| Colistin,also known as colistin sulfate or polymyxin E,is an alkaline trivial cyclic polypeptide antibiotic obtained from the culture medium of polymyxobacteria.Colistin sulfate is a multi-component polypeptide antibiotic,mainly composed of colistin A and B,which is considered to be the last resort for the treatment of infections caused by multidrug-resistant gram-negative bacteria.Colistin sulfate is also used worldwide for growth promotion,disease prevention and control in livestock and poultry.The widespread use of colistin sulfate in animal husbandry leads to a large number of colistin sulfate into the surrounding environment through animal urine and feces,which may lead to the prevalence of colistin-resistant bacteria and affect the ecological environment.At present,there are few studies on the metabolic fate and environmental behavior of colistin sulfate in livestock and poultry at home and abroad,which cannot make an objective assessment of the environmental risk of colistin sulfate.Therefore,based on the characteristics of high resolution,high sensitivity and accurate mass measurement of ultra-performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）,a method for determining the content of colistin sulfate in the gastrointestinal tract of swine and broiler was established by UPLC-MS/MS,and the metabolism and absorption of colistin sulfate in the gastrointestinal tract of swine and broiler,as well as the adsorption,desorption,migration and degradation behavior of colistin sulfate in different soil were studied.The results are as follows:1.Establishment of UPLC-MS/MS method for determination of colistin sulfate.Ultra performance liquid chromatography（UPLC）was performed on a Thermo Uti Mate 3000 UPLC system equipped with Acquity UPLC BEH C18column（1.7μm,2.1 mm×100 mm）,mobile phase A was 0.5%acetonitrile-0.2%formic acid solution,and mobile phase B was 100%acetonitrile,column temperature 20℃,the injection volume was 5μL.A quadrupole orbit-trap MS/MS system（QExactive）was used with HESI ion source,spray voltage of 3200V,capillary temperature of 320°C,sheath gas volume flow of 25psi and auxiliary gas volume flow of 15psi.Quantitative analysis was performed in selective reaction monitoring（SRM）scan mode.The reaction ions and retention time（t R）were monitored as follows:colistin sulfate A([M+3H]3+),m/z390.60>101.0696,241.1860,t R=7.30 min,colistin sulfate B([M+3H]3+),m/z385.90>101.0696,227.1710,t R=6.85 min.The m/z tolerance of fragment ions was set to 5 ppm,and the deviation in retention time was less than 5%.The pretreatment method of colistin sulfate in gastrointestinal contents and feces of swine and broiler was established.The sample was freeze-dried,then extracted twice with 10%trichloroacetic acid:acetonitrile（40:60,v/v）,and the p H of the extract was adjusted to9.0.The extract was extracted with WCX solid-phase extraction column.The eluent was concentrated by vacuum centrifugation at 50℃and then reconstituted with deionized water for analysis.The calibration curve was linear in the range of 10-500ng/m L for colistin sulfate.And the recoveries were in the range of 73.2-103.9%with the relative standard deviations of 4.45-7.98%.The detection limits of colistin sulfate in different matrices were 0.11-0.56 ng/g,and the limit of quantification was 0.37ng/g.The method can be used to determine the content of colistin in the gastrointestinal tract and feces of swine and broiler.2.Study on the absorption of colistin sulfate in gastrointestinal tract of swine and broiler.Weaned swine and Cobb broiler were fed a basal diet supplemented with 20mg/kg colistin sulfate for 14 days.The metabolism and absorption of colistin sulfate in gastrointestinal tract of swine and broiler were studied by measuring colistin sulfate content in gastrointestinal tract and feces of swine and broiler.The results showed that the colistin sulfate content in feces was the highest（1248.3 ng/g）compared with all the tested samples of gastrointestinal contents of swine,and the colistin sulfate content in duodenum and jejunum was low,which indicated that the absorption of colistin sulfate might occur in duodenum and jejunum.The high content of colistin sulfate in swine cecum contents might be due to the decreased absorption and accumulation of colistin sulfate in the cecum.The colistin sulfate content in caecum contents was the highest（4882.9 ng/g）,and the lowest colistin sulfate content was detected in the proventriculus and duodenum contents of broilers,indicating that the proventriculus and duodenum may be the main absorption sites.The mean concentrations of colistin sulfate in jejunum,ileum,and cecum increased progressively,indicating little or no absorption of colistin sulfate at these sites.3.Study on Characteristics of adsorption and desorption of colistin sulfate in three soils（sandy loam,loam and sandy soil）.The results showed that Freundlich model could well describe the adsorption behavior of colistin sulfate in three soils,and R2was 0.9139 to 0.9853.The adsorption strength（1/n）of the three soils to colistin sulfate ranged from 0.6897 to 1.333 3,with little difference.The adsorption affinity of three soils to colistin sulfate was moderate,and the adsorption capacity of sandy loam soil was the strongest,while the adsorption capacity of loam soil was the weakest.The adsorption of colistin sulfate by the three soils was almost irreversible.4.Study on the Migration Characteristics of colistin sulfate in the environment.Colistin sulfate in soil was detected by composting broilers manure containing colistin sulfate with sandy loam.The results showed that the sulfate colistin in compost could quickly enter the soil and remain in the soil for a certain time,and a small amount of colistin sulfate would migrate to 20 cm below the soil surface layer.5.Study on the degradation characteristics of colistin sulfate in sandy loam soil.The degradation behavior of colistin sulfate at three concentrations（10,20 and 40μg/g）in sandy loam soil was studied by simulating the soil micro-environment under natural conditions.The results showed that the degradation of colistin sulfate was relatively slow in sandy loam soil,and the higher the concentration of colistin sulfate,the slower the degradation.The half-lives of colistin sulfate at initial concentrations of10μg/g and 20μg/g were 13.15 d and 19.15 d,respectively,and that at initial concentration of 40μg/g was 29.65 d.Colistin sulfate with a concentration of 10-40μg/g could remain in sandy loam soil for a long time,and could be basically eliminated after 65.75-148.25 days..In conclusion,the established method can be used to determine the content of colistin sulfate in gastrointestinal tract,feces and soil of swine and broilers,which is helpful to understand the absorption of colistin sulfate in gastrointestinal tract of swine and broilers,as well as to study the adsorption,desorption,degradation and migration behavior of colistin sulfate in soil,and to provide reference information for environmental risk assessment of colistin sulfate. |
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