Food Chemistry 178 (2015) 18–25

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Novel molecular imprinted polymers over magnetic mesoporous silica microspheres for selective and efficient determination of protocatechuic acid in Syzygium aromaticum Lianwu Xie a,b,⇑, Junfang Guo b, Yuping Zhang b, Yunchu Hu a, Qingping You b, Shuyun Shi b,⇑ a b

College of Sciences, Central South University of Forestry and Technology, Changsha 410004, China College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China

a r t i c l e

i n f o

Article history: Received 4 July 2014 Received in revised form 4 November 2014 Accepted 13 January 2015 Available online 21 January 2015 Keywords: Molecularly imprinted polymers Mesoporous silica Magnetic microsphere Protocatechuic acid Syzygium aromaticum

a b s t r a c t Improving sites accessibility can increase the binding efficiency of molecular imprinted polymers (MIPs). In this work, we firstly synthesized MIPs over magnetic mesoporous silica microspheres (Fe3O4@mSiO2@ MIPs) for the selective recognition of protocatechuic acid (PCA). The resulting Fe3O4@mSiO2@MIPs were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectrometer (FT-IR), thermo-gravimetric analysis (TGA), Brunauer–Emmett–Teller (BET), and vibration sample magnetometer (VSM), and evaluated by adsorption isotherms/kinetics and competitive adsorption. The maximum adsorption capacity of PCA on Fe3O4@mSiO2@MIPs was 17.2 mg/g (2.3 times that on Fe3O4@ SiO2@MIPs). In addition, Fe3O4@mSiO2@MIPs showed a short equilibrium time (140 min), rapid magnetic separation (5 s) and high stability (retained 94.4% after six cycles). Subsequently, Fe3O4@mSiO2@MIPs were successfully applied for the selective and efficient determination of PCA (29.3 lg/g) from Syzygium aromaticum. Conclusively, we combined three advantages into Fe3O4@mSiO2@MIPs, namely, Fe3O4 core for quick separation, mSiO2 layer for enough accessible sites, and surface imprinting MIPs for fast binding and excellent selectivity, to extract PCA from complex systems. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Protocatechuic acid (PCA) is widely distributed in commonly consumed foods, fruits, and natural products. Meanwhile, PCA is the major in vivo metabolite of cyanidin-glucosides, which are present in fruits (Duthie, 2008). Pharmaceutical investigations indicate that PCA exhibits a wide variety of bioactivities, such as antioxidant, anticancer, anti-inflammatory and anti-carcinogenic properties (Kong, Mat-Junit, Ismail, Aminudin, & Abdul-Aziz, 2014; Masella et al., 2012; Yin, Lin, Wu, Tsao, & Hsu, 2009). Nowadays, PCA has attracted increasing interests as dietary supplement. Therefore, it is necessary to develop a sensitive, simple and rapid method to monitor PCA in complex matrices. Efficient extraction of target components from complex matrices relies on the development of specific adsorption materials for specifically capturing target components, especially for lowabundant components. Up to now, reversed-phase C18 or C8, normal-phase SiO2, carbon nanotubes, and macroporous materials ⇑ Corresponding authors at: College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China. Tel./fax: +86 731 88879616. E-mail addresses: [email protected] (L. Xie), [email protected] (S. Shi). http://dx.doi.org/10.1016/j.foodchem.2015.01.069 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

are usually used as robust pretreatment materials (Arrua, Causon, & Hilder, 2012; Herrera-Herrera, González-Curbelo, HernándeBorges, & Rodríguez-Delgado, 2012). However, they are based on physiochemical retention onto a functionalized surface, lack selectivity, and always lead to the co-extraction of matrices interference (Chianella, Karim, Piletska, Preston, & Piletsky, 2006). Molecularly imprinted polymers (MIPs) are relatively novel selective materials, which contain specific recognition sites in synthesized polymers with the memory of template molecule and their analogs due to shape, size and functionality recognition (Chen & Li, 2013). Other powerful features of MIPs are mechanical/chemical stability, low cost, easy preparation and reversible adsorption/release (Haupt & Mosbach, 2000). Recently, it has attracted wide attention and for the effective and selective separation of small bioactive components or macromolecules (e.g. proteins) from complex matrices (Davoodi, Hassanzadeh-Khayyat, Rezaei, & Mohajeri, 2014; Pardeshi, Dhodapkar, & Kumar, 2014; Sadeghi & Jahani, 2013; Tan, Huang, Peng, Tang, & Li, 2014). In most cases, MIPs showed specific adsorption of target components at very low concentrations (Davoodi et al., 2014; Pardeshi et al., 2014; Sadeghi & Jahani, 2013). However, MIPs prepared by conventional methods (such as bulk polymerization and

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precipitation polymerization) always led to poor binding capacity and low binding kinetics because of the existence of embedded binding sites (Xiao et al., 2013). Various forms of surface-imprinting techniques, imprinting MIPs on the surface of nano/micro solid support [e.g. SiO2 particles (Hu et al., 2014), Fe3O4 (Mehdinia, Kayyal, Jabbari, Aziz-Zajani, & Ziaei, 2013; Xie, Guo, Zhang, & Shi, 2014), Fe3O4@SiO2 (Shi, Guo, You, Chen, & Zhang, 2014; You, Peng, Zhang, Guo, & Shi, 2014), carbon nanotubes (Xiao et al., 2013), nanowires (Li, Yang, You, & Zhuang, 2006), and porous materials (Aboufazeli, Zhad, Sadeghi, Karimi, & Najafi, 2013; Liu et al., 2014)] have been successfully and widely used with promising results to address this problem. The results suggest that the surface MIPs increase the binding capacity and shorten the equilibrium time largely (Shi et al., 2014). Nevertheless, the binding capacity is severely dependent on the surface area of the solid support. Therefore, novel materials with large surface area have great potential for the design and application of surface MIPs. Ordered mesoporous/macroporous materials with absolute high surface to volume ratio have gained much attention in catalysis (Huang, Xu, & Lin, 2011) and separation (Deng, Qi, Deng, Zhang, & Zhao, 2008; Xue et al., 2014). We speculated that enough sites on the surface of mesoporous/microporous materials would be provided for MIPs imprinting with high binding capacity. Therefore, the integration of mesoporous/macroporous materials with surface MIPs is undoubtedly of great interest for practical applications. However, to the best of our knowledge, research in this area is limited. Liu at al. prepared macroporous poly(glycidyl methacrylate)@MIPs for the selective extraction and determination of 2,4dichlorophenoxyacetic acid from environmental water, and they found that synthesized MIPs exhibited shorter equilibrium time because of the excellent permeability of the surface recognition sites (Liu et al., 2014). Aboufazeli and coworkers synthesized lead ion imprinted MIPs on magnetic mesoporous silica nanoparticles (Fe3O4@mSiO2@MIPs) and then investigated their binding properties (Aboufazeli et al., 2013). Yang et al. synthesized and characterized surface MIPs over K2Ti6O13@mSiO2 to selectively extract dibenzothiophene (Yang, Zhou, Xu, Li, & Huang, 2012). However, they did not show the superiority of these materials, and little work has been done to investigate the adsorption properties of Fe3 O4@mSiO2@MIPs in comparison with MIPs imprinted on magnetic non-mesoporous silica nanoparticles (Fe3O4@SiO2@MIPs). Herein, we attempted to prepare and characterize Fe3O4@ mSiO2@MIPs using PCA as template, and investigate the adsorption isotherms, adsorption kinetics and competitive adsorption as well as applications for the selective enrichment of PCA from a type of spice, Syzygium aromaticum leaves. Meanwhile, the adsorption properties of prepared Fe3O4@mSiO2@MIPs were further compared with those of Fe3O4@SiO2@MIPs for the first time to understand their excellent molecular recognition ability. The results suggested that prepared Fe3O4@mSiO2@MIPs contained high binding capacity, fast binding kinetics, excellent selectivity, and quick separation ability because of the combination of mSiO2, surface MIPs and magnetic Fe3O4, which would demonstrate significant advantages for the fast, reliable, effective and specific extraction of bioactive components from complex mixtures.

2. Method and materials 2.1. Chemicals and reagents Iron (III) chloride hexahydrate (FeCl36H2O), polyethylene glycol 6000 (PEG 6000), 2,2-azobis(isobutyronitrile) (AIBN), cetyltrimethyl ammonium bromide (CTAB), dimethyl sulfoxide (DMSO), tetraethyl orthosilicate (TEOS), and HPLC grade methanol were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai,

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China). Ethylene glycol dimethacrylate (EGDMA), 4-vinylpyridine (4-VP), acrylamide (AM), methacrylamide (MAM), and 3-(trimethoxysilyl) propyl methacrylate (MPS) were acquired from Shaen Chemical Technology Co., Ltd (Shanghai, China). 4-Hydroxybenzoic acid (4-HBA), salicylic acid (SAA), caffeic acid (CA), gallic acid (GA), syringic acid (SYA), vanillic acid (VA), ferulic acid (FA), and PCA with a purity of over 99% were obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ultrapure water (18.2 MX), collected from a Milli-Q water purification system (Millipore, Bedford, MA, USA), was used to prepare aqueous solutions. All other reagents were of analytical grade and obtained from Kemiou Chemical Reagent Co., Ltd (Tianjin, China). 2.2. Apparatus and conditions Transmission electron microscopy (TEM) (JEM-2100F, JEOL, Japan) was used to observe the morphology of microspheres. Infrared spectra (4000–400 cm1) were recorded on a FT-IR (Nicolet 6700, Thermo Nicolet Co., Waltham, MA, USA). The encapsulation efficiency of microspheres was carried out by TGA (TGA SDTQ600, TA, USA). Magnetization was measured at room temperature in a VSM (VSM7407, Lake Shore, USA). Nitrogen sorption isotherms were performed at 77 K with a Monosorb Autosorb (Monosorb Autosorb, Quantachrome, USA). Chromatographic separation was performed on a ZORBAX SBC18 column (150 mm  4.6 mm, 5 lm, Agilent, Santa Clara, CA) in tandem with a Phenomenex C18 guard cartridge (4.0 mm  3.0 mm, Phenomenex, Torrance, CA). The eluent was delivered using an Agilent 1260 HPLC quaternary pump (Agilent Technologies, Santa Clara, CA) equipped with an online vacuum degasser, an autosampler, a thermostated column compartment, and a diode array detector. The mobile phase was methanol/water (V/V = 20/80) mixture containing 0.4% acetic acid. The flow rate was set at 0.8 ml/min while the temperature was controlled at 25 °C. Spectra were recorded from 190 to 400 nm (peak width 0.2 min and data rate 1.25 s1) while the chromatogram was acquired at 260 nm. 2.3. Procedures for preparation of Fe3O4@mSiO2@MIPs The process of preparing Fe3O4@mSiO2@MIPs is shown in Fig. 1. At first, Fe3O4 microspheres were synthesized according to our previous work (Shi et al., 2014; Zhang et al., 2014). Then, Fe3O4 microspheres were coated with mSiO2 layer through surfactant based sol–gel approach according to reported method with minor modifications (Deng et al., 2008). Typically, as-prepared Fe3O4 microspheres (50.0 mg) were mixed with CTAB (500.0 mg) in deioned water and ultrasonicated for 30 min. Then, the resultant homogenous solution was diluted with 1.0 mM NaOH aqueous solution (450.0 ml) and ultrasonically treated for another 5 min. Subsequently, the obtained basic dispersion was mechanically stirred at 60 °C for 30 min, and injected TEOS/ethanol (1/4, V/V) solution (2.5 ml) for stirring about 1 min, and then let stand for 12 h. The Fe3O4@CTAB/SiO2 microspheres were collected magnetically and then redispersed in acetone for refluxing at 80 °C twice of 24 h each to remove CTAB. Finally, Fe3O4@mSiO2 microspheres were collected, repeatedly washed by deioned water, and dried in vacuum. After that, Fe3O4@mSiO2 was modified by vinyl groups. Briefly, Fe3O4@mSiO2 (250.0 mg) was dissolved in MPS solution (150.0 ll of MPS dispersed in 40.0 ml of water containing 10% acetic acid). After stirring for 5 h at 60 °C, the vinyl-modified Fe3O4@mSiO2 was magnetically gathered, repeatedly washed with water, and dried in vacuum (You et al., 2014).

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Fig. 1. Schematics for the synthesis of Fe3O4@mSiO2@MIP.

Lastly, Fe3O4@mSiO2@MIPs were prepared via the surfaceimprinted polymerization method (Shi et al., 2014; You et al., 2014). Typically, PCA (0.25 mmol) and 4-VP (1.0 mmol) were dissolved in anhydrous acetonitrile (6.0 ml), purged with N2, and then stored in refrigerator at 4 °C for 12 h to prepare preassembly solution. Then, vinyl-modified Fe3O4@mSiO2 (50.0 mg), EGDMA (5.0 mmol) and AIBN (20.0 mg) were dissolved in acetonitrile (15.0 ml) and added into the above solution, purged with N2 on ice, and then allowed to proceed for 24 h at 60 °C under constant stirring. After polymerization, Fe3O4@mSiO2@MIPs were collected magnetically, rinsed with acetonitrile until the supernatant was clear, and then eluted with methanol–acetic acid (9/1, V/V) to remove the template absolutely. Finally, the Fe3O4@mSiO2@MIPs were washed with methanol to neutral pH and dried overnight under vacuum at 50 °C. As a control, the same procedures were applied for the preparation of Fe3O4@mSiO2@NIPs in the absences of template. Meanwhile, for comparison, Fe3O4@SiO2@MIPs without the addition of CTAB were prepared by the same method. 2.4. Adsorption experiments For the equilibrium experiments, Fe3O4@mSiO2@MIPs/Fe3O4@ mSiO2@NIPs (or Fe3O4@SiO2@MIPs) (10.0 mg) were suspended in a series of PCA acetonitrile solutions (3.0 ml) with initial concentrations ranging from 0.05 to 6.0 mg/ml. The series of mixtures were shaken for 3 h at 298 K, 308 K, and 318 K, and then the equilibrium concentrations of PCA were detected by HPLC. The equilibrium adsorption capacity Qe (mg/g) was calculated according to the following equation:

Q e ¼ ðC 0  C e ÞV=m

ð1Þ

where C0 (lg/ml) represents the initial concentration of PCA, and Ce (lg/ml) is the equilibrium concentration of PCA. V (ml) is the volume of PCA solution, while m is the mass of Fe3O4@mSiO2@ MIPs/Fe3O4@mSiO2@NIPs (or Fe3O4@SiO2@MIPs) (g). Similarly, for the kinetic experiments, Fe3O4@mSiO2@MIPs/Fe3 O4@mSiO2@NIPs (10.0 mg) were suspended in 1.0 mg/ml PCA acetonitrile solution (3.0 ml). The mixtures were then continuously shaken at 298 K, and the concentrations of PCA in the supernatant at a certain intervals (5, 10, 20, 30, 40, 50, 70, 100, 140, 180 and 220 min) were analyzed by HPLC, and then the adsorption capacity Qt (lg/g) at different contact times t was calculated as:

Q t ¼ ðC 0  C t ÞV=m

ð2Þ

where Ct (mg/ml) is the concentration of PCA at different contact times. The selectivity of adsorption was assessed using PCA and its seven structurally similar compounds (4-HBA, SAA, CA, GA, CA, SYA, VA and FA) in individual standard solutions with the same initial concentrations of 1 mg/ml. 2.5. Regeneration and reused experiments Fe3O4@mSiO2@MIPs with loaded PCA were separated magnetically, rinsed with acetonitrile to reduce the nonspecific adsorption, and then eluted with methanol–acetic acid (9/1, V/V) (1.0 ml) for 1 h for complete PCA desorption, and then the regenerated Fe3O4@ mSiO2@MIPs were reused for the next adsorption experiment. 2.6. Extraction and determination of PCA in S. aromaticum S. aromaticum leaves were purchased from local Chinese medical herbs shop in Changsha. The plant material was identified as S. aromaticum leaves by Prof. Mijun Peng, Key Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou University, Zhangjiajie, China. S. aromaticum leaves (100.0 g) were crushed and extracted by 800.0 ml of 75% (V/V) ethanol three times, each for 3 h, and the filtrates were concentrated on a rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) under reduced pressure at 40 °C to yield dried residue. The residues were then suspended in 100 ml of hot water and submitted to liquid–liquid extraction using equivalent volumes of petroleum ether and ethyl acetate, respectively. Ethyl acetate extract of S. aromaticum leaves (8.32 g) was stored at 4 °C for further experiments. Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPs (20.0 mg) were suspended in S. aromaticum extract (3.0 ml). After shaking for 140 min, Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPs were collected by a magnet, and then washed with acetonitrile followed by methanol–acetic acid (9/1, V/V) (1.0 ml) at 20 °C to enrich PCA. The eluates were analyzed by HPLC. 3. Results and discussion 3.1. Preparation of Fe3O4@mSiO2@MIPs The schematic preparation process of Fe3O4@mSiO2@MIPs is shown in Fig. 1. The thin layer of MIPs is prepared on the surface of the micro size of solid support, which benefits the enhancement

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of mass transfer and the complete removal of template (Xie, Zhang, Wang, Gao, & Liu, 2006). Furthermore, mesoporous materials with higher surface to volume ratio contain remarkable superiorities for preparing surface MIPs. At the beginning, Fe3O4 microspheres were synthesized by solvothermal reaction, which have higher magnetic response than those prepared by coprecipitation method (Shi et al., 2014; You et al., 2014). A layer of mesostructured CTAB/silica composites was then deposited on the Fe3O4 microspheres through a facile one-step sol–gel process using CTAB as a template with structure-directing property. After that, well-dispersed Fe3O4@ mSiO2 microparticles with magnetic core and ordered mesoporous silica shell were formed by removing CTAB in a mild way by acetone extraction (Liu et al., 2010). Notably, the mesoporous silica shell provided high surface area for the derivation of numerous functional groups, which then favored the adsorption of target components (Katz & Davis, 2000). Furthermore, our previous research indicated that the encapsulation of Fe3O4 with SiO2 should improve their dispersion in water, reduce agglomeration phenomenon in repetitious magnetic separation, and then increase their reusability (Zhang et al., 2014). After that, vinyl groups were introduced onto the surface of Fe3O4@mSiO2 with MPS for reaction with EDGMA to initiate the copolymerization of 4-VP and PCA in the presence of AIBN. Finally, Fe3O4@mSiO2@MIPs with surface binding sites were achieved by removal of templates. The binding properties of Fe3O4@mSiO2@MIPs depend on various factors. Then some essential factors were investigated during the preparation procedures to achieve high adsorption capacity. Functional monomer is a key factor for the powerful molecular recognition of MIPs because the molecular recognition is based on the intermolecular interaction between the template and the functional monomer (Sun, Schussler, Sengl, Niessner, & Knopp, 2008). Previously, articles have reported the preparation of PCA MIPs by bulk polymerization method and precipitation polymerization method, and they have both selected neutral AM as functional monomer (Chen, Wang, & Shi, 2011; Karasová, Lehotay, Sádecká, Skacˇáni, & Lzchová, 2005). Here, three kinds of functional monomers (4-VP, AM and MAM) with same amounts were selected to evaluate the specific recognition ability of Fe3O4@ mSiO2@MIPs for PCA (Supplementary Fig. S1a). Finally, 4-VP was considered as the optimum functional monomer because of the existence of strong electrostatic bonds and hydrogen-bonding interactions between pyridyl group of 4-VP and PCA as well as the p–p interactions between aromatic rings (Haupt, Dzgoev, & Mosbach, 1998). Thus, a more stable template–monomer complex between PCA and 4-VP can be formed in the imprinting process, which will lead to the formation of well-defined specific binding sites in Fe3O4@mSiO2@MIPs for high selectivity. Acetonitrile was selected as porogen, in which PCA and 4-VP was dissolved well. Moreover, the weak polarity solvent of acetonitrile was suitable for non-covalent molecular recognition of MIPs for strong polar molecule without any hydrophobic functional group (Shi et al., 2014). The molar ratio of template–functional monomer–crosslinker is a critical factor in a successful imprinting protocol due to their effect on the number of recognition sites in synthesized MIPs and the quality of MIPs. Different molar ratios of template–functional monomer–cross-linker (1:3:20, 1:4:20, 1:5:20, 1:4:16, 1:4:30, and 1:5:30) were evaluated and optimized. The results indicated that PCA-4-VP–EDGMA at 1:4:20 achieved highest adsorption capacity and molecular recognition ability of Fe3 O4@mSiO2@MIPs (Supplementary Fig. S1b). In addition, the amount of MIPs coated on the surface of Fe3O4@mSiO2 should also affect the binding capacity of Fe3O4@mSiO2@MIPs, and then different quantities of Fe3O4@mSiO2 (25, 50, 75 and 100 mg) were used when the concentration of PCA was set at 4.0 mg/ml. Eventually, 50 mg of Fe3O4@mSiO2 was evaluated as the best choice (Supplementary Fig. S1c).

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3.2. Characterization of Fe3O4@mSiO2@MIPs Particle size and morphology of Fe3O4@mSiO2 and Fe3O4@ mSiO2@MIPs can be observed by TEM (Fig. 2). It can be seen that the core–shell structure of Fe3O4@mSiO2 microspheres with diameter approximate at 400 nm were successfully prepared with regular spherical shape and relatively narrow size distribution. About 50 nm thick mSiO2 shell was clearly seen to be uniformly coated on Fe3O4 dark core (Fig. 2a). After imprinting process, an external polymer layer with 50 nm was clearly observed around Fe3O4@mSiO2 microparticles (Fig. 2b), which suggested that MIPs layer had been successfully grafted on the surface of Fe3O4@mSiO2 microparticles. There were hardly any free Fe3O4, mSiO2 and MIPs microspheres in TEM view. As the cylindrical mesoporous in mSiO2 were perpendicular to the Fe3O4 surface (Deng et al., 2008), the imprint precursor permeated into the channels and MIPs layer could be located on the internal surface of Fe3O4@mSiO2 (Li et al., 2013), which was very helpful for more recognition sites and higher adsorption capacity. It was also noted that the surface recognition sites could improve the mass transfer rate for adsorption and desorption of templates quickly. In the FT-IR spectra, the strong absorption peak at about 580 cm1 was characteristic of the Fe–O vibration (Supplementary Fig. S2a), and the strong peak around 1072 cm1 (Si–O asymmetric stretching vibration), and 800 cm1 (Si–O symmetric stretching vibration) indicated that SiO2 was successfully encapsulated onto the surface of Fe3O4 microspheres (Supplementary Fig. S2b). The characteristic absorption bands at 2922 cm1 and 2851 cm1 (C– H stretching vibration) were ascribed to CTAB (Supplementary Fig. S2b). The stretching vibration of the C@C bonds at 1632 cm–1 was attributed to the successful functionalization with vinyl groups (Supplementary Fig. S2c). Moreover, the disappearance of C–H stretching vibration peaks in Fig. 3c suggested the complete removal of CTAB. The new adsorption peak of C@O stretching band at 1720 cm1 for EDGMA showed that the MIPs was successfully coated on the surface of Fe3O4@mSiO2. The existence of C@C vibration indicated that not all of the EDGMA were crosslinked (Gam-Derouich et al., 2010). TGA was performed to quantify the encapsulation. As shown in Supplementary Fig. S3, the small weight loss of about 5% at the temperature less than 200 °C was attributed to the elimination of water. After that, Fe3O4@mSiO2/Fe3O4@SiO2 showed a weight loss of about 2% when heated to 800 °C probably due to some contaminations. And about 10% more weight loss was observed for vinylmodified Fe3O4@mSiO2 because of the decomposition of grafted MPS. Significant weight loss of Fe3O4@mSiO2@MIPs (17%) and Fe3 O4@mSiO2@NIPs (20%) could be observed. The slight difference may be come from the different grafting density caused by PCA. Comparatively, only about 4% and 8% weight loss were observed for vinyl-modified Fe3O4@SiO2 and Fe3O4@SiO2@MIPs, respectively, which were obviously lower than those for Fe3O4@mSiO2 and Fe3O4@mSiO2@MIPs. Therefore, a large surface to volume ratio of Fe3O4@mSiO2 provided more reaction sites for surface imprinting. The N2 adsorption–desorption isotherms at 77 K were studied to measure the surface area and pore volume. Supplementary Fig. S4 showed the characteristic adsorption–desorption isotherms for Fe3O4@mSiO2@MIPs, which were representative type IV curves with a sharp capillary condensation step at a relative pressure from 0.2 to 0.4, indicative of the existence of small cylindrical pores (Liu et al., 2009). Calculated from the adsorption isotherm by the Barrett–Joyner–Halenda (BJH) method, Fe3O4@mSiO2@MIPs had a pore size of 3.2 nm. The BET surface area and total pore volume for Fe3O4@mSiO2@MIPs were calculated to be 119.2 m2/g and 0.13 cm3/g, respectively, which were not obviously different from those for Fe3O4@mSiO2@NIPs (118.7 m2/g and 0.11 cm3/g). As a

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20

a

16

Fe 3O4@mSiO 2@MIPs

12

Fe 3O4@mSiO 2@NIPs

8

Fe 3O4@SiO 2@MIPs Fe 3O4@SiO 2@NIPs

4 0

0

1

2 3 4 5 6 Concentration (mg/ml)

Absorption capacity (mg/g)

Absorption capacity (mg/g)

Fig. 2. TEM images of Fe3O4@mSiO2 (a) and Fe3O4@mSiO2@MIPs (b).

318K

b

24 20

308K

16

298K

12 8 4 0

1

2 3 4 5 6 Concentration (mg/ml)

Fig. 3. Equilibrium adsorption curves of Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPs and Fe3O4@SiO2@MIPs/Fe3O4@SiO2@NIPs for PCA at 298 K (a), and equilibrium adsorption isotherms of Fe3O4@mSiO2@MIPs for PCA with the temperature at 298, 308 and 318 K (b).

result, the distinct adsorption properties of Fe3O4@mSiO2@MIPs and Fe3O4@mSiO2@NIPs could be attributed to the imprinting effect (Pan et al., 2010). However, the specific surface area and average pore diameter for Fe3O4@SiO2@NIPs were 93.9 m2/g and 0.14 nm. These results also indicated that the large surface of Fe3 O4@mSiO2 was efficiently imprinted. The magnetic saturation of Fe3O4@mSiO2@MIPs was about 38 emu/g (a remanence of 2.5 emu/g and a coercivity of 41.2 Oe), which was lower than that of Fe3O4 microspheres (56 emu/g, a remanence of 2.9 emu/g and a coercivity of 12.6 Oe) at the field of 10 KOe (Supplementary Fig. S5). This might be related to the magnetic inactive layer containing mSiO2 and imprinted polymer layers. However, the decrease did not seriously affect the magnetic separation of Fe3O4@mSiO2@MIPs, and they remained strongly magnetic and could be accumulated within 5 s in solution under conventional magnet and dispersed quickly with a slight shake once the magnetic field was removed, which indicated that Fe3O4@mSiO2@MIPs was successfully synthesized with a high magnetic responsivity. 3.3. Adsorption isotherms Adsorption isotherms of PCA on Fe3O4@mSiO2@MIPs/Fe3O4@ mSiO2@NIPs and Fe3O4@SiO2@MIPs/Fe3O4@SiO2@NIPs were determined at 298 K, 308 K and 318 K. Fig. 3a showed the characteristic adsorption isotherms at 298 K. It is can be seen that the equilibrium adsorption capacity of PCA on four adsorbents firstly increased sharply, then slightly with the increment of initial concentrations, and then became saturated when the concentration reached 4.0 mg/ml for Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPs, and 0.4 mg/ml for Fe3O4@SiO2@MIPs/Fe3O4@SiO2@NIPs. The

equilibrium adsorption capacity of PCA on Fe3O4@mSiO2@MIPs was 17.2 mg/g, 1.87 times that on Fe3O4@mSiO2@NIPs (9.2 mg/g), which might be resulted from the imprinting effect. Therefore, Fe3 O4@mSiO2@MIPs exhibited higher adsorption capability to PCA, and would be better to enrich trace PCA from complex system. It was noted that the equilibrium adsorption capacity of PCA on Fe3 O4@mSiO2@MIPs was 2.3 times that on Fe3O4@SiO2@MIPs (7.5 mg/g), and the imprinting factor IF for PCA on Fe3O4@mSiO2@ MIPs (1.87) was larger than that on Fe3O4@SiO2@MIPs (1.42). And MIPs for PCA prepared by precipitation polymerization method had even smaller adsorption capacity (0.19 mg/g) (Chen et al., 2011). Clearly, MIPs grafted on microparticles can take measure to ensure almost all the imprinting sites on surface for the accessibility of the template (Shi et al., 2014). Moreover, using Fe3O4@ mSiO2 as solid support, the abundant surface recognition sites would increase the binding capacity considerably. Fig. 3b showed adsorption isotherms of PCA on Fe3O4@mSiO2@ MIPs with the temperature at 298 K, 308 K and 318 K. The equilibrium adsorption capacities of PCA increased with the increment of temperature, which corresponded with previous investigations that MIPs prepared at higher temperatures tend to work better at higher temperatures because of the similar 3D structures (Lu, Li, Wang, Sun, & Xing, 2004). Moreover, lower viscosity and surface tension of the solvent at higher temperatures improved wetting of the Fe3O4@mSiO2@MIPs, which was then in certain extent led to higher binding capacity. To further estimate the binding properties of Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPs, two classical isotherm models, Langmuir and Freundlich equations, were selected to fit the experimental data. The Langmuir equation [1/Qe = 1/ (KLCeQm) + 1/Qm, where Qm is the maximum adsorption capacity (mg/g), and KL is a characteristic constant (ml/mg)] can be used

L. Xie et al. / Food Chemistry 178 (2015) 18–25

to describe a monolayer adsorption, whereas the Freundlich equation [log Qe = (log Ce)/n + log KF, where n and KF are Freundlich constants] can be used to describe a monolayer adsorption as well as a multilayer adsorption. Supplementary Table S1 summarizes the fitted parameters Qm, KL, KF, n and R2 (correlation coefficient), which revealed that Langmuir equation with R2 > 0.99 was found to better fit the isotherm data. Moreover, the saturated adsorption capacities of Fe3O4@mSiO2@MIPs for PCA at different temperatures were greatly higher than those of Fe3O4@mSiO2@NIPs, which indicated that Fe3O4@mSiO2@MIPs showed high binding affinity for PCA than Fe3O4@mSiO2@NIPs.

23

Farooq, & Knaebel, 1994). Supplementary Fig. S6 displays the plots of intra-particle diffusion model for adsorption of PCA onto Fe3 O4@mSiO2@MIPs, which was divided into two stages: a sharp rise step followed by a plateau. The initial step was ascribed to the intra-particle diffusion, stimulating further mass transfer of PCA from the fluid phase to the internal surface of Fe3O4@mSiO2@MIPs, while the plateau phase was attributed to the final equilibrium state. Two lines did not pass through the origin, which suggested that intra-particle diffusion may not be the only rate limiting mechanism in the adsorption process. 3.5. Selectivity adsorption for PCA

3.4. Adsorption kinetics

Absorption capacity (mg/g)

The kinetic curves of PCA adsorption on Fe3O4@mSiO2@MIPs/ Fe3O4@mSiO2@NIPs at 298 K are illustrated in Fig. 4. As we can see, adsorption capacities of PCA increased with the increment of adsorption time. It took more time for Fe3O4@mSiO2@MIPs to achieve binding equilibrium than Fe3O4@mSiO2@NIPs, which could be due to the specific molecular recognition process on tailored stereo-cavity and binding sites of Fe3O4@mSiO2@MIPs, and common adsorption (namely physical adsorption) on randomly distributed functional groups on the surface of Fe3O4@mSiO2@NIPs (Shi et al., 2014). The adsorption of PCA on Fe3O4@mSiO2@MIPs reached adsorption equilibrium at 140 min, suggesting that the adsorption is a fast process, and then 140 min was chosen as optimal extraction time. In comparison, traditionally imprinted materials would take 12–24 h to reach adsorption equilibrium (Cheong, Yang, & Ali, 2013). Therefore, surface imprinting technology provided binding sites at the surface of microparticles, which then showed the advantage of faster mass transfer and binding kinetics. Lagergren pseudo-first-order equation [ln(Qe  Qt) = ln Qe  K1t), where K1 is the pseudo-first-order rate constant (min1)] and pseudo-second-order equation [t/Qt = t/Qe + 1/(Qe2K2), where K2 is the pseudo-second-order rate constant] were used to analyze the kinetic data according to our previous report to determine the rate-controlling and mass transfer mechanisms (Shi et al., 2014). The corresponding parameters were calculated by two kinetic models and are displayed in Supplementary Table S2. It can be concluded that the pseudo-second-order rate equation was considered as the better-fit model from high R2 (>0.99) and similar data between the calculated Qe and experimental data were obtained. Similar results were reported for the adsorption of hydrobenzoic acids on magnetic MIPs (Shi et al., 2014). Then it was assumed that the chemical interactions of the rate-limiting step were possibly involved in the adsorption process for PCA. Intra-particle diffusion model [Qt = Kit0.5 + Ci, where Ki is the intra-particle diffusion rate constant (mg g1 min0.5), and Ci is the effect of the boundary layer thickness] was studied to determine the diffusion mechanism adsorption system (Ruthven,

14

Fe 3O4@mSiO 2@MIPs

12

3.6. Reusability of Fe3O4@mSiO2@MIPs

10 8

Fe 3O4@mSiO 2@NIPs

6 4 2

The molecular recognition ability of Fe3O4@mSiO2@MIPs was mainly dependent on the binding between Fe3O4@mSiO2@MIPs and adsorbed components, and then the binding ability was related to similarity between template and adsorbed components in functional groups, size and shape. It can be seen in Supplementary Fig. S7 that the adsorption capacities of eight similar components (PCA, 4-HBA, SAA, GA, CA, SYA, VA and FA) on Fe3O4@mSiO2@NIPs (non-specific adsorption) had no significant difference. In comparison, the adsorption capacities of eight selected components on Fe3O4@mSiO2@MIPs were much higher than those on Fe3O4@mSiO2@NIPs. The different results illustrated the success of the imprinting process. Although eight selected components had close structural homology, PCA with higher adsorption capacity was about 1.22, 1.80, 1.48, 1.60, 2.23, 2.06 and 1.96 times that for 4-HBA, SAA, GA, CA, SYA, VA and FA, which was because the specific sites in Fe3O4@mSiO2@MIPs were complementary in shape, size and spatial distribution to match PCA. The selectivity of Fe3O4@mSiO2@MIPs was further evaluated using three parameters: the distribution coefficient (Kd), selectivity coefficient (K), and relative selectivity coefficient (K0 ). As shown in Table 1, the K0 values were greater than 1, which showed a higher binding selectivity of Fe3O4@mSiO2@MIPs for PCA than other tested similar compounds. This could be attributed to the imprinting effect. The comparative results of the adsorption capacity were attributed to the molecular analog degree of detected compounds to the template. Although 4-HBA, SAA, GA, SYA and VA had close structural homology with PCA except for the numbers and positions of phenolic hydroxyl groups and the methylation of hydroxyl groups, the lower adsorption capacities were probably because there were no specific imprinted sites for compounds with different molecular size and stereochemistry. For CA, the steric effect of acrylic group presumably prevented it from entering into the binding cavities, and as a result, weak binding capacity was detected. However, for FA, the steric effect of acrylic group and the disappeared electrostatic interaction of methylation induced lower adsorption capacity. These data indicated that the imprinting effect of Fe3O4@mSiO2@MIPs could make sense for selective extraction of PCA from real complex samples. So the characteristic of Fe3O4@mSiO2@MIPs were superior to those traditional inefficient selective adsorbents.

0

40

80 120 160 Time (min)

200

Fig. 4. Kinetic adsorption curves of Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPs for PCA at 298 K.

Our previous report indicated that methanol–acetic acid (9/1, V/ V) could weaken the non-covalent interactions between hydroxybenzoic acids and MIPs, and was considered as the optimum desorption solvent to recover the template and regenerate MIPs. The reusability of Fe3O4@mSiO2@MIPs was investigated by monitoring the efficiency of adsorption of three batch samples for great cost benefit on extending its applications. As shown in Supplementary Fig. S8, after six consecutive adsorption–desorption cycles, the efficiency of adsorption was still as high as 94.4% of the first one,

24

L. Xie et al. / Food Chemistry 178 (2015) 18–25

Table 1 The selectivity parameters of Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPsa (n = 3).

Fe3O4@mSiO2@MIPs

Fe3O4@mSiO2@NIPs

a

Q (mg/g) Kd (ml/g) K Q (mg/g) Kd (ml/g) K K0

PCA

4-HBA

SAA

GA

CA

SYA

FA

VA

11.9 12.3 – 5.51 5.58 – –

8.11 8.26 1.49 5.13 5.17 1.08 1.38

5.72 5.78 2.12 3.69 3.74 1.49 1.42

7.53 7.68 1.60 4.91 4.97 1.12 1.43

6.26 6.36 1.93 3.64 3.67 1.52 1.27

5.34 5.47 2.25 3.72 3.77 1.48 1.52

5.78 5.92 2.08 3.65 3.69 1.51 1.38

6.07 6.21 1.98 3.89 3.93 1.42 1.39

Kd = binding amount/equilibrium concentration, K = Kd(PCA)/Kd(Similar

components),

K0 = KMIPs/KNIPs.

which manifested that Fe3O4@mSiO2@MIPs were stable and six cycles had little influence on its efficiency of adsorption. mAU

3.7. Method validation

200 150

c

100

b

The Fe3O4@mSiO2@MIPs as adsorbent for extraction of PCA and the HPLC–UV method for analysis of PCA was established based on the above experiments. Then the application of the method was evaluated by determining its performance characteristics regarding linearity, reproducibility, accuracy, precision, limit of detection, ruggedness and robustness. Using standard curve based method, the ratios of HPLC peak areas (A) with respect to corresponding concentrations of PCA (C) were found to be linear from 1 to 100 lg/ml with R2 value at 0.9995. The regression equation was A = 49.2C  40.8. The limit of detection (S/N = 3) for PCA was 0.5 lg/ml, which was sufficient for PCA determination in real samples. The intra-day and inter-day precisions were investigated by extraction and analysis of PCA spiked reference standard in S. aromaticum sample at low, medium and high concentration levels for five times per day and for consecutive six days. The RSD values of intra-day precisions ranged from 2.0% to 5.0% (n = 6), which were indicative of high reproducibility, and the RSD values of interday precisions over a six-day span were all less than 6.0%. Indeed, acceptable recovery values, ranging from 94% to 101% with RSD less than 5%, were obtained for PCA. The results showed that the proposed Fe3O4@mSiO2@MIPs extraction and HPLC analysis method was sufficiently accurate, selective and practical for the determination of PCA from complex mixtures.

was incorrect. After being dissolved in acetonitrile, S. aromaticum extract was extracted by Fe3O4@mSiO2@MIPs/Fe3O4@mSiO2@NIPs. PCA was selectively adsorbed and enriched onto Fe3O4@mSiO2@ MIPs (Fig. 5b), and the peak of PCA appeared distinctly, while no obvious PCA peak was observed in the eluted solution from Fe3 O4@mSiO2@NIPs (Fig. 5c), which also demonstrated the selectivity of Fe3O4@mSiO2@MIPs. Although there were some small peaks of PCA analogs in Fig. 5b (such as GA, CA, SAA and ellagic acid), the components with significantly different structures, and which eluted around PCA in the HPLC method were nearly eliminated (Fig. 5b). Therefore, the content of PCA in S. aromaticum was determined as 29.3 lg/g. The results indicated that the prepared Fe3O4@mSiO2@MIPs were very suitable to be directly exploited as a fast, efficient and selective enrichment of trace PCA in complex mixtures (e.g. food, natural product, biological sample).

3.8. Selective enrichment and determination of PCA in S. aromaticum

4. Conclusions

Natural products are very complex matrices with hundreds or thousands of compounds, which subsequently can cause matrix effect during the extraction process. For this reason, a selective pretreatment step was necessary to be applied to isolate target components before HPLC analysis. To demonstrate the amenability of our synthesized Fe3O4@mSiO2@MIPs in selective adsorption of PCA from complex mixtures, we enriched and quantified PCA in S. aromaticum leaves. S. aromaticum is a popular spice, which has several medicinal uses due to its antioxidant, anti-fungal, antiviral, anti-microbial, anti-diabetic, anti-inflammatory, antithrombotic and anesthetic properties; these have been mainly associated with their polyphenolic components (e.g. PCA, GA, CA, SAA, ellagic acid, and eugenol) which have been reported to be the main bioactive components (Chaieb et al., 2007; Sajjad, Khan, & Ahmad, 2012). PCA is a polar component with retention time at 5.52 min, and the peak area of PCA in S. aromaticum extract was very low (Fig. 5a). From 3D UV–vis spectra, we can see interferences at almost the same retention time. Therefore, quantitative analysis of trace PCA in S. aromaticum by HPLC–UV method without selective pretreatment process was difficult and the subsequently result

Novel Fe3O4@mSiO2@MIPs were successfully prepared and evaluated for the selective recognition of PCA from complex mixtures with excellent molecular recognition ability. Fe3O4@SiO2 and Fe3O4@mSiO2 had a high MIPs encapsulation efficiency and adsorption capacity because of the existence of larger surface area of mSiO2 for more recognition sites. The equilibrium data fitted well to Langmuir equation. The faster kinetic binding of PCA on Fe3 O4@mSiO2@MIPs than MIPs prepared by bulk polymerization were attributed to the thin MIPs thickness and surface recognition sites. The adsorption process could be described by pseudo-second order model. The magnetic separation materials could be dispersed into the solution directly and separated quickly within 5 s by an external magnet without a SPE column packing or filtration operation. In addition, Fe3O4@mSiO2@MIPs exhibited excellent reusability. Lastly, the proposed Fe3O4@mSiO2@MIPs were successfully applied to the enrichment of trace PCA from S. aromaticum with good recovery and high selectivity. Therefore, the Fe3O4@mSiO2@MIPs obtained in this study combined the advantage of a high surface area of mSiO2 for high binding capacity, surface MIPs for fast binding kinetics and excellent selectivity for easy and rapid separation ability.

PCA

50

a

0 0

5

10 15 20 25 30 35 40 Time (min)

Fig. 5. Chromatograms of S. aromaticum (a), the elutions with methanol–acetic acid (9/1, V/V) from Fe3O4@mSiO2@MIPs (b) and Fe3O4@mSiO2@NIPs (c).

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