Summary
Sulfonamides represent a wide range of synthetic compounds commonly used in veterinary therapy for the treatment of several bacterial and protozoan infections in cattle, swine and poultry. Trimethoprim is another antibacterial agent mainly used in fish culture and often combined with sulfonamides in commercial preparations. Residues of these drugs in foodstuffs are of concern because of their potential carcinogenic character. Consequently, the European Union (EU) and United States Food and Drug Administration set maximum residue limits for both sulfonamides (100 µg/kg either as a single molecule or as a sum of all detected compounds within the class) and trimethoprim (TMP) (50 µg and 100 µg/kg, according to the matrix) in chicken, fish muscle and eggs.On the other hand, these limits have made of concern the development of confirmatory methods for the analysis of these molecules. LC-MS/MS technique, in particular, resulted fit for the detection of these medium polarity compounds. An effective multi-residue method is presented for the simultaneous determination of certain sulfonamides (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) and TMP in products of animal origin (chicken muscle, fish muscle and eggs) by liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS) at levels in compliance with the legislation in force. The drugs were extracted with a mixture of dichloromethane/acetone (1:1, v/v) and clean-up was carried out by solid phase extraction (SPE) on a sulfonic acid column after addition of acetic acid to the extract, so as to allow for ionexchange. Sulfonamides and TMP were then eluted from the SPE column using a solution of ammonia in methanol. The chromatographic separation was performed on a C₁₈ column by using a mobile phase of methanol/5 mM aqueous ammonium acetate and 0.1% formic acid in gradient, and the LC-MS/MS analysis was performed in a triplequadrupole mass spectrometer equipped with a TurboIonSpray source and operated in positive ion mode. The multiple reaction monitoring (MRM) approach was adopted for the identification and quantification of the molecules of concern and was applied by selecting three specific diagnostic ions (one precursor ion and two product ions) for each analyte, so as to meet the criteria set by the EU both for the minimum required number of identification points and for the ion intensity ratio tolerances. Calculated detection limits for sulfonamides (signal/noise ratio 3:1) ranged from 0.1 µg to 1.7 µg/kg, whereas the recovery rates varied between 69.5% and 94.2% throughout the different compounds and matrices. The corresponding values for TMP ranged from 0.2 µg to 0.4 µg/kg and between 51.9% and 52.8%, according to the matrix. The easy sample preparation procedure and the specific and selective mass spectrometric detection make the present method reliable and suitable for the unambiguous identification and quantitation of the analytes taken into account in chicken, fish muscle and eggs. Moreover, method application has successfully extended to other sulfonamides, such as sulfaguanidine, sulfapyridine, sulfamoxole and sulfamethizole.

Keywords
Chicken, Fish muscle and eggs, LC-MS/MS, SPE, Sulfonamides, Trimethoprim.