Study On Ivermectin And Its Metabolites In Goat Milk And Establishment Of Avermectin Drug Database Based On High Resolution Mass Spectrometry

Avermectins（AVMs）,classified as highly potent but toxic natural products isolated from Streptomyces avermitilis,are widely used as antihelminthic and antiparasitic agents in aquaculture,animal husbandry and agriculture.AVMs and their metabolites eventually accumulate in the environment through runoff,drug spraying,field drainage,aquaculture medication,or excretion,causing toxic hazards to nontarget aquatic and terrestrial organisms and generating residues in animal derived foods,causing food safety concerns.In this study,we focused on AVMs as target compounds to establish a rapid screening database,establish an analytical detection method based on high-resolution mass spectrometry for AVMs,and analyze the drug metabolized forms of ivermectin（IVM）and its metabolites to further fill the gap in the residues of IVM veterinary drugs regarding their metabolites.The main research content is as follows:（1）An ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry detection method with five AVMs signature residues as target compounds was established.A target screening database was created to explore the cleavage rule in electronic source for its target compounds.Qu ECh ERS method was utilized as the pretreatment method to optimize the pretreament conditions with ammoniated acetonitrile as the extraction agent.The AVMs were monitored and analyzed quantitatively by an external standard method with anhydrous Mg SO₄ and Na₂SO₄ as the water removers and C₁₈ as the adsorbent.The results indicated that the five AVMs were in the linear range of20-1000 ng/m L,with the detection limits of the instrument ranging from 5 to 10 ng/m L,the blank amniotic milk spiked recoveries were 60.53-105.9%,and the RSDs were 3.19-6.71%.（2）An ultra performance liquid chromatography tandem mass spectrometer was developed for the determination of five AVMs in amniotic milk.The samples were purified by SPE using C₁₈ as the stationary phase packed SPE cartridge with acetonitrile as activating agent,acetonitrile water（30:70,v/v）solution containing 0.02%triethylamine as washing solution,and isooctane as eluent.Quantitative analytical detection of AVMs was performed by multiple reaction monitoring and quantification by external standard method according to the characteristic fragment ions of target compounds.The results indicated accurate linearity of MOX,DOR,AVM B1a,and EPR B1a in the mass concentration range of 2-200 ng/m L,IVM B1a in the range of 0.5-50 ng/m L,and the instrumental detection limits were 0.2-1.0 ng/m L,with blank amniotic milk spiked recoveries of 76.9-104%and RSDs of 2.3-6.9%.（3）Analysis of the drug metabolism pathways of IVM in amniotic milk.The unknown metabolites were screened and identified by IVM using oxidation and demethylation as the first phase metabolic reactions.The structures of target metabolites,metabolic reactions,and conversion sites were identified and confirmed by control group experiments,mass defect filtration techniques,chromatograms,and secondary mass spectrograms.Final identification of four first phase IVM metabolites.