Analysis of sulfonamides in animal feeds by liquid chromatography with fluorescence detection.

Two analytical methodologies for the simultaneous analysis of eight sulfonamide antibiotics in animal feeds were developed. Analytes were extracted in a simple and rapid procedure by manual shaking with an ethyl acetate/ultrapure water mixture (99:1, v/v) without further sample cleanup. Mean recoveries ranging from 72.7% to 99.4% with relative standard deviations below 9% were achieved from spiked animal feed samples. Determination was carried out by high-performance liquid chromatography using fluorometric detection with precolumn derivatization. The separation of the derivatized compounds was performed using two different chromatographic columns: a conventional C(18) column and a recently available core-shell particle Kinetex C(18) column. Both methods were validated in-house in six different feed matrices, and the two approaches were compared. The experiments showed that the method using the Kinetex column was superior with regard to speed of analysis and precision, both under repeatability and intermediate reproducibility conditions. The limits of detection and quantification were also greatly improved, below 0.10 and 0.34 µg/g, respectively. Finally, this novel approach was successfully applied to the analysis of real feed samples.

Validation of an enzyme-linked immunosorbent assay for detecting sulfonamides in feed resources.

An enzyme-linked immunosorbent assay (ELISA) to screen sulfadiazine and sulfamethazine residues in feeds has been developed and validated according to Commission Decision 2002/657/EC criteria. Sulfonamides are easily extracted with a 95:5 acetonitrile/water mixture, obtaining recoveries between 80 and 100%. Accuracy, precision, selectivity, robustness, limit of detection (LOD), and detection capability (CCbeta) of the assay have been assessed during the validation process. LOD values in pig feed samples were 0.2 microg/g for sulfadiazine and 0.04 microg/g for sulfamethazine without any sample treatment other than extraction, dilution with the assay buffer, and filtering of the resulting solution. Furthermore, a new strategy for the determination of CCbeta in an ELISA screening method is proposed; this gave CCbeta values of 0.8 microg/g for sulfadiazine and 0.1 microg/g for sulfamethazine. Besides sulfadiazine and sulfamethazine, other sulfonamides can be detected with this immunoassay; this has been verified calculating their LOD values and cross-reactivities. Finally, real feed samples were analyzed with the ELISA methodology and a previously developed liquid chromatography (LC) method, and results confirmed the utility of this new immunoassay for screening purposes.