Analytical Methods Validation of antibiotics in catfish by on-line solid phase extraction coupled to liquid chromatography tandem mass spectrometry Jorge Hurtado de Mendoza a,1 , Luana Maggi c,1 , Liliana Bonetto a , Beatriz Rodríguez Carmena b , Alicia Lezana b , Francisco A. Mocholí a , Manuel Carmona c,d,⇑ a Sailab, Argenters, 5 Ed. I. Bajos D, Parc tecnológic del Vallès, 08290 Cerdanyola del Vallès, Spain b Laboratorio de Salud Pública – Madrid Salud, c/ Emigrantes 20, 28043 Madrid, Spain c Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain d Albacete Science and Technology Park, Paseo de la Innovación 1, 02006 Albacete, Spain article info Article history: Received 6 June 2011 Received in revised form 2 February 2012 Accepted 19 February 2012 Available online 3 March 2012 Keywords: Antibiotics Residues Catfish XLC–MS/MS abstract For the first time automated on-line solid phase extraction coupled to liquid chromatography tandem mass spectrometry was developed for the simultaneous determination of 13 antibiotics (sulfonamides and tetracyclines) in catfish. The method proposed was validated according to Commission Decision 2002/657/EC, showing good linearity between 2 and 350 lg kg À1 , high recovery (80–99%) and reproduc- ibility (13–20%) values, lower detection limits than 0.1 lg kg À1 , and quantification limits under 2.4 lg kg À1 (between 39 and 84 times lower than the MRL fixed by the EU). Moreover, the proposed method was also used to determine sulfonamides and tetracyclines in 16 out of 107 samples, all previ- ously analysed by microbiological screening that gave positive results. Five out of 13 antibiotics were found, having tetracycline the higher occurrence (10 samples); in all cases the concentrations were lower than the MRL established. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction In the last 20 years, a large increase in the demand for fish and seafood products coupled with a decrease from natural sources has stimulated the intensive development of fish farming and aquacul- ture techniques to enhance production. Antibiotics have been used on a large scale in current ranching practices, especially in veteri- nary medicine at therapeutic levels to prevent and treat several diseases, as well as to promote growth in food-producing animals. Despite the positive effects of these drugs, inappropriate use of antibiotics poses a potential health risk to consumers. This is due to the increasing incidence of microbial resistance and the risk of allergic reactions to antibiotic residues and their metabolites (Juan-García, Font, & Picó, 2007; Lee, Chung, Chung, & Lee, 2007; Samanidou & Evaggelopoulou, 2007). To ensure human food safety, the European Union has set maximum residue limits (MRLs) for veterinary drug residues in foods of animal origin (Council Regula- tion EEC No. 2377/90) that are based on the acceptable daily intake (ADI) of each drug while taking into account maximum food in- takes. In December 2009, the European Commission published Regulation No. 37/2010 for pharmacologically active substances and their classification regarding MRLs in foodstuffs of animal ori- gin (Commission Decision 2002/657/EC; Commission Regulation EU No 37/2010). Recently, a fish known as white catfish (Pangasius hypopthalmus), also called Vietnamese River Cobbler, industrially farmed in Vietnam along the Mekong River, has been introduced into the European market. In a short time, P. hypopthalmus has grown in popularity in France and Spain because its cost is low and it is sold in filets with no bones, while also having a neutral fla- vour and texture. Due to these neutral sensory characteristics, these catfish filets have been copiously consumed by children, thus requiring more exhaustive controls. Immunoassay techniques are widely used as screening methods to detect residues in food and tissues because they do not require sample pre-concentration and clean-up steps. Although they provide only a semi-quantitative estimation of ‘‘total’’ residues and do not distinguish between members of a class of antibiotics, they continue to be used because of their simplicity and low cost (Althaus, Berruga, Montero, Roca, & Molina, 2009; Granelli & Branzell, 2007). When they give a positive sample, other techniques such as high perfor- mance liquid chromatography (HPLC) (Juan-García et al., 2007; Lee et al., 2007; Samanidou & Evaggelopoulou, 2007), capillary electro- phoresis (CE) (Castro-Puyana, Crego, & Marina, 2008; Juan-Garcia, Font, & Picó, 2006; Juan-García et al. 2007) or capillary electrochro- matography (CEC) (Lu et al., 2005), must be applied to confirm and 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.02.108 ⇑ Corresponding author at: Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain. Tel.: +34 967 599310; fax: +34 967 599238. E-mail address: Manuel.Carmona@uclm.es (M. Carmona). 1 These authors contribute equally to this work. Food Chemistry 134 (2012) 1149–1155 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem