Research And Application Of Analysis Method For Teicoplanin And Ramoplanin In Aquaculture Water And Aquatic Products

Antibiotics are effective methods for treating various bacterial infections.Among glycopeptide antibiotics,teicoplanin（TEC）and ramoplanin（RAM）are commonly used and studied antibiotics in clinical settings.They are used to treat a variety of severe Gram-positive bacterial infections,particularly methicillin-resistant Staphylococcus aureus infections and vancomycin-resistant clostridium difficile infection.Due to the good antibacterial effect,they may be used in aquaculture for the prevention and treatment of bacterial infections.Among them,some glycopeptide antibiotics have been used at home and abroad in food animals,such as livestock and poultry and they are detected in the food.On the other hand,they can also enter aquaculture through medical wastewater,pharmaceutical waste water and other indirect channels.Their residues in environmental water and aquatic products can threaten human health and ecological balance.Therefore,it is urgent to establish the detection methods for the determination of teicoplanin and ramoplanin in environmental water and aquatic products,so as to provide technical support for the government supervision and risk assessment in environmental water and aquatic products.In this paper,a simple,sensitive and simultaneous method was established and validated for active clinical components of teicoplanin and ramoplanin in environmental water by LC-MS/MS coupled with cascade elution.Gradient elution was performed by using a C₁₈ column separated with 0.1%formate acetonitrile and 0.1%formic acid water as two mobile phases.Multiple reaction monitoring mode（MRM）was selected,and the internal standard method was used for determining all targets.Samples were filtered by glass microfiber membrane and enriched by HLB solid phase extraction box.Then the seven structurally different targets were classified by their polarity,the cascade elution technique was used to elute these targets according to similar compatibility principle.TA2-1,TA2-2,TA2-3,TA2-4 and TA2-5 were eluted by methanol,and RA2 and TA3-1 were eluted by 20%（v/v）methanol aqueous solution（containing 0.5%formic acid）.Under the optimized conditions,the method showed excellent linearity with correlation coefficient（R²）more than 0.998 in the range of 1ng/L to 100 ng/L.By spiked in two different matrix samples（river water and nearshore seawater）at low（LOQ）,medium（5 LOQ）and high（10 LOQ）level,the recoveries of the target ranged from 86.0%to 114.5%,and the RSDs is less than 3.0%.In these two sample matrix,the low matrix effects were obtained for most targets.The limits of detection and limit of quantification were 0.1 ng/L-1.3 ng/L and 0.3 ng/L-4.0 ng/L,respectively.This method was successfully applied for the determination of teicoplanin and ramoplanin in forty water samples（six types）from pearl river basin and other places.TA2-2,3 was quantified in only one sample with the concentration of 8.0 ng L^-1.In brief,a sample pretreatment method for synchronous elution of seven different polar compounds were successfully proposed,realizing the simultaneous determination of seven trace components of teicoplanin and ramoplanin in environmental water.A developed Qu ECh ERS method combined with LC–MS/MS was developed for the simultaneous determination of TEC and RAM residues in aquatic products.The experiment optimized the type of extraction agent,the extraction solvent volume,the purification agent type and the amount of purification agent to determine the optimal sample pretreatment method.The new green adsorbent-chitosan was used as the modified Qu ECh ERS purification agent.Compared to PSA,C₁₈,and GCB,it was evaluated by the purification ability for teicoplanin and ramoplanin from complex aquatic product matrix.In comparison with PSA,C₁₈ and GCB,chitosan has a relatively better purification capacity for the targets（the recoveries ranged from 90.3%to 93.5%）.The purification capacity was sorted from large to small to:chitosan>PSA>C₁₈>GCB.In addition,derived from natural waste resources such as shrimp shells,crab shells and others,chitosan is a green,renewable,and biodegradable material.Thus,Chitosan has a good purification capacity for complex aquatic product matrix,and it is a potential green purifying agent for Qu ECh ERS.Under the optimal conditions,the limits of detection and quantification ranged from 0.4μg/kg～0.8μg/kg and 1.0μg/kg～3.0μg/kg,respectively.Calibration curves showed correlation coefficient values higher than 0.998 in rang between 1 ng/m L and 100 ng/m L.The recoveries ranged from89.8%to 110.1%with relative standard deviation less than 7.2%for this method.The matrix effect of all targets is-2.6%～-18.5%,less than±20%,and which is low in nine different aquatic products,such as carp,tilapia fish,grass carp,silver carp,bighead carp,turbot,prawn,sea cucumber,and eel.The method was successfully applied to 90aquatic products randomly selected from aquaculture fish ponds and local supermarkets in Guangzhou.These targets were not detected in the samples.Several blank samples were selected for low-concentration spiked experiment（3.0μg/kg）,and the recoveries of all samples were between 90.0%and 106.8%,and the RSD ranged from 0.9%to5.6%.The proposed method proved to be a green,economical,sensitive,efficient,accurate,reliable,and rapid determination for multitudinous samples,indicating their applicability for detection and monitoring of TEC and RAM residues in aquatic products.