Determination Of Sulfonamides Antibiotics By Dispersive Liquid-liquid Microextraction/Molecularly Imprinted Solid-phase Extraction Combined With High Performance Liquid Chromatography

Sulfonamides（SAs）antibiotics are a kind of synthetic antibiotics with p-aminobenzene sulfonamide structure.SAs are widely used in animal husbandry and aquaculture for the prevention and treatment of animal-borne diseases due to their wide antibacterial spectrum,low price,easy availability and good efficacy.However,the extensive use or even abuse of SAs will lead to its long-term residue in animal body and environmental samples,causing great harm and thereby requiring sensitive determination.Chromatography is a widely used qualitative and quantitative detection technology in SAs detection,among which high performance liquid chromatography（HPLC）is more widely used because of its advantages of high separation efficiency,fast analytical speed,high detection sensitivity,good reproducibility and multi-component separation and analysis.However,SAs residues are characterized by low residual amount,complex residual matrix and multi-category presence,which poses great challenges to the sensitivity and accuracy of analysis methods.Sample pretreatment technology can effectively enrich the targets and reduce or even eliminate matrix interferences before instrumental analysis,thus improving the sensitivity and accuracy of analysis.Dispersive liquid-liquid microextraction（DLLME）is a sample pretreatment technology developed based on liquid-liquid extraction technology.DLLME has the advantages of low consumption of organic solvents,simple convenient and quick operation,high enrichment efficiency and so on.Dispersive solid-phase extraction（DSPE）is a sample pretreatment technology developed based on solid-phase extraction（SPE）technology.The adsorbent is fully dispersed in the sample solution,therefore,the extraction efficiency is obviously improved,and the operation is more convenient without washing.In recent years,molecularly imprinted polymers（MIPs）have become popular adsorbents of DSPE because of their recognition specificity,structure predictability,easy preparation and low cost,etc.In this thesis,DLLME and MIPs-DSPE were developed,respectively,and combined with HPLC-UV for the simultaneous enrichment,separation and detection of multi-component SAs in environmental water and/or seafood samples.The main research contents and results are as follows:1.A research on simultaneous enrichment and separation of seven SAs in environmental water and seafood by UA-DLLME.Using tetrachloroethane as extractant and acetonitrile as dispersant,UA-DLLME was developed for simultaneous enrichment and separation of seven SAs in environmental water and seafood,and combined with HPLC-UV determination.The extraction of sulfamethoxyl（SCT）,sulfamethoxine（SMR）,sulfamepyridine（SPD）,sulfamethoxine（SDZ）,sulfamethoxine（SMM）,sulfamethoxazole（SMX）and sulfamethoxine（SDM）were optimized by one-variable-at-a-time（OVAT）method,including the type and volume of extractant,the type and volume of dispersant,the pH of sample solution,the ionic strength of sample solution and the type of desorption agent.Under the optimal conditions,the separation and detection of seven SAs could be realized within 14.5 min.The analytical performance of the method was evaluated in actual samples（seawater,lake water,aquaculture wastewater,shrimp,pomfret）.The linear range of the method was wide（5-5000 or 10-5000μg/L）,and the correlation coefficient r=0.9999.The LODs of seven SAs in water samples and seafood were 1.1-7.8μg/L and 0.7-5.9μg/L,respectively.LOQs were 3.6-26.0μg/L and 2.4-19.6μg/L,respectively.The intraday precision of retention time and peak area for seven SAs are 0.3-1.2%and 0.3-1.5%,respectively,and the interday precision is 0.9-2.7%and 0.8-2.4%,respectively,showing good precision.The matrix effect（ME）of the method was evaluated in seawater,lake water and shrimp,and the ME%was within a reasonable range（-20%-20%）.The results showed that UA-DLLME eliminated ME and improved the accuracy,sensitivity and reliability of detection.The spiked recoveries of the seven kinds of SAs were tested with three concentrations.High recoveries ranging from 80.0-116.0%and low relative standard deviations（RSD,%）within 0.1-8.1%were obtained.It should be noted that the SDM was detected in seawater sampled near Chang Island,with an average concentration of 24.49μg/L.Consequently,the developed UA-DLLME-HPLC-UV method proved facile,accurate and reliable,and practical applicable for multiresidue analysis of antibiotics.2.A research on preparation of dummy template MIPs and enrichment of four SAs by DSPE in environmental water samples.The dummy template molecularly imprinted polymers（DMIPs）were prepared by one-step precipitation polymerization with sulfanobenzene（SZ）as dummy template based on dummy template imprinting strategy.The structure characterization and adsorption performance of DMIPs were fully evaluated.The adsorption capacities of SMM,SFZ,SMX and SDM were 30.24,28.61,21.97 and 22.37 mg/g,respectively,and the imprinting factors were all greater than 2.1.The factors affecting the extraction efficiency of DMIPs-DSPE（DMIPs）,such as dosage of DMIPs,extraction time,pH of sample solution,volume of desorption,time of desorption)were optimized.The linear range of the method was 1-200μg/L,r≥0.9995,LODs 0.27-0.64μg/L and LOQs 0.99-2.14μg/L in seawater,lake water and tap water.The precision of the method was excellent,for example,the intraday precision of retention time and peak area of four SAs were 0.07-0.12%and 0.53-1.13%,respectively,and the interday precision was 0.23-0.28%and 0.95-1.33%,respectively,all lower than 15%.The ME of the method was evaluated in the above water sample,ME%≤6%,indicating that the established method can effectively eliminate the interference of complex matrix.The recoveries of four SAs in seawater,aquaculture wastewater and lake water were 93.8-99.8%,94.8-100.1%and 95.6-102.6%,respectively,with corresponding RSD%values of 2.6-7.0%,2.8-5.4%and 2.8-5.2%.The established method has high sensitivity,precision and satisfactory recovery,which has been applied to the determination of actual samples and has great potential in the accurate quantitative analysis of trace SAs in complex matrix.3.Preparation and characterization of SZ surface imprinted MIPs.In order to improve the performance of MIPs in adsorption recognition,a combination of several molecular imprinting techniques and preparation strategies was explored.Through the combination of dummy template imprinting strategy and surface imprinting technology,the surface imprinted DMIPs was prepared by precipitation polymerization using SZ as dummy template and SiO₂ as core carrier.Combining the above two imprinting strategies with magnetic response imprinting strategy,surface imprinted magnetic MIPs（MMIPs）was expected to be prepared.Fe₃O₄@SiO₂ grafted with double bonds on the surface was synthesized,which could be used as magnetic core carrier to prepare magnetic DMIPs for magnetic SPE（MSPE）in the later stage.