UNCORRECTED PROOF Analytica Chimica Acta xxx (2003) xxx–xxx Extraction procedure for sulfachloropyridazine in porcine tissues and detection in a sulfonamide-specific enzyme-linked immunosorbent assay (ELISA) 3 4 5 P. Cliquet a , E. Cox a,∗ , W. Haasnoot b , E. Schacht c , B.M. Goddeeris a,d 6 a Laboratory of Veterinary Immunology, Faculty of Veterinary Medicine, Universityof Ghent, 7 Salisburylaan 133, Merelbeke B-9820, Belgium 8 b State Institute for Quality Control of Agricultural Products (RIKILT), Bornsesteeg 45, 6708 PD Wageningen, The Netherlands 9 c Laboratory of Organic Chemistry, Faculty of Science, University of Ghent, Krijgslaan 281 (S4), Gent B-9000, Belgium 10 d Laboratory of Physiology and Immunology of Domestic Animals, Faculty of Agricultural and Applied Biological Science, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, Heverlee B-3001, Belgium 11 12 Received 1 July 2003; accepted 4 August 2003 13 14 Abstract 15 Sulfonamide-specific polyclonal rabbit antibodies were obtained after immunization with a sulfathiazole derivative (N1- [4-(carboxymethyl)-2-thiazolyl]sulfanilamide = TS) coupled to keyhole lympet hemocyanin. Using these antibodies, two sulfonamide-specific enzyme-linked immunosorbent assays (ELISAs) were developed differing in coating antigen: TS-oval- bumin (TS-ova) and PS-ovalbumin (PS-ova, PS = N1-[4-methyl-5-[2-4-carboxyethyl-1-hydroxyphenyl]-azo-2-pyridyl]sul- fanilamide). The detection of sulfamethazine, sulfamerazine, sulfadimethoxine, sulfadiazine, sulfathiazole, sulfapyridine, sulfachloropyridazine and sulfisoxazole in buffer was studied. Higher antibody titers were obtained in the ELISA coated with TS-ova (TS-ciELISA) as compared to the ELISA coated with PS-ova (PS-ciELISA), but the detection of sulfonamides was more sensitive in the PS-ciELISA, allowing the detection of all tested sulfonamides at the maximum residue level (MRL) value (100 ng ml -1 ). 16 17 18 19 20 21 22 23 24 In a subsequent step, an extraction procedure was developed for the detection of sulfonamides in muscles, kidney, liver and fat by both ELISAs using sulfachloropyridazine as a model. As extraction buffer a carbonate/hydrogen carbonate buffer (pH 10) was chosen in which sulfonamides are highly soluble. Differences in homogenizing techniques (high-speed mixer (Ultraturax) versus vortex) and the effect of kaolin (hydrated aluminum silicate) treatment, to diminish the background signal in the ELISA, were evaluated. The best extraction procedure was the one using a vortex mixer as homogenizer and no kaolin treatment. Sulfachloropyridazine was easily detected at the MRL in all tissues. 25 26 27 28 29 30 © 2003 Published by Elsevier B.V. 31 Keywords: Sulfonamides; ELISA; Extraction procedure; Porcine tissues 32 33 ∗ Corresponding author. Tel.: +32-9-264-7398; fax: +32-9-264-7496. E-mail address: eric.cox@UGent.be (E. Cox). 1. Introduction 34 Sulfonamides are chemotherapeutics widely used 35 in veterinary medicine for the treatment of bacterial 36 infections and as feed additives [1]. As a result, sul- 37 fonamides can occur in food products of animal origin 38 1 0003-2670/$ – see front matter © 2003 Published by Elsevier B.V. 2 doi:10.1016/j.aca.2003.08.010 ACA 224771 1–8