Sulfonamide derivatization studies. Development and validation of the methods simultaneous determination of sulfonamides by means of high pressure liquid chromatography in biological fluids and foods of animal origin

In this study, two new, sensitive and selective LC methods have been developed and validated for the simultaneous determination of several sulfonamides, in the presence of putrescine as internal standard, after precolumn derivatization with fluorescamine. In the first method, the derivatives of eight sulfonamides (sulfaguinidine, sulfanilamide, sulfisomidine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine and sulfamethoxazole) were separated on a reversed-phase column, Inertsil ODS-3 (250 x 4 mm I.D., 5μm) using a 20 min gradient elution with a binary system of methanol– 0.05 mol L-1 acetate buffer (pH 3.4) and fluorescence detection at excitation and emission wavelengths of 406 and 496 nm respectively. Fluorescamine was chosen as the derivatization reagent. The following conditions that influence the pre-column derivatization of sulfonamides, such as: reaction medium pH-value, fluorescamine concentration and reaction time were examined by varying one parameter at a time, while keeping the others constant. Linearity of derivatization was obtained for concentrations from 1.0 to 300 μg L-1 in standard solutions. The within- and between-day relative standard deviations (RSDs) ranged from 0.8 to 3.4% and 1.0 to 3.8% respectively. The method was applied to the determination of sulfonamides in human serum and urine and in poultry muscle and eggs, after solid-phase extraction and subsequent derivatization of the extracts. The whole procedure was evaluated and fully validated according to the European Union Decision 2002/657/EC for the determination of sulfonamides in foods of animal origin. The detection limits varied from 0.25 to 0.30 μg L-1 in serum, 0.10 to 0.25 μg L-1 in urine samples, 1.0 το 8.0 μg kg-1 in poultry muscle and 1.0 to 5.0 μg kg-1 in egg samples and were much lower than the Maximum Residue Limits (MRLs). In the second method, the derivatives of twelve sulfonamides (sulfaguinidine, sulfanilamide, sulfisomidine, sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) were separated on a monolithic silica column, Chromolith Performance RP-18 (100 x 4.6 mm) using the same binary system (methanol-0.05 mol L-1 acetate buffer pH 3.4) as above, but with different gradient elution programme. In order to maximize the reaction yield and the sensitivity of the method, the critical derivatization parameters (medium pH, fluorescamine concentration and reaction time) were studied using the univariate approach and optimized. Linearity of derivatization was obtained for concentrations from 3.0 to 300 μg L-1 in standard solutions. The withinand between-day relative standard deviations (RSDs) ranged from 0.6 to 3.2% and 0.9 to 3.3% respectively. The whole procedure was evaluated and fully validated, according to the European Union Decision 2002/657/EC, for the determination of sulfonamides in poultry muscle and eggs following solid-phase extraction. The method was also applied in human serum and urine. The detection limits varied from 2.0 to 17.0 μg kg-1 in poultry muscle, 2.0 to 15.0 μg kg-1 in egg, 0.30 to 5.0 μg L-1 in human serum and 0.25 to 2.5 μg L-1 in urine samples. In terms of sensitivity, the conventional column, Inertsil ODS-3, offers lower detection limits for all sulfonamides. On the other hand, correlation coefficients are higher with the monolithic column, indicating better linearity. Seperation efficiency is also superior with the monolithic column, which enables analytical runs under lower back pressure at shorter time. Both analytical procedures were new, fast, simple, selective and highly sensitive. Additionally, the combined use of a monolithic column with fluorescence detection in order to determine sulfonamides by pre-column derivatization with fluorescamine, in human biological fluids, poultry muscle and egg samples, was applied here for the first time in the literature