Development Of Methods For Determination And Confirmation Of Chloramphenicol And Thiamphenicol Residues In Food Derived From Animals

Chloramphenicol and thiamphenicol belong to phenicols broad-spectrum antibiotic and were widely used to prevent and treat infectious disease of animals effectively in clinic. But their residues in food can do harm to human health because of the toxic-effect and side-effect to blood system. Many countries and organizations set down MRLs of chloramphenicol and thiamphenicol in food derived from animals respectively. Illogical application and misuse of them bring about the residues in food in fact and the methods for determination were not coincidence with the requirements of residues control criterion at home now. So it is necessary to develop determination and confirmation methods separately to find out the absolute mass and avoid the false-positive proportion. So GC and GC/MS/MS methods using solid phase extraction were developed on the basis of studies home and overseas and the applicability of GC/MS/MS method was tested via animal experiment.The samples (β -Glucuronidase and pH6.8 PBS were first added to the liver samples at 37℃ to hydrolyze) were extracted with ethyl acetate via ultrasonic, Then the extract was centrifuged and the organic layer was collected and evaporated to nearly dryness. The residue was dissolved in sodium chloride solution. Then the extract was defatted with hexane and cleaned up with C18 solid phase extraction cartridges that were preconditioned with water and acetonitrile. The cartridges were washed with water and eluted with acetonitrile. The eluant was collected and evaporated to dryness under a stream of nitrogen. Then acetonitrile and Sylon BFT were added to the test tube .The mixture reacted at 70 ℃ for 30min and then was evaporated to dryness with nitrogen, The residue was redissolved in hexane and cyclohexane (6:4) , centrifuged and the upper solution was analysed byGC/ECD and GC/MS/MS.The limits of detection of chloramphenicol and thiamphenicol were 0.5μg/kg and 1 .0μg/kg and the limits of quantitation were 1.0μg/kg and 2.0μg/kg respectively in all tissues in determination method. The recoveries of chloramphenicol in the range of 1-4μg/kg and thiamphenicol in the range of 2~8μg/kg were in the range of 60.1%~ 104.9% and coefficients of variation were less than 16.9%. The calibration curves of standard solution of chloramphenicol and thiamphenicol were linear in the range of 10.0~640.0μg/L with an correlation coefficients over 0.9990.The confirmation method was based on the MS/MS mode. The parent ion of chloramphenicol was 225 and the daughter ions were 208 and 178. The parent ion of thiamphenicol was 330 and the daughter ions were 315, 225 and 178. GC/MS/MS results showed that the limits of detection of chloramphenicol in milk and eggs were 0.1μg/kg and 0.3μg/kg in swine, poultry, fish and poultry liver. The limits of quantitation of chloramphenicol in milk and eggs were 0.3μg/kg and 0.5μg/kg in swine, poultry and fish. The limits of detection of thiamphenicol in milk and egg were 1.0μg/kg and 2.0μg/kg in swine, poultry and fish muscle. The limit of quantitation of thiamphenicol in milk and eggs were 2.0μg/kg and 3.0 μg/kg in swine, poultry and fish muscle The recoveries of chloramphenicol in the range of 0.3~2.0μg/kg and thiamphenicol in the range of 2.0~8%g/kg were in the range of 61.7%~91.2% and coefficients of variation were less than 15.2%. The calibration curve of chloramphenicol standard solution was linear with correlation coefficient 0.9990 by ion 208 in the range of 5.0~80.0μg/L and thiamphenicol was linear with correlation coefficient 0.9980 by ion 315 in the range of 50.0~800.0μg/L.Hens were fed a cereal diet supplemented with chloramphenicol at 1000mg/kg for 5 consecutive days. Eggs of 1st, 6th, 16th .day and livers and muscle of 1st, 7th, 15th day after dosing ceased were collected. Chloramphenicol residues of these samples were determined with confirmation method. There were no interfering peaks during the retention time of chloramphenicol in extract ion chromatogram.The confirmation method quantified with MS/MS is the first method in homeland and could be used to determine residues of animals after the cessation of medication. The key is it provided methodology and analytic demonstration for other confirmation methods. The twomethods are very simple, sensitive and their precision and accuracy according with analytic methodology of the residue methods and suitable for a large number of samples in routine analysis. They could be used as reference method for monitoring the residues of animal drugs in food derived from animals.