Bulletin USAMV-CN, 63 - 64/2007 OPTIMIZATION OF HPLC METHOD FOR QUANTIFYING TETRACYCLINE RESIDUE IN HONEY Bonta Victorita, L. Al. Mărghitaş, D. Dezmirean, Adela Moise, Otilia Bobiş, O. Maghear University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, 3-5 Manastur Str., 400 372 Cluj-Napoca, Romania Key words: tetracycline, oxytetracycline, honey, HPLC Abstract. Tetracyclines are used for the treatment or prevention of American and European fouldbrood in bee colonies which are caused especially by two species of bacteria: Paenibacillus larvae larvae and Melissococcus pluton, respectively. In the present study a method for determination of residual tetracyclines in honey has been optimized and validated. It was used the reversed-phase high performance liquid chromatography (HPLC) with UV detection. Tetracyclines were extracted from honey with 0.01M sodium succinate buffer. For the clean up step, solid phase extraction using metal chelate resin was applied. Tetracyclines were eluted with buffer containing EDTA. Linearity, precision, recovery and sensitivity were satisfactory. The limit of detection was established at 5 μg/kg. INTRODUCTION Tetracyclines are chemically characterized by a partially conjugate four-ring structure with a carboxyamide functional group. Chlortetracycline, oxytetracycline and tetracycline are representative members of this antibiotic class. They have the ability to form strong complexes with multivalent cation [1]. For this reason, in extraction and analysis methodologies for tetracyclines determination we used aqueous solution containing chelating agents to decrease the tendency of tetracycline to bind to cations in the matrix. The metal chelating affinity column was used to clean-up the succinate buffer solution of honey. The mini-column has been previously charged with copper ions. Tetracyclines were specifically absorbed from honey solution due to their chelating to copper ions reversibly bound, to iminodiacetic acid epoxy-activated resin. The analytics were eluted from column using another chelating agent: McIlvaine-EDTA buffer. Honey is generally considered as a natural and healthy product [2]. Addition of additives or conserving agent to honey is not allowed. Antibiotics are mainly used in apiculture for the treatment of bacterial brood diseases. Antibiotics used in the colony can contaminate also honey and royal jelly [3, 4]. Lately, in many publications the problem of residues of antibiotics in honey was mentioned [5]. In 1990, the Commission of the European Union laid down the procedure for establishing maximum residue limits (MRLs) of veterinary drugs in foodstuffs of animal origin. However, no MRLs have been fixed for use with bee products. Antibiotics such as tetracyclines have had MRLs imposed for their use in animal’s nutrition, but are illegal for use in apiculture [6]. The European Union countries do not permit honey to contain drugs above the corresponding detection limit, while others, e.g. Switzerland, UK and Belgium have established action limits, which generally lie between 10 to 50 μg/kg for each antibiotic group. brought to you by CORE View metadata, citation and similar papers at core.ac.uk