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Trakia Journal of Sciences, Vol. 4, No. 1, pp 33-36, 2006 ISSN 1312-1723
Original Contribution
M. Saridaki-Papakonstadinou 1, S. Andredakis 1, A. Burriel *2, I. Tsachev 3
1Veterinary Laboratory Service, Ministry of Agriculture, Chania 73100, Crete, Greece 2Laboratory of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of 3 Department of Microbiology, Infectious and Parasitic Diseases, Veterinary Faculty, Trakia ABSTRACT
Liquid chromatography was used to detect tetracycline-derived residues in Greek honey. Of the
examined samples 29% had these drug residues. 20.3% of these contained more than one of the
observed tetracycline derivatives. Majority of samples contained residues to a proportion of 0.018-
0.055 mg/kg of honey while some others had residues in excess of 0.100mg/kg. The findings show
that tetracycline drug residues in Greek honey are present and possibly influence the naturally
occurring protective properties of this medicinal food.

Key Words:
honey, tetracycline, liquid chromatography
protective properties in honey and may consequently affect adversely its antimicrobial Honey is historically an essential ingredient of activity. Specifically, these residues may Mediterranean diet (Skidas and Lascaratos, contribute to microbial resistance thereby 2001) and is also promoted currently around altering the beneficial effects of the honey. the world as a naturally protective product The antimicrobial properties of honey appear against various infections (Basson et al. 1994; Drouin, 1999; Taormina et al. 2001) or burns “inhibines” (Taormina et al. 2001; Zaghloul et and wounds (Molan, 2002)1 The antimicrobial al. 2001) that have intrinsic antibacterial effects of honey, attributed to the presence of actions, including activities against bacterial antioxidant power (Taormina et al. 2001), appear to be influenced by the type of honey. 2000; Ceyhan and Ugur, 2001; Dixon, 2003). The dark coloured varieties of honey are This honey protective property is making it a thought to possess more inhibitory properties preferred natural product for studying less on selected pathogens than the light coloured intrusive alternatives for protection of ones. Other properties of honey, among which is its anti-inflammatory activity, contribute to rapid pain, oedema and exudate reductions. of honey have also increased interest in the maceration and adherence of dressings to the determining honey quality, purity is the most important. Various chemical contaminants or residues of chemicals, including drugs used strength of these properties, including the researched vigorously and detection methods are continuously improving (Vinas et al. * Correspondence to: Burriel Angie DVM, MSc., MSc.,
naturally give rise to more classes of drug and PhD., MRCVS; Mavromihali 154, Athens 11472, Greece; Tel.: 0030-6972-314842; E-mail: Trakia Journal of Sciences, Vol. 4, No.1, 2006 SARIDAKI-PAPAKONSTADINOU M., et al. Verzegnassi et al., 2003; Wang, 2004), with the concomitant increase in proportion of honey samples reported as positive (Heering et al., 1998; Verzegnassi et al., 2003; Vinas et passed through glass wool (SUPELCO) filters before it was further purified through a Baker organisms, suffer the consequence of disease, column 10C18 (SUPELCO) suitable for and beekeepers often use either preventive or separating organic compounds. The column therapeutic antimicrobials to protect bee was washed with 10 ml distilled water and health and levels of honey production. The further cleaned by the passing of high speed tetracyclines, among other agents, have been used consistently for treating foulbrood were removed by passing through the column caused by Paenibacillus larvae (Evans, 2003). of 5 ml of ethyl acetate having 10% of MeOH. The collected solutions were passed again detectable amounts, possibly having through a 10 COOH Baker column, which considerable consequences to the consumer. purification was achieved with the through objective of state food inspecting agencies passing of 0.01M and pH of 3.0 Oxalic acid charged with the responsibility of ensuring the safety level of honey. Various methods used Merck) and Acetonitrile (LiChrosolv, Merck) in detecting residual antimicrobials in foods in a ratio of 5:4:1 respectively. The final (Mitchell et al. 1998) may not be suitable for honey, a product having naturally occurring chromatographer using as positive controls bacteriostatic substances (Ceyhan and Ugur, samples having 0.30, 0.20, 0.05 and 0.100 2001; Zaghloul et al. 2001), but technological improvements in available methods are giving promising results (Vinas et al. 2004). Greek honey, in order to be exported, must conform Twenty nine percent of the examined samples had drug residues of at least one of the substances. To meet this requirement, the tetracyclines. Of them 17 (20.3%) samples Veterinary Laboratory Services of the Greek had residues of more than one derivative. The Ministry of Agriculture in Chania, Crete, usually tests honey for tetracycline residues before exportation. This effort, which started samples was 0.018-0.057, 0.023-0.335, 0.018- officially in 2003, is revealing interesting 0.190 and 0.013-0.393 for TC, OTC, DC and results. The preliminary results reported here CTC respectively. With an intra-laboratory were produced using the slightly modified reproducibility of 97%, 94%, 90% and 96% method of Oka et al. (1987) for detecting for TC, OTC, CTC DC, the positive samples minute amounts of tetracycline derivatives. detected and the areas they have originated from are detailed in Table 1. The highest
proportion of positive samples had originated Two hundred and fifty one samples of honey from Thrace (53%), with the islands second (37.5%) and Macedonia third (30.9%). Most certification for exportation were mailed of the positive samples were positive for OTC between 2003 and 2004 to this laboratory. (34 samples) with DC second (27 samples). Producers wanting state permission to export their product submitted these samples for 0.393 mg/kg of OTC and CTC respectively. evaluation following government controls on Honey from the islands appears to have the the use of antibiotics for treating bee diseases. lowest concentration of drugs and that of Most samples were either pine tree or spring classes used OTC residue was found in 14% oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DC) were detected according to Oka et al (1987) by a Hewlett DISCUSSION
Packard 1100 liquid chromatographer (Hewlett Packard Inc) with a Diode Array Very little to no information is available in the column (25 cm x 4.6 mm, 5µm) at a light tetracycline residues in honey, and this is the Trakia Journal of Sciences, Vol. 4, No.1, 2006 SARIDAKI-PAPAKONSTADINOU M., et al. internationally set acceptable amounts of evidence of a rising problem with possible tetracycline residues or standard methods for sensitivity of the method used in the detection Thus, one understands that, in the absence of
Table 1: Number of positive samples to each antimicrobial agent, area of origin and range of drug residues
Pos (+)
Neg (-)
Antimicrobial (+)
Area No
Total 251
12 36 25 21
The present findings compare well with those suitable drugs or dose of various drugs used in of others (Vinas et al. 2004) using a similar combating bee infections or parasitic diseases. methodology. The method used here detects minute amounts of drug; thus it has high REFERENCES
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excessive use of the observed substances, of which OTC and DC are more often detected. The reason for this frequent use could have arisen from easier access connected with pricing, flexibility of use or the need to go Dixon, B., 2003. Bacteria can't resist honey. dwindling efficacy. These scenarios Drouin, E., 1999. Helicobacter pylori: novel inexorably give rise to residue accumulation microbial resistance when the honey is used against microorganisms (Heering et al. 1998; tetracycline resistance in the honey bee bacterial pathogen Paenibacillus larvae. Journal of Invertebrate Pathology 83, 46- findings and methods used for drug detection in honey point to the need for more work. Heering, W., Usleber, E., Dietrich, R. & This work should show the spread and levels screening for antimicrobial drug residues governments to further regulate the use of antibiotic and antiparasitic drugs in apiculture. Kaufmann, A, Roth, S., Ryser, B., Widmer, Current EU restrictions of common antibiotics M. & Guggisberg, D. 2002. Quantitative in apiculture call for more research on the LC/MS-MS determination of sulfonamides and some other antibiotics in honey. Trakia Journal of Sciences, Vol. 4, No.1, 2006 SARIDAKI-PAPAKONSTADINOU M., et al. Tichy, J. & Novak, J., 2000. Detection of antimicrobials in bee products with activity Mitchell, J. M., Griffiths, M. W., McEwen, S. against viridans streptococci. Journal of Verzegnassi, L., Royer, D., Mottier, P. & regulations, tests, and test performance. Molan, P. C., 2002. Re-introducing honey in management 48, 28-40.
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