What are the methods for detecting chloramphenicol residues?
Chloramphenicol was originally prepared by extracting the culture solution of Streptomyces veneris, and is now mass produced by chemical synthesis. It is a broad-spectrum antibiotic. It has inhibitory effects on Gram-positive and Gram-negative bacteria. Among them, typhoid bacillus, influenza bacillus and pertussis are stronger than other antibiotics, and rickettsial infections such as typhus are also effective, but the effect on Gram-positive cocci is less than that of penicillin and tetracycline.
1. Physical and chemical properties: stable in nature, can preserve drug efficacy for more than 2 years in dry state, soluble in organic solvents, only dissolve 25mg in 100ml water at 25°C. The aqueous solution is neutral, stable in acidic and neutral solutions, and easily decomposed in the presence of alkalis. Its synthetic product has a left-handed optical property, also known as Laevomycetin.
2. Chemical structure: Chloramphenicol is a diacetamide derivative of a class of 1-phenyl-2amino-1 propanol. These unique antibiotics are usually composed of three compounds: chloramphenicol (CAP), sulfone (chloro) (THA), and fluoromethylsulfone (chloro) (FLR). All of the above three antibiotics have a common main chain frame, and their structures are shown as follows: they are all called "phenols".
Compound name
R1
R2
R3
CAP
NO
OH
COCHCl2
THA
CH3SO2
OH
COCHCl2
FLR
CH3SO2
F
COCHCl2
"Multiple Substituted 1-Phenyl-2-amino-1-propanol Main Chain"
3. Antibacterial mechanism: Chloramphenicol is a bacteriostatic agent. It is bactericidal at high concentration or when it acts on bacteria that are highly sensitive to this product. It is fat-soluble, diffuses into bacterial cells, and can be reversely combined in bacteria. The 50S subunit of the ribosome hinders the growth of the peptide chain, thus inhibiting the formation of the peptide chain, thereby preventing the synthesis of the protein.
Second, the status and impact of chloramphenicol residues
In the first half of 2002, a series of trade disputes between China and the EU were triggered around animal-derived food. On January 28, 2002, the European Union announced a total ban on the import of animal-derived food from China on the grounds of chloramphenicol residues. The "ban" has blocked China's annual export of about 700 million US dollars of related products. The Chinese side quickly responded to this. Gao Yan, spokesman of the Ministry of Foreign Trade and Economic Cooperation, said in a speech that China has paid serious attention to this matter. He also pointed out that it is unfair and unreasonable for the EU to make a unilateral decision based solely on the investigation report without consulting the Chinese side. The EU is China's traditional honey export market, with an annual import of 150,000 tons of honey, of which 3-4 million tons imported from China, accounting for about 27% of its total imported honey. According to China's customs statistics, in 2001 I exported honey to the EU 4.3. Ten thousand tons, accounting for 41% of the total global exports. On February 7, the United Kingdom stopped selling Chinese honey on the grounds that chloramphenicol exceeded the standard. In the middle and late February, the British Food Standards Agency found out that the honey contained in the market contained the EU banned drug, chloramphenicol residue, in the sampling test on the market. A vicious incident in which listed Chinese honey was recalled. It has also affected the export of Chinese honey to Japan, Canada and other countries. The General Administration of Quality Supervision, Inspection and Quarantine of China issued two urgent notices on the volume of chloramphenicol (National Quality Inspection Letter [2001] No. 510, National Quality Inspection Letter [2001] No. 578), prohibiting the export of aquatic products. Chloramphenicol is used in the production of breeding (including disinfection of environment and equipment), processing, preservation, packaging and transportation, and local inspection and quarantine institutions are required to strengthen exports and rectify within a time limit. Enterprises that fail to pass the inspection will be cancelled. Eligibility for export registration, prohibiting the export of its products On August 21, 2002, the Ministry of Agriculture and the General Administration of Quality Supervision, Inspection and Quarantine issued a joint notice stating that China's aquatic products will be specially rectified to remove the harm of drugs to Chinese aquatic products as soon as possible.
Third, the harm and impact
1. Bone marrow hematopoietic disorder: Chloramphenicol has an inhibitory effect on bone marrow blood production, which can cause thrombocytopenic purple hall, agranulocytosis, aplastic anemia, hemolytic, etc., most of which are used in long-term or multiple administrations. Happened in. The incidence rate is approximately 1/100000-1/5000. The toxicity of bone marrow is divided into two categories: one is reversible inhibition. Mainly affect the formation of red blood cells, platelets and white blood cells. The second is aplastic anemia. The former is related to the dose, and the blood concentration of the drug can be 5ug/ml; the latter is rare, and it occurs late and extremely severe, and has no direct relationship with the daily dose and total amount of chloramphenicol.
2. Toxicity to premature infants and neonates: Some enzymes in the premature and neonatal livers are not fully developed, and the ability to bind glucuronic acid is poor, thus affecting the detoxification process of chloramphenicol in the liver; Kidney excretion ability is also weak, which can lead to drug accumulation and poisoning.
3, gastrointestinal symptoms, oral symptoms: gastrointestinal reactions are mainly abdominal distension, diarrhea, loss of appetite, nausea, vomiting is rare. Oral symptoms such as congestion of the oral mucosa, pain, erosion, angular cheilitis and glossitis.
4, other adverse reactions: can cause optic neuritis, visual impairment, polyneuritis, neurological deafness and severe insomnia. Sometimes toxic psychosis occurs, mainly manifested as hallucinations, auditory hallucinations, loss of orientation, and mental disorders.
Fourth, some national inspection and quarantine requirements:
â— On September 13, 1999, the Ministry of Agriculture of the People's Republic of China issued a notice on the Maximum Residue Limits of Veterinary Drugs in Animal Foods, stipulating that chloramphenicol should not be detected in the edible tissues of all food animals.
â— The Ministry of Agriculture of China has removed chloramphenicol from the 2000 edition of the Chinese Veterinary Pharmacopoeia as a banned drug.
â— In January 2002, the US Food and Drug Administration (FDA) announced a ban on the use of chloramphenicol in imported animal-derived foods.
◠The EU's import food hygiene standards stipulate that “the chloramphenicol content standard is not detectable.†The meaning of “not detectable†is that the chloramphenicol content is below 1 ppb, that is, the content is below one billionth.
â— The special testing standard for some German states is 0.2ppb.
â— China's method detection limit value:
Milk 0.25ng/ml meat 1.5ng/g urine 1.0ng/ml
Serum 0.5ng/ml honey (quick) 1.5ng/g honey (18 columns) 0.1ng/g
5. Detection method of chloramphenicol residue:
(1) Liquid chromatography:
After extracting with ethyl acetate, a portion of ethyl acetate extract was taken, and ethyl acetate was evaporated from nitrogen, and the residue was dissolved in perchloric acid. The fat was removed with n-hexane, and determined by a liquid chromatograph equipped with an ultraviolet detector, and quantified by an external standard method.
(2) Gas chromatography:
The tissue was digested with β-glucuronidase, extracted with ethyl acetate, and then concentrated with ethyl acetate. A 4% sodium chloride solution was added and the remaining ethyl acetate was dried with nitrogen. The salt solution was passed through a C18 extraction cartridge, washed with 20% methanol and eluted with methanol. The eluate was evaporated to dryness and solidified, and chloramphenicol was determined using a gas chromatograph equipped with an electron capture detector. Isochlormycin was used as an internal standard for quantification. The detection limit of the method in bovine muscle tissue is 1.0 ng/g.
(C) enzyme-linked immunosorbent assay
At present, domestic detection of clenbuterol is screened by ELISA and confirmed by GS-MS or HPLC. The ELISA assay is an antigen-antibody reaction performed by an enzyme-labeled antibody or an anti-antibody. Due to the specific catalytic action of the enzyme, the original colorless substrate can be displayed by hydrolysis, oxidation or reduction reaction, so the advantages of the method are as follows: 1. The color can be used for rapid qualitative analysis; Strong; 3, high sensitivity; 4, the enzyme label can be used for multiple samples at one time.
The principle of ELISA is to combine the plant horseradish peroxidase (HRP) with chloramphenicol to form an enzyme-coupled chloramphenicol by chemically combining the specific binding of immunological antigens and antibodies and the high-efficiency catalysis of the enzyme. The coated antibody (goat anti-rabbit lgG antibody) on the solid phase carrier is combined with a specific anti-Clenbuterol antibody, and then the chloramphenicol to be tested and the enzyme-conjugated chloramphenicol are added, which are competitive with the chloramphenicol The antibody is bound, washed, and the substrate is added, and the amount of chloramphenicol to be measured is measured according to the change of the colored substance. If the chloramphenicol to be tested is large, the bound enzyme is less coupled with chloramphenicol, and the amount of colored material is less. The chloramphenicol content in the sample is determined by visual or colorimetric methods. The optimal wavelength for colorimetric is 450 nm and the reference wavelength should be greater than 650 nm.
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