| Macrolide and polypeptide antibiotics,as two of the widely used drugs in the antibiotic family,have played an important role in the therapy,prophylaxis and growth promotion for food animals.The long-time addition of these drugs at sub-therapeutic dose in animal feed could cause drug residues in the animal,further posing a threat to human health through food chain and increasing the risk of drug resistance.Due to complex matrices in biological samples（e.g.feed and animal tissue）and the absence of selectivity in traditional sample pretreatment techniques,it is of great significance to synthesize specific adsorption material for the selective extraction,purification and enrichment of trace amounts of macrolides and polypeptide antibiotics from complex biological matrices.In this paper,the molecularly imprinted microspheres,which have class-specific adsorption towards macrolide and polypeptide antibiotics,were prepared using surface imprinting technique based on silica gel and precipitation polymerization method.The morphology and structure of imprinted microspheres were characterized by several methods.Combined with the static and dynamic adsorption experiments,the possible adsorption mechanism was speculated.The imprinted microspheres were successfully applied to the analysis and detection of biological samples.The main research contents of this paper are as follows:The molecularly imprinted microspheres based on silica gel were prepared by surface imprinting technique using silica gel which was modified by methacryloxy propyl trimethoxyl silane（γ-MPS）as the carrier,acetonitrile as the porogen,tilmicosin as the template,methacryclic acid（MAA）as the functional monomer and ethylene glycol dimethacrylate（EGDMA）as the crosslinker.The scanning electron microscopy and infrared spectra analyses showed that the tilmicosin-molecularly imprinted polymers were successfully grafted onto the surface of silica gel（Si O2-MPS@MIP）.The specific surface area of Si O2-MPS@MIP could reach 161.6 m2/g and the imprinting factor was 3.7.The static adsorption experiments showed that Si O2-MPS@MIP had high adsorption capacity and two different binding sites.The Langmuir model was more suitable to describe the static adsorption process and the adsorption mechanism was the monolayer surface adsorption.The dynamic adsorption of Si O2-MPS@MIP was well described by the pseudo-second-order kinetic model,suggesting that the adsorption was mainly controlled by chemical adsorption.Selective adsorption experiments showed that Si O2-MPS@MIP had high adsorption capacity towards macrolide antibiotics such as tilmicosin and tulathromycin,indicating the class-specific adsorption performance.The Si O2-MPS@MIP microspheres prepared were used as the solid phase extraction sorbents,and based on specific molecularly imprinted solid phase extraction,the High performance liquid chromatography-evaporative light scattering detection and liquid chromatography tandem mass spectrometry（LC-MS/MS）methods were established to detect macrolide antibiotics and to analyze their residues in complex matrices such as feed,plasma and animal tissues.The recoveries of seven macrolide antibiotics including tilmicosin in feed were 62.9%～98.6%and the relative standard deviation was less than 13.5%;the recoveries in plasma and animal muscle were 61.9%～96.6%and the relative standard deviation was less than 16.8%.The limit of detections of macrolide antibiotics in feed,plasma and animal tissues were 0.3～0.6 mg/kg,0.3～0.6 ng/m L and 0.2～0.3μg/kg,respectively.Compared with the traditional solid-phase extraction cartridge,the molecularly imprinted cartridge exhibited better purification effect.The polymyxin E-imprinted microspheres were synthesized using polymyxin E as the template,MAA and hydroxyethyl methacrylate as the double-monomer,EGDMA and divinylbenzene as the double-crosslinker.The scanning electron microscopy and specific surface area analyses showed that morphology and structure of imprinted microspheres and non-imprinted microspheres were similar.The static adsorption experiment showed that the adsorption capacity of imprinted microspheres was higher than non-imprinted microspheres,which was well described by the Langmuir model.The selective experiment showed that polymyxin E-imprinted microspheres had class-specific adsorption to polymyxin antibiotics.The imprinted microspheres were used as the solid phase extraction material,and a HPLC method based on molecularly imprinted solid phase extraction was developed for the determination of polymyxin B and polymyxin E in environmental water.The recoveries were65.9%～90.1%with the relative standard deviation less than 9.7%and the detection limits of polymyxin antibiotics were from 1.0 to 2.0μg/L.The polymyxin E-imprinted microspheres were used as the stationary phase and packed into a chromatographic empty column as an imprinted chromatography column.Under the optimal chromatographic conditions,the imprinted column had high column efficiency and good stability.The chromatographic separation experiment showed that the drugs,which are similar to the template in size or spatial structure,could be retained well in the imprinted column,indicating the class-specificity of the imprinted column.Accordingly,based on the online purification and enrichment of the molecularly imprinted microsphere column,a LC-MS/MS method was developed for the determination of six polypeptide antibiotics in animal edible tissues.The recoveries of polypeptide antibiotics were 66.7%～94.5%with the relative standard deviation less than 16.0%and the detection limits of six polypeptide antibiotics were3.0～8.0μg/kg.The imprinted column could effectively remove impurities and reduce matrix effects,and thus improve analysis accuracy. |
