Validation of an Enzyme-Linked Immunosorbent Assay for Detecting Sulfonamides in Feed Resources

Abstract

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.