Detection Method Reseach Of Antibiotics And Sadatives In Biological Samples

Veterinary drugs are widely used in animal for the prevention,treatment of diseases and the promotion of animal growth,and used as preanaesthetic and muscle relaxant in animals to prevent mortality and loss of meat quality during the transportation to the slaughterhouse.These veterinary drugs include antibiotics,tranquilizers,hormones and so on.Antibiotics usually used in animal feeding process is the main purpose of antimicrobial drugs,but studies have shown that antibiotics in the antimicrobial drugs can promote gastrointestinal absorption of nutrients to indirectlypromoteanimalgrowth.Thereforechlortetracycline,oxytetracycline and virginiridae are often used as animal additives.Tranquilizers have a sedative and hypnotic effect that can be used during animal husbandry to reduce animal activity and energy consumption and promote growth.In the process of animal transport,the use of tranquilizers reduces the alarm reaction of animals and avoids the sudden death.These drugs often include benzodiazepines,barbiturates and?-blockers.Although the widespread use of veterinary drugs has contributed to the development of livestock husbandry,it has also brought harm to human diet and health.The use of antibiotics has reached the point of abuse in the past century.Antibiotics were absorbed into animals by the blood circulates and caused drug residues in muscles.Such residuals may lead to bacterial resistance.There are reports that antibiotic abuse lead to super bacteria cannot be ignored.Some antibiotics with animal excrement into the environment caused the environment and drinking water were polluted.Eating animal food with residue sedatives will lead to increased liver burden,long-term drowsiness,impaired memory,and inhibition of motor and muscle functions.In order to ensure the health and safety of human food,the relevant provisions of the EU prohibit the use of antibiotics as growth promoters in the process of animal husbandry.China has also established relevant testing standards and LOD.Chinese Ministry of Agriculture No.176 and 235prohibits the use of sedatives in drinking water and cannot be detected in animal-derived foods.In this paper,UPLC-MS/MS method was established for the screening of 15 antibiotics in bovine muscle,urine and blood and 20sedatives in bovine muscle on the bias of screening and optimization of sample pretreatment conditions,instrument conditions and analytical methods.The result shows that the methods have high sensitivity and good reproducibility and accuracy.Part I Separately detection of 15 antibiotics in bovine muscle, bovine blood and bovine urine by UPLC-MS/MSObjective:A novel method was established to detect 15 antibiotics in bovine muscle,bovine biological samples including blood and urine.Methods:For the sample of bovine muscle and blood,the analytes were respectively extracted with 1 mL 0.1 mol/L EDTA-2Na and 9 mL acetonitrile,10 mL of ACN-water(90:10,V/V).A total of 10 mL extraction was pipetted in a 15 mL glass tube concentrated to dryness under gentle nitrogen stream below 40?.The residue was reconstituted with 1 mL ACN-water(3:7,V/V).The target drugs in urine were extracted by adding 1mL 0.1 mol/L EDTA-2Na solution and adjusting the pH to 5.2 and shake for 5 min;then transfer sample to HLB column for purification.Wash with3 mL methanol-water(10:90,V/V);then eluted the analyte with methanol-ethyl acetate(1:1,V/V).The eluent was collected and dried under nitrogen at 40?.The residue was reconstituted with 1 mL of acetonitrile-water(3:7,V/V).The target drugs were separated on ACQUITY UPLC~?BEH C18(2.1×100 mm,1.7?m),mobile phase component A is acetonitrile,and B is a 2 mmol/L ammonium acetate solution containing 0.2%formic acid.The flow rate was 0.3 mL/min;the column temperature was 40?.Mass spectrometry conditions:positive ion mode(ESI+):electrospray voltage 5.5KV;atomization gas pressure0.45MPa;air curtain gas pressure 0.21MPa;auxiliary air pressure 0.41MPa;temperature 650°C;collision chamber inlet 10V.Matrix-matched calibration curves were used for reducing disturb and quantification.Results:The mean recovery at three spiked levels are respective74.6%-119.3%,75.7%-110.6%and 75.9%-119.2%in bovine muscle,blood,and urine.RSD%range from1.1%to 14.1%in spiked bovine muscle,RSD%range from 1.0%to 13.6%in spiked bovine blood,and RSD%range from 1.1%to 14.7%in spiked bovine urine.Conclusion:The method is rapid,sensitive and simple pretreatment.It provides a theoretical basis for the detection of antibiotic growth promoters in bovine muscle,blood and urine.Part II Simultaneous determination of sedatives and?-receptor blocker in bovine muscle using UPLC-MS/MSObjective:To establish an UPLC-MS/MS method for the simultaneous determination of sedatives and?-blockers in bovine muscle.Methods:15mL 0.26 mol/L ammonia-acetonitrile(8:92,V/V)extraction and 40?L?-glucuronidase/sulfatase were added into the sample,enzymatic hydrolysis 16 h in shaker under the condition of 37°C and 110 r/min.After that,2 g anhydrous magnesium sulfates were added to the centrifuge tube and shake for 30 min on the shaker.Pipette the supernatant and concentrate to dryness.The residue was dissolved with 3 mL 0.1mol/L hydrochloric acid and purified with HLB cartridge.The analytes rinsed with 3mL methanol-water(5:95,V/V)solution and then elute with 3 mL methanol and 3 mL acetonitrile.The eluent was concentrated to dryness and with 1mL acetonitrile-water(1:9,V/V)to reconstitute the residue.The analytes were separated on an Acquity UPLC~TMM BEH C18 column.Positive ion electrospray mode was eluted by gradient with acetonitrile and 2 mmol/L ammonium acetate solution(containing 0.2%formic acid).Negative ion electrospray mode was eluted by gradient with acetonitrile and water.The flow rate was 0.3 mL/min;the column temperature was 40?.Mass spectrometry conditions of positive ion electrospray mode:electrospray voltage 5.5KV;atomization gas pressure 0.4MPa;air curtain gas pressure0.4MPa;auxiliary air pressure 0.5MPa;collision chamber inlet 10V;temperature 500?;negative ion mode(ESI-):electrospray voltage 4.5KV;atomization gas pressure 0.4MPa;air curtain gas pressure 0.4MPa;auxiliary air pressure 0.5MPa;temperature 500?;collision chamber inlet-10V.Matrix-matched calibration curves were used for reducing disturb and quantification.Results:Twenty compounds showed a good linear relationship in the range of 0.5-50?g/L(r~2?0.99)with LOD ranged from0.012 to 0.604?g/kg and the limit of quantification in the range of0.021-0.216?g/kg.The mean recovery percentages at three spiked levels(0.5,1.0,5.0?g/kg)were in the range of 71.2%-123.8%with relative standard deviations(RSD)of 1.1%-14.9%.Conclusion:The established method was accuracy,sensitivity and reproducibility can meet the requirements of veterinary drug residue analysis and provide a reliable and accurate technical support for the detection of sedatives and?-blockers in animal-derived foods.