Determination of sulfonamides in animal tissues using cation exchange reversed phase sorbent for sample cleanup and HPLC–DAD for detection

A new extraction method for 12 sulfonamides (sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethizole, sulfadimidine, sulfisoxazole, sulfamethoxazole, sulfatroxazole, sulfachlorpyrazine, sulfaphenazole and dapsone) in muscle, liver and kidney was developed. Fortified tissues of muscle, liver and kidney from bovine, pig and chicken were studied. A 10 g tissue and 10 ml acetonitrile were homogenised with an Ultra-Turrax. Next, the tissue was defatted with 5 ml n-hexane, centrifuged and filtered into flasks. Then the extracts were acidified with 10 ml hydrochloric acid and diluted with deionised water. Next, the samples were loaded on OASIS® MCX columns, which were conditioned with 5 ml methanol and 5 ml water. The columns were washed with 5 ml hydrochloric acid and 5 ml methanol. Then the samples were eluted with 5 ml ammonia solution/acetonitrile (v/v 1/19), allowed to dry under nitrogen and reconstituted in 200 μl acetonitrile/water-mixture (v/v 1/4). The analyses were carried out on HPLC–DAD. Mobile phase was 0.01 M ammonium acetate pH 4.6 (A) and acetonitrile (B). Chromatographic separation was obtained by gradient elution (5% B to 40% within 16 min, back to 5% in 1 min, equilibration for 3 min). The sulfonamides were detected at 260 nm and dapsone at 294 nm. The detection limits of the HPLC method were 1 ppb for all analytes.