Study On The Detection Methods For Amphemicols Residue In Animal-derived Food

Amphemicols is a kind of broad-spectrum antibiotic,including chloramphenicol(CAP) and its representative derivatives,such as thiamphnicol(TAP) and florfenicol(FF).They have a strong inhibition to Gram-negative and Gram-positive bacteria,in particular stronger for the former.At present, amphemicols have been widely used in the production of animal-derived foods.However,they are harmful for human's health.Chloramphenicol residues can inhibit on hematopoietic function of human bone marrow,and can cause aplastic anemia.Therefore,they have been listed as prohibited drugs in many countries and regions.Thiamphenicol and florfenicol are substitute products of chloramphenicol. Florfenicol amine is a major metabolite of florfenicol.In recent years,animal-derived foods in our country have been frequently detected residues of chloramphenicol,which have become a major obstacle in the international trade.In order to protect human healthn and our country's normal export trade,it is urgent to control drugs residues in animal-derived foods.The foundation is to establish sensitive and reliable detection methods for drug control.The detection methods of amphemicols include microbiological method,immunoassay method, chemical analysis and other methods.Among them,microbiological method is easy influenced by other antibiotic residues in samples,and specificity and sensitivity are low.Fasle positive results are easy to appear in immunoassay,samples needs complex pretreatment before detection in chromatographic analysis. Chromatography-mass spectrometry with high sensitivity is an important qualitative and quantitative detection method.Nowadays the detection object is almost single in the available methods for amphemicol drugs,and no report on mass spectrometry of florfenicol amine can be found.However,many kinds of amphemicol drugs frequently exist in animal-derived foods,it is necessary to develop simpler,highly sensitive detection techniques for multi-residues.Based on the properties of amphemicol drugs,the conditions of sample extraction and purification were optimized for CAP,TAP,FF and FFA,and established simultaneous detection methods for the four drugs by using gas chromatography(GC),high performance liquid chromatography(HPLC),gas chromatography-mass spectrometry(GC-MS) and liquid chromatography-tadem mass spectrometry (LC-MS/MS),and the main results obtained were summarized as follows.1.Optimization of sample extraction:pork and casing as research objects,the four organic solvents such as methanol,acetonitrile,acetone and ethyl acetate were used to extract amphemicol drugs,and the extraction results were compared by different solvents.The results showed that the extraction rates were high using alkalizing ethyl acetate containing 2%of ammonia,and the recoveries were all obove 90%.2.Optimization of purification conditions:Good purification results could be found by using liquid-liquid extraction and solid-phase extraction.The samples can be directly detected after liquid-liquid extraction by LC-MS/MS with high sensitivity.However,the samples need be extracted by liquid-liquid extraction and solid-phase extraction before detection by HPLC,GC and GC-MS methods.Otherwise,the detection would be influenced by the matrix interference.Amphemicol drugs could be retained by MCX, C₁₈ and HR-X columns.Among them,the desired retention results for the four drugs could be obtained by using MCX coulm,and all of the recoverie were over 95%.3.Optimization of derivation conditions:The samples need be transformed to the derivants with highly thermal stability and easy to volatize before detection by using GC and GC-MS methods.The optimal derivation conditions were obtained,i.e.the derivation temperature was 50-70℃,and the time was about 40 min.It could be found that the recoveries were low and the operation was difficult to control by drying the derivatization reagent.This problem could be solved by constant volume with the addition of toluene and inactivation with NaOH solution detection.4.Establishment of simultaneous detection methods for multi-residues of amphemicols:FF and FFA residues in pork and casing could be simultaneously detected by using HPLC method with UV detector, and the low limits of detection of the two drugs were both 100μg/kg.CAP,TAP,FF and FFA in pork and casing could be simultaneously detected by using HPLC-MS/MS method,and the low limits of detection of the four drugs were 0.1μg/kg,0.1μg/kg,0.1μg/kg and 8.0μg/kg,respectively.The four drugs in pork and casing could be simultaneously detected by using GC method with ECD detector,and the low limits of detection of the four drugs in pork were 0.1μg/kg,2.0μg/kg,2.0μg/kg and 2.0μg/kg,respectively,and that on casing were 0.2μg/kg,4.0μg/kg,4.0μg/kg and 4.0μg/kg,repectively.The four drugs in pork and casing could be simultaneously detected by using GC-MS method,and the low limits of detection of the four drugs in pork were all 0.1μg/kg,and that in casing were 0.2μg/kg,0.2μg/kg,0.2μg/kg and 2.0μg/kg, repectively.