Establishment Of Detection Method For Antibacterial Active Ingredients In Different Matrices And Toxicity Studies

Antibacterial active ingredients can be added to many disinfecting and cleaning products.As the use of such products increases year by year,especially during the spread of new coronavirus disease(COVID-19),the negative impact on the environment has gradually appeared.Lack of proper use of disinfection and cleaning products can lead to direct or indirect toxicity of antibacterial active ingredients to animals and plants.At present,some antibacterial active ingredients have been regarded as potential endocrine disruptors.It can be seen that the antibacterial active ingredients had posed a threat to the environment and human health.Antibacterial active ingredients can pollute the ecological environment through various ways such as domestic sewage,medical wastewater and industrial sewage,and can further harm the human health through food and water.Therefore,the establishment of detection methods for antibacterial active ingredients in different matrices in this paper can provide reliable support for the fields of environmental forensic medicine,environmental science and food science.In addition,because of the convenient use and easy purchase of disinfection and cleaning products,users often ignore their toxicity.In view of this,the toxicities of 4-chloro-3-methylphenol and benzethonium chloride were studied by metabolomics using zebrafish as model organism in this paper,which provides data for the toxic effect and mechanism of action of antibacterial active ingredients on organisms.The main contents are as follows:Part 1 Magnetic Nanomaterials Combined with HPLC-MS/MS to Detect Multiple Antibacterial Active Ingredients in Different MatricesObjective:Based on magnetic nanomaterials,the detection methods for11 multi-type antibacterial active ingredients in surface water,and 5quaternary ammonium antibacterial active ingredients in infant fruit and vegetable puree had been established.Method:Fe₃O₄@PPy magnetic nanoparticles and Fe₃O₄@Si O₂-NH₂-G2magnetic nanoparticles were synthesized by different methods.Based on the optimized mass spectrometry parameters and chromatographic conditions of the analytes,the relevant conditions affecting the magnetic solid phase extraction process,such as adsorption and desorption conditions,were optimized by single factor evaluation method.Results:The recoveries of 11 antibacterial active ingredients in surface water by Fe₃O₄@PPy magnetic nanoparticles were 80.21%?105.80%.And the recoveries of 5 quaternary ammonium antibacterial active ingredients in infant fruit and vegetable puree by Fe₃O₄@Si O₂-NH₂-G2 magnetic nanoparticles were greater than 80%.Both methods showed good linearities,low detection limits and quantitative limits,and high sensitivities and accuracies.Conclusion:Two new methods for the detection of antibacterial active ingredients in different matrices based on magnetic solid phase extraction were established.Both methods were sensitive,reliable and stable,and can be used for the determination of actual samples with satisfactory results.Part 2 Based on Plackett Burman and Box Behnken design optimization QuEChERS Technology to Detect Triclosan,Triclocarban and Cloflucarban in SedimentsObjective:Based on the optimization QuEChERS technology of multi-factor experimental design,a method for the detection of triclosan,triclocarban and cloflucarban in sediments was established.Method:After determining the mass spectrum parameters and chromatographic separation conditions of three analytes,the influencing factors of sample pretreatment process were first selected by single factor evaluation method;then,the Plackett Burman design was used to screen out the significant factors affecting the extraction efficiencies;finally,the values of significant factors for pretreatment were optimized and determined by Box Behnken design and response surface analysis method.Results:In this study,single factor evaluation method,Plackett Burman design,and response surface analysis method were used to optimize pretreatment conditions for triclosan,triclocarban,and cloflucarban in river floodplain sediments,including acetonitrile 10.35 m L,30.5?ultrasonic treatment 13 min,0.1 g Mg SO₄ and 0.3 g PSA,and performed one time extraction.Methodological verification showed that this method has good linearity,high intra-day and inter-day precision,low limit of determination and low limit of quantitation.It can be used for the simultaneous determination of triclosan,triclocarban and cloflucarban in actual sediment samples.Conclusion:The optimized QuEChERS technique combined with HPLC-MS/MS could be used for the successful determination of triclosan,triclocarban and cloflucarban in the river floodplain sediments.Part 3 Analysis of the Toxic Effect and Mechanism of Antibacterial Active Ingredients Based on the Non-targeted Metabolomics Technology of High Resolution Mass SpectrometryObjective:UPLC-QTOF-MS-based non-targeted metabolomics technology was used to analyze the toxic effects of 4-chloro-3-methylphenol and benzethonium chloride on zebrafishMethod:According to the acute toxicity test of 4-chloro-3-methylphenol to adult zebrafish,the semi-lethal concentration of 4-chloro-3-methylphenol was determined,and 10%of semi-lethal concentration was selected for metabolomics analysis of adult zebrafish.The same method was used to determine the semi-teratogenic concentration and the semi-lethal concentration of benzethonium chloride on zebrafish embryos and adult male zebrafish respectively.The high and low exposure concentrations were selected for metabolomics analysis of zebrafish at different developmental stages.All the metabolomics data were determined by UPLC-QTOF-MS,and the obtained data were screened out and analyzed by multiple software such as XCMS,SIMCA 14.1,HMDB,mz Cloud and Metaboanalyst 5.0.Results:Both 4-chloro-3-methylphenol and benzethonium chloride could have toxic effects on adult zebrafish,and mainly affected the metabolism of glycerophospholipids.Benzethonium chloride could also cause significant toxic effects on zebrafish embryos,mainly interfering with 7 metabolic pathways including aminoacyl t RNA biosynthesis,alanine,aspartate and glutamate metabolism,and arginine biosynthesis.Conclusion:Both 4-chloro-3-methylphenol and benzethonium chloride have toxic effects on zebrafish.