| Coccidiosis is a kind of common parasitic disease in the raising industry caused by single-celled parasite enter the intestinal, and its easily outbreak in large-scale farms. In order to prevent and treat coccidiosis, triazine coccidiostats-diclazuril(DIC) and toltrazuil(TOL) are often added to animal feed and drinking water because of its high efficiency, low toxicity and broad-spectrum characteristics. But if use drugs excessively or blindly, or not in compliance with the withdrawal period and so on, will not only affect the quality of poultry meat, endanger human body health, and even will pollute the environment. Therefore, the study of monitoring and analysis method of coccidiostats is very necessary.The aim of this study was to use two kinds of triazine coccidiostats- diclazuril and toltrazuil for the composite templates, studied the proportion of the two template molecules and the synthetic method of the two template molecules imprinting polymers, prepared a new type of composite templates molecules imprinting polymers. With the polymers as filler material, prepared of composite templates molecular imprinting solid-phase extraction column, and studied the method of this molecular imprinting solid-phase extraction column. Build a diclazuril and toltrazuil composite template molecules imprinting polymers solid-phase extraction- high performance liquid chromatography method (MISPE-HPLC), for simultaneous detection of the two kinds of coccidiostats provide a novel sample pretreatment and detection technology.The specific study contents were as follows:1. Synthetic and evaluation of MIP with composite templates- diclazuril and toltrazuil (DIC&TOL MIPs)The composite template molecules imprinting polymers was prepared using diclazuril and toltrazuil as template molecules, a-methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent, and azobisisobutyronitrile(AIBN) as initiator by mass polymerization and suspension polymerization. By investigating the effect of the type of solvent (porogen) and the ratio between the composite template molecules, the functional monomer and the cross-linking agent, as well as to compare the adsorption quantity of target molecules by the composite template molecules imprinting polymers by prepared in different polymerizations, the optimal preparation condition of diclazuril and toltrazuil composite template moleculars imprinted polymers were:using the mass polymerization, taking N,N- dimethylformamide as solvent (porogen), the molar ratio of two kinds of template molecule, functional monomer, cross-linking agent and initiator was 0.5:0.5:4:20:3, the adsorption quantity for diclazuril and toltrazuil were more than 90% and 80% respectively.The structure and morphology of the composite template molecules imprinting polymers were characterized using infrared spectroscopy and scanning electron microscopy, the polymers adsorption performance for target molecules was studied by using the static adsorption and dynamic adsorption experiments. Scachard analysis showed that this composite template molecules imprinting polymers has high apparent combine quantity for diclazuril and toltrazuil.2. Preparation of the MISPE column with composite templates and evaluation of the MISPE methodThe prepared composite template molecules imprinting polymers as adsorbent (filling materials) was mixed with diatomite at different ratios in the empty SPE column. By comparing the flow rate of sample solution crossing the MISPE column as well as the adsorption effect of MISPE column, the ratio between composite template molecules imprinting polymers and diatomite chose 1:1.The method of MISPE was established by studying the effect of washing agents and eluting agents to MISPE column. Chose hexane as washing agents, acetonitrile:water: acetic acid solution (volume ratio of 6:3:1) as eluting agents, the recovery of this method was more than 94%.3. Establishment of the method of simultaneous detection of diclazuril and toltrazuil by HPLCTo establish a high performance liquid chromatography (HPLC) method for simultaneous detection two kinds of triazine coccidiostats-diclazuril and toltrazuil. By studying some factors such as the detection wavelength, the proportion of mobile phase and its flow rate. The optimal chromatographic conditions were:detection wavelength of 240 nm and 280nm, acetonitrile:water (65:35) as mobile phase, the flow rate of 1.0 mL/min, sample volume 10 μL, column temperature 30℃. The results showed that, the two drugs could separate completely in 11 minutes, and the concentration of the two drugs had good linear relationship with the peak area, the correlation coefficient r2 were 0.9998～0.9999, the relative standard deviation of the peak area RSD (n=6) were below 1.5%; The migration time of RSD (n=6) were below 0.15%.4. The application of triazine composite template moleculars imprinted polymers in detection of real sampleTaking chicken feed from farms round He Fei as experimental material, the prepared composite template MISPE column was applied to pre-treatment of samples, the results showed the good effect of purification and enrichment. At the same time, the method of HPCE was applied to simultaneous detection the two triazine coccidiostats in the chicken feed. Standard addition recovery rate could reach 96.6% and 77.5% respectively. |
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