the sorbent and the sample was obtained by pounding with the pestle for some minutes to produce a homogenous packing material for MSPD. The homogenized mixture was then added into a 6 mL solid-phase extraction (SPE) column, which contained a polyethylene frit at the bottom and compacted by another frit on the top. After being packed for 5 min, 0.3 mL of methanol-water (1 : 2, v/v) and 0.1 mL of 10% magnesium chloride (MgCl2) were added into column; the column was shelved and incubated for a period of 3 h at room temperature. Then, the column was rinsed with 5.0 mL of methanol-water (1 : 9, v/v) and eluted with 6.0 mL of acetonitrile-trifluoroacetic acid (99 : 1, v/v). The eluent was then evaporated to dryness under nitrogen and dissolved in 1.0 mL dichloromethane of for further GC analysis.
To test the accuracy of the MIP-GC method, the samples of blank carrot and yacon were, respectively, spiked with standard substance of four phosphorothioate pesticides, which all were collected from farmland of Huazhong Agricultural University. Prior to spiking, the blank carrot and yacon were ensured to be free of the four phosphorothioate pesticides. Briefly, 0.01 mL of mixed standard solution (0.01 and 0.05 mg·L−1) containing 0.1 and 0.5 ng of phosphorothioate pesticides in acetonitrile were added into 0.2 g of homogenated blank carrot and yacon sample, respectively. After being incubated for a period of 1.0 h, the spiked samples were extracted and cleaned according to the above sample preparation procedure. The treatment for each sample was repeated for five times. Then the resulting extractions were detected by GC.
In addition, as a comparison with MIPs, 100 mg commercial N-ethylenediamine propyl silane was also used as dispersant of MSPD to clean and enrich the above four pesticides in blank carrot and yacon samples by above same spiked sample procedure. Furthermore, the treatment for each sample was also repeated for five times.
An adequate molar ratio of template to functional monomer is essential to successful imprinting [
The effect of added amount of crosslinking agent EGDMA on adsorption amount of MIPs towards template molecule tolclofos-methyl was investigated with different adding quantity. And the added amounts of tolclofos-methyl, MAA, and activated nanosilica gel were, respectively, 1.0 mmol, 3.0 mmol, and 5 g among them. As is shown in Figure
Effect of the amount of cross-linker on the adsorption capacity of MIPs.
Under the optimized synthesis conditions, MIPs were produced in a mold, such as a glass bottle or tube. The polymers were subsequently filtered and cleared either manually or mechanically. This method is simple and fast, yielding polymers that can be used as extracted sorbents in matrix solid-phase dispersion. Figure
Micrographs of the molecularly nonimprinted microspheres (a) and molecularly imprinted nanomicrospheres (b, c).
Figure
FT-IR spectra of molecularly imprinted nanomicrospheres (a), molecularly nonimprinted microspheres (b), and gamma-(methacryloyloxy) propyl trimethoxy silane-silicon dioxide (SiO2) (c).
To investigate the adsorption kinetics of MIPs and NIPs, equilibrium time was investigated in this work [
Adsorption kinetics of MIPs and NIPs to phosphorothioate pesticides (A, B, C, D, E, F, G, and H—adsorption kinetics of tolclofos-methyl (A), parathion-methyl (B), chlorpyrifos (C), phoxim (D) on MIPs (A, B, C, and D) and tolclofos-methyl (E), parathion-methyl (F), chlorpyrifos (G), and phoxim (H) on NIPs (E, F, G, and H)).
The isothermal adsorptions of the imprinted and no-imprinted polymer nanomicrospheres are plotted in Figure
Adsorbance of imprinted polymer nanomicrospheres of three group (A, C-added magnesium chloride, B-no addition of magnesium chloride).
Adsorbant | Treated group | Concentration (mg·L−1) | 0.04 | 0.2 | 0.4 | 1.0 | 2.0 | 4.0 |
Pesticides | Actual adsorbance ( | |||||||
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Imprinted polymer microspheres | A | Tolclofos-methyl | 7.2 | 36.1 | 77.3 | 140.8 | 162.4 | 173.2 |
Chlorpyrifos | 7.4 | 33.4 | 53.2 | 123.8 | 137.4 | 143.5 | ||
Parathion-methyl | 6.7 | 37.6 | 61.0 | 147.2 | 158.3 | 165.9 | ||
Phoxim | 7.7 | 38.9 | 48.1 | 118.6 | 147.2 | 161.3 | ||
B | Tolclofos-methyl | 4.6 | 28.3 | 43.1 | 107.3 | 122.9 | 129.4 | |
Chlorpyrifos | 4.4 | 21.5 | 47.6 | 76.4 | 87.3 | 116.2 | ||
Parathion-methyl | 5.3 | 22.6 | 40.4 | 90.7 | 127.4 | 122.8 | ||
Phoxim | 4.2 | 25.2 | 44.7 | 82.7 | 96.4 | 111.7 | ||
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Nonimprinted polymers microspheres | C | Tolclofos-methyl | 6.6 | 18.0 | 32.3 | 47.2 | 50.7 | 51.3 |
Chlorpyrifos | 4.4 | 22.0 | 32.3 | 45.3 | 54.0 | 57.7 | ||
Parathion-methyl | 4.2 | 19.7 | 32.7 | 51.3 | 59.0 | 63.1 | ||
Phoxim | 5.1 | 23.3 | 29.0 | 39.2 | 49.3 | 68.2 | ||
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Theoretical adsorbance ( |
8.0 | 40.0 | 80.0 | 200.0 | 400.0 | 800.0 |
MIPs: molecularly imprinted polymers; NIPs: molecularly nonimprinted polymers.
Adsorption isotherms curves of four pesticides on molecularly imprinted nanomicrospheres (MIPs) and molecularly nonimprinted microspheres (NIPs) (A, B, C, D, E, F, G, and H—adsorption curves of tolclofos-methyl (A), phoxim (B), chlorpyrifos (C), parathion-methyl (D) on MIPs (A, B, C, and D) and phoxim (G), parathion-methyl (F), chlorpyrifos (E), and tolclofos-methyl (H) on NIPs (E, F, G, and H)).
In addition, magnesium chloride was used in determination of the adsorbance of MIPs and NIPs for the four pesticides at group A and group C. By contrast, magnesium chloride was not added at group B. Then, the adsorbance of three groups was shown in Table
The structurally similar compound iprobenfos was used as a typical species that compared with representational four pesticides (phoxim, tolclofos-methyl, chlorpyrifos, and parathion-methyl) in a recognition study. The distribution coefficient (
Compared loading of four pesticides and iprobenfos by imprinted and nonimprinted polymers.
Pesticides | Sorbents | Initial solution (mg·L−1) | Final solution (mg·L−1) |
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Tolclofos-methyl | Imprinted | 5 | 3.75 | 166.7 | 2.2 | 2.8 | 385 |
Nonimprinted | 5 | 4.52 | 53.1 | 0.8 | |||
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Parathion-methyl | Imprinted | 5 | 3.82 | 154.5 | 2.0 | 1.9 | 270 |
Nonimprinted | 5 | 4.36 | 73.4 | 1.1 | |||
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Chlorpyrifos | Imprinted | 5 | 4.43 | 64.3 | 0.8 | 1.6 | 120 |
Nonimprinted | 5 | 4.67 | 35.3 | 0.5 | |||
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Phoxim | Imprinted | 5 | 3.94 | 134.5 | 1.7 | 2.0 | 262 |
Nonimprinted | 5 | 4.46 | 60.2 | 0.9 | |||
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Iprobenfos | Imprinted | 5 | 4.33 | 77.4 | 30 | ||
Nonimprinted | 5 | 4.39 | 69.5 |
Four pesticides (phoxim, tolclofos-methyl, chlorpyrifos, and parathion-methyl).
The tolclofos-methyl imprinted polymer adsorbed twice to fourfold as much four pesticides as iprobenfos. The
As expected, iprobenfos was retained to some extent in the active centers of MIPs, but not in a quantitative way and the repeatability was poor. There were specific and nonspecific binding sites in the MIPs, but in the NIPs there were only nonspecific binding sites. The MIPs show a higher affinity for four pesticides owing to the specific sites. It is also obvious that the specific recognition sites are mainly complementary to the template in terms of size and shape. Furthermore, the
The analytical figures presented in this method for the simultaneous determination of four phosphorothioate pesticides were estimated under optimal conditions. This study defined the lowest concentration used in the calibration curve, 10 ng·g−1, to be the limit of detection (LOD) for each pesticide. Detection limits were verified by injection of the samples prepared at 10 ng·g−1 to ensure that discernible peaks had a signal-to-noise ratio ≥3. The results indicated that the limit of detection (LOD) (
Equation, correlation coefficients (
Pesticide | Equation |
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LOD (ng·g−1) | Linear range (ng·g−1) |
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Phoxim |
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0.9971 | 0.026 | 0.09~16.0 |
Tolclofos-methyl |
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0.9993 | 0.017 | 0.07~13.0 |
Chlorpyrifos |
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0.9996 | 0.021 | 0.09~17.0 |
Parathion-methyl |
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0.9983 | 0.012 | 0.05~10.0 |
Chromatograms of the standard mixture solution of four pesticides in 25 ng·mL−1 (a), the spiked sample in 2.5 ng·g−1 of four pesticides after preconcentration by N-ethylenediamine propyl silane sorbent (b), the spiked sample in 2.5 ng·g−1 of four pesticides after preconcentration by MIPs sorbent (c), the actual carrot sample (d), and the actual yacon sample (e). Peak identification: 1, tolclofos-methyl; 2, parathion-methyl; 3, chlorpyrifos; 4, phoxim.
At present, only several relevant literatures have been reported [
For another example, some other scholars such as Wong et al. employed capillary gas chromatography-mass spectrometry and flame photometric detection to build a method for the determination of organophosphorus pesticides in ginseng root in 2007 [
To evaluate the applicability of the this method, the selectivity and enrichment of MIP matrix solid-phase dispersion and N-ethylenediamine propyl silane matrix solid-phase dispersion were compared by the spiking of four phosphorothioate pesticides at the levels of 0.5 and 2.5 ng·g−1 in the food samples; and then the spiked samples were extracted and analyzed (Figures
For each concentration, five replicate measurements were performed, and good recovery rates between 85.4 and 105.6% were obtained by MIPs when MIPs were used for extracting pesticides in carrot and yacon (Tables
Recovery rates of four pesticides in the spiked blank carrot samples when molecularly imprinted nanomicrospheres were used for extracting pesticides and results of actual samples (mean ± SD,
Pesticides | Samples (ng·g−1) | Spiked level = 0.5 ng·g−1 | Spiked level = 2.5 ng·g−1 | ||
Carrot | Detected content | Recovery (%)/RSD (%) | Detected content | Recovery (%)/RSD (%) | |
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Phoxim | 0.83 | 0.431 ± 0.026 | 86.2/(6.0) | 2.29 ± 0.22 | 91.6/(9.6) |
Tolclofos-methyl | 0.69 | 0.445 ± 0.028 | 89.0/(6.3) | 2.64 ± 0.23 | 105.6/(8.7) |
Chlorpyrifos | N.D | 0.452 ± 0.029 | 90.4/(6.4) | 2.25 ± 0.19 | 90.0/(8.4) |
Parathion-methyl | N.D | 0.464 ± 0.034 | 92.8/(7.3) | 2.43 ± 0.18 | 97.2/(7.4) |
N.D: no detection; RSD: relative standard deviation.
Recovery rates of four pesticides in the spiked blank yacon samples when molecularly imprinted nanomicrospheres were used for extracting pesticides and results of actual samples (mean ± SD,
Pesticides | Samples (ng·g−1) | Spiked level = 0.5 ng·g−1 | Spiked level = 2.5 ng·g−1 | ||
Yacon | Detected content | Recovery (%)/RSD (%) | Detected content | Recovery (%)/RSD (%) | |
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Phoxim | N.D | 0.447 ± 0.025 | 89.4/(5.6) | 2.38 ± 0.22 | 95.2/(9.2) |
Tolclofos-methyl | 0.47 | 0.427 ± 0.021 | 85.4/(4.9) | 2.31 ± 0.19 | 92.4/(8.2) |
Chlorpyrifos | 1.26 | 0.486 ± 0.024 | 97.2/(4.9) | 2.28 ± 0.14 | 91.2/(6.1) |
Parathion-methyl | N.D | 0.506 ± 0.031 | 101.2/(6.1) | 2.36 ± 0.16 | 94.4/(6.8) |
N.D: no detection; RSD: relative standard deviation.
The developed method was then applied for the extraction and determination of four pesticide residues in actual carrot and yacon samples (Figures
Furthermore, compared with the published methods for the determination of organophosphorous pesticides in food, this developed method has easier popularization, less time consumption, lower LOD value, better linearity range, and easier peak identification. Thus, this method can facilitate the effective and efficient analysis of the phosphorothioate pesticides in food such as carrot and yacon samples.
An efficient method with high sensitivity, namely, molecularly imprinted matrix solid-phase dispersion coupled with GC, was developed for the rapid extraction and determination of four phosphorothioate organophosphorus pesticides in carrot and yacon. Under the optimized conditions, a high extraction efficiency was obtained for the four pesticides with low LODs (0.012~0.026 ng·g−1). Meanwhile, a good linearity of phosphorothioate pesticides was observed in a range of 0.05~17.0 ng·g−1, and the spiked recovery rates at two spiked levels were in a range of 85.4–105.6%. This method will provide a new tool for the rapid determination of multipesticide residues in the complicated food samples, which will facilitate the studies of food safety concerning carrot and yacon.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors acknowledge the financial support from the Projects in the National Science and Technology Pillar Program during the Twelfth Five-Year Plan Period (No. 2011BAI06B03). The authors also acknowledge Professor Zuoxiong Liu for amending paper.