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Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples
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Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples Minjuan Zhao , Hua Shao , Jun Ma , Hui Li , Yahui He , Miao Wang , Fen Jin , Jing Wang , A. M. Abd El-Aty , ˘ , Feiyan Yan , Yanli Wang , Yongxin She Ahmet Hacımuft ¨ uo ¨ glu PII: DOI: Reference:
S0021-9673(19)31196-3 https://doi.org/10.1016/j.chroma.2019.460751 CHROMA 460751
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Journal of Chromatography A
Received date: Revised date: Accepted date:
24 October 2019 28 November 2019 28 November 2019
Please cite this article as: Minjuan Zhao , Hua Shao , Jun Ma , Hui Li , Yahui He , ˘ , Feiyan Yan , Miao Wang , Fen Jin , Jing Wang , A. M. Abd El-Aty , Ahmet Hacımuft ¨ uo ¨ glu Yanli Wang , Yongxin She , Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460751
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Highlights
a novel MMIP with specific adsorption of patulin was successfully synthesized.
The magnetic dispersion solid-phase extraction (MDSPE) method was established.
This pretreatment method saves time and reduces the use of organic reagents.
This study provides a new pretreatment method for the detection of patulin in juice.
Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples Minjuan Zhaoa, Hua Shaoa*, Jun Maa, Hui Lia, Yahui Hea,b, Miao Wang a, Fen Jina, Jing Wanga, A. M. Abd El-Atyd,e,f, Ahmet Hacımüftüoğluf, Feiyan Yanc, Yanli Wangc and Yongxin She1* a
Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R China Chinese Academy of Agricultural Sciences Institute of Agro-Products Quality Standards and Testing Technology b
Beijing Technology and Business University, 100048, P.R. China
c
Institute of Quality Standards & Testing Technology for Agro-Products, Guangxi Academy of Agricultural Sciences, Nanning 530000, P.R. China d
State Key Laboratory of Biobased Material and Green Papermaking, College of Food Science and Engineering, Qilu University of Technology, Shandong Academy of Science, Jinan 250353, China e
Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211-Giza, Egypt
f
Department of Medical Pharmacology, Medical Faculty, Ataturk University, 25240-Erzurum, Turkey
Abstract: In this study, a novel magnetic molecularly imprinted polymer (MMIP) was prepared by surface imprinting technology using 2-oxin and 6-HNA as dual virtual templates and 4-vinyl pyridine (4-VP) as the functional monomer for extraction of patulin (PAT) from the surface of magnetic nanoparticles. MMIPs were characterized by fourier transformed infrared (FT-IR) spectroscopy, X-ray diffraction, and vibrating sample magnetometry. The results showed that the molecularly imprinted polymer (MIP) was successfully coupled with magnetic nanoparticles and could be used as a magnetic selective recognition material. Moreover, MMIPs have a greater adsorption capacity for PAT than conventional MIPs. The magnetic dispersion solid-phase extraction procedure was optimized and then combined with liquid chromatography-tandem mass spectrometry (MDSPE-LC-MS/MS) for detection of PAT in juice samples. The method showed excellent analytical performance in terms of linearity (ranged between 0.5 μg L −1 and 100 μg L−1with correlation coefficients (r) higher than 0.999) and limit of detection (LOD) (0.1 μg L−1, S/N = 3). At three spiking concentrations (1, 10, and 50 μg L-1), the mean recoveries were ranged between 79.4% and 97.9% with relative standard deviations (RSDs) less than 4.7% (n = 3). Keywords: Patulin; Magnetic molecularly imprinted polymers; Juice; Magnetic dispersion solid-phase extraction; LC-MS/MS; HPLC-DAD 1. Introduction Patulin (PAT) is a toxic mycotoxin produced by species belonging to genera of Penicillium, Byssochlamys, and Aspergillus [1-3]. PAT has been found in various mildewed fruits. It is a water soluble molecule with high thermal stability. Additionally, it is more stable in acidic solution and may be difficult to be removed by general methods, such as heating and drying [4,5]. Some studies indicate that PAT is teratogenic, carcinogenic, and mutagenic in laboratory animals [6,7]. Toxicity assessment (acute, subacute, and chronic) due to consumption of patulin shows restlessness, twitching, shortness of breath, , congestion of the lungs, fester, dropsy, extravasated blood, and gastrointestinal diseases [8-10]. Furthermore, PAT has been shown to exhibit adverse effects on the development of fetus in rodents [11,12]. Due to its potential toxicity and other reported adverse effects, most countries and organizations have established standards for maximum residue limits (MRLs). Specifically, European Commission has set a maximum limit of 50 ppb for PAT in fruit juices and other fermented beverages containing apple or apple juice, 25 ppb in solid apple products, and 10 ppb in apple puree and solid apple products for infants and children [13]. In China, the MRL was stipulated to be 50 ppb in fruit products, fruit and vegetable *Corresponding authors. E-mail addresses: [email protected] (Y. SHE), [email protected](H. SHAO).
juices, and wines (except sweetened rolls) with apple and hawthorn as raw materials [14]. The most effective way to prevent/control mold contamination and ensure food safety is to develop a rapid and effective detection method for patulin quantification in agricultural products. The overall sample preparation process used for extraction and enrichment of PAT, include liquid-liquid extraction (LLE) [15], dispersion solid-phase extraction [16], and solid-phase extraction (SPE) [17]. Among them, the SPE technology is the most widely used pre-processing method, yielding high enrichment and concentration effect [18]. However, the SPE column may suffer from a lack of selectivity, the purification effect is not ideal, and the co-extracted impurities may alter the ionization efficiency of target analytes owing to the matrix effect [19, 20]. This may affects the instrumental analysis which is the last step of analyte identification. Therefore, it is necessary to develop a pretreatment method with good selectivity, purification, and enrichment ability. Recently, the application of molecularly imprinted polymers (MIPs) has attracted much attention, because it could solve the aforementioned pitfalls [21-23]. Molecularly imprinted polymers (MIPs) are novel biomimetic materials (with strong molecular recognition ability), which exhibited excellent reusability and reproducibility, cheap, easy to prepare, and high specificity [24,25]. With the development of molecularly imprinted solid-phase extraction, magnetic molecularly imprinted solid-phase extraction has attracted considerable attention because of its simple operation, good enrichment effect, and fast separation. A magnetic molecularly imprinted polymer (MMIP) prepared by surface molecular imprinting technique and nanotechnology exhibits large specific surface area, distinct superparamagnetism, molecular imprinting specificity, and adsorption performance; the characters which are superior than that of ordinary polymers. As an adsorbent for dispersive solid-phase extraction (d-SPE), MMIPs can be directly dispersed in the sample solution, and the target can be quickly separated from the matrix by applying an external magnetic field, which simplifies the experimental operation, reduces the use of organic reagents, and achieves the purpose of separating and enriching the target [26-30]. In this context, Cheng et al. prepared an MMIP with a core-shell structure for extraction and enrichment of quercetin using surface molecular imprinting technique coupled with nanotechnology [31]. Additionally, Gholami et al. synthesized MMIPs to adsorb and enrich melamine in milk samples [32]. On the other hand, Liu et al. prepared MMIP microspheres by synthesizing MIPs on the surface of Fe3O4@Ag for SERS-based sensing of molecular species and applied the designed MMIPs for simple, rapid, ultrasensitive, and label-free SERS detection of sibutramine [33]. Notably, there have been no (scarce) studies on the preparation of MMIPs based on the MDSPE for detecting patulin and their application in real samples. In this study, core-shell MMIPs were prepared by surface imprinting technology using SiO 2 layered Fe3O4 nanoparticles as carriers to develop a fast and selective adsorbent for patulin during the pre-processing procedure. Application of an external magnetic field enables the facile separation of MMIPS that was used as an adsorbent for MDSPE. The characterization of patulin-MMIPs and the MDSPE procedure was conducted systematically. Consequently, combined with LC-MS/MS, the MDSPE was successfully used to enrich and detect patulin in fruit juice. 2. Experimental 2.1 Materials and Instruments All chemical reagents and instrumental conditions used in the experimental works are listed in the supplemental materials. 2.2 Preparation of core-shell Fe3O4@SiO2@MIPs We prepared magnetic Fe3O4@SiO2@MIP nanoparticles using a laboratory method and the procedure is shown in Figure 1. The specific method was as follows. First, we used the chemical co-precipitation method to prepare Fe3O4 nanoparticles. A total of 10 mL deionized water was used to gradually dissolve FeCl3·6H2O (2.35 g) and FeCl2·4H2O (0.86 g). The two solutions were transferred to a three-necked flask containing 80 mL deionized water, and the mixture was stirred until the temperature reaches to 70 ℃. Thereafter, 10 mL of a 25% ammonium hydroxide solution was added, and the mixture was stirred for 30 min at 80℃. To increase the surface dispersibility of magnetic particles, 100 mg of trisodium citrate dihydrate was added and the mixture was stirred for 30 min. After the reaction was completed, Fe3O4 was separated by external magnetic field and washed with ethanol until neutral. The prepared nanoparticles were stored in 50 mL ethanol pending analysis. Second, Fe3O4@SiO2 was synthesized according to the hydrolysis of a silylation reagent. Specifically, 25 mL of magnetic nanoparticles dissolved in ethanol (approximately 0.5 g of Fe3O4) was transferred to a three-necked flask containing 75 mL ethanol and 20 mL deionized water; the solution was mixed for 30 min under ultrasonication. Next, 1 mL of a 25% ammonium hydroxide solution and 2 mL tetraethoxysilane were added and the mixture was stirred for 24 h at
room temperature. The prepared Fe3O4@SiO2 microspheres were separated by external magnetic field and washed with ethanol until neutral. The third step involved the preparation of Fe3O4@SiO2 functionalized with vinyl groups. Fe3O4@SiO2 was transferred to a 250 mL three-necked flask containing 100 mL of a 10% acetic acid water. Then, 150 μL of MPS was added and the mixture was stirred for 5 h at 60 ℃. Fe3O4@SiO2-CH=CH2 nanoparticles were magnetically separated and washed multiple times with water and methanol and stored in 50 mL methanol. Finally, the molecularly imprinted polymer (MIP) was coupled to magnetic nanoparticles by precipitation polymerization. Specifically, 0.25 mmol of 2-oxin and 6-HNA as virtual templates and 1 mmol of 4-VP were dissolved in 30 mL methanol and pre-polymerized for 30 min to allow the templates to fully interact with a functional monomer. Then, 25 mg of Fe3O4@SiO2-CH=CH2 particles were added. Thereafter, 2 mmol of TRIM and 20 mg of AIBN were added. The mixture was ultrasonically dispersed for 20 min and nitrogen was blown for 5 min. Then, the mixture was reacted by heating in a water bath for 24 h at 60°C. After polymerization, MMIPs were collected by external magnetic field. The template molecules were extracted by Soxhlet extraction with methanol-acetic acid (90:10, v/v) until 2-oxin and 6-HNA could not be detected at 248 nm and 258 nm by UV–vis absorption spectroscopy. Subsequently, magnetic nanoparticles were washed with methanol and dried under vacuum at 55 ℃. The magnetic non-imprinted polymer (MNIP) was synthesized using the same method without adding the templates. 2.3 Adsorption capacity of MMIPs MMIPs or MNIPs (10 mg) were weighed and put into a 1.5 mL vial and mixed with 1 mL of different concentrations of PAT aqueous solution. The mixture was shaken at room temperature for 2 h at room temperature under a horizontal shaking and then separated by external magnetic fields. The free concentrations of PAT were measured by HPLC equipped with a diode array detector (DAD) at 276 nm. The amount of PAT that binds to the polymer was calculated by subtracting the free amount of PAT from the initially added amount. 2.4 MDSPE procedure First, 20 mg MMIPs was added into 1 mL juice samples and mixed for 8 min. MMIPs were separated by a magnetic field and water was completely removed. Then, the analyte was eluted from MMIPs with 1 mL acetonitrile by shaking for 15 min. Finally, the eluate was filtered and 5 μL was taken for LC-MS/MS analysis. 2.5 Sample preparation Apple, pear, and grape juice samples were purchases from local markets in Beijing (China). All juice samples were filtered (to remove particulate impurities) and stored (in the dark) in a refrigerator at 4 ℃ until use. 3. Results and discussion 3.1 Characterization of PAT-MMIPs To demonstrate the successful coupling of magnetic nanoparticles with molecularly imprinted polymers, the FT-IR spectra of Fe3O4, Fe3O4@SiO2, vinyl-functionalized Fe3O4@SiO2, and MMIPs were characterized. As shown in Figure 2 (a), the peak at 573 cm-1 was attributed to the characteristic absorption vibration of Fe-O. In Figure 2 (b), the peak at 1088 cm−1 originates from the characteristic absorption vibration of Si-O-Si, which indicates that SiO2 was efficiently coupled onto the surface of Fe3O4 nanoparticles. Peak at 1632 cm-1 in Figure 2 (c) was related to the C=C stretching absorption vibration, which indicates that the vinyl groups were modified onto the surface of Fe3O4@SiO2 nanoparticles. In Figure 2 (d), peaks at 1734 cm-1 and 2976 cm-1 were originated from the characteristic vibrations of C=O and C-H in TRIM, respectively. This observation indicates that MIPs were successfully coupled onto the surfaces of magnetic spheres. When X-rays are irradiated onto the surface of crystals, characteristic diffraction peaks that correspond to the crustal structure (analyzed by X-ray diffraction) are observed. Figure 3 shows the XRD patterns of prepared Fe3O4, Fe3O4@SiO2, vinyl-functionalized Fe3O4@SiO2, and MMIPs. It can be readily observed that Fe3O4 particles exhibit typical diffraction peaks, which means that the produced material was highly crystalline and had an anti-spinel structure. The main diffraction peaks were also found in Fe3O4@SiO2, Fe3O4@SiO2-CH=CH2, and MMIPs, which indicates that the crystal form of Fe3O4 particles did not change during the modification and surface synthesis of nanoparticles. However, the difference in peak width of the particles reflects the change in the average particle size. Figure 3 (d) shows that the intensity of the diffraction peak of MMIP particles was considerably reduced, the finding which means that the size of the particles changed.
As the surface of Fe3O4 nanoparticles was coated with SiO 2, modified with CH=CH2, and a polymer was synthesized on the surface, the measured and calculated saturation magnetization was gradually decreased ( Figure 4). The saturation magnetization saturation values were 69.85, 33.55, 29.90, and 4.28 emu g -1, respectively. Additionally, MMIPs were separated rapidly from the solution using an external magnetic field, and the magnetic molecularly imprinted polymer was uniformly re-distributed in the solution when the magnetic field is removed. Therefore, the prepared surface molecularly imprinted polymer had a good paramagnetism and could be used for d-SPE. This means it could simplify the sample pretreatment steps (by magnetic separation), thus saving the pretreatment time, decreases the use of organic reagents, and finally achieving the purpose of environmental protection and rapid detection. 3.2 Evaluation of adsorption capacity of polymers The binding amount of PAT to imprinted and non-imprinted polymers was determined by static adsorption experiments. The static adsorption experiments data are shown in Figure 5. The synthesized polymer had shown good adsorption performance to PAT in aqueous solution; and the adsorption capacity increases with increasing the initial concentration of PAT. This finding indicates that a binding site with specific adsorption was formed in the MIP, and specificity was shown at high concentrations. From the Scatchard equation (Figure 6), different slopes of MIP indicate the presence of different binding site. The linear regression equations in the linear region were q/c = -0.01802X + 168.7 (r=0.9998) and q/c = -0.0009017X + 114.7 (r=0.9556). From the slope and intercept of the fitting line, the Kd values were determined to be 55.49 μg.mL−1 and 119.02 μg.mL−1, and the Qmax values were 9.32 mg.g-1 and 127.20 mg.g-1, respectively, the values which are higher than that reported elsewhere [34]. Hence, the prepared MMIPs had good specific adsorption and hydrophilicity to PAT. 3.3 Optimization of MDSPE To determine the best conditions during the experimental work, 20 mg of MMIPs was used, and the following parameters were optimized: loading solution, sample pH, adsorption time, elution solution, and elution time. 3.3.1 Loading solution Polymer has the maximum adsorption capacity to target analytes in aqueous solution, and herein juice was the tested sample. Therefore, 1 mL was selected as a sample solution to ensure the adsorption of the target. 3.3.2 Sample pH The activity of PAT in an alkaline media was reduced. Thus, a pH ranged from 1 to 7 was assessed. As shown in Figure 7, the recovery of the target analyte was high and remained unchanged within the pH range. This finding might be attributed to hydrogen bonding interaction between the target and MMIPs in an acidic environment. As the pH of the juice sample was below 7, there was no need to adjust the pH throughout the experimental work. 3.3.3 Extraction time Extraction time is an important factor that could affect the extraction efficiency of the tested analyte during the MDSPE procedure. In this experiment, extraction time was evaluated in the range of 1-30 min while maintaining the other conditions. It has been found that the recovery gradually increased within 1-10 min and remained unchanged thereafter (Figure 8a). Therefore, 15 min was selected as the best extraction time. 3.3.4 Elution solvent and time In this study, we evaluated the elution effect of different volumes of methanol and acetonitrile on PAT. The results showed that the elution strength of acetonitrile was greater than that of methanol, and when the volume was 1 mL, a higher recovery rate was obtained. Thus, we have chosen 1 mL acetonitrile as an eluent. Afterward, the elution time was optimized in the range of 3-25 min. Figure 8b illustrates that 20-min elution enables the maximum elution of analyte in MMIPs. Therefore, 20 min was selected as an optimal elution time. 3.4 Method validation To evaluate the applicability of the developed MDSPE sample pretreatment procedure, the MDSPE-LC-MS/MS method for detecting PAT in juice samples was established. The linearity range, sensitivity, and limit of detection (LOD) were evaluated under the optimized conditions (Table 1).
To eliminate the matrix effect and obtain accurate results, the quantification was performed using matrix-matched standard curves [35]. The results are shown in Table 1. The method showed a good linear relationship between 0.5 μg.L-1 and 100 μg.L-1 with a satisfactory correlation coefficient (r ≥ 0.999). The LOD (S/N = 3) and LOQ (S/N = 10) were 0.1 μg.L−1 and 0.3 μg.L−1, respectively. Blank samples (n=3) were spiked with PAT at three concentration levels (i.e., 1, 10, and 50 μg.L−1) to determine the accuracy of the developed method. As shown in Table 1, the average recovery of PAT was ranged between 79.4% and 97.9%, and the RSD was between 0.8% and 4.7%. The extracted ion chromatograms for PAT are shown in Figure 9. The results showed that MMIPs can be used as highly efficient and specific adsorbents for selective extraction and enrichment of PAT in juice matrix during the MDSPE pretreatment process. The developed MDSPE-LC-MS/MS was accurate and can be used to detect PAT residues in real samples. 3.5 Detection of real samples The MDSPE-LC-MS/MS methodology was used for enrichment and detection of PAT in three juice samples purchased from local markets. The results showed that PAT residues were not detected in any of the samples. Conclusions In this research, a new type of pretreatment material, MMIPs was prepared by surface imprinting and nanotechnology using 2-oxin and 6-HNA as dual virtual templates. The polymer has good selectivity and adsorption for PAT and can be used as an adsorbent during the MDSPE pretreatment process. The pretreatment technique was evaluated by LC-MS/MS to detect PAT in juice samples.
Author Contribution Dear editors, The following are the specification of contribution of each author. (Title: Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples) Minjuan Zhao carried out experiments and wrote the paper; Associate Professor Hua Shao, Professor Jing Wang, Professor Yongxin She, Professor Fen Jin and Professor Feiyan Yan designed the experiments, provided useful suggestions and solved the problems in the experiments; Jun Ma, Yahui He, Miao Wang and Yanli Wang helped to synthesis magnetic molecularly imprinted polymers; Hui Li provided the experiment tools and helped to used the instrument; Professor A. M. Abd El-Aty and Ahmet Hacımüftüoğlu revised the paper. Thank you and best regards. Yours sincerely, Corresponding author: Professor Yongxin She, E-mail: [email protected] Associate Professor Hua Shao, E-mail: [email protected]
Conflict of interest Authors have declared no conflicts of interest. Acknowledgements This work was supported by the National Key Research and Development Special Plan (2017YFF0210201). The authors are grateful to the National Natural Science Foundation of China (contract No. 31772071), the Guangxi Innovation-driven Development Project (AA17204043-2), and Agriculture Research System of China
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Figure captions
Figure 1. Schematic diagram for synthesis of magnetic molecular imprinted polymers (MMIPs) and its application toward extraction, elution, and clean-up of patulin from juice with an external magnetic field. The MMIPs were synthesized by surface imprinted technology using 2-oxin and 6-HNA as dual dummy templates.
Figure 2. FT-IR spectra of Fe3O4 (a), Fe3O4@SiO2 (b), Fe3O4@SiO2-CH=CH2 (c) and magnetic molecular imprinted polymers (d)
Figure 3. XRD patterns of Fe3O4 (a), Fe3O4@SiO2 (b), Fe3O4@SiO2-CH=CH2 (c) and magnetic molecular imprinted polymers (d).
Figure 4. Hysteresis loop of Fe3O4 (a), Fe3O4@SiO2 (b), Fe3O4@SiO2-CH=CH2 (c) and magnetic molecular imprinted polymers (d)
Figure 5. Adsorption isotherms of patulin on MIPs and NIPs
Figure 6. Scatchard equation for MIP in aqueous solution
Figure 7. Effect of sample pH on the recovery of patulin
Figure 8. Effect of extraction and elution time on the recovery of patulin Formatted: Font: Times New Roman, 9 pt
Figure 9. Representative chromatograms of patulin in apple juice samples spiked with 50 μg L−1
Table 1 Recovery, precision, linearity and sensitivity characteristics of the MDSPE-LC -MS/MS method for patulin in juices (n = 3) Linearity Mean
Spiked Matrix
（μg L−1）
recovery
RSD (%)
Linearity range
(%)
r
LOD (μg L−1)
LOQ (μg L−1)
0.1
0.3
(μg L−1)
Grape juice
Orange juice
Apple juice
1
96.1
3.5
10
97.9
4.7
50
81.1
0.8
1
86.5
2.5
10
79.4
1.4
y=8.88e6x+-1.52e3 0.9993
y=6.4e6x+-849 0.5-100 0.9996 50
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Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples Minjuan Zhao , Hua Shao , Jun Ma , Hui Li , Yahui He , Miao Wang , Fen Jin , Jing Wang , A. M. Abd El-Aty , ˘ , Feiyan Yan , Yanli Wang , Yongxin She Ahmet Hacımuft ¨ uo ¨ glu PII: DOI: Reference:
S0021-9673(19)31196-3 https://doi.org/10.1016/j.chroma.2019.460751 CHROMA 460751
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
24 October 2019 28 November 2019 28 November 2019
Please cite this article as: Minjuan Zhao , Hua Shao , Jun Ma , Hui Li , Yahui He , ˘ , Feiyan Yan , Miao Wang , Fen Jin , Jing Wang , A. M. Abd El-Aty , Ahmet Hacımuft ¨ uo ¨ glu Yanli Wang , Yongxin She , Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460751
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Highlights
a novel MMIP with specific adsorption of patulin was successfully synthesized.
The magnetic dispersion solid-phase extraction (MDSPE) method was established.
This pretreatment method saves time and reduces the use of organic reagents.
This study provides a new pretreatment method for the detection of patulin in juice.
Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples Minjuan Zhaoa, Hua Shaoa*, Jun Maa, Hui Lia, Yahui Hea,b, Miao Wang a, Fen Jina, Jing Wanga, A. M. Abd El-Atyd,e,f, Ahmet Hacımüftüoğluf, Feiyan Yanc, Yanli Wangc and Yongxin She1* a
Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R China Chinese Academy of Agricultural Sciences Institute of Agro-Products Quality Standards and Testing Technology b
Beijing Technology and Business University, 100048, P.R. China
c
Institute of Quality Standards & Testing Technology for Agro-Products, Guangxi Academy of Agricultural Sciences, Nanning 530000, P.R. China d
State Key Laboratory of Biobased Material and Green Papermaking, College of Food Science and Engineering, Qilu University of Technology, Shandong Academy of Science, Jinan 250353, China e
Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211-Giza, Egypt
f
Department of Medical Pharmacology, Medical Faculty, Ataturk University, 25240-Erzurum, Turkey
Abstract: In this study, a novel magnetic molecularly imprinted polymer (MMIP) was prepared by surface imprinting technology using 2-oxin and 6-HNA as dual virtual templates and 4-vinyl pyridine (4-VP) as the functional monomer for extraction of patulin (PAT) from the surface of magnetic nanoparticles. MMIPs were characterized by fourier transformed infrared (FT-IR) spectroscopy, X-ray diffraction, and vibrating sample magnetometry. The results showed that the molecularly imprinted polymer (MIP) was successfully coupled with magnetic nanoparticles and could be used as a magnetic selective recognition material. Moreover, MMIPs have a greater adsorption capacity for PAT than conventional MIPs. The magnetic dispersion solid-phase extraction procedure was optimized and then combined with liquid chromatography-tandem mass spectrometry (MDSPE-LC-MS/MS) for detection of PAT in juice samples. The method showed excellent analytical performance in terms of linearity (ranged between 0.5 μg L −1 and 100 μg L−1with correlation coefficients (r) higher than 0.999) and limit of detection (LOD) (0.1 μg L−1, S/N = 3). At three spiking concentrations (1, 10, and 50 μg L-1), the mean recoveries were ranged between 79.4% and 97.9% with relative standard deviations (RSDs) less than 4.7% (n = 3). Keywords: Patulin; Magnetic molecularly imprinted polymers; Juice; Magnetic dispersion solid-phase extraction; LC-MS/MS; HPLC-DAD 1. Introduction Patulin (PAT) is a toxic mycotoxin produced by species belonging to genera of Penicillium, Byssochlamys, and Aspergillus [1-3]. PAT has been found in various mildewed fruits. It is a water soluble molecule with high thermal stability. Additionally, it is more stable in acidic solution and may be difficult to be removed by general methods, such as heating and drying [4,5]. Some studies indicate that PAT is teratogenic, carcinogenic, and mutagenic in laboratory animals [6,7]. Toxicity assessment (acute, subacute, and chronic) due to consumption of patulin shows restlessness, twitching, shortness of breath, , congestion of the lungs, fester, dropsy, extravasated blood, and gastrointestinal diseases [8-10]. Furthermore, PAT has been shown to exhibit adverse effects on the development of fetus in rodents [11,12]. Due to its potential toxicity and other reported adverse effects, most countries and organizations have established standards for maximum residue limits (MRLs). Specifically, European Commission has set a maximum limit of 50 ppb for PAT in fruit juices and other fermented beverages containing apple or apple juice, 25 ppb in solid apple products, and 10 ppb in apple puree and solid apple products for infants and children [13]. In China, the MRL was stipulated to be 50 ppb in fruit products, fruit and vegetable *Corresponding authors. E-mail addresses: [email protected] (Y. SHE), [email protected](H. SHAO).
juices, and wines (except sweetened rolls) with apple and hawthorn as raw materials [14]. The most effective way to prevent/control mold contamination and ensure food safety is to develop a rapid and effective detection method for patulin quantification in agricultural products. The overall sample preparation process used for extraction and enrichment of PAT, include liquid-liquid extraction (LLE) [15], dispersion solid-phase extraction [16], and solid-phase extraction (SPE) [17]. Among them, the SPE technology is the most widely used pre-processing method, yielding high enrichment and concentration effect [18]. However, the SPE column may suffer from a lack of selectivity, the purification effect is not ideal, and the co-extracted impurities may alter the ionization efficiency of target analytes owing to the matrix effect [19, 20]. This may affects the instrumental analysis which is the last step of analyte identification. Therefore, it is necessary to develop a pretreatment method with good selectivity, purification, and enrichment ability. Recently, the application of molecularly imprinted polymers (MIPs) has attracted much attention, because it could solve the aforementioned pitfalls [21-23]. Molecularly imprinted polymers (MIPs) are novel biomimetic materials (with strong molecular recognition ability), which exhibited excellent reusability and reproducibility, cheap, easy to prepare, and high specificity [24,25]. With the development of molecularly imprinted solid-phase extraction, magnetic molecularly imprinted solid-phase extraction has attracted considerable attention because of its simple operation, good enrichment effect, and fast separation. A magnetic molecularly imprinted polymer (MMIP) prepared by surface molecular imprinting technique and nanotechnology exhibits large specific surface area, distinct superparamagnetism, molecular imprinting specificity, and adsorption performance; the characters which are superior than that of ordinary polymers. As an adsorbent for dispersive solid-phase extraction (d-SPE), MMIPs can be directly dispersed in the sample solution, and the target can be quickly separated from the matrix by applying an external magnetic field, which simplifies the experimental operation, reduces the use of organic reagents, and achieves the purpose of separating and enriching the target [26-30]. In this context, Cheng et al. prepared an MMIP with a core-shell structure for extraction and enrichment of quercetin using surface molecular imprinting technique coupled with nanotechnology [31]. Additionally, Gholami et al. synthesized MMIPs to adsorb and enrich melamine in milk samples [32]. On the other hand, Liu et al. prepared MMIP microspheres by synthesizing MIPs on the surface of Fe3O4@Ag for SERS-based sensing of molecular species and applied the designed MMIPs for simple, rapid, ultrasensitive, and label-free SERS detection of sibutramine [33]. Notably, there have been no (scarce) studies on the preparation of MMIPs based on the MDSPE for detecting patulin and their application in real samples. In this study, core-shell MMIPs were prepared by surface imprinting technology using SiO 2 layered Fe3O4 nanoparticles as carriers to develop a fast and selective adsorbent for patulin during the pre-processing procedure. Application of an external magnetic field enables the facile separation of MMIPS that was used as an adsorbent for MDSPE. The characterization of patulin-MMIPs and the MDSPE procedure was conducted systematically. Consequently, combined with LC-MS/MS, the MDSPE was successfully used to enrich and detect patulin in fruit juice. 2. Experimental 2.1 Materials and Instruments All chemical reagents and instrumental conditions used in the experimental works are listed in the supplemental materials. 2.2 Preparation of core-shell Fe3O4@SiO2@MIPs We prepared magnetic Fe3O4@SiO2@MIP nanoparticles using a laboratory method and the procedure is shown in Figure 1. The specific method was as follows. First, we used the chemical co-precipitation method to prepare Fe3O4 nanoparticles. A total of 10 mL deionized water was used to gradually dissolve FeCl3·6H2O (2.35 g) and FeCl2·4H2O (0.86 g). The two solutions were transferred to a three-necked flask containing 80 mL deionized water, and the mixture was stirred until the temperature reaches to 70 ℃. Thereafter, 10 mL of a 25% ammonium hydroxide solution was added, and the mixture was stirred for 30 min at 80℃. To increase the surface dispersibility of magnetic particles, 100 mg of trisodium citrate dihydrate was added and the mixture was stirred for 30 min. After the reaction was completed, Fe3O4 was separated by external magnetic field and washed with ethanol until neutral. The prepared nanoparticles were stored in 50 mL ethanol pending analysis. Second, Fe3O4@SiO2 was synthesized according to the hydrolysis of a silylation reagent. Specifically, 25 mL of magnetic nanoparticles dissolved in ethanol (approximately 0.5 g of Fe3O4) was transferred to a three-necked flask containing 75 mL ethanol and 20 mL deionized water; the solution was mixed for 30 min under ultrasonication. Next, 1 mL of a 25% ammonium hydroxide solution and 2 mL tetraethoxysilane were added and the mixture was stirred for 24 h at
room temperature. The prepared Fe3O4@SiO2 microspheres were separated by external magnetic field and washed with ethanol until neutral. The third step involved the preparation of Fe3O4@SiO2 functionalized with vinyl groups. Fe3O4@SiO2 was transferred to a 250 mL three-necked flask containing 100 mL of a 10% acetic acid water. Then, 150 μL of MPS was added and the mixture was stirred for 5 h at 60 ℃. Fe3O4@SiO2-CH=CH2 nanoparticles were magnetically separated and washed multiple times with water and methanol and stored in 50 mL methanol. Finally, the molecularly imprinted polymer (MIP) was coupled to magnetic nanoparticles by precipitation polymerization. Specifically, 0.25 mmol of 2-oxin and 6-HNA as virtual templates and 1 mmol of 4-VP were dissolved in 30 mL methanol and pre-polymerized for 30 min to allow the templates to fully interact with a functional monomer. Then, 25 mg of Fe3O4@SiO2-CH=CH2 particles were added. Thereafter, 2 mmol of TRIM and 20 mg of AIBN were added. The mixture was ultrasonically dispersed for 20 min and nitrogen was blown for 5 min. Then, the mixture was reacted by heating in a water bath for 24 h at 60°C. After polymerization, MMIPs were collected by external magnetic field. The template molecules were extracted by Soxhlet extraction with methanol-acetic acid (90:10, v/v) until 2-oxin and 6-HNA could not be detected at 248 nm and 258 nm by UV–vis absorption spectroscopy. Subsequently, magnetic nanoparticles were washed with methanol and dried under vacuum at 55 ℃. The magnetic non-imprinted polymer (MNIP) was synthesized using the same method without adding the templates. 2.3 Adsorption capacity of MMIPs MMIPs or MNIPs (10 mg) were weighed and put into a 1.5 mL vial and mixed with 1 mL of different concentrations of PAT aqueous solution. The mixture was shaken at room temperature for 2 h at room temperature under a horizontal shaking and then separated by external magnetic fields. The free concentrations of PAT were measured by HPLC equipped with a diode array detector (DAD) at 276 nm. The amount of PAT that binds to the polymer was calculated by subtracting the free amount of PAT from the initially added amount. 2.4 MDSPE procedure First, 20 mg MMIPs was added into 1 mL juice samples and mixed for 8 min. MMIPs were separated by a magnetic field and water was completely removed. Then, the analyte was eluted from MMIPs with 1 mL acetonitrile by shaking for 15 min. Finally, the eluate was filtered and 5 μL was taken for LC-MS/MS analysis. 2.5 Sample preparation Apple, pear, and grape juice samples were purchases from local markets in Beijing (China). All juice samples were filtered (to remove particulate impurities) and stored (in the dark) in a refrigerator at 4 ℃ until use. 3. Results and discussion 3.1 Characterization of PAT-MMIPs To demonstrate the successful coupling of magnetic nanoparticles with molecularly imprinted polymers, the FT-IR spectra of Fe3O4, Fe3O4@SiO2, vinyl-functionalized Fe3O4@SiO2, and MMIPs were characterized. As shown in Figure 2 (a), the peak at 573 cm-1 was attributed to the characteristic absorption vibration of Fe-O. In Figure 2 (b), the peak at 1088 cm−1 originates from the characteristic absorption vibration of Si-O-Si, which indicates that SiO2 was efficiently coupled onto the surface of Fe3O4 nanoparticles. Peak at 1632 cm-1 in Figure 2 (c) was related to the C=C stretching absorption vibration, which indicates that the vinyl groups were modified onto the surface of Fe3O4@SiO2 nanoparticles. In Figure 2 (d), peaks at 1734 cm-1 and 2976 cm-1 were originated from the characteristic vibrations of C=O and C-H in TRIM, respectively. This observation indicates that MIPs were successfully coupled onto the surfaces of magnetic spheres. When X-rays are irradiated onto the surface of crystals, characteristic diffraction peaks that correspond to the crustal structure (analyzed by X-ray diffraction) are observed. Figure 3 shows the XRD patterns of prepared Fe3O4, Fe3O4@SiO2, vinyl-functionalized Fe3O4@SiO2, and MMIPs. It can be readily observed that Fe3O4 particles exhibit typical diffraction peaks, which means that the produced material was highly crystalline and had an anti-spinel structure. The main diffraction peaks were also found in Fe3O4@SiO2, Fe3O4@SiO2-CH=CH2, and MMIPs, which indicates that the crystal form of Fe3O4 particles did not change during the modification and surface synthesis of nanoparticles. However, the difference in peak width of the particles reflects the change in the average particle size. Figure 3 (d) shows that the intensity of the diffraction peak of MMIP particles was considerably reduced, the finding which means that the size of the particles changed.
As the surface of Fe3O4 nanoparticles was coated with SiO 2, modified with CH=CH2, and a polymer was synthesized on the surface, the measured and calculated saturation magnetization was gradually decreased ( Figure 4). The saturation magnetization saturation values were 69.85, 33.55, 29.90, and 4.28 emu g -1, respectively. Additionally, MMIPs were separated rapidly from the solution using an external magnetic field, and the magnetic molecularly imprinted polymer was uniformly re-distributed in the solution when the magnetic field is removed. Therefore, the prepared surface molecularly imprinted polymer had a good paramagnetism and could be used for d-SPE. This means it could simplify the sample pretreatment steps (by magnetic separation), thus saving the pretreatment time, decreases the use of organic reagents, and finally achieving the purpose of environmental protection and rapid detection. 3.2 Evaluation of adsorption capacity of polymers The binding amount of PAT to imprinted and non-imprinted polymers was determined by static adsorption experiments. The static adsorption experiments data are shown in Figure 5. The synthesized polymer had shown good adsorption performance to PAT in aqueous solution; and the adsorption capacity increases with increasing the initial concentration of PAT. This finding indicates that a binding site with specific adsorption was formed in the MIP, and specificity was shown at high concentrations. From the Scatchard equation (Figure 6), different slopes of MIP indicate the presence of different binding site. The linear regression equations in the linear region were q/c = -0.01802X + 168.7 (r=0.9998) and q/c = -0.0009017X + 114.7 (r=0.9556). From the slope and intercept of the fitting line, the Kd values were determined to be 55.49 μg.mL−1 and 119.02 μg.mL−1, and the Qmax values were 9.32 mg.g-1 and 127.20 mg.g-1, respectively, the values which are higher than that reported elsewhere [34]. Hence, the prepared MMIPs had good specific adsorption and hydrophilicity to PAT. 3.3 Optimization of MDSPE To determine the best conditions during the experimental work, 20 mg of MMIPs was used, and the following parameters were optimized: loading solution, sample pH, adsorption time, elution solution, and elution time. 3.3.1 Loading solution Polymer has the maximum adsorption capacity to target analytes in aqueous solution, and herein juice was the tested sample. Therefore, 1 mL was selected as a sample solution to ensure the adsorption of the target. 3.3.2 Sample pH The activity of PAT in an alkaline media was reduced. Thus, a pH ranged from 1 to 7 was assessed. As shown in Figure 7, the recovery of the target analyte was high and remained unchanged within the pH range. This finding might be attributed to hydrogen bonding interaction between the target and MMIPs in an acidic environment. As the pH of the juice sample was below 7, there was no need to adjust the pH throughout the experimental work. 3.3.3 Extraction time Extraction time is an important factor that could affect the extraction efficiency of the tested analyte during the MDSPE procedure. In this experiment, extraction time was evaluated in the range of 1-30 min while maintaining the other conditions. It has been found that the recovery gradually increased within 1-10 min and remained unchanged thereafter (Figure 8a). Therefore, 15 min was selected as the best extraction time. 3.3.4 Elution solvent and time In this study, we evaluated the elution effect of different volumes of methanol and acetonitrile on PAT. The results showed that the elution strength of acetonitrile was greater than that of methanol, and when the volume was 1 mL, a higher recovery rate was obtained. Thus, we have chosen 1 mL acetonitrile as an eluent. Afterward, the elution time was optimized in the range of 3-25 min. Figure 8b illustrates that 20-min elution enables the maximum elution of analyte in MMIPs. Therefore, 20 min was selected as an optimal elution time. 3.4 Method validation To evaluate the applicability of the developed MDSPE sample pretreatment procedure, the MDSPE-LC-MS/MS method for detecting PAT in juice samples was established. The linearity range, sensitivity, and limit of detection (LOD) were evaluated under the optimized conditions (Table 1).
To eliminate the matrix effect and obtain accurate results, the quantification was performed using matrix-matched standard curves [35]. The results are shown in Table 1. The method showed a good linear relationship between 0.5 μg.L-1 and 100 μg.L-1 with a satisfactory correlation coefficient (r ≥ 0.999). The LOD (S/N = 3) and LOQ (S/N = 10) were 0.1 μg.L−1 and 0.3 μg.L−1, respectively. Blank samples (n=3) were spiked with PAT at three concentration levels (i.e., 1, 10, and 50 μg.L−1) to determine the accuracy of the developed method. As shown in Table 1, the average recovery of PAT was ranged between 79.4% and 97.9%, and the RSD was between 0.8% and 4.7%. The extracted ion chromatograms for PAT are shown in Figure 9. The results showed that MMIPs can be used as highly efficient and specific adsorbents for selective extraction and enrichment of PAT in juice matrix during the MDSPE pretreatment process. The developed MDSPE-LC-MS/MS was accurate and can be used to detect PAT residues in real samples. 3.5 Detection of real samples The MDSPE-LC-MS/MS methodology was used for enrichment and detection of PAT in three juice samples purchased from local markets. The results showed that PAT residues were not detected in any of the samples. Conclusions In this research, a new type of pretreatment material, MMIPs was prepared by surface imprinting and nanotechnology using 2-oxin and 6-HNA as dual virtual templates. The polymer has good selectivity and adsorption for PAT and can be used as an adsorbent during the MDSPE pretreatment process. The pretreatment technique was evaluated by LC-MS/MS to detect PAT in juice samples.
Author Contribution Dear editors, The following are the specification of contribution of each author. (Title: Preparation of core-shell magnetic molecularly imprinted polymers for extraction of patulin from juice samples) Minjuan Zhao carried out experiments and wrote the paper; Associate Professor Hua Shao, Professor Jing Wang, Professor Yongxin She, Professor Fen Jin and Professor Feiyan Yan designed the experiments, provided useful suggestions and solved the problems in the experiments; Jun Ma, Yahui He, Miao Wang and Yanli Wang helped to synthesis magnetic molecularly imprinted polymers; Hui Li provided the experiment tools and helped to used the instrument; Professor A. M. Abd El-Aty and Ahmet Hacımüftüoğlu revised the paper. Thank you and best regards. Yours sincerely, Corresponding author: Professor Yongxin She, E-mail: [email protected] Associate Professor Hua Shao, E-mail: [email protected]
Conflict of interest Authors have declared no conflicts of interest. Acknowledgements This work was supported by the National Key Research and Development Special Plan (2017YFF0210201). The authors are grateful to the National Natural Science Foundation of China (contract No. 31772071), the Guangxi Innovation-driven Development Project (AA17204043-2), and Agriculture Research System of China
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Figure captions
Figure 1. Schematic diagram for synthesis of magnetic molecular imprinted polymers (MMIPs) and its application toward extraction, elution, and clean-up of patulin from juice with an external magnetic field. The MMIPs were synthesized by surface imprinted technology using 2-oxin and 6-HNA as dual dummy templates.
Figure 2. FT-IR spectra of Fe3O4 (a), Fe3O4@SiO2 (b), Fe3O4@SiO2-CH=CH2 (c) and magnetic molecular imprinted polymers (d)
Figure 3. XRD patterns of Fe3O4 (a), Fe3O4@SiO2 (b), Fe3O4@SiO2-CH=CH2 (c) and magnetic molecular imprinted polymers (d).
Figure 4. Hysteresis loop of Fe3O4 (a), Fe3O4@SiO2 (b), Fe3O4@SiO2-CH=CH2 (c) and magnetic molecular imprinted polymers (d)
Figure 5. Adsorption isotherms of patulin on MIPs and NIPs
Figure 6. Scatchard equation for MIP in aqueous solution
Figure 7. Effect of sample pH on the recovery of patulin
Figure 8. Effect of extraction and elution time on the recovery of patulin Formatted: Font: Times New Roman, 9 pt
Figure 9. Representative chromatograms of patulin in apple juice samples spiked with 50 μg L−1
Table 1 Recovery, precision, linearity and sensitivity characteristics of the MDSPE-LC -MS/MS method for patulin in juices (n = 3) Linearity Mean
Spiked Matrix
（μg L−1）
recovery
RSD (%)
Linearity range
(%)
r
LOD (μg L−1)
LOQ (μg L−1)
0.1
0.3
(μg L−1)
Grape juice
Orange juice
Apple juice
1
96.1
3.5
10
97.9
4.7
50
81.1
0.8
1
86.5
2.5
10
79.4
1.4
y=8.88e6x+-1.52e3 0.9993
y=6.4e6x+-849 0.5-100 0.9996 50
86.4
1.4
1
95.0
4.1
10
96.4
2.2
50
83.2
2.7
y=6.84e6x+-64.6 0.9990
Notes: e means ×10a (a:
1, 2,3, ......)