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2014/SCSC/WKSP2/009 Session: 6.2
Monitoring of Residual Content of Antibiotics in Food
Submitted by: Russia
Food Safety Cooperation Forum Partnership Training Institute Network Proficiency Testing
WorkshopBeijing, China
10-11 September 2014
MONITORING OF RESIDUAL CONTENT OF ANTIBIOTICS IN FOODProfessor, D.Sc. TYRSIN Yu.
Moscow State University of Food Production, Federal Agency on the Technical Regulation and Metrology, RUSSIA
ANTIMICROBIAL AGENTS IN VETERINARY
• Agricultural production includes someantimicrobial agents in veterinarymedicine for prevention and treatmentof livestock and poultry. However, dueto their lack of efficiency, in somecases, manufacturers should applyantibiotic substances used for thetreatment of humans (derivatives oftetracycline, penicilline, levomicetine,sulfonamides, furaxane, furazolidonand so on).
PROBLEMS OF ANTIMICROBIAL AGENTS
• The use of these drugs may lead to thesubsequent development of asustainable data substances of humanmicroflora consuming foods thatcontain antibiotics. A person developsa goiter, and appointing him theantibiotic treatment is a high risk oftreatment failure. Possibility of suchmeat products while importing, as wellas the development of intensivelivestock and poultry makes theproblem of control highly relevant.
Chloramphenicol (levomicetine)• Synthetic broad-spectrum antibiotic,
which is prohibited for use in animalhusbandry. The relative cheapness ofproduct and high antibacterial efficacyleading to his unauthorized use of afairly large scale, therefore in meats,liver, kidneys, eggs and other productsfrequently detected residues oflevomicetine at concentrations rangingfrom 0.02 - 0.50 units/g of sample.
• 1 unit of activity corresponds to 1 mgof pure substance.
TETRACYCLINE• A broad-spectrum antibiotic that is highly
effective and used in medicine for thetreatment of various diseases, as well asin veterinary medicine due to the highantimicrobial efficiency(chlortetracycline, oxytetracycline) that isunsafe in terms of sustainability of themicroflora to the human antibiotic,consuming food products contaminatedtetracyclines. MPC of tetracycline groupsantibiotics residue in meat is 0.01 units/g.1 unit corresponds to 1 μg. RussianFederation safety requirements ofantibiotic residue content in meatproducts is not permitted and regulatedat the level of 0,01 unit of the antibioticactivity (μg) in one gram of the sample.
SULFONAMIDES• Sulfonamides is a group of chemicals
derived from para-aminobenzolsulfamid.Para-aminobenzolsulfamid is the simplestconnection class — also called whitestreptocide and is used in medicine so far.The result of sulfonamides action is aviolation of the synthesis of nucleic acidsin bacteria.▫ Many side effects of sulfonamides
(sulfamethoxazole, sulfadimidine(synonyms sulfamethazine, sulfamezathine),sulfaquinoxaline) in human organism.
CIPROFLOXACIN• Ciprofloxacin (according UPAC: 1-
cyclopropyl-6-fluor-4-oxo-7-pyperasine-1-quinoline-3-carbonic acid) a syntheticantibacterial effective against manygram-positive and gram-negative bacteria.
• A synthetic broad-spectrum antibiotic ofthe fluoroquinolone class that inhibitsenzyme deoxyribonucleic acid (DNA)gyrase needed for replication of DNA.▫ Many side effects of Ciprofloxacin in
human organism.
RUSSIAN LEGISLATION
• In Russian Federation there is The State Standart (GOSTR 55481-2013, introduced the 01.07.2014) “Meat andmeat products. Qualitative method for detection ofantibiotics residues and other antimicrobialchemotherapeutic agents”. This standard applies to alltypes of animal slaughter meat, poultry meat, offal andsets the microbiological method for qualitativedetermination of residues of antibiotics and otherantimicrobial chemotherapy substances.
• There is The State Standart (GOST R 51447-99 (ISO3100-1-91), introduced the 05.12.2009) “Meat and meatproducts. Methods of sampling” as well, where thequantification of residual antibiotics carried out by theinternal standard peak area according to the amount ofsampls.
RUSSIAN PRACTICE• Currently for analytical determination of
residues of antibiotic drugs they usemicrobiological methods based onregistration growth test cultures of micro-organisms in the presence of the standardquantities of antibiotics or extracts; highperformance liquid chromatography (HPLC);liquid chromatography with massspectrometry (LCMS); thin-layerchromatography (TLC), which allows toregister the appearance of individual spots ofthe test material; fluorescent analysis basedon the formation of fluorescent complexantibiotic with special organic chromophore.▫ Gas chromatographic method was not used
because of the complexity of the transfer ofantibiotics in the volatile state.
THERE ARE A NUMBER OF PROBLEMS• First, the chromatographic identification involves the
review of specific substances on the residence time ofchromatographic peak. We know a significant number ofreasons why the retention times may vary and evenmatch for certain substances. In the case of very lowconcentrations of substances, this feature is ofteninsoluble problem, even with the use of internal standarddefined substances. Leading manufacturers ofchromatographic equipment offer to secure the doubleidentity of a substance in accordance with peak use, forexample, the UV-spectrum or mass spectrum ofsubstance followed by comparison with a standarddatabase of computer data. This approach to thedetermination of the residue of dangerous impurities inthe raw food is reliable enough, but requires veryexpensive analytical equipment and could not berecommended for mass analysis.
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)• Taking into account the requirements of
rapid methods for monitoring food(sensitivity, selectivity of the method,results, speed of execution, cost ofanalyses), the most preferred method isimmune-enzyme analysis ELISA - methodthat meets all the requirements formethods of routine control.
• For the analysis of chemical compoundsin food and biological objects they usemainly a variant of the "competing"immune-enzyme analysis, therefore, it isonly on this version of the ELISA we stay.
SCHEMATIC PROCESS OF ENZYME IMMUNOASSAY (ELISA)
• Ready made kits (sets) for ELISA in thecomplete supply manufacturertypically includes all the necessarymaterials to perform the analysis,buffer and standard solutions.Antibodies to a particular chemicalcompound (antigen) blood serumobtained from living organisms areadsorbed on a solid surface of theTablet (media), usually at 96 holes.
SCHEMATIC PROCESS OF ENZYME IMMUNOASSAY (ELISA)
SCHEMATIC PROCESS OF ENZYME IMMUNOASSAY (ELISA)
• a-the start of immunosorbtion process; b- thecompletion of the immunosorbtion; c- the tabletwashing; d- the chain reaction development.
• During the contact of media activated surfacewith a solution containing antigens (e.g.,tetracycline), part of the antibodies specificallyinteracts with molecules of controlledcompounds, i.e. deactivated. In a competitiveELISA version controlled solution is mixeddirectly into the hole of the tablet with a solutionof the so-called conjugate (antigen molecules),which is chemically linked (labeled) withmolecules of the enzyme. Over a period ofincubation of the tablet (from 30 minutes to 2hours) at a certain temperature antibodies on thesurface of the media are deactivated as a resultof the immunosorbtion of labeled and no labeledantigens (fig.c).
SCHEMATIC PROCESS OF ENZYME IMMUNOASSAY (ELISA)
• After the procedure of the tablet washingfollowing the incubation, on the surface ofsupporter there are only adsorbed antigens,and the ratio of labeled and no labeledantigens depends on the concentration ofantigens in the controlled solution (fig. c).
• At the stage of developing a solution of theso-called substrate is added into tablet holes.
• The enzyme molecule fragment adsorbedfrom solution conjugate together with thelabeled antigen on the surface of the holes,catalyzes a chemical reaction making thesubstrate in the colored compound (fig. d).
SCHEMATIC PROCESS OF ENZYME IMMUNOASSAY (ELISA)
• After a certain time the color reactiondevelopment a fixative reagent is added intotablet holes and the optical density of thecontents of each hole is measured . After theoptical density measuring in holes it’s easyto build a calibration curve automatically ormanually and then you can calculate theconcentration of the controlled testcompound in the controlled trial.
• Applied analytical methods for determinationof antibiotics distinguish by the minimumdetectable level of different substancesdepending on the antibiotic properties (table1). For analytical determination of chemicaltoxicants complex in food using methodsdiffer in complexity and sensitivity.
THE MINIMUM LEVELS OF ANTIBIOTICS IN MEAT PRODUCTS DETERMINED BY DIFFERENT METHODS
Method Determined substance
Detection limit
MPC, no more
Analysis time, h
TLC Tetracycline 0,1 µg/g 0,01 units/g
5
fluorescence spectroscopy
Tetracycline 1 µg/g 0,01 units/g
4
ELISA Tetracycline 6 pg/g 0,01 units/g
3
ELISA Levomicetine
1 ng/g (µg/kg)
0,01 units/g
1
CONCLUSION• The most successful combination of
sensitivity, speed and cost is ELISAmethod that allows fast enough to holdscreening of food products by themaximum number of indicators. Thestandard methods loose to the method ofELISA for simplicity and minimaldetectable level of the test material. Thismethod enables you to determine thecontent of harmful impurities at a level of0.1 ng/ml.
• The most justified is the application ofthe method of ELISA to determine thecomplex organic toxicants, includinghormones and antibiotics.
