Quantitative determination of ceftiofur-related residues in bovine raw milk by LC-MS/MS with electrospray ionization

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of ceftiofur-related metabolites that retain the ß-lactam structure, expressed as desfuroylceftiofur free acid has been developed and validated for raw bovine milk. The method described is based on the cleavage of the disulfide and/or thioester bonds between the metabolites and their sulfur-containing moiety, using dithioerythritol to yield desfuroylceftiofur and further stabilization to desfuroylceftiofur-acetamide (DCA) using iodacetamide. The final extract containing the DCA derivative was cleaned up by a simple solid-phase extraction step prior to separation on a phenylether reversed phase HPLC-column. Chromatography was performed using a formic acid/methanol gradient. Collision induced dissociation with argon was used for fragmentation of the pseudomolecular ion [M+H]⁺ to achieve the required specificity of the method. The new EU guidelines for identification and quantification were fulfilled.