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

Abstract

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 C 18 column (100 mm × 2.1 mm i.d., 5 μm) 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 Ccα (lower than 3 ng g −1), Ccβ (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).