Identification And Enrichment Release Of Pleuromutilin In Carassius Auratus

Pleuromutilin is a broad-spectrum antibiotic that is a tricyclic diterpene compound produced by higher fungi.The antibiotics synthesized by the pleuromutilin precursors mainly include valnemulin（VLM）and tiamulin（TML）.The above antibiotics have a five-membered ring and an eight-membered ring.A group such as a carbonyl group,an acyl group at the C-14 position,and a hydroxyl group at the C-11 position.Valnemulin mainly binds to the 50S subunit of pathogenic microbial ribosomes in poultry,aquatic products,etc.,and interacts with the V-site of pathogen 23SrRNA to prevent the correct localization of the pathogen’s peptidyl transferase at the CCA-terminus.,thereby inhibiting the production of proteins and achieving the purpose of killing pathogens.The tiamulin has good effects on mycoplasma,porcine sputum dysentery,ureaplasma infection,porcine actinomycetin pleuropneumonia,intestinal spirochete,and porcine pneumonia.Valnemulin has low toxicity,no carcinogenic,teratogenic,mutagenic and reproductive toxicity,and no immune system damage.Because pleuromutilin can effectively act on intestinal spirochetes,mycoplasma and Gram-positive bacteria,they are widely used in the prevention and treatment of aquatic products such as fish and terrestrial poultry diseases such as pigs and cattle.The test animals herein are Carassius auratus.detectioning methods for the residues of valnemulin and tiamulin in fish were established under experimental conditions.The enrichment and release of these two pleuromutilin antibiotics in the Carassius auratus were further explored.The main research contents are as follows:1.A detection method suitable for the residues of valnemulin and tiamulin in fish was established by comparing the extraction reagents,optimization of purification conditions,chromatographic conditions and optimization of mass spectrometry conditions.The sample of each tissue of Carassius auratus were ultrasonically extracted with 2%formic acid-acetonitrile,and then purified by Oasis MCX SPE cartridge and determined by ultra performance liquid chromatography-tandem mass spectrometry（UPTM-MS/MS）.Both the mycobacterin and the valnemulin were quantified by the internal standard method and the external standard method.The aqueous solution of ammonium acetate containing 0.05%formic acid was used as a mobile phase,separated by gradient elution,and detectioned by electrospray positive ion multiple reaction detectioning mode.The results showed that the two pleuromutilin antibiotics showed a good linear relationship in the mass range of5¹000 ng/mL.The detection and quantification limits of valnemulin and tiamulin were0.03μg/kg and 0.1μg/kg,respectively.Under the conditions of 0.1,1.0,5.0,10.0,50.0μg/kg,The average recovery of valnemulin and tiamulinin muscle was 96.7%¹13.78%,the relative standard deviation was 1.02%³.68%;the average recovery rate of valnemulin and tiamulinin the liver was 99.2%¹15.43%,the relative standard deviation was2.60%⁷.06%;the average recovery rate of valnemulin and tiamulinin intestinal was80.35%¹11.41%,the relative standard deviation was 1.68%⁷.24%;the relative standard deviation between batches is 2.26%⁹.10%,both less than 10.0%,which meets the requirements of the experiment.The method has the advantages of simple operation,less time consumption,good repeatability and high sensitivity,and the test results show that the method is suitable for the actual determination of valnemulin and tiamulinin the squid.2.The squid tissues were taken at different time points by the intraperitoneal injection method,and analyzed by ultra performance liquid chromatography-tandem quadrupole and time-of-flight mass spectrometry（UPLC/Q-TOF MS）,combined with the accurate quality of each metabolite and related literature,to determine the production of tiamulin in the Carassius auratus.The five metabolites are M1(510.2908,C₂₈H₄₈NO₅S⁺),M2(510.2908,C₂₈H₄₈NO₅S⁺),M3(466.2750,C₂₆H₄₄NO₄S⁺),M4(482.2663,C₂₆H₄₄NO₅S⁺)and M5(482.2663,C₂₆H₄₄NO₅S⁺).M1 and M2 are isomers;M4 and M5 are also isomers,but are also metabolites of M3,which are secondary metabolites of tiamulin.Valnemulin produces three metabolites V1(581.3182,C₃₁H₅₂N₂O₆S),V2(581.3182,C₃₁H₅₂N₂O₆S)and V3(597.0903,C₃₁H₅₂N₂O₇S)in the Carassius auratus,and V1 and V2 are isomers;Due to the limitations of the literature and experimental instruments,the specific structure has not been determined.The target organs of valnemulin and tiamulinare livers and are excreted through the gallbladder.Metabolites are also present in other tissues.The three metabolites of valnemulin are more likely to appear in the gallbladder and skin.In addition,there are different metabolites produced by different species between valnemulin and tiamulin.3.Explore the enrichments and metabolism of tiamulin and valnemulin,and their metabolites in the Carassius auratus fry by the medicated bath metho.The results showed that the main metabolic production of tiamulin was M1（8α-OH-TML）,M3（N-deethly-TML）and M4（8α-OH-N-deethly-TML）.The main metabolites of valnemulin were V1（8α-OH-VLM or 2β-OH-VLM）and V2（2β-OH-VLM or8α-OH-VLM）,and the main metabolic pathway was hydroxylation of the mother nucleus.Determined from this,the potential marker residues of tiamulin in Carassius auratus are8α-OH-TML,N-deethly-TML and 8α-OH-N-deethly-TML;the potential marker residue of valnemulin in Carassius auratus is 8α-OH-VLM and 2β-OH-VLM.In the enrichment stage,the content of valnemulin and tiamulin exhibit a zigzag rise,exhibit a stable wave during the saturation phase,and exhibit a downward trend in the final release phase.The whole process shows a trend of rising first and then falling.The bioconcentration factors of valnemulin and tiamulin in the Carassius auratus fry were 1.23 and 3.01,respectively.The bioconcentration factor of tiamulin was greater than the bioconcentration factor of valnemulin,and the tiamulin was rich.The set rate is faster,but both substances can reach saturation within 2 days.The half-life of valnemulin and tiamulin in Carassius auratus is4.28 days and 3.74 days,respectively,and the elimination rate of tiamulin is faster than that of valnemulin.And the second week of the purification of the two drugs,did not eliminate completely;intraperitoneal injection of two drugs,the drug in each tissue can be completely eliminated within one day.The results showed that the metabolism of the two drugs in different ways was different in different ways of administration.The route of administration by intraperitoneal injection was much faster than that after the drug was stopped on the seventh day.