The Establishment Of An LC-MS/MS Method For The Determination Of Rifampicin Residues In Aquatic Products And The Eliminating Regularity Of Rifampicin In Tilapia

Rifampicin,a broad-spectrum antibiotic for human use,is used to treat varieties of bacterial infectious diseases.The drug is not approved for animal use.However,it might be illegally added in aquaculture due to its low price,wide range of sources and good antibacterial effect.Rifampicin is prone to drug resistance and toxic side effects.If rifampicin was abused in aquaculture,the remaining drug in the edible aquatic animals would be accumulated in the human body through the food chain,posing heavy security risk to consumers'lives and health.However,the technical standards for the determination of rifampicin in aquatic products or related animal foods have not yet been established in our country,so the rifampicin remaining in aquatic products would not be monitored.Therefore,the research on the determination of rifampicin residues in aquatic products and the residual eliminating regularity of rifampicin in tilapia would be of great theoretical and practical significance for regulating the illegal use of rifampicin in aquaculture and guaranteeing the quality and safety of aquatic products.A LC-MS/MS method for the determination of rifampicin residues in aquatic products was established by optimizing the pretreatment methods of samples,the chromatographic conditions and the mass spectrometry conditions.And the eliminating regularity of rifampicin in tilapia was explored.The main contents and results of the research were shown as follows:1. Determination of rifampicin residues in aquatic products by QuEChERS-HPLC-MS/MS:The sample was extracted with acetonitrile after adding anhydrous sodium sulfate and internal standard rifampicin-D4.The analyte mixture was vortexed,sonicated,and dried by nitrogen.The residue was reconstituted with the initial mobile phase and then performed by using 30 mg C₁₈and 30 mg PSA during the dispersive solid-phase extraction procedure.The analytes were separated on a Inert Sustain Swift C₁₈column using binary mobile phase gradient with water containing 3 mmol/L ammonium acetate solution?containing 0.05%formic acid?and acetonitrile.The targeted compounds were detected under a multiple reaction monitoring?MRM?mode with positive electrospray ionization.As the results shown,the good linear relationship in the concentration range of 1.0?250?g/kg for rifampicin was shown with the correlation coefficients?r?of over 0.9994.The detection limit of the method was 0.3?g/kg,and the limit of quantification was 1.0?g/kg.The average recoveries for Oreochromis mossambicus,Penaeus Vannamei Boone,and Pelodiscus sinensisr at three spiked levels?1,20 and 200?g/kg?ranged from 93.6%?105.2%with the intraday precision between 3.15%and 5.94%and the daytime precision under 9.31%.The method could be applied in the determination and confirmation of rifampicin in aquatic products due to its simplicity,rapidity,sensitivity and high accuracy.2. The establishment of analytical method for rifampicin in tilapia tissuesThe analytical methods of rifampicin in the tilapia tissues were established.The detection limits of rifampicin for the tilapia plasma,muscle,liver and kidney were3.0?g/L,0.3?g/kg,0.3?g/kg,0.6?g/kg,respectively.The quantification limits were10.0?g/L,1.0?g/kg,1.0?g/kg and 2.0?g/kg,respectively.The average recovery of rifampicin for tilapia tissues ranged from 95.0%to 105.2%with the intraday precision and daytime precision less than 5.21%and 9.31%,respectively.The method was simple,stable,sensitive and accurate,and it could be applied in the confirmation and quantitative analysis of rifampicin in the tilapia tissues of plasma,muscle,liver and kidney.3. Study on the elimination of rifampicin in tilapiaAfter the tilapias were given a single oral dose of rifampicin?40 mg/kg?,the plasma concentration-time curve,liver concentration-time curve,kidney concentration-time curve were best described by a one-compartment model with first-order absorption,and the muscle concentration-time curve was best described by a two-compartment model with first-order absorption.The main pharmacokinetic parameters of RIF in each tissue were:the T_maxin plasma,muscle,liver and kidney tissues were 6.750,6.539,6.062,6.680 h,respectively.The T_1/2?were 8.653,10.542,7.908,8.509 h,respectively.The C_maxwere:1.566,0.087,2.691,1.181?g/m L or?g/g,respectively.After the tilapias were given a multiple oral dose of rifampicin?40 mg/kg per day?for 5 consecutive days,the concentration-time curve of plasma,muscle,liver and kidney were best described by the non-compartmental model.The main pharmacokinetic parameters of rifampicin in tissues were:The T_maxin plasma,muscle,liver and kidney tissues were at 6^thh;T_1/2?was 9.372,11.816,8.692and9.315 h,respectively;C_maxwas 2.438?g/m L,0.141,3.953,1.664?g/g,respectively.After single-dose and multi-dose administration,the concentration of drug in plasma,muscle,liver and kidney tissues could reached the maximum concentration between the 6^thand 7^thh,and the residual time in the body was a little long.There was no significant difference in the T_1/2?and C_maxof the the same tissue between the single-dose group and the multi-dose group.A well-established LC-MS/MS method for the determination of rifampicin residues in aquatic products and the research on the eliminating regularity of rifampicin in tilapia would provide technical support for ensuring the quality and safety of aquatic products and a theoretical basis for regulating the illegal use of rifampicin in aquaculture as well.