Simultaneous Determination Of Four Kinds Of Typical Antibiotics In Soil And Method Optimization

Antibiotics have been widely used for people and animals to prevent and cure diseases. In addition, antibiotics are also added in animal feed in order to improve the feed efficiency as well as promote animal growth. In recent years, with the expansion of the livestock breeding industry, more and more antibiotics are put into use. The abuse of Antibiotics become more and more serious. Antibiotics cannot be absorbed or metabolized absolutely in vivo, and some antibiotics are excreted into the environment via feces. Generally, manure amended to soil without processing will result in soil pollution and lead to serious environmental problems including ecological risk and human health damage.As a new environmental pollutants, antibiotics has caused high attention at home and abroad.Therefore,it is necessary to establish a high sensitive and stabilized analytical method for simultaneous determination of multiple compounds. This study intends to establish a mothed for simultaneous determination of multiple compounds at high sensitivity and stability and optimize the method.This method have important theoretical and practical significance for soil antibiotics pollution research.Objective1. Optimize main parameters, extraction slovent and dispersing agent of ASE mthod, establish ASE method as sample preparation method.2. Develope and optimize the accelerated solvent extraction(ASE)-solid-phase extraction (SPE)-high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for Simultaneous, determination of sulfonamides(SAs), fluoroquinolones(FQs), tetracyclines(TCs) and macrolides(MLs) antibiotics in soils.3.Compare ASE method with SAE method in the same experimental condition, provide reference for the soil antibiotics extraction research. Method1. Extraction:In ASE mthod,acetonitrile-phosphate buffer (pH=3) was used as extraction slovent,EDTA washed diatomite was used as dispersing agent.Soil was extracted by ASE with the parameter conditions:pressure1500psi, temperature70℃,static10min,1circle.In SAE mthod, acetonitrile-phosphate buffer (pH=3) was used as extraction slovent, soil was extracted with vibration, sonication-assisted extraction and centrifugation.Soil residue was extracted twice again.2. Purification and enrichment:Extraction slovent was purified and concentrated by SAX-HLB tandem cartridges, eluted with6ml methanol, evaporated with nitrogen and reconstituted with1ml methanol-water(50:50,v/v).3. Detection:Separation of the target antibiotics was done by gradient elution. mobile phase was0.1%formic acid(A) and acetonitrile(B), flow rate was0.4mL/min, column temperature was20℃.10ul of the redissolved extract was injected into the chromatographic system.Sample acquisition was performed in the multiple reaction monitoring (MRM) mode.Result1. In ASE-SPE-HPLC-MS/MS method, the recoveries of SAs,FQs,TCs and MLs were86.3%～97.4%,63.2%～90.2%67.3%～87.4%and68.4%～78.3%. RSD<10%, r>0.99. Limits of detection was0.5～0.9μg/kg for SAs,0.2～1.1μg/kg for TCs and0.2～0.3μg/kg for MLs.2.Extracted soil sample by SAE mthod, Spiking recovery of antibiotics were51.6%～80.6%, RSD<11%. Extracted Spiked soil(200μg/kg) by ASE and SAE mthod,used the same extraction slovent with their best conditions.In the first method,96minutes and18ml extraction slovent were used, recovery were50%～80%.In the second method,18minutes and18ml extraction slovent were used, recovery were60%～85%.Conclusion1. Optimized ASE method extracted antibiotics from soil in16min. Optimized ASE-SPE-HPLC-MS/MS method determined15kinds of antibiotics in15min, the detect accuracy can meet the actual sample requirements. 2. ASE mthod is better than SAE mthod in automation, extraction efficiency and health effect of experimenter. However, SAE mthod use less time when deal with large number of samples.