| Clenbuterol hydrochloride(CLB), commonly known as “leanness enhancer”, is a β2-adrenergic agonist. It can break down animal fats, accelerate metabolism, increase lean mass and the efficiency of feed conversion, and thus it is usually misused as a feed additive. However, the CLB residues in animal tissues are poisonous to human beings, especially to the cardiovascular and central nervous systems. Therefore, it is necessary to develop a rapid, accurate and sensitive detection method for CLB. Due to the complexity of the sample matrix, sample pretreatment is a must before instrumental analysis. The sample pretreatment procedures in our national standard for CLB analysis include repeated solvent extraction, ultrasonication, centrifugation and solid-phase extraction, which are cumbersome, time-consuming and selectively limited. In addition, the pretreatments consume large amounts of toxic and hazardous solvents. Magnetic solid phase extraction(MSPE) is a kind of solid phase extraction technology based on magnetic adsorbents. The separation process in MSPE can be performed directly in crude samples containing suspended solid materials by applying an external magnet without centrifugation or filtration, which makes the separation easier and faster. Magnetite/reduced graphene oxide(MRGO) nanocomposites are excellent adsorbents for MSPE, which possess high adsorption capacity, good stability and recyclability. The sensitivity and accuracy of the method can be improved by combining MSPE with instrumental analysis.In this paper, three kinds of MRGO nanocomposites were prepared by solvothermal, hydrothermal and co-precipitation methods, respectively, and the as-prepared nanocomposites were characterized and compared in detail. Thereinto, the solvothermal method was selected for the following study, and the preparation conditions were optimized. An effective sample pretreatment method was determined for pork sample before MSPE after six sample pretreatment methods were compared. Then, MRGO nanocomposite, prepared under the optimum conditions, was applied as MSPE adsorbent to isolate CLB from pork. Consequently, a magnetic solid phase extraction- high performance liquid chromatography(MSPE-HPLC) method was established for detecting CLB residues in pork. The main contents in this thesis are described as follows: 1. Synthesis, characterization and comparison of MRGO prepared by different methodsThree kinds of MRGO nanocomposites were prepared by solvothermal, hydrothermal and co-precipitation methods, respectively. The as-prepared nanocomposites were characterized and compared by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction and zeta potential analysis. The results showed that MRGO made by different methods differed in surface functional groups, crystal structure, particle size, surface morphology and surface charge. Due to their unlike features, these nanocomposites displayed dissimilar performances when they were used to adsorb drugs, dyes and metal ions. The MRGO prepared by co-precipitation method showed special adsorption ability to negative ions, but those synthesized by solvothermal method obtained the best extraction ability and reusability to the others and showed a good prospective in magnetic solid-phase extraction. Therefore, it is highly recommended to use the right preparation method before application in order to attain the best extraction performance. 2. Synthesis and characterization of MRGO prepared by solvothermal methodGraphite oxide was prepared by modified Hummers method, and the reaction conditions were optimized, including the reaction time of high temperature stage and the mass ratio of graphite and KMn O4. And then, graphite oxide was used as raw materials together with Fe Cl3, DEG and Na OAc to synthesize MRGO nanocomposites via solvothermal method. The mass ratio of precursors and the solvothermal reaction time were optimized as well. In the end, seventeen kinds of nanocomposites were obtained. Then the as-prepared MRGO were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray diffraction. Clenbuterol hydrochloride, sulfadiazine and amoxicillin were used as the target analytes to study the effect of preparation conditions on the adsorption ability of MRGO. At last, the mass ratio of graphite and KMn O4 was set as 1:2, the time of high temperature stage was chosen as 30 min, the solvothermal reaction time was selected as 8h, and the mass ratio of graphite oxide and Fe Cl3 was chosen as 1:1. The prepared MRGO was decorated with uniform and small Fe3O4 nanoparticles, which laid the foundation for MRGO as a good adsorbent for MSPE of CLB. 3. Development of sample pretreatment methods for pork before MSPESince pork is a complicated matrix, it should be pretreated before MSPE. Six different methods were used to deal with pork samples according to references, and the Agriculture Industry Standard of People’s Republic of China standard-NY/T 468-2006 was selected. Then some detailed conditions were further optimized to enhance the recovery, such as the treatment of sample solution, extraction times by ethyl acetate and the vortex time, and ultimately the extraction times was set as three, the vortex time was chosen as 30 s, and the sample was rotarily evaporated to dryness, dissolved by deionized water, and then used for MSPE. 4. Application to the detection of CLB in pork based on MRGO nanocompositeThe optimum MRGO nanocomposite was used as an adsorbent for MSPE to absorb CLB from pork samples prior to HPLC detection. Several parameters affecting the extraction efficiency were optimized, including extraction time, adsorbent dosage, ionic strength and desorption conditions. The adsorption of CLB was fitted by pseudo-second-order kinetic model. Subsequently, the method was validated under the optimum conditions. Wide linearity(1-100 ng/m L), low limit of detection(0.542 μg/kg), good repeatability(with a RSD of 5.11%), satisfactory recoveries(87.38%-92.87%) and high enrichment factor(166.32-197.76) were obtained. The established method was successfully applied to the preconcentration and detection of CLB in pork. |
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