Study On The Determination Of CAP And TAP Residue In Milk By High Performance Liquid Chromatography-Mass Spectrometry

Objective Antibiotic have been widely used in animal husbandry for a long time.To monitor and detect antibiotic residues in animal derived foods is not only significant for the food safety but also can provide technical support for the export and import of animal derived foods.The method of detecting the CAPs residues in milk was not been mentioned in GB/T 22338-2008.The pretreatment methods reported in papers were complicated,the SPE coloums were expensive and the usage of organic reagents were large.A rapid method of detecting CAP and TAP in milk was explored in this study,and would provide a technical support for detecting drug residues in other samples for the next step.Methods Liquid-liquid Extraction?LLE?was used to extract TAP and CAP from milk.0.45?m syringe filters made by polyether sulphone was used for purify to accelerate the speed of progress.Impact of fat and other impurities in milk were reduced,then the sensitivity of the detection was improved.ZORBAX Eclipse Plus C18 chromatographic column was used to separate TAP and CAP,single quadrupole mass spectrometer was used for qualitative and quantitative analysis at the same time.Parameters of LLE,liquid chromatography and mass spectrometry were optimized,and parameters of methodology such as linear range,limit of detection,limit of quantification,accuracy,precision,stability and ability of practical application were tested confirmed that the method could meet the requirment of methodology and practical application.Results 5mL acetonitrile was added to 1.00g milk then supersonic treatment for 5min and centrifugated for 10min at the speed of 4000r/min.4.5mL supernatant liquid was volumed to 5mL by adding 0.5mL acetonitrile,and then was sufficiently mixed before going through a 0.45?m polyether sulphone syringe filters which were saturation with acetonitrile.The mobile phase was ammonium acetate-water and acetonitrile with a flow of 0.300mL/min.TAP and CAP separated perfectly and the shape of peak was good when ammonium acetate-water vs acetonitrile is 40 vs 60 in the gradient elution.Mode of single ion and scan monitoring were all in negative.Capillary voltage was 4500v,drying gas flow was 10.0L/min,nebulizer pressure was 35psig,drying temperature was 350 degrees Celsius and fragmentor voltage was 70v.Quantitation ion of CAP was m/z 321.0 and TAP was m/z 354.0.A good linear relationship of TAP and CAP was showed at range from 0.1ng/mL to 100ng/mL,linear regression equations were y=82.276x+141.31 and y=299.2x+267.31 while the related coefficients were R_TAP=0.9996 and R_CAP=0.9999.The detection limit of CAP was 0.25?g/kg and TAP was 0.27?g/kg by calculating 3 times of the noise-to-signal ratio.Recovery experiment were done in order to check the accuracy,revealing the average recoveries of TAP and CAP in high,low,middle three range of 91.3%to 97.3%and 89.0%to 98.2%,respectively.The relative standard deviations of day precision and day-to-day precision were 0.58%to 1.47%,1.52%to 0.77%and 6.17%to 4.90%,2.06%-6.90%.The samples could be stored for two weeks in 4 degrees Celsius exposure to light which was showed by stability tests.Conclusions A liquid chromatography-mass spectrometry method was established in this study for the rapidly detection of chloramphenicol and thiamphenicol residues in milk.The method was feasible and could be used to determine the CAPs residues in milk by doing actual test with milk bought from market.The method of purified through syringe filter after LLE then detected by mass spectrometry was fast,convenient,accurate,sensitive and could reach the requirements claimed by the methodology,and met the requirements of current laws and regulations to detect CAP and TAP residues in milk.Other more sophisticated equipments such as triple-quadrupole mass spectrometer and ion-trap mass spectrometry could be used to improve the precision and lower the detection limit of the method in future.