Determination Of Eleven Anabolic Steroid In Edible Tissues Of Porcine And Chicken By UPLC-MS/MS

Anabolic hormone is a kind of steroids with strong protein assimilation,which can inhibit dissimilation or oxidative metabolism and improve protein deposition,and has important residual toxicological significance.Among anabolic steroid,Testosterone,Methyltestosterone,Progesterone,Trenbolone,Boldenone,Nandrolone,Methandrostenolone,Stanozolol,Nandrolone 17-propionate,Testosterone Propionate and Nandrolone Phenylpropionate were abuse most in animal husbandry.Announcement No.250 issued by the Ministry of agriculture clearly indicates that Methyltestosterone,Trenbolone and some other drugs shall not be added to food animals.But nowadays,there still report the illegally using of this kind of drugs in livestock and poultry and aquaculture.In order to provide a detection technology for anabolic steroid residues in animal derived foods,this study establish a reliable and sensitive ultra performance liquid chromatography-tandem mass spectrometry method for the detection of eleven anabolic steroid in edible tissues(muscle,kidney,liver,egg)of porcine and chicken.Considering the composition of tissue sample and egg has difference,this study establish two methods for the pretreatment of sample.The anabolic hormones in muscle,liver and kidney of animal tissue samples were extracted with 10 ml methanol-acetonitrile(1:1,v/v)solution,then placed it at-20 ℃,frozen for 2 h.Then centrifuged it at 12000 R/min for 10 min,the extracted supernatant was rotated and evaporated to nearly dry in a 37 ℃ water bath.Dissolved the residue in 4 ml 25%acetonitrile,then added reconstitution fluid to the C18 solid-phase extraction column after centrifugation for purification,then dried in nitrogen under water bath at 37 ℃.Using 1 m L30% acetonitrile water(30:70,v/v)for re-dissolution,and vortex for 2min.The supernatant was filtered through 0.22 μm filter membrane,then detect by UPLC-MS/MS.Using 0.1% formic acid water and 0.1% acetonitrile as mobile phase and gradient elution.The eggs were extracted with 10 ml methanol-acetonitrile(80:20,v/v),dried with nitrogen,and dissolved again with 0.1% formic acid-30%acetonitrile water(30:70,v/v),other steps were the same as the pretreatment of pig and chicken tissues.In this method,the limit of detection of eleven anabolic steroid in porcine muscle,liver and kidney were 0.1～0.5 μg/kg,0.2～1.0 μg/kg and 0.1～0.5 μg/kg respectively;the limit of detection of eleven anabolic hormones in chicken muscle,liver and kidney were 0.1～0.5 μg/kg,0.1～1.0 μg/kg and 0.1～1.5 μg/kg respectively;the limit of detection in eggs were 0.1～0.5 μg/kg.The limits of quantification of eleven anabolic steroid in pig muscle,liver and kidney were 0.3～1.0 μg/kg,0.6～3.0μg/kg and 0.3～1.5 μg/kg respectively;the limits of quantification of eleven anabolic steroid in chicken muscle,liver and kidney were 0.3～1.0 μg/kg,0.3～3.0 μg/kg and0.4～1.5 μg/kg respectively;the limits of quantification of eleven anabolic steroid in egg were 0.4～1.5 μg/kg.The linear range of eleven anabolic steroid in pig muscle,liver and kidney was made to 1～100.0 μg/kg,3～150 μg/kg,1.5～100 μg/kg,the correlation coefficient was greater than 0.99,which showed good linearity.Besides,the linear range of anabolic hormones in chicken muscle,liver,kidney and egg was made to 1～100 μg/kg,3～150μg/kg,1.5～100 μg/kg,1.5～100 μg/kg,the correlation coefficient was greater than0.992,also showed good linearity.In order to investigate parameters such as repeatability and reproducibility,he research add three concentrations of eleven anabolic steroid(10 μg/kg,20 μ g/kg and50 μg/kg)to the blank muscle,kidney,liver and egg of pig and chicken.The average recoveries of eleven anabolic steroid in muscle,liver and kidney of pig were74.3%～101.1%,70.8%～100.1%,70.5%～99.8%,the coefficients of variation(CV)within-run were found to be about 2.7%～14.1%,1.1%～7.9%,0.9%～6.1%,respectively.The coefficients of variation between-run were found to be about6.0%～13.7%,2.2%～13.5%,3.4%～12.6%,respectively.Besides,the average recoveries of eleven anabolic steroid in muscle,liver,kidney and egg of chicken were 70.1%～97.4%,73.5%～102.3%,72.4%～98.0%,72.7%～97.0%,the coefficient of variation within-run were found to be 2.4%～14.1%,0.8%～6.8%,2.6%～7.1%,2.4%～8.9%.Besides,the coefficient of variation between-run were found to be6.0%～13.7%,2.1%～9.1%,2.1%～9.7%,2.6%～8.6%,respectively.Results showed that the method established by ultra performance liquid chromatography-tandem mass spectrometry for the determination of eleven kinds of anabolic steroid in edible tissues of porcine and chicken is sensitive,reproducible and has great operability,which conform to the requirements of the analysis of veterinary drug residue.