Validation of an Analytical Methodology for Determination of Oxytetracycline and Tetracycline Residues in Honey by HPLC with Fluorescence Detection A. PENA,* ,† N. PELANTOVA, ‡ C. M. LINO, † M. I. N. SILVEIRA, † AND P. SOLICH ‡ Group of Bromatology, Centre of Pharmaceutical Studies, Faculty of Pharmacy, University of Coimbra, 3000 Coimbra, Portugal, and Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Prague, Czech Republic An analytical method for the determination of OTC and TC residues in honey was developed. Sample treatment involves an extraction in EDTA-McIlvaine buffer, followed by a solid-phase cleanup step. With regard to the cleanup procedure, different SPE cartridges were evaluated and the results presented. The method was validated according to the guidelines laid down by the 2002/657/EC European Decision parameters: decision limit (CcR) and detection capability (CC) were 20 and 21 µg/Kg and 49 and 50 µg/Kg for OTC and TC, respectively, and recoveries of OTC and TC from spiked samples, at three fortification levels, were higher than 87% for both compounds. The analytical method was applied to 57 honey samples. KEYWORDS: Antibiotics; Tetracyclines; Honey; Solid-phase extraction; High-performance liquid chro- matography 1. INTRODUCTION In apiculture, antibiotics are mainly used for the treatment of bacterial brood diseases, American and European foulbrood (AFB and EFB) in honeybees (Apis melifera), which are caused by two species of bacteria, Paenibacillus (Bacillus) larVae and Melissococcus pluton, respectively (1). Oxytetracycline (OTC) has been used in the United States of America since the early 1950s for the prevention and control of these diseases. Because of the easy availability and relatively low cost of OTC, this method has been widely adopted by beekeepers (2). The occurrence of antibiotic residues in human food, arising from its veterinary use, is a cause of concern to consumers worldwide, because of possible toxic or allergic reactions and the possibility that pathogenic organisms could become resistant to these drugs (3-4). Under European Community (EC) legislation, antibiotics are not authorized for the use on honeybees, since no maximum residue levels (MRLs) have been fixed for antibiotic residues in honey in the EU Council Regulation (5). In recent years, several initiatives have been launched to establish or strengthen surveillance systems, both in the EC member states and at the international level, to control the presence of antibiotic residues in honeys, and these have revealed levels of antibiotic residues in honey samples. Tetracyclines (TCs) can be successfully determined in various biological matrices, using high-performance liquid chromatog- raphy (HPLC) in the reverse-phase mode, with different detection modes, such as spectrophotometry, fluorescence, and mass spectrometry (6). The UV detection has low sensitivity, while mass spectrometry still requires costly instruments. In general, fluorescence detection is sensitive and selective (7). The present study describes the development and validation of an analytical methodology for the specific and sensitive determination of OTC and tetracycline (TC) in honey by HPLC with fluorescence detection. They were extracted with Na 2 - EDTA-McIlvaine buffer (pH 4.0), and different solid-phase extraction (SPE) cartridges were evaluated in order to find the most efficient cleanup method for TCs in honey samples. To enhance the precision and accuracy of the analytical method, the validation was compliant with European Com- munity (EC) Decision 2002/657/EC (8). Finally, the method was applied to the analysis of OTC and TC residues in different types of honeys from Portugal and Spain. 2. EXPERIMENTAL PROCEDURES 2.1. Apparatus. The HPLC system consisted of Gilson Model 302 and 307 pumps (Gilson, Medical Electronics, Villiers-le-Bel, France) and a Model 7125 loop injector (Rheodyne, Cotati, CA). The fluorescence detector was a Model LC 305 instrument (LabAlliance, LabAlliance, State College, PA) operating at an excitation wavelength of 385 nm and an emission wavelength of 500 nm. The spectral bandwidth was 10 nm for both excitation and emission. The results were recorded on a Model 3390 A integrator (Hewlett-Packard, Philadelphia, PA) and a Unipoint Gilson data system (Gilson, Medical * To whom correspondence should be addressed. Tel: 00 351 239859994. Fax: 00 351 239827126. E-mail: apena@ci.uc.pt. † University of Coimbra. ‡ Charles University. 3784 J. Agric. Food Chem. 2005, 53, 3784-3788 10.1021/jf050065r CCC: $30.25 © 2005 American Chemical Society Published on Web 04/19/2005 Downloaded by PORTUGAL CONSORTIA MASTER on July 7, 2009 Published on April 19, 2005 on http://pubs.acs.org | doi: 10.1021/jf050065r