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In this paper, European food safety legislation is presented, and special attention is devoted to monitoring residues of veterinary drugs in foodstuffs of animal origin. After a short review of the state of the art of analytical methodology for antibiotic residue analysis, the paper focuses on validation of analytical methods, with Decision 657/2002/EC as reference document. Finally, the main issues of the quality control of the analytical data, i.e. analysis of reference materials and participation in proficiency tests, are briefly addressed.

The effect of mobile-phase constituents (pH and ionic strength) and chromatographic behaviour of ten aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, apramycin, paramomycin, kanamycin A, gentamycin C1, gentamycin C2/C2a, gentamycin C1a and neomycin) in the bare silica, amino, amide and zwitterionic phases of hydrophilic interaction chromatography (HILIC) were studied systematically. Among the stationary phases studied, the zwitterionic phase provided the best separation of aminoglycosides. The effect of pH, ionic concentration and column temperature on retention time, peak shape and sensitivity was studied using a central composite design. pH affected sensitivity of the detection of analytes but not the retention time. High ionic strength in the mobile phase was necessary to control the ionic interactions between ionised aminoglycosides and the hydrophilic phase, thereby influencing peak shape and retention time. Column temperature affected sensitivity of the detection but not the retention time. During method development, crosstalk between the MS/MS channels of the analytes was observed and resolved.

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.