Analytica Chimica Acta 594 (2007) 211–218 Synthesis of haptens and development of a sensitive immunoassay for tetracycline residues Application to honey samples Nuria Pastor-Navarro, Sergi Morais, ´ Angel Maquieira ∗ , Rosa Puchades ∗ Departamento de Qu´ ımica, Universidad Polit´ ecnica de Valencia, Camino de Vera s/n, 46071 Valencia, Spain Received 13 April 2007; received in revised form 24 May 2007; accepted 24 May 2007 Available online 29 May 2007 Abstract A new synthesis way of haptens for tetracyclines (tetracycline, chlortetracycline and oxytetracycline) based on the formation of their carboxamido and diazo derivatives that maintain the tetracyclic nucleus and possess an aromatic or aliphatic spacer arm – in order to create a degree of heterology in the chemical structure – is described. The haptens were used to successfully raise polyclonal antibodies and a reliable and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for tetracycline was set up. After the optimization of physical and chemical parameters (pH, salt, detergent concentration, and time), a detection limit of 0.4 ng mL -1 for tetracycline was reached. The optimized ELISA showed cross- reactivity to rolitetracycline (91%), oxytetracycline (30%), methacycline (14%), and chlortetracycline (10%). The analytical potential of the immunoassay was demonstrated by the excellent recoveries obtained (mean value ranging between 79% and 108%) when determining tetracycline residues in honey samples with a simple preparation procedure. The obtained results confirmed the suitability of the assay as screening method for either food monitoring or laboratory quantification of tetracycline residues. © 2007 Elsevier B.V. All rights reserved. Keywords: Tetracyclines; Hapten synthesis; Immunoassay; Honey analysis 1. Introduction Tetracyclines (TCs) are a group of broad-spectrum antibiotics used against a wide range of Gram-negative and Gram-positive microorganisms. The use of these antibiotics in animal health- care has raised concerns as the presence of residues in food may lead to increase microbial resistance in humans. Current studies support that the emergence of resistance is a relatively modern event related with the introduction of these antibiotics for clinical, veterinary, and agricultural use [1]. Tetracyclines are also prescribed in aquaculture to con- trol infections in salmon, catfish, and lobsters [2–4], sprayed onto fruit trees and other plants to treat infection by Erwinia amylovara, injected into palm trees to treat mycoplasma infec- tions (lethal yellow), and used to control infection of seeds by ∗ Corresponding authors. Tel.: +34 96 387 73 42; fax: +34 96 387 93 49. E-mail addresses: nupasna@doctor.upv.es (N. Pastor-Navarro), amaquieira@qim.upv.es ( ´ A. Maquieira), rpuchades@qim.upv.es (R. Puchades). Xanthomonas campestris. They have also been applied in the treatment of bee livestock to foulbrood disease of the honey- bee, which is caused by either Bacillus larvae or Streptococcus pluton [4]. The use of tetracyclines for treatment of bacterial infec- tions and their presence in the environment are a big concern in many countries. In the United States, the maximum residue limits (MRLs) of tetracycline (TC) for all food producing species are 2 mg kg -1 in muscle, 6 mg kg -1 in liver, and 12 mg kg -1 in kidney [5]. The European Commission had set MRLs for tetracyclines – as sum of tetracycline, oxytetracycline (OTC), chlortetracycline (CTC) and their 4-epimer – in mus- cle (100 g kg -1 ), kidney (600 g kg -1 ), egg (200 g kg -1 ) and milk (100 g kg -1 ). For other products such as honey no MRL are established. Several methods have been reported for the determination of tetracyclines using a variety of techniques, including high performance liquid chromatography with ultraviolet detection (UV-HPLC) [6,7], and liquid chromatography–mass spec- trometry (LC-MS) methods [8,9]. Wen et al. [10] reported 0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.05.045