Analytical MethodsDevelopment and validation of an HPLC method for the determination of sixpenicillin and three amphenicol antibiotics in gilthead seabream (SparusAurata) tissue according to the European Union Decision 2002/657/ECEvaggelia N. Evaggelopoulou, Victoria F. Samanidou⇑Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 541 24, Greecea r t i c l e i n f oArticle history:Received 14 December 2011Received in revised form 1 September 2012Accepted 12 September 2012Available online 26 September 2012Keywords:HPLCPenicillinsAmphenicolsGilthead seabreamSolid phase extractiona b s t r a c tA confirmatory high performance liquid chromatography method for the determination of six penicillinantibiotics and three amphenicol antibiotics in gilthead seabream (Sparus Aurata) tissue was developed.Ampicillin (AMP), penicillin G (PG), penicillin V (PV), oxacillin (OXA), cloxacillin (CLO), dicloxacillin(DICLO), thiamphenicol (TAP), florfenicol (FFC) and chloramphenicol (CAP) were separated on an Inertsil,C 8 (250 ? 4 mm, 5 l m) column by gradient elution with a mobile phase consisting of ammonium acetate0.05 M and acetonitrile at 25 ?C. Diode array detection with monitoring at 225 nm (for the determinationof AMP, PG, PV, TAP and FFC), 240 nm (for OXA, CLO and DICLO) and 278 nm (for CAP) was applied. Exam-ined antibiotics were isolated from gilthead seabream tissue by liquid–liquid extraction and furtherclean-up was performed by solid phase extraction using Oasis HLB (200 mg/6 mL) cartridges. The devel-oped method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sen-sitivity according to the European Union Decision 2002/657/EC.? 2012 Elsevier Ltd. All rights reserved.1. IntroductionAntibiotics are chemical substances produced by microorgan-isms that either destroy (bactericidal) or inhibit the growth of othermicroorganisms (bacteriostatic). Antibiotics can be either broadspectrum, active against a wide range of microorganisms or narrowspectrum active against a specific group of microorganisms beingable to interfere with a metabolic process specific to those organ-isms. In general, antibiotics work by: (i) preventing the synthesisof bacterial cell wall components (e.g. penicillins); (ii) damagingthe bacterial cytoplasmic membrane; (iii) interfering with proteinor nucleic acid synthesis (Samanidou & Evaggelopoulou, 2007;Samanidou, Evaggelopoulou, & Papadoyannis, 2006). In aquacul-ture, antibiotics have been used mainly for therapeutic purposesand as prophylactic agents. Their extensive administration to fish,destinated for human consumption, has become a serious prob-lem because their residues can persist in edible animal tissues.Antibiotics may be directly toxic or be the source of resistant hu-man pathogens representing a possible risk to human health. Theycan produce allergic hypersensitivity reactions or toxic effects. Forthese reasons regulatory agencies have enacted decisions that keepthese substances under control (Samanidou & Evaggelopoulou,2007; Samanidou et al., 2006).Penicillins are beta (b)-lactam antimicrobial agents used againstvarious organisms by inhibiting the synthesis of the peptidoglycanlayer of bacterial cell walls. The peptidoglycan layer is importantfor cell wall structural integrity. Broad-spectrum penicillins in-clude ampicillin (AMP), penicillin G (PG), penicillin V (PV), oxacillin(OXA), cloxacillin (CLO) and dicloxacillin (DICLO). Their chemicalstructures are quite similar as shown in Fig. 1. These are bacterici-dal in action, effective against Gram-positive and Gram-negativebacteria, but not very effective against Pseudomonas (Samanidou& Evaggelopoulou, 2007; Samanidou et al., 2006).Amphenicols, such as thiamphenicol (TAP), florfenicol (FFC) andchloramphenicol (CAP) are synthetic broad-spectrum antibiotics.Their use in food animals is illegal in most countries. Their chem-ical structures are shown in Fig. 2.Chloramphenicol acts primarily by binding reversibly to the 50Sribosomal subunit and also can inhibit mitochondrial protein syn-thesis in mammalian cells. Additionally, chloramphenicol is abroad-spectrum antibiotic exhibiting activity against both Gram-positive and Gram-negative bacteria as well as other groups of mi-cro-organisms. It exerts its action through protein inhibition and iseffective in the treatment of several infectious diseases. This, to-gether with its low cost and ready availability, has made it exten-sively used since the 1950s in the treatment of food-producing fishall over the world. However, CAP is, in certain susceptible individ-uals, associated with serious toxic effects in humans in the form ofbone marrow depression, particularly severe in the form of fatal0308-8146/$ - see front matter ? 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2012.09.044⇑ Corresponding author. Tel.: +30 231997698, fax: +30 2310997719.E-mail address: samanidu@chem.auth.gr (V.F. Samanidou).Food Chemistry 136 (2013) 1322–1329Contents lists available at SciVerse ScienceDirectFood Chemistryjournal homepage: www.elsevier.com/locate/foodchem
