Study On Determination Of Nosiheptide In Feeds And Animal Tissues

As a new animal feed additive,nosiheptide has been widely used in animal husbandry and aquaculture industries.However,the irregular or excessive use of nosiheptide may lead to serious or even excessive residue of nosiheptide in animal-derived foods,which might be potentially harmful to human health.In this study,on basis of dispersive solid-phase extraction and alkaline hydrolysis strategies,efficient,sensitive and reliable methods for the determination of nosiheptide content in feeds and nosiheptide residue in animal tissues were developed,which could provide new techniques and means for the analysis of nosiheptide.A method based on dispersive solid-phase extraction was developed for the determination of nosiheptide in animal feeds by high-performance liquid chromatography coupled with fluorescence detection（HPLC-FLD）.Feed samples were extracted with acetonitrile-0.1%formic acid aqueous solution（8:2,v/v）and then purified via a novel dispersive solid-phase extraction procedure using silica gel as the sorbent.After centrifugation,the supernatant was filtered through a 0.22μm filter prior to HPLC-FLD analysis.Based on the combination of 5 mmol/L ammonium acetate aqueous solution（containing 0.1%formic acid）and acetonitrile as the mobile phase,nosiheptide was separated well on an Agilent Poroshell 120 EC-C₈ column（250 mm×4.6 mm i.d.,4μm）with isocratic elution program.The experimental results showed that the calibration curve for nosiheptide revealed good linearity（r≥0.999）in the concentrations range of 50～1000μg/L.The intra-day and inter-day recoveries of nosiheptide obtained from three spiked levels of 0.5,2.5 and 5.0 mg/kg in five types of feeds were from 78.5%to 96.8%and84.9%to 94.2%,respectively,with relative standard deviations（RSDs）below 15%.Limits of detection（LODs）and limits of quantification（LOQs）of nosiheptide in feed samples ranged from 17 to 35μg/kg and 50 to 100μg/kg,respectively.The developed method showed good selectivity,high accuracy and precision,which could be suitable for the detection and monitoring of nosiheptide in real feed samples.A new analytical method based on alkali hydrolysis was developed for the indirect determination of nosiheptide residue in animal tissues by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.Nosiheptide could not be ionized under both electrospray ionization and atmospheric pressure chemical ionization conditions,while the hydrolysis of nosiheptide under alkaline conditions could produce a variety of small molecular compounds.Among them,a specific small molecular fragment,named as 4-hydroxymethy-3-methyl-1H-indole-2-carboxylic acid（HMIA）,had a good mass spectrometry signal under electrospray ionization mode.In this case,the quantification of nosiheptide was achieved through analyzing HMIA.Animal tissue samples were digested with 1 mol/L sodium hydroxide solution.After a degreasing treatment with n-hexane,hydrolysis solution was purified and enriched using a mixed-mode anion-exchange（MAX）solid phase extraction cartridge.The eluent was evaporated to dryness and the residue was re-dissolved with 50%methanol in water.Finally,the solution was passed through a 0.22μm filter for LC-MS/MS analysis.Target compound（HMIA）was separated well on Phenomenex Luna C₁₈ column（150 mm×2.1 mm i.d.,5μm）using acetonitrile and water as mobile phases with a gradient elution program.Multi-reflection monitoring analysis was performed under electrospray ionization negative ion mode.The results showed excellent linearity（r≥0.99）within the nosiheptide concentrations of 2～500μg/kg.Average recoveries of nosiheptide at four spiked concentration levels of 2（4）,10,20 and 30μg/kg in five kinds of animal tissue samples including pork,chicken,fish,chicken liver and chicken kidney were ranged from 75.3%to 108.0%,with the intra-day and inter-day RSDs less than 15%.The LODs and LOQs of the method were ranged from 0.7～1.5μg/kg and 2～4μg/kg,respectively.The proposed method is scientific,sensitive,reliable,high selective and practical for the effective monitoring of nosiheptide residue in animal-derived foods,which will contribute to the studies of pharmacokinetics and residue elimination of nosiheptide.