# Preparation of Magnetic Molecularly Imprinted Polymers for Selective Recognition and Determination of Clenbuterol in Pork Samples

Authors: Yingjuan Zhao; Zufei Feng; Sijin Li; Yan Lu; Yangfan Hu; Hua Fan; Xinyue Zhai
Journal: Journal of Chemistry
 (2020)

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## Abstract

Magnetic molecularly imprinted polymer (MMIP) was successfully synthesized with acrylamide as a functional monomer and clenbuterol (CLB) as a template molecule. The synthesized MMIPs were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectrometry (FT-IR). MMIPs were used to identify and bind CLB as a solid phase extraction material. The experiment data were fitted by the Freundlich isotherm adsorption model. The results show that MMIPs have excellent recognition performance for CLB. MMIPs were successfully applied as adsorbents to preconcentrated CLB in pork samples and detected by HPLC with UV. The limit of detection (LOD) and limit of quantification (LOQ) were 4.27μg/L and 14.2 μg/L, respectively. The spiked recovery rates ranged from 94.44% to 102.29%. Therefore, the prepared MMIPs can be used for selective preconcentration of CLB content in complex animal-derived food samples.


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## Body

## 1. Introduction

Clenbuterol (CLB) is one of the most commonβ-adrenergic agonists that can be used in the treatment of diseases such as bronchial asthma and chronic bronchitis as a bronchodilator agent in the clinic treatment [1–3]. Moreover, a large amount of CLB can be used as feed additives to significantly promote the growth of the animal and increase the lean rate. It promotes the synthesis of proteins in muscles, especially skeletal muscles, and inhibits the synthesis of fat by changing the metabolic pathways in animals, thereby accelerating the growth rate, increasing the lean meat and improving the carcass quality. Therefore, CLB is also known as a lean meat agent [4–6].As a feed additive, CLB can be used to increase the lean rate by using a large dose, which is usually more than 10 times that of human medicine. However, a large dosage, long use time, and slow metabolism will lead to a problem which is undesirable drug residues in animal-originated foodstuffs [7]. Long-term ingesting CLB from animal foods can cause abnormal physiological reactions such as dizziness, vomiting, muscle tremors, and even death [8]. In addition, illegal and uncontrolled use of CLB can also increase the environmental burden and cause environmental pollution [9]. The Chinese government clearly stipulates that the use of clenbuterol in pig breeding is prohibited. According to the Chinese government standard, clenbuterol should be no more than 0.01 mg kg−1. Although many countries and regions have forbidden the use of CLB in livestock production, it has been still added in the fodder for animals to seek benefits [10–12]. Therefore, it is urgent to establish a simple, rapid, and efficient method to detect the residue of CLB to ensure food safety.Currently, there have been several reports on methods for determining residue of CLB, such as biosensors, immunology, and chromatography [13]. However, all of the above methods have more or fewer shortcomings. The limitation of biosensors [14] is the lack of stability and reproducibility. Immunological methods, such as flow immunoassay [15], immunochromatographic assay (ICA) [16], enzyme-linked immunosorbent assay (ELISA) [17], and time-resolved fluoroimmunoassay (TRFIA) [18], are less sensitive while the preparation, stability, and storage of antibodies always imply high costs [13]. Although the sample could be separated by chromatography and detected accurately by MS, time-consuming pretreatment, complex operation, and the high cost of the whole experiment are often cited as problems. Also, chromatography-mass spectrometry suffers from drawbacks of matrix effects which affect the selectivity, detection limits, maintenance frequency, and quantitative aspects. The most important shortcoming of these procedures is the lack of selectivity for target molecules. Moreover, the pretreatment of samples by these methods are mostly complicated.In order to avoid the above weakness and promote advantages, researchers have been trying to establish new methods. Recently, molecular imprinting technology (MIT) has received more and more attention and is used in many fields, especially in solid phase extraction [19, 20]. It is a technique for adsorbing target molecules by preparing molecularly imprinted polymers (MIPs) with specificity and selectivity [21].MIP is a three-dimensional network polymer with specific recognition and selective adsorption, which is usually synthesized by free-radical polymerization. First, the template molecule interacts with the functional monomer to form tailor-made binding sites; and then crosslinkers are distributed around the template molecules, which polymerize the highly crosslinked polymers. After the crosslinking reaction, the template molecules are eluted with a special solvent, the binding cavities which are capable of specifically recognizing the target molecules are revealed in the crosslinked material [22–26].Alternatively, magnetic separation technology has also received a lot of attention due to its being fast, economical, and efficient [27–29]. When the magnetic separation technology and molecular imprinting technology are combined, the magnetic molecularly imprinted polymers (MMIPs) are formed. These MMIPs not only show excellent specific selective binding for target analytes but also can be quickly separated and easily recovered from the samples by an external magnet without centrifugation or filtration.In this work, the MMIPs were synthesized and characterized as selective adsorbents for CLB. The Fe3O4 nanoparticles are encapsulated in the polymer to give it magnetic properties.The kinetic studies and adsorption capacity on CLB adsorption by MMIPs were evaluated in detail. Finally, CLB was successfully preconcentrated by prepared MMIPs and detected with HPLC-UV in actual pork samples.


## 2. Materials and Methods

### 2.1. Materials and Reagents

Clenbuterol was purchased from Alfa Aesar (Tianjin, China). Ferric chloride (FeCl3·6H2O) and ferrous chloride (FeCl2·4H2O) were obtained from Fuchen Chemical Reagents Factory (Tianjin, China). Methacrylic acid (MAA), acrylamide (AM) styrene (St), ethylene glycol dimethacrylamide (EGDMA), and 2,2′-azobisissobutyronitrile (AIBN) were from Alfa Aesar (Tianjin, China). Polyethylene glycol (PEG-6000) and ammonium hydroxide were obtained from Tianjin Chemical Reagent Co. (Tianjin, China). Chromatographic grade methanol and acetonitrile were acquired from Merck Co. (Darmstadt, Germany). All other organic solvents and inorganic reagents were of analytical reagent grade and provided by local suppliers. Pock was purchased from the local supermarket (Xi’an, China). All HPLC solutions were filtered using a filter (HA-0.45 μm) before being injected into the HPLC system.


### 2.2. Instrumentation

The HPLC analyses system consisted of a Shimadzu LC-20A HPLC system, CBM-102 UV/Vis detector (Shimadzu, Japan) and a chromatographic workstation (Shimadzu, Japan). A Sino-Chrom ODS-AP column (4.6 mm × 150 mm, 5μm) was applied. The mobile phase was 0.3% of triethylamine in aqueous solution (pH = 3 with phosphoric acid) and acetonitrile (85 : 15, v/v). The wavelength of detection, flow rate, and column oven temperature were 243 nm, 1.0 mL/min, and 30°C, respectively.The micromorphology of MMIPs and MNIPs was detected by field emission scanning electron microscopy (SEM, JSM-6700F, TESCAN Co., Ltd., Czech). The infrared spectrum was measured by Fourier Transform Infrared spectrometer IR-960 (Rui'an Technology Co., Ltd., Tianjin, China). X-ray diffraction was carried out using XRD-6100 (Shimadzu, Japan). The shaken bed used the KYC 100B rocking incubator. The ultrasonic cleaner uses KQ-300B (Ultrasonic Instrument Co., Ltd., Kunshan, China).


### 2.3. Preparation of Fe3O4 Magnetic Particles

Fe3O4 magnetic particles were synthesized by improved chemical coprecipitation method according to the previous study [30, 31]. Briefly, FeCl3∙6H2O (5.11 g) and FeCl2∙4H2O (1.83 g) were dissolved in 80 mL of deoxygenated water in a 250 mL four-necked flask, and the mixture was constantly stirred under nitrogen protection.Then 60 mL of ammonium hydroxide solution (5%) was added drop by drop when the temperature was raised to 80°C. The mixture was stirred vigorously for 60 min at 80°C. Subsequently, the Fe3O4 nanoparticles were collected by using an external magnet and washed with deionized water several times until the pH = 7.The PEG-modified Fe3O4 was prepared according to previous work with some minor modifications. The prepared Fe3O4 (2.0 g) and PEG (10.0 g) were dissolved in deoxygenated water (30 mL) by stirring for 30 min and sonicating for 20 min to obtain the homogeneously dispersed solution, which was stored in a dark place for later use.


### 2.4. Preparation of the MMIPs

The MMIPs were prepared as follows: firstly, an appropriate amount of clenbuterol (0.5 mmol), as the template molecule, and AM (4 mmol), as the functional monomer, were added to a glass flask containing 25 mL acetonitrile. Then, the mixture was stored in the dark for 18 h at room temperature to obtain the prepolymerization solution. After that, the prepolymerization solution and PEG-Fe3O4 particles were dispersed in 80 mL of doubly distilled water and copolymer monomer (St, 87.4 mmol), crosslinker (EDGMA, 30 mmol), and initiator (AIBN, 0.6 mmol) were added to the mixture in a 250 mL three-neck flask. The processed mixture was placed in a water bath and stirred for the polymerization at 70°C under nitrogen protection for 12 h. After polymerization, MMIPs were collected by an extra magnetic field and washed by a mixture of methanol-acetic acid (9 : 1, v/v) to remove the templates, and then rinsed with methanol. The synthesized MMIP (30 mg) was added to methanol (1 mL). After shaking for 15 min, HPLC was used to detect whether the template molecules were removed. Finally, the MMIPs were dried in a vacuum. For comparison, the nonimprinted polymers (NIPs) were also prepared as the same method mentioned above in the absence of the template.


### 2.5. Adsorption Capacity Experiment

To evaluate adsorption properties of MMIPs for clenbuterol, the batch adsorption test was performed as follows: 30 mg MMIPs or MNIPs were immersed into the 2 mL centrifuge tube containing methanol solution of clenbuterol (100–300μg L−1, 1 mL) followed by shaking for 15 min at room temperature. MMIPs or MNIPs were separated from the solution by using a magnet. The concentration of clenbuterol in the supernatant was analyzed by HPLC-UV after ﬁltration through a 0.45 μm microporous membrane. In order to evaluate the binding parameters of MMIP and MNIP, the Freundlich isotherm (FI) model was further used to further process the data from the adsorption experiments.In adsorption kinetics experiments, 30 mg of MMIPs and MNIPs were added to 1 mL of clenbuterol methanol solution (160μg L−1). The mixture was shaken mechanically for 5, 10, 15, 20, 30, 45, and 60 min in a shaken bed at room temperature to choose the optimal time of competitive adsorption.


### 2.6. Determination of CLB in Pork Samples

One hundred grams of pork (obtained from a local market) was broken with a tissue shredder in 100 mL pure water. Then MMIPs (30 mg) were added to the real sample homogenate solution (1 mL), followed by being shaken at 25°C for 15 min. And then, MMIPs were separated from the solution using an external magnet. The CLB was eluted from the MMIPs using 1 mL of methanol-acetic acid (9 : 1, v/v) solution. The eluted solution was filtered by a membrane filter (0.25μm), dried by nitrogen flow, and dissolved in 0.1 mL of methanol for the detection by HPLC-UV analysis.


## 2.1. Materials and Reagents

Clenbuterol was purchased from Alfa Aesar (Tianjin, China). Ferric chloride (FeCl3·6H2O) and ferrous chloride (FeCl2·4H2O) were obtained from Fuchen Chemical Reagents Factory (Tianjin, China). Methacrylic acid (MAA), acrylamide (AM) styrene (St), ethylene glycol dimethacrylamide (EGDMA), and 2,2′-azobisissobutyronitrile (AIBN) were from Alfa Aesar (Tianjin, China). Polyethylene glycol (PEG-6000) and ammonium hydroxide were obtained from Tianjin Chemical Reagent Co. (Tianjin, China). Chromatographic grade methanol and acetonitrile were acquired from Merck Co. (Darmstadt, Germany). All other organic solvents and inorganic reagents were of analytical reagent grade and provided by local suppliers. Pock was purchased from the local supermarket (Xi’an, China). All HPLC solutions were filtered using a filter (HA-0.45 μm) before being injected into the HPLC system.


## 2.2. Instrumentation

The HPLC analyses system consisted of a Shimadzu LC-20A HPLC system, CBM-102 UV/Vis detector (Shimadzu, Japan) and a chromatographic workstation (Shimadzu, Japan). A Sino-Chrom ODS-AP column (4.6 mm × 150 mm, 5μm) was applied. The mobile phase was 0.3% of triethylamine in aqueous solution (pH = 3 with phosphoric acid) and acetonitrile (85 : 15, v/v). The wavelength of detection, flow rate, and column oven temperature were 243 nm, 1.0 mL/min, and 30°C, respectively.The micromorphology of MMIPs and MNIPs was detected by field emission scanning electron microscopy (SEM, JSM-6700F, TESCAN Co., Ltd., Czech). The infrared spectrum was measured by Fourier Transform Infrared spectrometer IR-960 (Rui'an Technology Co., Ltd., Tianjin, China). X-ray diffraction was carried out using XRD-6100 (Shimadzu, Japan). The shaken bed used the KYC 100B rocking incubator. The ultrasonic cleaner uses KQ-300B (Ultrasonic Instrument Co., Ltd., Kunshan, China).


## 2.3. Preparation of Fe3O4 Magnetic Particles

Fe3O4 magnetic particles were synthesized by improved chemical coprecipitation method according to the previous study [30, 31]. Briefly, FeCl3∙6H2O (5.11 g) and FeCl2∙4H2O (1.83 g) were dissolved in 80 mL of deoxygenated water in a 250 mL four-necked flask, and the mixture was constantly stirred under nitrogen protection.Then 60 mL of ammonium hydroxide solution (5%) was added drop by drop when the temperature was raised to 80°C. The mixture was stirred vigorously for 60 min at 80°C. Subsequently, the Fe3O4 nanoparticles were collected by using an external magnet and washed with deionized water several times until the pH = 7.The PEG-modified Fe3O4 was prepared according to previous work with some minor modifications. The prepared Fe3O4 (2.0 g) and PEG (10.0 g) were dissolved in deoxygenated water (30 mL) by stirring for 30 min and sonicating for 20 min to obtain the homogeneously dispersed solution, which was stored in a dark place for later use.


## 2.4. Preparation of the MMIPs

The MMIPs were prepared as follows: firstly, an appropriate amount of clenbuterol (0.5 mmol), as the template molecule, and AM (4 mmol), as the functional monomer, were added to a glass flask containing 25 mL acetonitrile. Then, the mixture was stored in the dark for 18 h at room temperature to obtain the prepolymerization solution. After that, the prepolymerization solution and PEG-Fe3O4 particles were dispersed in 80 mL of doubly distilled water and copolymer monomer (St, 87.4 mmol), crosslinker (EDGMA, 30 mmol), and initiator (AIBN, 0.6 mmol) were added to the mixture in a 250 mL three-neck flask. The processed mixture was placed in a water bath and stirred for the polymerization at 70°C under nitrogen protection for 12 h. After polymerization, MMIPs were collected by an extra magnetic field and washed by a mixture of methanol-acetic acid (9 : 1, v/v) to remove the templates, and then rinsed with methanol. The synthesized MMIP (30 mg) was added to methanol (1 mL). After shaking for 15 min, HPLC was used to detect whether the template molecules were removed. Finally, the MMIPs were dried in a vacuum. For comparison, the nonimprinted polymers (NIPs) were also prepared as the same method mentioned above in the absence of the template.


## 2.5. Adsorption Capacity Experiment

To evaluate adsorption properties of MMIPs for clenbuterol, the batch adsorption test was performed as follows: 30 mg MMIPs or MNIPs were immersed into the 2 mL centrifuge tube containing methanol solution of clenbuterol (100–300μg L−1, 1 mL) followed by shaking for 15 min at room temperature. MMIPs or MNIPs were separated from the solution by using a magnet. The concentration of clenbuterol in the supernatant was analyzed by HPLC-UV after ﬁltration through a 0.45 μm microporous membrane. In order to evaluate the binding parameters of MMIP and MNIP, the Freundlich isotherm (FI) model was further used to further process the data from the adsorption experiments.In adsorption kinetics experiments, 30 mg of MMIPs and MNIPs were added to 1 mL of clenbuterol methanol solution (160μg L−1). The mixture was shaken mechanically for 5, 10, 15, 20, 30, 45, and 60 min in a shaken bed at room temperature to choose the optimal time of competitive adsorption.


## 2.6. Determination of CLB in Pork Samples

One hundred grams of pork (obtained from a local market) was broken with a tissue shredder in 100 mL pure water. Then MMIPs (30 mg) were added to the real sample homogenate solution (1 mL), followed by being shaken at 25°C for 15 min. And then, MMIPs were separated from the solution using an external magnet. The CLB was eluted from the MMIPs using 1 mL of methanol-acetic acid (9 : 1, v/v) solution. The eluted solution was filtered by a membrane filter (0.25μm), dried by nitrogen flow, and dissolved in 0.1 mL of methanol for the detection by HPLC-UV analysis.


## 3. Results and Discussion

### 3.1. Preparation and Characterization of MMIPs

Figure1 shows that the preparation process of MMIPs is mainly divided into two steps. The first is the synthesis of Fe3O4 nanoparticles. At present, the chemical coprecipitation method is widely used to prepare magnetic nanoparticles because of its simple operation and good reproducibility. Therefore, Fe3O4 nanoparticles are prepared by this method with superparamagnetism in this experiment. However, the prepared Fe3O4 nanoparticles are prone to magnetic flocculation, so that the result is that nanoparticles lack monodispersity. Therefore, the surface of Fe3O4 particles was modified with PEG to avoid electrostatic agglomeration.Figure 1
Schematic preparation procedure for CLB MMIPs.In noncovalent molecular imprinting, the interaction between functional monomers and template molecules affects the recognition performance of MMIPs directly. Therefore, acrylamide (AM) and methacrylic acid (MAA) were selected as functional monomers to evaluate optimal one. In theory, both AM and MAA could produce hydrogen bonds with clenbuterol, but the experimental results show that the specific recognition ability of MMIPs synthesized with AM as a functional monomer is stronger. Further, styrene was used as a comonomer in this experiment because the unsaturated bond in its structure contributes to the formation of a crosslinked structure and increases the structural stability of the polymers.SEM was used to measure the morphological features of MMIPs and MNIPs in Figure2. As can be seen from the figure, the surface of the MMIPs is rough and porous, while the surface of the MNIPs is relatively regular. As a result, the porous surface and imprinting sites of the MMIPs are more conducive to specific adsorption than MNIPs.Figure 2
Scanning electron micrographs of the MMIPs and MNIPs: (a) MMIPs; (b) surface of MMIPs; (c) surface of MNIPs.X-ray diffraction patterns of MMIPs and Fe3O4 nanoparticles are shown in Figure 3. The curve shows five strong diffraction peaks at 20–80°, (220), (311), (400), (440), (511) crystal planes, which correspond with the Inorganic Crystal Structure Database (ICSD). The diffraction peaks of MMIPs are similar to Fe3O4 nanoparticles, proving that the polymers are compounds of Fe3O4 nanoparticles.Figure 3
XRD patterns of the Fe3O4 (1) and MMIPs (2).FT-IR was used to analyze the main functional groups of synthesized results. Figure4 shows the infrared spectrum of Fe3O4 nanoparticles, MMIPs, and MNIPs. The typical bands at 3028 and 2933 cm−1 can be attributed to the C-H aromatic stretching vibrations of styrene units. The absorption peak of C = O at 1723.9 cm−1 is due to the crosslinking agent EGDMA reacted with AM. Compared with NIP, weak absorption peaks appeared at 1632.8 and 1599.8 cm−1, which attributed to carbonyl and bending vibration of the imino group in CONH2. It indicated that most AM and crosslinking agent had crosslinking polymerized during the preparation of MIPs, and only some AM residues were found. Compared to curve 3 (black), the band of Fe-O at 547.1 cm−1 was observed in MMIPs, which confirmed that Fe3O4 was implanted into the polymer network successfully.Figure 4
FT-IR spectra of MMIP (red), MNIP (blue), and Fe3O4 magnetic nanoparticles (black).


### 3.2. Adsorption and Analytical Kinetic Evaluation

An adsorption kinetics experiment was performed on MMIPs to select the best extraction time. Figure5(a) shows that the amount of adsorption increased sharply in the first 15 min. The adsorption equilibrium was reached at 15 min, and the maximum adsorption amount was obtained, so 15 min was selected as the extraction time.Figure 5
(a) Effect of adsorption time; (b) effect of desorption time.
(a)(b)Figure5(b) shows the desorption kinetics of CLB by MMIP, and the desorption time was optimized. After 15 min, the desorption ratio is close to 100%. After desorption, MMIPs can be quickly separated and recovered from the sample solution by an external magnetic field.


### 3.3. Adsorption Isotherm Equation

To assess the binding energy, binding site type and distribution between MMIPs or MNIPs and imprinted molecules, adsorption isotherms were adopted using different concentrations of CLB. Figure6(a) shows the adsorption isotherms of MMIPs and MNIPs. When the adsorption reaches equilibrium, there is a certain relationship between the adsorption amount B and the residual amount F. It is possible to observe an increase of adsorption amount of polymer materials with increasing of the initial concentration of CLB, and the adsorption capacity of MMIPs for CLB is greater than that of MNIPs. Some studies have shown that the Freundlich equation has good applicability to noncovalently imprinted materials [30, 32]. Therefore, the Freundlich isothermal (FI) adsorption model was adopted to analyze the data with adsorption isotherms.Figure 6
(a) CLB adsorption isotherms for MMIPs and MNIPs with the corresponding experimental FI for MMIPs and MNIPs; (b) affinity distributions of MMIPs (black curve) and MNIPs (red curve).
(a)(b)The FI equation is expressed as follows [32]:(1)BF=aFm,where B and F are the concentrations of bound and free analyte, respectively; a and m are characteristic parameters of the Freundlich isotherm equation: a is the total number of binding sites, which represents the adsorption capacity of the adsorbent; m is the heterogeneity index with a value from 0–1. The value of m closes to 0 or 1 indicates that the adsorption sites are heterogeneous and homogeneous, respectively. And the value of a and m can be obtained from the following equation:(2)logB=mlogF+loga.The number of binding sites with given affinity (Nk) of a polymer can be calculated by equation (3), where K is the affinity constant, K=1/F.(3)NK=2.303am1−m2K−m.The binding siteNkmin−kmax and the apparent average binding constant Kkmin−kmax per gram of polymer can be calculated from equation (4) and equation (5), respectively.Nkmin−kmax is the number of binding sites per gram of polymer. Kkmin−kmax is apparent average binding constant. They can be calculated from equation (4) and equation (5), respectively, where Kmin=1/Fmax; Kmax=1/Fmin. Fmax and Fmin are the maximum and minimum solute concentrations of the liquid phase at adsorption equilibrium, respectively; a and m are the characteristic parameters of the Freundlich isothermal equation.(4)Nkmin−kmax=a1−m2Kmin−m−Kmax−m,(5)Kkmin−kmax=mm−1Kmin1−m−Kmax1−mKmin−m−Kmax−m.The FI affinity distribution model was further used to analyze the adsorption data. The affinity distribution curve (Figure6(b)) was obtained by plotting the value of Nk and logK, indicating the number of binding sites with binding ability. The value of logK represents the binding energy as the abscissa. It can be seen from the figure that the number of binding sites of MMIPs is always higher than that of MNIPs when given the binding energy within the experimental concentration range. It indicates that there are specific adsorption sites in the MMIPs material, and the imprinting effect is evident.The results are exhibited in Table1. The total number of binding sites and binding constants of MMIPs is higher than MNIPs. The binding sites N, K of MMIPs per gram of polymer are larger than MNIPs. These results indicate that the target molecule plays an important role in the formation of specific sites during the imprinting process.Table 1
Characteristic parameters of the Freundlich isothermal equation.
Fitting parametersMMIPsMNIPsa0.5680.432m0.4260.985Krange (L μmol−1)2.541–6.4421.799–3.184NK (μmol g−1)0.206–0.3070.009–0.017Nkmin−kmax (μmol g−1)0.2200.003Kkmin−kmax (L μmol−1)4.0672.358


### 3.4. Method Validation

In order to verify the accuracy of the method, the linear relationship, correlation coefficient (r), the limit of detection (LOD), the limit of quantification (LOQ), and spiked recovery experiment were investigated. By drawing the standard curve, the linear regression equation is y = 7.724x + 278 (r = 0.9694) (Figure 7). The values of LOD and LOQ were measured to be 3 and 10 times the signal-to-noise ratio (S/N), respectively. Their values were 4.27 and 14.2 μg L−1.Figure 7
The standard curve with linear regression.At the same time, the spiked sample recovery experiment was used to assess the reproducibility and accuracy of the method. Three different concentrations of samples were added to the CLB samples, and the recovery was measured and calculated. The concentration of the base solution is 0.25μmol L−1. The recovery values were calculated by the formula below:(6)Recovery=Found−baseadded×100%.The results were shown in Table2. The recoveries ranged from 94.44 to 102.29%, and the relative standard deviation (RSD) ranged from 3.48 to 7.14%. The same sample was measured 5 times and the RSD was calculated to be 4.93%.Table 2
Accuracy of the method for sample solutions spiked at different concentrates (n = 3).
SamplesAdded (μmol L−1)Found (μmol L−1)Recovery (％a)Average (％)RSD (％)CLB0.180.45111.1194.447.140.180.3977.770.180.4294.440.290.55103.4497.705.380.290.5293.100.290.5396.550.360.5891.6696.293.480.360.62102.770.360.5994.44(7)aFound−baseadded100%.


### 3.5. Analysis of Real Samples

In order to investigate the extraction and enrichment ability of MMIPs on actual samples, we extracted and detected CLB in pork according to the method of section2.5 and realized the analysis and detection of trace components from the matrix.The chromatogram of the pork sample extracted by MMIPs (Line a) and MNIPs (Line b) and direct injection of the sample without enrichment (Line c) are shown in Figure8. Figure 8(a) shows that CLB cannot be detected directly because the concentration is low without enrichment. Figure 8(b) also shows the sample could not be extracted by MNIPs, which proves that its enrichment is nonspecific adsorption. Figure 8(c) shows that a higher peak appeared and highly selective adsorption was achieved after the sample was extracted by MMIPs. The content of CLB in pork was calculated resulting in 9.87 ± 0.48 μg kg−1. The result showed that MMIPs could be used to enrich and detect CLB in actual samples.Figure 8
HPLC-UV chromatograms of pork samples without enrichment (a), enrichment by MNIPs (b) and MMIPs (c).


## 3.1. Preparation and Characterization of MMIPs

Figure1 shows that the preparation process of MMIPs is mainly divided into two steps. The first is the synthesis of Fe3O4 nanoparticles. At present, the chemical coprecipitation method is widely used to prepare magnetic nanoparticles because of its simple operation and good reproducibility. Therefore, Fe3O4 nanoparticles are prepared by this method with superparamagnetism in this experiment. However, the prepared Fe3O4 nanoparticles are prone to magnetic flocculation, so that the result is that nanoparticles lack monodispersity. Therefore, the surface of Fe3O4 particles was modified with PEG to avoid electrostatic agglomeration.Figure 1
Schematic preparation procedure for CLB MMIPs.In noncovalent molecular imprinting, the interaction between functional monomers and template molecules affects the recognition performance of MMIPs directly. Therefore, acrylamide (AM) and methacrylic acid (MAA) were selected as functional monomers to evaluate optimal one. In theory, both AM and MAA could produce hydrogen bonds with clenbuterol, but the experimental results show that the specific recognition ability of MMIPs synthesized with AM as a functional monomer is stronger. Further, styrene was used as a comonomer in this experiment because the unsaturated bond in its structure contributes to the formation of a crosslinked structure and increases the structural stability of the polymers.SEM was used to measure the morphological features of MMIPs and MNIPs in Figure2. As can be seen from the figure, the surface of the MMIPs is rough and porous, while the surface of the MNIPs is relatively regular. As a result, the porous surface and imprinting sites of the MMIPs are more conducive to specific adsorption than MNIPs.Figure 2
Scanning electron micrographs of the MMIPs and MNIPs: (a) MMIPs; (b) surface of MMIPs; (c) surface of MNIPs.X-ray diffraction patterns of MMIPs and Fe3O4 nanoparticles are shown in Figure 3. The curve shows five strong diffraction peaks at 20–80°, (220), (311), (400), (440), (511) crystal planes, which correspond with the Inorganic Crystal Structure Database (ICSD). The diffraction peaks of MMIPs are similar to Fe3O4 nanoparticles, proving that the polymers are compounds of Fe3O4 nanoparticles.Figure 3
XRD patterns of the Fe3O4 (1) and MMIPs (2).FT-IR was used to analyze the main functional groups of synthesized results. Figure4 shows the infrared spectrum of Fe3O4 nanoparticles, MMIPs, and MNIPs. The typical bands at 3028 and 2933 cm−1 can be attributed to the C-H aromatic stretching vibrations of styrene units. The absorption peak of C = O at 1723.9 cm−1 is due to the crosslinking agent EGDMA reacted with AM. Compared with NIP, weak absorption peaks appeared at 1632.8 and 1599.8 cm−1, which attributed to carbonyl and bending vibration of the imino group in CONH2. It indicated that most AM and crosslinking agent had crosslinking polymerized during the preparation of MIPs, and only some AM residues were found. Compared to curve 3 (black), the band of Fe-O at 547.1 cm−1 was observed in MMIPs, which confirmed that Fe3O4 was implanted into the polymer network successfully.Figure 4
FT-IR spectra of MMIP (red), MNIP (blue), and Fe3O4 magnetic nanoparticles (black).


## 3.2. Adsorption and Analytical Kinetic Evaluation

An adsorption kinetics experiment was performed on MMIPs to select the best extraction time. Figure5(a) shows that the amount of adsorption increased sharply in the first 15 min. The adsorption equilibrium was reached at 15 min, and the maximum adsorption amount was obtained, so 15 min was selected as the extraction time.Figure 5
(a) Effect of adsorption time; (b) effect of desorption time.
(a)(b)Figure5(b) shows the desorption kinetics of CLB by MMIP, and the desorption time was optimized. After 15 min, the desorption ratio is close to 100%. After desorption, MMIPs can be quickly separated and recovered from the sample solution by an external magnetic field.


## 3.3. Adsorption Isotherm Equation

To assess the binding energy, binding site type and distribution between MMIPs or MNIPs and imprinted molecules, adsorption isotherms were adopted using different concentrations of CLB. Figure6(a) shows the adsorption isotherms of MMIPs and MNIPs. When the adsorption reaches equilibrium, there is a certain relationship between the adsorption amount B and the residual amount F. It is possible to observe an increase of adsorption amount of polymer materials with increasing of the initial concentration of CLB, and the adsorption capacity of MMIPs for CLB is greater than that of MNIPs. Some studies have shown that the Freundlich equation has good applicability to noncovalently imprinted materials [30, 32]. Therefore, the Freundlich isothermal (FI) adsorption model was adopted to analyze the data with adsorption isotherms.Figure 6
(a) CLB adsorption isotherms for MMIPs and MNIPs with the corresponding experimental FI for MMIPs and MNIPs; (b) affinity distributions of MMIPs (black curve) and MNIPs (red curve).
(a)(b)The FI equation is expressed as follows [32]:(1)BF=aFm,where B and F are the concentrations of bound and free analyte, respectively; a and m are characteristic parameters of the Freundlich isotherm equation: a is the total number of binding sites, which represents the adsorption capacity of the adsorbent; m is the heterogeneity index with a value from 0–1. The value of m closes to 0 or 1 indicates that the adsorption sites are heterogeneous and homogeneous, respectively. And the value of a and m can be obtained from the following equation:(2)logB=mlogF+loga.The number of binding sites with given affinity (Nk) of a polymer can be calculated by equation (3), where K is the affinity constant, K=1/F.(3)NK=2.303am1−m2K−m.The binding siteNkmin−kmax and the apparent average binding constant Kkmin−kmax per gram of polymer can be calculated from equation (4) and equation (5), respectively.Nkmin−kmax is the number of binding sites per gram of polymer. Kkmin−kmax is apparent average binding constant. They can be calculated from equation (4) and equation (5), respectively, where Kmin=1/Fmax; Kmax=1/Fmin. Fmax and Fmin are the maximum and minimum solute concentrations of the liquid phase at adsorption equilibrium, respectively; a and m are the characteristic parameters of the Freundlich isothermal equation.(4)Nkmin−kmax=a1−m2Kmin−m−Kmax−m,(5)Kkmin−kmax=mm−1Kmin1−m−Kmax1−mKmin−m−Kmax−m.The FI affinity distribution model was further used to analyze the adsorption data. The affinity distribution curve (Figure6(b)) was obtained by plotting the value of Nk and logK, indicating the number of binding sites with binding ability. The value of logK represents the binding energy as the abscissa. It can be seen from the figure that the number of binding sites of MMIPs is always higher than that of MNIPs when given the binding energy within the experimental concentration range. It indicates that there are specific adsorption sites in the MMIPs material, and the imprinting effect is evident.The results are exhibited in Table1. The total number of binding sites and binding constants of MMIPs is higher than MNIPs. The binding sites N, K of MMIPs per gram of polymer are larger than MNIPs. These results indicate that the target molecule plays an important role in the formation of specific sites during the imprinting process.Table 1
Characteristic parameters of the Freundlich isothermal equation.
Fitting parametersMMIPsMNIPsa0.5680.432m0.4260.985Krange (L μmol−1)2.541–6.4421.799–3.184NK (μmol g−1)0.206–0.3070.009–0.017Nkmin−kmax (μmol g−1)0.2200.003Kkmin−kmax (L μmol−1)4.0672.358


## 3.4. Method Validation

In order to verify the accuracy of the method, the linear relationship, correlation coefficient (r), the limit of detection (LOD), the limit of quantification (LOQ), and spiked recovery experiment were investigated. By drawing the standard curve, the linear regression equation is y = 7.724x + 278 (r = 0.9694) (Figure 7). The values of LOD and LOQ were measured to be 3 and 10 times the signal-to-noise ratio (S/N), respectively. Their values were 4.27 and 14.2 μg L−1.Figure 7
The standard curve with linear regression.At the same time, the spiked sample recovery experiment was used to assess the reproducibility and accuracy of the method. Three different concentrations of samples were added to the CLB samples, and the recovery was measured and calculated. The concentration of the base solution is 0.25μmol L−1. The recovery values were calculated by the formula below:(6)Recovery=Found−baseadded×100%.The results were shown in Table2. The recoveries ranged from 94.44 to 102.29%, and the relative standard deviation (RSD) ranged from 3.48 to 7.14%. The same sample was measured 5 times and the RSD was calculated to be 4.93%.Table 2
Accuracy of the method for sample solutions spiked at different concentrates (n = 3).
SamplesAdded (μmol L−1)Found (μmol L−1)Recovery (％a)Average (％)RSD (％)CLB0.180.45111.1194.447.140.180.3977.770.180.4294.440.290.55103.4497.705.380.290.5293.100.290.5396.550.360.5891.6696.293.480.360.62102.770.360.5994.44(7)aFound−baseadded100%.


## 3.5. Analysis of Real Samples

In order to investigate the extraction and enrichment ability of MMIPs on actual samples, we extracted and detected CLB in pork according to the method of section2.5 and realized the analysis and detection of trace components from the matrix.The chromatogram of the pork sample extracted by MMIPs (Line a) and MNIPs (Line b) and direct injection of the sample without enrichment (Line c) are shown in Figure8. Figure 8(a) shows that CLB cannot be detected directly because the concentration is low without enrichment. Figure 8(b) also shows the sample could not be extracted by MNIPs, which proves that its enrichment is nonspecific adsorption. Figure 8(c) shows that a higher peak appeared and highly selective adsorption was achieved after the sample was extracted by MMIPs. The content of CLB in pork was calculated resulting in 9.87 ± 0.48 μg kg−1. The result showed that MMIPs could be used to enrich and detect CLB in actual samples.Figure 8
HPLC-UV chromatograms of pork samples without enrichment (a), enrichment by MNIPs (b) and MMIPs (c).


## 4. Conclusions

In summary, this study synthesized MMIPs of CLB for the first time. The synthesized MMIPs were characterized by SEM, FT-IR, and XRD. Adequate Fe3O4 nanoparticles are coated in the MMIPs, which ensures that the MMIPs can be easily and quickly separated from the complex solution through an external magnetic field without centrifugation or filtration. Through isothermal adsorption experiments, it was confirmed that MMIPs have good specific recognition and selective adsorption ability. The high recovery of spiked recovery experiments demonstrates that this approach is effective. Therefore, the method can be used for the residue detection of clenbuterol in animal products such as pork or pig urine.



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*Source: 8820262-2020-11-17.xml*