Journal of Chromatography A, 1157 (2007) 281–288 Validation of a confirmatory method for the determination of macrolides in liver and kidney animal tissues in accordance with the European Union regulation 2002/657/EC Houda Berrada a,∗ , Francesc Borrull b , Guillermina Font a , Juan Carlos Molt ´ o a , Rosa Maria Marc´ e b a Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, Universitat de Val` encia, Avda Vicent Andres Estell´ es s/n, 46100 Burjassot, Spain b Department of Analytical Chemistry and Organic Chemistry, University Rovira i Virgili, Sescelades Campus, Marcel l´ ı Domingo s/n, 43007 Tarragona, Spain Received 27 November 2006; received in revised form 2 May 2007; accepted 7 May 2007 Available online 13 May 2007 Abstract This study proposes a simple multiresidue liquid chromatography–diode array detector (LC–DAD) method capable of determining seven macrolide antibiotics in samples of liver and kidney animals at concentrations lower than those allowed by current legislation. Samples were prepared by homogenizing the tissue with EDTA–McIlvaine’s buffer and extracted with an Oasis HLB cartridge. The consumption of organic solvent during extraction was minimum. The analytes were detected by LC–DAD and also by liquid chromatography–mass spectrometry with electrospray ionization (LC–(ESI)MS). The method was specific, stable and robust enough for the required purposes. The DAD method was validated in accordance with the European Commission Decision 657/2002. Recovery data were also satisfactory with values higher than 67% for most macrolide antibiotics extracted from liver and kidney samples spiked at 200 g/kg, the lowest MRL established for the macrolides studied. The relative standard deviations (RSD (%), (n = 3)) were lower than 13% and 15% for intra-day and inter-day assays. The method was applied to investigate the occurrence of the studied macrolides in 31 beef and kidney animal samples. The results obtained by LC–DAD for positive samples were compared to those obtained by LC–(ESI)MS. Therefore, the method with simpler instrumentation than a LC–(ESI)MS can be used as a control method and the results of the validation process demonstrate that this method is suitable for application in a European Union program for monitoring residues of veterinary drugs. © 2007 Elsevier B.V. All rights reserved. Keywords: Macrolides; Liver; Kidney; Liquid chromatography-DAD; ESI-MS; SPE 1. Introduction Macrolides are a group of antibacterial compounds that are active against Gram-positive and some Gram-negative bacteria. They are basic and lipophilic molecules that consist of macro- cyclic lacton rings containing 14–18 atoms with sugars linked via glycosidic bonds. They are widely used in human and vet- erinary medicine to prevent microbial infections. As veterinary drugs they are used in food-producing animals with either a ∗ Corresponding author. Tel.: +34 96 3544958; fax: +34 96 3544954. E-mail address: houda.berrada@uv.es (H. Berrada). curative and prophylactic aim or added directly to human food to prolong its freshness [1,2]. The incorrect use of these drugs can leave residues in food products and this can have such undesirable effects on consumer health as the development of allergic reactions, the appearance of resistant bacteria and even cross-resistance to other antibiotics with similar structures or mechanisms of action [3]. Therefore, the application of these antibiotics in feeding stuffs is regulated by Council Directive 70/524/EC [4]. Maximum residue limits (MRLs) have been established for these substances in animal products. The European Parlia- ment decided to ban the use in humans of related antibiotics that were administered to promote animal growth, so some 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.05.021