Preparation And Application Of Molecularly Imprinted Solid-phase Extraction Column For Sulfadiazine

Sulfonamides (SAs) are a kind of commonly-used veterinary drugs. The harm of the residual SAs in livestock and environment, caused by the overuse of the drugs, has drawn increasing attention from home and abroad. Generally, high performance liquid chromatography (HPLC) method and liquid chromatography-mass spectrometry (LC-MS) techniques have been used for monitoring SAs residues in official control programs. Sample preparation is the key part of the analytical process, and solid-phase extraction is one of the most frequently-used preparation methods. However, the nonspecific solid-phase extraction column, which is widely used, has low efficiency in extracting trace amount of SAs. After purification, the samples are still compound-mixed, which would have a negative effect on the subsequent chromatographic analysis. Molecularly imprinted solid-phase extraction (MISPE) column has been successfully used for the isolation and preconcentration of trace analytes from the complex matrix, which takes molecularly imprinted polymer (MIP) as selective sorbent materials. It is able to improve the accuracy and sensitivity of the detection through eliminating the strong matrix effects.In this study, MIP was firstly synthesized by using sulfadiazine (SDZ) as the template. Then, the MISPE column was prepared, and the MIP was used as adsorbent. The MISPE column finally was coupled with HPLC for the efficient determination of SAs. The details are shown as follows:1. Synthesis and characterization of MIPTo begin with, acrylamide was chosen as the appropriate functional monomer by ultraviolet scanning and nuclear magnetic resonance to synthetise MIP. Then the MIP was synthesized by ultraviolet initiated. During the process,1mmol of SDZ were used as template,4mmol of acrylamide as functional monomer,18mmol of ethylene glycol dimethacrylate as cross linker,10mL of acetonitrile as porogen and0.05g of azodiisobutyronitrile as initiator. The absorbability of MIP for SDZ reached6.73mg/g, higher than the nonimprinted polymer, and the average rate of adsorption could reach0.449mg/min. Meanwhile, the absorbability of MIP for SDZ and its analogues (sulfamethazine, sulfamonomethoxine, sulfamethoxazole and sulfaquinoxaline) was stronger than that of the drugs (oxolinic acid, enrofloxacin and tetracycline), which were not analogues of SDZ. Finally, dense pores could be identified on the surface of the MIP through scanning electron microscope, which indicated that the MIP had generated tailor-made binding sites with the memory of the template, while the surface of the nonimprinted polymer was rough, sparse and irregular.2. Preparation of MISPE column and optimization of solid-phase extractionMISPE column was prepared by using100mg dried MIP as sorbents. Then the procedures of loading, washing and eluting conditions were optimized. The final extraction conditions were listed as follows:dimethyl sulfoxide-water (1:9, V/V) as loading solvent;2mL of methanol-water (1:9, V/V) as washing solution;3mL of acetonitrile-water (4:6, V/V) as eluting solution; a flow rate of1mL/min for loading, washing and eluting steps. Under the conditions, the recovery quality exceeded92%after20mL10ng/mL SDZ were concentrated by the MISPE column. The extraction capacity of the column reached1.1mg/g and it could be reused for more than10times.3. Establishment of a MISPE-HPLC method to detect SAs in feeds and milkA method was developed to monitor five SAs in feeds and milk. MISPE column, coupled with HPLC, was applied for sample clean-up and detection. For the determination of feeds, the detection limit range was0.15-0.22mg/kg and the quantification limit range was0.38-0.47mg/kg. The recoveries of five SAs, extracted from feeds sample at5-10mg/kg spike level, were in the range of71.6%-90.1%with the intra-assay and inter-assay standard deviations less than8.5%and9.0%, respectively. It showed that the MISPE column decreased the matrix effect and had a better selectivity than Alumina B column. For the determination of milk, the detection limit range was15.0-22.5μg/kg and the quantification limit range was33.1-43.2μg/kg. The recoveries of five SAs, extracted from milk sample at80-120μg/kg spike level, were in the range of64.4%-80.5%with the intra-assay and inter-assay standard deviations less than10.8%and14.1%, respectively. It showed that the MISPE column decreased the matrix effect and had a better selectivity than HLB column.The MISPE-HPLC method was applied to analyze SAs of twenty feed samples. The results showed that five samples contained SAs at the concentration of0.15-0.51mg/kg. This method also was used to analyze SAs of twenty milk samples. It found that four samples contained SAs at the concentration of15.1-31.1μg/kg. Among them, one sample contained sulfamethazine exceeding the standard level. The SAs content were not out of limit in the rest of the samples.