Unauthorised antibiotic treatments in beekeeping Development and validation of a method to quantify and confirm tylosin residues in honey using liquid chromatography–tandem mass spectrometric detection

A liquid chromatographic–tandem mass spectrometric (HPLC-MS/MS) method is proposed for the identification and quantification of tylosin in honey. Sample treatment involves an extraction in a Tris buffer at pH 10.5, followed by a solid-phase clean up step on an Oasis HLB column. Roxithromycin was used as the internal standard. Chromatographic separation of tylosin and roxithromycin was performed on an XTerra MS C18 column (100 mm × 2.1 mm i.d., 5 (mu)m) using a gradient of aqueous 0.01 M ammonium acetate pH 3.5 and acetonitrile as the mobile phase, at a flow rate of 0.25 ml min^-1. The method was validated according to the guidelines laid down by the Commission Decision 2002/657/EC. Tylosin residues were confirmed by MS/MS experiments considering the appropriate identification points. All validation parameters such as Cc(alpha) (lower than 3 ng g^-1), Cc(beta) (lower than 5 ng g^-1), recovery and precision were assessed on the basis of the "critical ion” (less intense ion permitting unambiguous identification of the analyte).