Determination of ractopamine in pork using a magnetic molecularly imprinted polymer as adsorbent followed by HPLC

Food Chemistry 201 (2016) 72–79

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Determination of ractopamine in pork using a magnetic molecularly imprinted polymer as adsorbent followed by HPLC Yiwei Tang a,⇑, Jingwen Gao a, Xiuying Liu a,c, Jianxing Lan a, Xue Gao b,⇑, Yong Ma b, Min Li a, Jianrong Li c,⇑ a

College of Food Science and Project Engineering, Bohai University, Jinzhou 121013, China Food Safety Key Lab of Liaoning Province, Bohai University, Jinzhou 121013, China c National & Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products, Bohai University, Jinzhou 121013, China b

a r t i c l e

i n f o

Article history: Received 12 June 2015 Received in revised form 11 January 2016 Accepted 18 January 2016 Available online 18 January 2016 Keywords: Ractopamine Magnetic molecularly imprinted polymer Pork Extraction HPLC

a b s t r a c t A new magnetic molecularly imprinted polymers (MMIPs) for separation and concentration of ractopamine (RAC) were prepared using surface molecular imprinting technique with methacryloyl chloride as functional monomer and RAC as template. The MMIPs were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometer. The results of re-binding experiments indicated that the MMIPs had fast adsorption kinetics and could reach binding equilibrium within 20 min, and the adsorption capacity of the MMIPs was 2.87-fold higher than that of the corresponding non-imprinted polymer. The selectivity of the MMIPs was evaluated according to its recognition to RAC and its analogues. The synthesized MMIPs were successfully applied to extraction, followed by high performance liquid chromatography to determine RAC in real food samples. Spiked recoveries ranged from 73.60% to 94.5%, with relative standard deviations of <11.17%. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Ractopamine (RAC), a b-adrenergic agonist, is not only used in the treatment of asthma, respiratory infection and heart failure in human and veterinary medicine, but also used as a growth promoter to improve livestock growth with leaner meat and less fat (Shishani, Chai, Jamokha, Aznar, & Hoffman, 2003). However, serious side effects on human health, such as muscle tremor, headache, emesis, heart palpitations, have been observed due to the abuse of RAC in agri-food products (Thompson et al., 2008; Zhang, Ni, & Kokot, 2010). China, Russia and most European countries enacted laws to ban or restrict the use of RAC as feed additive during animal production (Nielen et al., 2008). Efficient methods including high performance liquid chromatography with fluorescence detection (HPLC-FD) (Shishani et al., 2003; Ying et al., 2006), liquid chromatography–mass spectrometry (LC–MS) (Antignac, Marchand, Le Bizec, & Andre, 2002; Blanca et al., 2005; Juan, Igualada, Moragues, León, & Mañes, 2010), gas chromatography–mass spectrometry (GC–MS) (Bocca, Fiori, Cartoni, & Brambilla, 2003; He, Su, Zeng, Liu, & Huang, 2007), enzyme-linked immunosorbent assay (ELISA) (Shelver & ⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Tang), [email protected] (X. Gao), [email protected] (J. Li). http://dx.doi.org/10.1016/j.foodchem.2016.01.070 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

Smith, 2002; Shelver, Smith, & Berry, 2000), electrochemical detection (Shen & He, 2007; Wang, Zhang, Wang, Shi, & Ye, 2010), immunochromatographic test strip assay (Gao et al., 2014; Jin, Lai, Xiong, Chen, & Liu, 2008; Zhang et al., 2009), and biomimetic enzyme-linked immunosorbent assay (Fang et al., 2011) to determine RAC have been reported. Due to the complexity of the food matrices and extremely low concentration of the analyte, a sample pretreatment step for analyte separation and enrichment is critical before instrumental analysis. Solid phase extraction (SPE) has been extensively applied to separate and enrich RAC from environmental, clinical, and agri-food samples (Antignac et al., 2002; Shishani et al., 2003). Nevertheless, some interference is not separated from RAC because of the non-specificity of traditional SPE towards the target molecule, which may affect the accuracy and precision of the analysis. Molecularly imprinted polymers (MIPs) have recently gained tremendous attention due to their ideal performance of predetermination, specific recognition and practicability (Zeng, Wang, Nie, Kong, & Liu, 2012). MIPs towards RAC have been prepared and used as SPE sorbents for selective separation and concentration of RAC from different types of samples (Liu et al., 2013; Tang, Fang, Wang, & Li, 2011; Wang, Liu, Fang, Zhang, & He, 2009; Zhang et al., 2011), indicating that MIPs could be a powerful molecular receptor to further improve the selectivity of the recognition material. Recently, magnetic separation technology based

Y. Tang et al. / Food Chemistry 201 (2016) 72–79

upon magnetic nano- and micro-particles has been applied for sample pretreatment (Hiratsuka, Funaya, Matsunaga, & Haginaka, 2013). The magnetic molecular imprinting polymers (MMIPs) containing the superparamagnetic particles can be directly added to crude samples or solution containing the target analyte and perform the magnetic separation process in an easy manner (Gao et al., 2011; Kong, Gao, He, Chen, & Zhang, 2012; Su et al., 2015). This separation process does not require packing the MMIPs into a solid phase extraction (SPE) cartridge. Further, the phase separation is easy and fast by employing a magnetic field without comprehensive centrifugation and/or filtration. In the current work, a novel magnetic Fe3O4@MIPs with a good recognition capability towards RAC was prepared with a uniform core–shell structure by integrating surface imprinting and nanotechnology. Herein, Fe3O4 nanoparticles were prepared using a novel co-precipitation method, and the vinyl and carbonyl groups were then grafted onto the 3-aminopropyltrimethoxysilane (APTS)-modified Fe3O4 surface with methacryloyl chloride (MC). The MIPs shells were synthesized around Fe3O4 nanoparticles using RAC as the template, ethylene glycol dimethacrylate (EGDMA) as the cross-linking agent and azobisisobutyronitrile (AIBN) as the initiator. The characterization and adsorption property of Fe3O4@MIPs and Fe3O4@NIPs (non-imprinted polymers, NIPs) were studied by transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), vibrating sample magnetometer (VSM), and rebinding experiments. The major factors impacting the clean-up and preconcentration of the analyte based upon Fe3O4@MIPs were optimized, followed by HPLC to determine trace level of RAC in real food samples. 2. Materials and methods 2.1. Reagents and chemicals RAC (>98% purity) was obtained from Changzhou Huaren Chemical Co. (Jiangsu, China). Isoproterenol (ISOP), isoxsuprine (ISOX), and terbutaline (TER) were purchased from Sigma (Sigma–Aldrich, St. Louis, MO, USA), which were used as the analogues of RAC in the cross-selectivity experiment. MC and APTS were purchased from Aladdin (Shanghai, China) to modify Fe3O4 nanoparticles. The polymerization reagents EGDMA and AIBN were obtained from Sigma–Aldrich (St. Louis, MO, USA). A RAC ELISA kit was obtained from Bonna-Agela Technologies (Tianjin, China). Other reagents were purchased from Tianjin Chemical Reagent Factory (Tianjin, China). All reagents were of the highest available purity and at least of analytical reagent grade. Stock solutions of RAC (500.0 mg L
1) and the analogues (500.0 mg L
1) were prepared in acetonitrile and stored at 4 °C. The working solutions at different concentrations were freshly prepared by diluting the stock solution in acetonitrile. 2.2. Instrumentation An Agilent 1260 HPLC system with fluorescence detector (excitation: 225 nm, emission: 306 nm) and a reversed-phase C18 analytical column (4.6 mm  150 mm, 5 lm, Agilent, USA) were employed for the determination of RAC in food samples. The mobile phase was aqueous solution containing 0.087% (w/v) sodium pentanesulfonate and 2% acetic acid and acetonitrile (80:20, v/v) and the flow rate of mobile phase was 1.0 mL min
1 (column oven temperature: 40 °C). FT-IR spectra (4000–400 cm
1) were collected using a Vector 22 FT-IR spectrometer (Bruker, Germany) and KBr pellet-based method. Morphological characteristics of Fe3O4 and Fe3O4@MIPs

73

were examined using TEM (FEI Tecnai G20, USA) at 200 kV. XRD patterns (10–80°) of the prepared samples were determined using a Rigaku Ultima IV X-ray diffractometer (Rigaku, Japan) with Cu Ka radiation source at a rate of 2° min
1. Magnetic measurement was carried out using a model MPMS-XL-7 vibrating sample magnetometer (Quantum Design, USA) at 22 °C. An SZCL-4A electric mixer (Beijing STWY Equipment Co., Ltd, China) was also used in this study. 2.3. Preparation of RAC MMIPs The preparation process of MMIPs is shown in Scheme 1. Fe3O4 nanoparticles were prepared by the co-precipitation method. The resultant Fe3O4 nanoparticles were functionalized with APTS by a simple coordination reaction, and then the amino groups of the APTS monomer were grafted onto the surface of Fe3O4 nanoparticles. Fe3O4 modified with APTS were further reacted with MC to introduce methacrylamide (MAC) as functional monomer for the pre-polymerization with template molecule (i.e., RAC). MIPs were finally coated onto the surface of Fe3O4 nanoparticles by copolymerization. After the removal of RAC, the MMIPs were obtained. 2.3.1. Preparation of Fe3O4 nanoparticles Fe3O4 nanoparticles were synthesized using co-precipitation method according to Zhang and Shi (2012) and Mo, Zhang, Guo, Meng, and Zhang (2011) with some minor modifications. Briefly, FeCl36H2O (0.06 mol) and FeCl24H2O (0.03 mol) were dissolved in 300.0 mL of deoxygenated water, and the mixture was then stirred using an electric mixer at 250 rpm in a nitrogen condition. Subsequently, 60.0 mL of ammonium hydroxide solution (28%, weight percent) was added dropwise over a 30 min period at 65 °C. One hour later, the mixture was allowed to cool down gradually to room temperature, and the black Fe3O4 nanoparticles were obtained. Finally, the products were washed for several times with purified water until the pH of the washings became neutral, followed by drying in a vacuum oven at 45 °C for 24 h. 2.3.2. Preparation of APTS-modified Fe3O4 The surface of Fe3O4 nanoparticles was grafted with APTS according to the work by Zhu et al. (2013). Fe3O4 nanoparticles (1.5 g) were dispersed in 200.0 mL of ethanol–water (1:1, v/v) in a three-neck 500.0 mL flask using an ultrasonic oscillator. After ten minutes, the pH of the solution was adjusted to 4 using acetic acid with constant stirring. APTS (3.268 mL) was then added and the mixture was stirred for 10 h at 60 °C in a nitrogen environment. The products were collected using an external magnetic field and then rinsed thoroughly with deionized water, ethanol, and diethyl ether until no oily suspension was determined. Finally, the APTSmodified Fe3O4 nanoparticles were dried in a vacuum oven at 45 °C for 24 h. 2.3.3. Preparation of MC-modified Fe3O4 The obtained APTS-modified Fe3O4 nanoparticles were further modified with MC. APTS-modified Fe3O4 nanoparticles (400.0 mg) and K2CO3 (0.4 g) were dispersed in toluene (200.0 mL) in an ice/ water bath for 30 min under constant stirring. Then, 4 mL of MC were added dropwise over a 20 min period at 0 °C. Subsequently, the mixture was warmed to room temperature and the reaction was carried out for 12 h under continuous stirring. Lastly, the MC-modified Fe3O4 was separated using an external magnetic field and then dried in a vacuum oven at 45 °C for 24 h. 2.3.4. Preparation of Fe3O4@MIPs and Fe3O4@NIPs For the preparation of Fe3O4@MIPs, RAC (0.1 mmol) and MCmodified Fe3O4 (100.0 mg) were dispersed in 20.0 mL of acetonitrile and the mixture was stirred for 3 h for self-assembly between

74

Y. Tang et al. / Food Chemistry 201 (2016) 72–79

Scheme 1. Scheme of the preparation of Fe3O4@MIPs.

the template and the MC-modified Fe3O4. EGDMA (2.0 mmol) and AIBN (40.0 mg) were then added and the mixture was purged by nitrogen for 3 min at 0 °C to completely remove the dissolved oxygen. The flask was sealed and placed in an oil bath at 65 °C for 24 h. After polymerization, the resultant Fe3O4@MIPs was crushed using a mortar and pestle, washed with methanol-dichloromethane (4:1, v/v), and then dried in a vacuum oven at 45 °C for 24 h. The template molecule was removed using 300 mL of methanol-acetic acid (9:1, v/v) in a Soxhlet apparatus for 72 h until no RAC was detected using HPLC. MMIPs were dried at 45 °C. For comparison, Fe3O4@NIPs were prepared using the same procedure in the absence of the template molecules (i.e., RAC).

separated using a magnet and the concentration of RAC in the supernatant was measured using HPLC with fluorescence detector after filtration through 0.22 lm microporous membrane. The adsorption capacity (Q mg g
1) was calculated using the following equation

2.4. Adsorption properties of Fe3O4@MIPs

2.4.2. Static adsorption test To evaluate the binding capacity of Fe3O4@MIPs towards RAC, 20.0 mg of Fe3O4@MIPs or Fe3O4@NIPs were equilibrated with different concentrations (0, 0.01, 0.1, 1, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160 mg L
1) of RAC in 10.0 mL acetonitrile solution, which were shaken at 300 rpm for 60 min at 22 °C. The concentration of RAC in the supernatant was determined as described in the uptake kinetics test.

2.4.1. Uptake kinetics test To evaluate the binding kinetics of MMIPs, Fe3O4@MIPs (140.0 mg) were suspended in seven separate 50.0 mL glass flasks containing 10.0 mL of RAC acetonitrile solution (100.0 mg L
1), which were then shaken at 300 rpm for 5, 10, 20, 40, 60, 80 and 120 min at 22 °C, respectively. Subsequently, the mixture was

Q¼

ðC 0
C 1 Þ  V m

ð1Þ

where C0 (mg L
1) is the initial concentration of RAC solution, C1 (mg L
1) is the concentration of RAC in the supernatant solution after adsorption, V (mL) is the volume of the initial RAC solution, and m (g) is the mass of Fe3O4@MIPs.

Y. Tang et al. / Food Chemistry 201 (2016) 72–79

2.4.3. Selectivity test To certify the selectivity of Fe3O4@MIPs, the structural analogs (i.e., ISOP, ISOX and TERB) (Fig. S1 for structures) to RAC were used to carry out the selective adsorption experiment. Twenty milligram of Fe3O4@MIPs or Fe3O4@NIPs were added into a 10.0 mL acetonitrile solution (100.0 mg L
1) of RAC, ISOP, TERB, or ISOX, with shaking (300 rpm) for 60 min at 22 °C. The mixture was filtered using a membrane filter (pore size, 0.22 lm) and the filtrate was analyzed for non-extracted RAC, ISOP, TERB and ISOX using HPLC. 2.5. Extraction procedure Fe3O4@MIPs (20.0 mg) was added into a 10.0 mL of RAC acetonitrile solution. The mixture was shaken at 300 rpm for 40 min at 22 °C. Fe3O4@MIPs were then separated from the solution by an external magnetic field. RAC were eluted from the Fe3O4@MIPs with 10.0 mL a mixed solvent of methanol and acetic acid (7:3; v/v) for 30 min. The eluent was collected and evaporated to dryness. The residues were redissolved in 0.5 mL of methanol for further HPLC analysis after filtration with a membrane filter (pore size, 0.22 lm). 2.6. Sample analysis Fresh pork purchased from a local grocery store was selected for the evaluation of the performance of Fe3O4@MIPs. The non-spiked pork sample was first confirmed free of RAC using HPLC-MS. Briefly, minced pork samples (10.0 g) were spiked with 12.5, 50.0 and 200.0 lL of the RAC solution (1.0 mg L
1) separately. The spiked samples were then homogenized using a stirrer at 300 rpm for 5 min and then kept at 4 °C for 12 h. The spiked sample was subsequently extracted with 20.0 mL of acetonitrile and 1.0 mL of potassium carbonate aqueous solution (4.0 mol L
1) using ultrasonic agitation for 30 min. After centrifugation (8157g for 20 min at 4 °C), the supernatant was collected. The extraction procedure was repeated twice and the two-part supernatants were combined and dried using a rotary evaporator. The residues were redissolved in 10.0 mL of acetonitrile solution, and then subjected to the extraction procedure by Fe3O4@MIPs or Fe3O4@NIPs. 3. Results and discussion 3.1. Synthesis of Fe3O4@MIPs The preparation of Fe3O4@MIPs towards RAC included grafting MC onto the surface of magnetic particulates, polymerization of Fe3O4@MIPs, and removal of RAC. The preparation process of Fe3O4@MIPs is shown in Scheme 1. Fe3O4 nanoparticles were synthesized and then modified with APTS and MC, which introduced functional group carbonyl that can bind to RAC through hydrogen bonding and double bonds and further co-polymerize with cross-linker EGDMA. These two chemical groups offer specificity and stability of Fe3O4@MIPs. To investigate the influence of the amount of cross-linker on specific adsorption capability of Fe3O4@MIPs towards RAC, molar ratios of 1:5, 1:10, 1:20, 1:30, and 1:40 between template molecular (RAC) and cross-linker (EGDMA) were selected for the preparation of Fe3O4@MIPs. Fe3O4@NIPs were prepared using the same procedure in the absence of template RAC. To evaluate the property of the polymers obtained, 20.0 mg of Fe3O4@MIPs or Fe3O4@NIPs were suspended in 10.0 mL of RAC acetonitrile solution (100.0 mg L
1), which was then mechanically shaken at 300 rpm for 24 h at 22 °C. The filtrate was measured for the unextracted RAC by HPLC after filtration with a membrane filter (pore size, 0.22 lm).

75

The adsorption capacities of Fe3O4@MIPs and Fe3O4@NIPs (Fig. S2) showed that the molar ratio of template to cross linker of 1:20 was the best and the adsorption capacity of Fe3O4@MIPs (10.05 mg g
1) was 2.87-fold higher than that of the corresponding Fe3O4@NIPs. The results suggested that the cross-linkers play an important role during the polymerization process, which can firmly fix the binding sites for template molecules in a threedimensional structure of the polymer. Nevertheless, excessive amounts of cross-linkers also result in challenging removal of the template from the polymer matrices due to steric hindrance (Tang et al., 2015). Therefore, the optimum ratio of template (RAC) to cross-linker of 1:20 was selected to synthesize Fe3O4@MIPs. 3.2. Characterization of Fe3O4@MIPs

3.2.1. FT-IR spectroscopic analysis FT-IR spectra of Fe3O4 (a), APTS-modified Fe3O4 (b), MCmodified Fe3O4 (c) and Fe3O4@MIPs (d) are shown in Fig. S3. The absorption peak 582 cm
1 (a) is attributed to the vibration of Fe–O band in Fe3O4 magnetic nanoparticles. The absorption band of Fe–O–Si at 588 cm
1, the band of N–H at 3442 cm
1 and 1635 cm
1 were found in APTS-modified Fe3O4 (b), and the stretching vibration absorption of the band of C@C at 1618 cm
1, the band of C@O at 1396 cm
1, the band of N–H at 1548 cm
1 were found in MC-modified Fe3O4 (c). These results indicated that MC was grafted on the surface of Fe3O4 nanoparticles. In addition, the band at 588 cm
1 was also observed in Fe3O4@MIPs spectrum (d), indicating that the magnetic molecularly imprinted polymers were successfully synthesized. 3.2.2. VSM analysis To evaluate the magnetic properties of Fe3O4@MIPs, VSM was employed in this study and the magnetic hysteresis loops are shown in Fig. S4 (A). Obviously, there was no magnetic hysteresis, suggesting that Fe3O4 nanoparticles and Fe3O4@MIPs were superparamagnetic (Wang, Wang, He, Zhang, & Chen, 2009). The saturation magnetization of Fe3O4@MIPs (16.64 emu g
1) decreased markedly in comparison to that of Fe3O4 nanoparticles (81.86 emu g
1) due to shielding effect of the polymers shell on the surface of Fe3O4. Fig. S4 (B) showed that the Fe3O4@MIPs remained strong and sufficient magnetism to meet the need of fast separation of the adsorbent from samples by a strong magnet. Further, this superparamagnetism of Fe3O4@MIPs is good to regenerate following adsorption of targeted molecules (Xu et al., 2012). 3.2.3. XRD analysis XRD spectra of the synthesized Fe3O4 nanoparticles, APTSmodified Fe3O4, MC-modified Fe3O4 and Fe3O4@MIPs are shown in Fig.1. There were six relatively strong diffraction peaks in the 2h region of 20–80°, 220, 311, 400, 422, 511 and 440, which matched well to the magnetite data in the database of JCPDS (JCPDS Card: 19-629) file (Lu et al., 2012). The peak positions of APTS-modified Fe3O4, MC-modified Fe3O4 and Fe3O4@MIPs were similar to that of Fe3O4, validating that APTS-modified Fe3O4, MC-modified Fe3O4 and Fe3O4@MIPs were composed of Fe3O4, and the synthesized procedure did not result in any variations in Fe3O4 core crystal structure (Li et al., 2010). 3.2.4. TEM analysis The morphologies of Fe3O4 and Fe3O4@MIPs were obtained using TEM (Fig. 2). The diameter of Fe3O4 nanoparticles ranged between 10 and 25 nm (Fig. 2A) and the diameter of Fe3O4@MIPs ranged between 15 and 40 nm (Fig. 2B). Fe3O4 core and MIPs shell

76

Y. Tang et al. / Food Chemistry 201 (2016) 72–79

Fig. 1. XRD patterns of Fe3O4 (a), APTS-modified Fe3O4 (b), MC-modified Fe3O4 (c), and Fe3O4@MIPs (d).

Fig. 2. TEM images of Fe3O4 nanoparticles (A) and Fe3O4@MIPs (B).

can be clearly distinguished from the image. These results suggested that the paramagnetic MIPs were successfully synthesized. 3.3. Adsorption properties of Fe3O4@MIPs 3.3.1. Uptake kinetics The results of uptake kinetics of Fe3O4@MIPs to RAC in acetonitrile are shown in Fig. 3(A). The adsorption capacity of Fe3O4@MIPs increased rapidly during the first 10 min, and the binding equilibrium reached within 20 min. The rapid adsorption kinetics is advantageous in saving time for the extraction process. The pseudo-second-order kinetic model was used to describe the adsorption process according to the equation:

t 1 t ¼ þ Q t kQ 2e Q e

ð2Þ

where Qt is the adsorption capacity (mg g
1) at a specific time, Qe is the adsorption capacity at equilibrium (mg g
1), and k is the rate constant of second-order sorption (mg g
1 s
1). The correlation coefficient (R2) of the fitting curve obtained by t/Qt versus t (Fig. 3 (A)) was 0.9974. In addition, the Qe value (11.1 mg g
1) calculated from the pseudo-second-order kinetic model agreed well with the experimental Qe value (10.0 mg g
1). Thus, the adsorption reaction of Fe3O4@MIPs towards RAC can be estimated to be pseudosecond-order kinetic model, and the adsorption process was controlled by chemical adsorption mechanism by sharing electrons between MMIPs and analyte (Hu et al., 2014).

3.3.2. Static adsorption The results of static adsorption of Fe3O4@MIPs and Fe3O4@NIPs towards RAC with different initial concentrations are shown in

Y. Tang et al. / Food Chemistry 201 (2016) 72–79

77

where Cfree is the equilibrium concentration of RAC (mg L
1), Q is the adsorption amount at equilibrium (mg g
1), Qmax is the maximum adsorption capacity (mg g
1), Kd is the equilibrium dissociation constant (mg L
1), m is the adsorption intensity or surface heterogeneity, a is the adsorption capacity of RAC (mg g
1), KL is the Langmuir adsorption equilibrium constant (L mg
1). The relationship between Q/Cfree and Q could be expressed using two straight lines from Scatchard analysis, indicating that two different types of binding sites existed in MMIPs (Fig. S5). Kd and Qmax values were determined from the slopes and intercepts of the linear regression formulas. Qmax and Kd of the high affinity sites were 13.23 mg g
1 and 32.669 mg L
1, while Qmax and Kd of the low affinity sites were 0.82 mg g
1 and 0.084 mg L
1, respectively. The isotherm parameters obtained from the linear analysis of Freundlich and Langmuri models are listed in Table S1. The results indicated that the binding process of RAC onto the Fe3O4@ MIPs fitted well the Freundlich isotherm model (R2 = 0.9807). Thus, MMIPs possessed a heterogeneous binding site distribution. 3.3.3. Selectivity of the Fe3O4@MIPs ISOP, ISOX, TERB and RAC were selected to test the selectivity of Fe3O4@MIPs. The adsorption capacity of Fe3O4@MIPs towards RAC was much higher than the adsorption capacity towards ISOP, ISOX, and TERB (Fig. S6 and Table S2). The parameters of Kd, K, and K0 were used to estimate the competitive selective capability of MIPs and NIPs. Kd indicated the distribution coefficient. The larger the value of Kd is, the stronger the adsorption capability of a substance would be. Kd was calculated to be 0.103 mL g
1 of RAC for Fe3O4@ MIPs and 0.031 mL g
1 of RAC for Fe3O4@NIPs. K suggested the selectivity between the target molecule and its structural analogues. K value of ISOP, ISOX, and TERB for Fe3O4@MIPs was 2.943, 14.714, and 1.471, respectively while K value of ISOP, ISOX, and TERB for Fe3O4@NIPs was 1.938, 31.000, and 0.517, respectively (Table S2). K0 value (the relative selectivity coefficient) of ISOP, ISOX, and TERB was 1.519, 0.475, and 2.845, respectively. These results validated that RAC Fe3O4@MIPs were successfully synthesized by the surface imprinting approach and had specific recognition capability towards the target molecules. Fig. 3. Uptake kinetics plot of Fe3O4@MIPs and pseudo-second-order kinetic model for the adsorption of RAC onto Fe3O4@MIPs (A), adsorption isotherms of Fe3O4@MIPs and Fe3O4@NIPs (B).

Fig. 3(B). Fe3O4@MIPs had a stronger memory function and a higher adsorption capacity towards RAC than Fe3O4@NIPs. The adsorption capacity of Fe3O4@MIPs and Fe3O4@NIPs increased along with the increase of initial concentration of RAC and reached saturation at a higher concentration (>100.0 mg L
1). The binding capacity (Qmax) of Fe3O4@MIPs was determined to be 10.85 mg g
1. Obviously, the equilibrium adsorption amounts of Fe3O4@MIPs towards RAC were always higher than that of Fe3O4@NIPs, indicating that the special binding sites were created during polymerization. The Scatchard (Xiao et al., 2013), Freundlich (Chen, Xie, & Shi, 2013), and Langmuir (Zhu et al., 2013) isotherm models were used to evaluate the adsorption properties of MMIPs in the current study. The linear form of Freundlich, Langmuir, and Scatchard isotherm model was expressed by the following equations, respectively,

Q Q Q ¼ max
C free Kd Kd

ð3Þ

lg Q ¼ m lg C free þ lg a

ð4Þ

C free C free 1 þ ¼ Q Q max Q max K L

ð5Þ

3.4. Optimization of extraction conditions To apply Fe3O4@MIPs for the extraction and enrichment of RAC in an optimal condition, extraction solvent and incubation time were firstly optimized. Fe3O4@MIPs had a satisfactory adsorption rate in acetonitrile (99%), which was better than using water, ethanol, or methanol as the extraction solvent. The recovery of RAC showed no obvious improvement when the incubation time was over 40 min (Fig. 4A). To study the effect of eluting solvent, different eluents (i.e., methanol, acetonitrile, chloroform, ethanol, toluene, hexane, and acetone) were evaluated. Methanol was more effective in extracting RAC from Fe3O4@MIPs than other solvents. To receive the highest RAC recovery, a mixture of methanol/acetic acid (9:1, 8:2, 7:3, 6:4, 5:5; v/v) was employed because acetic acid can reduce the binding of template to MIPs (Stafiej, Pyrzynska, & Regan, 2007). Satisfactory recovery (98%) was obtained when a mixture of methanol/acetic acid (7:3; v/v) was adopted as elution solvent (Fig. 4B). Different desorption times (5, 10, 15, 20, 30, 45, 60 min) were investigated and 30 min was sufficient to accomplish desorption of RAC from Fe3O4@MIPs (Fig. S7). 3.5. Method validation Method validation was carried out under USP (USP 29/NF 24, 2006) and ICH (ICH Q2, 2005) guidelines, including linearity, range,

78

Y. Tang et al. / Food Chemistry 201 (2016) 72–79

3.5.2. Accuracy and precision The recovery studies were carried out to determine the accuracy. Each sample was spiked with RAC at three different concentration levels. The recoveries of RAC ranged from 73.60% to 94.50% with relative standard deviations (RSDs) ranging from 3.86% to 10.10% (Table 1). Further, the accuracy of the developed method was validated by parallel analysis of the spiked samples using enzyme linked immunosorbent assay (ELISA) (Table 1). Precision of the method was also evaluated by measuring RSD of intra- and inter-day tests. RSDs of intra-day tests ranging from 5.72% to 10.23% were obtained by analyzing spiked pork samples (1.25, 5.0 and 20.0 lg kg
1) six times per day. RSDs of interday tests ranging from 4.51% to 11.17% were obtained by analyzing spiked pork samples (1.25, 5.0 and 20.0 lg kg
1) over seven days. 3.5.3. Specificity and robustness The specificity of this method was determined by observing interferences from the pork samples such as protein, fat and carbohydrate. The test results indicated a good selectivity of the developed method to determine of RAC in pork samples without interferences. To evaluate the robustness of the proposed method, deliberate change in chromatographic condition were carried out. The assay results were found to be non-affected by changes in flow rate (±5%), column temperature (±3 °C), and mobile-phase composition (±2%).

Fig. 4. Recoveries of extraction at different extraction time (A) and different elute solvent (B).

accuracy, precision, specificity, detection limit and quantitation limit.

3.5.4. Detection and quantification limit Limit of detection (LOD) and limit of quantification (LOQ) were determined using the formula based on the standard deviation of the response and the slope of the calibration curve. LOD and LOQ, defined standard deviation of the response-to-slope ratio of three and ten, were 0.05 and 0.17 lg kg
1, respectively. Compared with the results obtained from the method of SPE (using Oasis MCX cartridge) coupled with HPLC-UV detector for the analysis of RAC (Table S3), our currently developed method had a lower detection limit and wider linear range. The recovery and (or) LOD was in similar to SPE based on the MIP coupled with different detection techniques. In addition, our sample preparation process was convenient due to the use of magnetic separation. 4. Conclusions

3.5.1. Linearity and range Standard solutions containing 1.0 mg L
1 of RAC was prepared in acetonitrile and eight different concentrations of the RAC standard solutions were spiked into pork samples (0.5–100.0 lg kg
1), which could be used for MMIPs extraction coupled with HPLC determination. Calibration curve of RAC concentration in spiked samples versus peak area was plotted. The regression equation, y = 0.998x + 0.039, where y is the peak area (excitation: 225 nm, emission: 306 nm) and x is the concentration of RAC (lg kg
1), was obtained. The linearity range was determined to be 0.5– 100.0 lg kg
1 with the correlation coefficient of 0.9963.

In the current study, Fe3O4@MIPs were prepared using surface imprinting approach based upon magnetic Fe3O4 nanoparticles modified with APTS and MC. The synthesized polymers had high adsorption capacity, good selectivity, and fast binding kinetics towards RAC, and can be easily collected using an external magnetic field without extensive centrifugation and/or filtration. Further, an analytical method for measuring RAC in pork samples by using RAC Fe3O4@MIPs as sorbent along with HPLC-FD detection was developed with good recovery and high reproducibility.

Table 1 Determination of RAC in pork samples by MMIPs-HPLC and ELISA (n = 3). Sample

Pork

Spiked levels (lg kg
1)

1.25 5.00 20.00

MMIPs-HPLC

ELISA

Found (lg kg
1)

Recovery (%)

RSD (%)

Found (lg kg
1)

Recovery (%)

RSD (%)

0.92 4.58 18.90

73.60 91.62 94.50

10.10 8.40 3.86

0.95 4.51 19.01

76.00 90.20 95.05

9.56 5.23 5.18

Y. Tang et al. / Food Chemistry 201 (2016) 72–79

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31201370), Scientific Research Foundation for Doctor of Science and Technology Department of Liaoning Province (Grant No. 20121080) and the National Key Technologies R&D Program of China during the 12th Five-Year Period (2012BAD29B06). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2016. 01.070. References Antignac, J. P., Marchand, P., Le Bizec, B., & Andre, F. (2002). Identification of ractopamine residues in tissue and urine samples at ultra-trace level using liquid chromatography-positive electrospray tandem mass spectrometry. Journal of Chromatography B, 774, 59–66. Blanca, J., Muñoz, P., Morgado, M., Méndez, N., Aranda, A., Reuvers, T., et al. (2005). Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by liquid chromatography tandem mass spectrometry. Analytica Chimica Acta, 529, 199–205. Bocca, B., Fiori, M., Cartoni, C., & Brambilla, G. (2003). Simultaneous determination of zilpaterol and other beta agonists in calf eye by gas chromatography/tandem mass spectrometry. Journal of AOAC International, 86, 8–14. Chen, F. F., Xie, X. Y., & Shi, Y. P. (2013). Preparation of magnetic molecularly imprinted polymer for selective recognition of resveratrol in wine. Journal of Chromatography A, 1300, 112–118. Fang, G. Z., Lu, J. P., Pan, M. F., Li, W. W., Ren, L., & Wang, S. (2011). Substitution of antibody with molecularly imprinted film in enzyme-linked immunosorbent assay for determination of trace ractopamine in urine and pork samples. Food Analytical Methods, 4, 590–597. Gao, H. F., Han, J., Yang, S. J., Wang, Z. X., Wang, L., & Fu, Z. F. (2014). Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol. Analytica Chimica Acta, 840, 68–74. Gao, R., Kong, X., Wang, X., He, X., Chen, L., & Zhang, Y. (2011). Preparation and characterization of uniformly sized molecularly imprinted polymers functionalized with core–shell magnetic nanoparticles for the recognition and enrichment of protein. Journal of Materials Chemistry, 21, 17863–17871. He, L., Su, Y., Zeng, Z., Liu, Y., & Huang, X. (2007). Determination of ractopamine and clenbuterol in feeds by gas chromatography–mass spectrometry. Animal Feed Science Technology, 132, 316–323. Hiratsuka, Y., Funaya, N., Matsunaga, H., & Haginaka, J. (2013). Preparation of magnetic molecularly imprinted polymers for bisphenol A and its analogues and their application to the assay of bisphenol A in river water. Journal of Pharmaceutical Biomedical Analysis, 75, 180–185. Hu, C. H., Deng, J., Zhao, Y. B., Xia, L. S., Huang, K. H., Ju, S. Q., et al. (2014). A novel core–shell magnetic nano-sorbent with surface molecularly imprinted polymer coating for the selective solid phase extraction of dimetridazole. Food Chemistry, 158, 366–373. ICH, Q2 (R1), Harmonised tripartite guideline (2005). Validation of analytical procedure: Text and methodology. In: International Conference on Harmonization, Geneva. Jin, J., Lai, W., Xiong, Y., Chen, Y., & Liu, W. (2008). Colloidal gold-based immunochromatographic assay for detection of ractopamine in swine urine samples. Journal of Biotechnology, 136, S754. Juan, C., Igualada, C., Moragues, F., León, N., & Mañes, J. (2010). Development and validation of a liquid chromatography tandem mass spectrometry method for the analysis of b-agonists in animal feed and drinking water. Journal of Chromatography A, 1217, 6061–6068. Kong, X., Gao, R., He, X., Chen, L., & Zhang, Y. (2012). Synthesis and characterization of the core–shell magnetic molecularly imprinted polymers (Fe3O4@MIPs) adsorbents for effective extraction and determination of sulfonamides in the poultry feed. Journal of Chromatography A, 1245, 8–16. Li, Y., Li, X., Chu, J., Dong, C., Qi, J., & Yuan, Y. (2010). Synthesis of core–shell magnetic molecular imprinted polymer by the surface RAFT polymerization for the fast and selective removal of endocrine disrupting chemicals from aqueous solutions. Environmental Pollution, 158, 2317–2323. Liu, H. L., Liu, D. R., Fang, G. Z., Liu, F. F., Liu, C. C., Yang, Y. K., et al. (2013). A novel dual-function molecularly imprinted polymer on CdTe/ZnS quantum dots for highly selective and sensitive determination of ractopamine. Analytica Chimica Acta, 762, 76–82. Lu, F., Sun, M., Fan, L., Qiu, H., Li, X., & Luo, C. (2012). Flow injection chemiluminescence sensor based on core-shell magnetic molecularly imprinted nanoparticles for determination of chrysoidine in food samples. Sensors and Actuators B Chemical, 173, 591–598.

79

Mo, Z. L., Zhang, C., Guo, R. B., Meng, S. J., & Zhang, J. X. (2011). Synthesis of Fe3O4 nanoparticles using controlled ammonia vapor diffusion under ultrasonic irradiation. Industrial and Engineering Chemistry Research, 50, 3534–3539. Nielen, M., Lasaroms, J., Essers, M., Oosterink, J., Meijer, T., Sanders, M., et al. (2008). Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography eletrospray ionisation tandem mass spectrometry. Analytical and Bioanalytical Chemistry, 391, 199–210. Shelver, W. L., & Smith, D. J. (2002). Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals. Journal of Agricultural and Food Chemistry, 50, 2742–2747. Shelver, W. L., Smith, D. J., & Berry, E. S. (2000). Production and characterization of a monoclonal antibody against the b-adrenergic agonist ractopamine. Journal of Agricultural and Food Chemistry, 48, 4020–4026. Shen, L., & He, P. (2007). An electrochemical immunosensor based on agarose hydrogel films for rapid determination of ractopamine. Electrochemistry Communications, 9, 657–662. Shishani, E. I., Chai, S. C., Jamokha, S., Aznar, G., & Hoffman, M. K. (2003). Determination of ractopamine in animal tissues by liquid chromatographyfluorescence and liquid chromatography/tandem mass spectrometry. Analytica Chimica Acta, 483, 137–145. Stafiej, A., Pyrzynska, K., & Regan, F. (2007). Determination of anti-inflammatory drugs and estrogens in water by HPLC with UV detection. Journal of Separation Science, 30, 985–991. Su, X. M., Li, X. Y., Li, J. J., Liu, M., Lei, F. H., Tan, X. C., et al. (2015). Synthesis and characterization of core–shell magnetic molecularly imprinted polymers for solid-phase extraction and determination of Rhodamine B in food. Food Chemistry, 171, 292–297. Tang, Y. W., Fang, G. Z., Wang, S., & Li, J. L. (2011). Covalent imprinted polymer for selective and rapid enrichment of ractopamine by a noncovalent approach. Analytical and Bioanalytical Chemistry, 401, 2275–2282. Tang, Y. W., Gao, Z. Y., Wang, S., Gao, X., Gao, J. W., Ma, Y., et al. (2015). Upconversion particles coated with molecularly imprinted polymers as fluorescence probe for detection of clenbuterol. Biosensors and Bioelectronics, 71, 44–50. Thompson, C. S., Haughey, S. A., Traynor, I. M., Fodey, T. L., Elliott, C. T., Antignac, J. P., et al. (2008). Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry. Analytica Chimica Acta, 608, 217–225. USP 29/NF 24, the United States Pharmacopoeia (2006). 29 th Rev. and the National Formulary (pp. 1964–1966). United States Pharmacopoeial Convention Inc.: Rockville, MD. Wang, S., Liu, L., Fang, G. Z., Zhang, C., & He, J. X. (2009). Molecularly imprinted polymer for the determination of trace ractopamine in pork using SPE followed by HPLC with fluorescent detection. Journal of Separation Science, 32, 1333–1339. Wang, X., Wang, L., He, X., Zhang, Y., & Chen, L. (2009). A molecularly imprinted polymer-coated nanocomposite of magnetic nanoparticles for estrone recognition. Talanta, 78, 327–332. Wang, W., Zhang, Y., Wang, J., Shi, X., & Ye, J. (2010). Determination of b-agonists in pig feed, pig urine and pig liver using capillary electrophoresis with electrochemical detection. Meat Science, 85, 302–305. Xiao, D., Dramou, P., Xiong, N., He, H., Li, H., Yuan, D., et al. (2013). Development of novel molecularly imprinted magnetic solid-phase extraction materials based on magnetic carbon nanotubes and their application for the determination of gatifloxacin in serum samples coupled with high performance liquid chromatography. Journal of Chromatography A, 1274, 44–53. Xu, L., Pan, J., Dai, J., Li, X., Hang, H., Cao, Z., et al. (2012). Preparation of thermalresponsive magnetic molecularly imprinted polymers for selective removal of antibiotics from aqueous solution. Journal of Hazardous Materials, 233–234, 48–56. Ying, Y., Pi, X., Wu, P., Chen, H., Zhu, C., & Ren, Y. (2006). Determination of ractopamine residues in animal tissues by solid phase extraction-high performance liquid chromatography. Chinese Journal of Analytical Chemistry, 34, 143. Zeng, H., Wang, Y., Nie, C., Kong, J., & Liu, X. (2012). Preparation of magnetic molecularly imprinted polymers for separating rutin from Chinese medicinal plants. Analyst, 137, 2503–2512. Zhang, Q. L., Ni, Y., & Kokot, S. (2010). Molecular spectroscopic studies on the interaction between ractopamine and bovine serum albumin. Journal of Pharmaceutical and Biomedical Analysis, 52, 280–288. Zhang, H. F., & Shi, Y. P. (2012). Magnetic retrieval of chitosan: Extraction of bioactive constituents from green tea beverage samples. Analyst, 137, 910–916. Zhang, Q. J., Su, Y. J., He, Q. Q., Shen, X. G., He, L. M., Zhang, N., et al. (2011). Molecularly imprinted solid-phase extraction for the selective HPLC determination of ractopamine in pig urine. Journal of Separation Science, 34, 3399–3409. Zhang, M. Z., Wang, M. Z., Chen, Z. L., Fang, J. H., Fang, M. M., Liu, J., et al. (2009). Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine. Analytical and Bioanalytical Chemistry, 395, 2591–2599. Zhu, S., Gan, N., Pan, D., Li, Y., Yang, T., Hu, F., et al. (2013). Extraction of tributyltin by magnetic molecularly imprinted polymers. Microchimica Acta, 180, 545–553.