Development of Radiometric and Allied Analytical Methods to Strengthen National Residue Control Programs for Antibiotic and Anthelmintic Veterinary Drug Residues

Under the project “Strengthening Analytical Capabilities to the Effective Monitoring of Florfenicol Residues in Fish”, 2 methods were developed and validated two methods, namely
Determination of Florfenicol and Florfenicol Amine in Fish Muscle by Radioimunnoassay (RIA) and Confirmation of positive results by LC-MS/MS
Determination of Florfenicol in Fish Muscle by Liquid Chromatography (HPLC-UV)
A radioimmunoassay kit was also developed and antibodies developed in Brazil found to be capable of detecting drug residues in question florfenicol and florfenicol-amine within recommended levels
Methods were applied to Brazil’s official residue monitoring program
Two standard operating procedures were developed
Under the IAEA Technical cooperation program (ANG5009), the institute trained a fellow from Angola and therefore contributed to the initiation and strengthening of residue testing in Angola.
The CRP perhaps contributed in some way to the following at the CSI (Microbioticos in Brazil):
ISO/IEC 17025:2005 in 2011 by ACLASS/ANSI (Now ANAB) of the United States of America
Service to 9 countries (besides Brazil): USA, Mexico, Argentina, Paraguay, Uruguay, Peru, Chile, South Africa and Guatemala.

Under the project “Development of multi-dot-ELISA for Enrofloxacin and Ciprofloxacin residues in chicken tissue (and LC-MSMS method development and validation)”, the following occurred
A multi-parametric Dot ELISA was developed for the detection of enrofloxacin and ciprofloxacin in chicken meat including depletion studies.
Specific factors contributing to successful production of antibodies were studied and challenges reported. Thus experiences gained and challenges encountered will be shared with and used to inform other Member States through the TECDOC.
A complementary accurate, reproducible and highly sensitive LC-MS/MS method for testing enrofloxacin and ciprofloxacin in chicken matrices was also developed, validated and published.
This is suitable monitoring residues and meets European community requirements. While it is relevant to Member States especially those trading with the European Union, we cannot verify how widely it has been applied to national residue monitoring since publication.
A Post-Doctoral research undertaken under the project

Under the research project entitled “The Development of Microbiological Multi-Residue and Allied LC-MS-MS Methods to Monitor the Veterinary Drug Residues in the Environment”
A number of analytical methods were developed, validated and applied, including a new multi-residue screening/confirmatory methods including use of state-of-the-art LC-MS/MS techniques with stable isotopes (e.g. 13C labelled sulfa compounds) for drug residue analysis in animal products and environmental samples were developed.
Methods were useful for the Quarantine and Inspection Agency and the Ministry of Food and Drug Safety in the national residue monitoring programs
In additional to a number of published methods, short courses conducted and proficiency testing undertaken to ensure performance to international standards, an SOP was prepared in English and will be shared
As reported by the CSI, the CRP also contributed to the development of a “Handbook of Residue Analytical Methods for Veterinary Drugs and Contaminants in Food of Animal Origin, Fish and Fishery Products” prepared in the Korean language [(http://www.mfds.go.kr/index.do?x=0&searchkey=title:contents&mid=695&sear...); (http://www.mfds.go.kr/eng/index.do;jsessionid=NvTyOfAIubm3Lm4DWOcHaX4Itr...)]
Technology developed and expertise enhanced under the CRP by a CSI in Republic of Korea was transferred/disseminated to Mongolia through a TCP MON5019 (including expert missions). This has strengthened the institutional capability and efficiency of Mongolia’s State Central Veterinary Laboratory, a key partner in ensuring food security/safety in Mongolia.
Proficiency testing undertaken

Under the project “Development of methods for aminoglycosides (LC-MS/MS and HPTLC)”
A robust confirmatory and quantitative LC-MS/MS method was developed for simultaneous determination of sixteen Aminoglycoside antibiotics in porcine tissues.
The method was applied by Shenzhen Centre for Disease Control and Prevention to residue monitoring in Guangdong Province.
The method was transferred to Botswana National Veterinary Laboratory (BNVL) and the laboratory in China also trained 2 BNVL scientists (under an IAEA Technical Cooperation Project) in this and other methods of analysis. This is important to Botswana as the country strives to reduce costs of outsourcing analytical tests. It is envisioned, a memorandum of understanding may be developed between the 2 institutions in China and Botswana to continue food safety partnerships.
Note: The FEPL played an important role in the method validation process and trained another Scientist from BNVL, who would implement the method transferred from China.

Under the project “Screening and confirmatory methods for detecting multi-benzimidazoles, probenzimidazoles and their metabolites in animal products”
Two methods were developed, validated and applied to residue monitoring through the Institute of Quality Standards and Testing Technology for Agro-Products to enhance food safety in the country
Two SOPs have been developed is in place to share knowledge developed through IAEA technical cooperation and other avenues
Completion of an MSc Thesis under the project reported

Under the project “Development and Validation of a Multiresidue Method for Simultaneous Determination of Benzimidazole and Avermectin Anthelmintics Residues in Bovine Milk by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)”
Two multianalyte methods developed and 2 standard operating procedures developed under the general title: “Determination of benzimidazoles and avermectins residues in bovine milk by liquid chromatography with tandem mass spectrometry (LC-MS/MS) and fluorescence detection”
The methods have been applied to Peru’s residue monitoing programme by the Servicio Nacional de Sanidad Agraria (SENASA) Centro de Control de Insumos y Residuos Tóxicos, Avenida La Molina 1915 Lima
The methods and the validation process helped SENASA, Peru obtain accreditation by ANSI-ASQ accreditation body, USA. This achievement supports and facilitates MS’s agro-exports - a major economic activity – and ensures public health;
The methods/technology were transferred (including through group training) through an ARCAL-IAEA regional project RLA/5059 to other Member States., thus contributing to regional institutional capacity building and harmonization of analytical methods for residue monitoring. The project consisted of 15 countries and a number of these countries have either obtained or are in the process of accreditation.

Under the project “Development of HPTLC screening method for antimicrobial veterinary drug residues”
Methods were developed for analysis of sulphonamides (and tetracyclines) in chicken muscle
An SOP was developed for 5 sulfonamides and method found to be transferable to analysis of related residues in Shrimps
Method was applied to national residue testing
Research “innovated” a semi-automated Thin Layer Chromatography spotter (using an old microscope); this could be reproduced elsewhere
Laboratory accreditations of the implementing institution, Food Safety & Quality Assurance Laboratory (FSQAL), Faculty of Veterinary Medicine & Animal Science, University of Peradeinya, Sri Lanka by the Sri Lanka Accreditation Board (SLAB) on 15th September 2010 and in 2013. It is believed this CRP and perhaps previous IAEA collaboration contributed to the accreditation process.
Completion of MPhil Degree programme at University of Peradeniya

Under the project “Confirmatory Analysis on Chloramphenicol Presence in Certain Number of Pasture Grass in Mongolia”
A method was developed and used in Mongolia to investigate natural occurrence of the antibiotic Chloramphenicol in animal products and environmental samples (pasture and soil) based on a tailored rapid screening technique supported by an LC-MS/MS method.
Relevance: Chloramphenicol is banned by many trading partners and presence of the antibiotic in food samples (though drug may not have been used in treating animals) previously led to ban exports. Certain plants and environmental samples have been found to contain such compounds as Chloramphenicol
A relatively large number of root samples contain Chloramphenicol, and that plants belonging to different families can contain Chloramphenicol and the levels depend on geography.
The study has influenced others around the world to pay more attention to and do more research on natural sources of chemical contaminants that have implications on trade
An SOP has been prepared

Under the project “Development of immunoassays for selected antibiotics for the detection and monitoring of the drug residues in livestock and livestock products”
A method was developed by the then Kenya Trypanosomiasis Research Centre (now Kenya Agricultural and Livestock Research Organization for testing oxytetraycline in animal products
This is relevant to given the collaboration with Kenya Meat Commission of Kenya in the monitoring veterinary drug residue levels in meat products collected from various parts of the country and where the research institute develops in-house methods.
Research highlighted various bottlenecks in antibody production and related method development and validation and this experience is relevant to other Member States.

Under the project “Development of microbiological screening method for the detection of multi-residues of antimicrobial substances”
Investigation was undertaken to evaluate microbiological screening method that would be inexpensive, reliable, practicable and more affordable to developing countries and therefore of great benefit to residue monitoring plans.
The protocol used by investigator indicates, for this in-house method to have comparative advantages over commercially available methods, modifications are required, such as sample concentration (or use un-extracted samples, addition of
This information and experience is relevant to other Member States that wish to develop related in-house methods

Under the project “Development and Implementation of Radio-Chemical Techniques for the Detection of Authorized Antibiotics Using Radioactive Tracers for the Effective Monitoring of Veterinary Drugs Residues in the Aquaculture Production”
The Disposition of 14C-flumequine in Sea Bream (Sparus Auratus) after a Single Intraperitoneal Administration was studied adding new knowledge on pharmacokinetic profile in Sea Bream when using whole body autoradiography and liquid scintillation counting.
Such knowledge is useful in understanding residue depletion and the determination of maximum recommended residue limits and withdrawal periods, parameters used to set national or international standards for residues in foods.