Determination of bromhexine and its metabolites in equine serum samples by liquid chromatography – Tandem mass spectrometry: Applicability to the elimination study after single oral dose

[Display omitted] •Time curses of BH and its metabolites concentrations determined for the first time in equine serum.•Fully validated LC-MS/MS method for determination of BH and its metabolites in equine serum samples.•Simple sample preparation utilizing acetonitrile protein precipitation. Bromhexine (BH), expectorant used in the treatment of respiratory disorders associated with viscid or excessive mucus, is not permitted for use in the competing horse by many authorities in horseracing and Olympic disciplines. Metabolic studies are of the great importance in anti-doping field because they allow for updating the selection of the most appropriate markers for prohibited substances, such as metabolites present at higher concentration levels and/or lasted for a longer period of time in biological samples than a parent drug. This study describes LC-MS/MS-based method for simultaneous determination of BH and its metabolites, including 4-(2-amino-3,5-dibromobenzylamino)cyclohexanol (4-HDMB), 3-(2-amino-3,5-dibromobenzylamino)cyclohexanol (3-HDMB), in equine serum samples. The 2-(2-amino-3,5-dibromobenzylamino)cyclohexanol (2-HDMB) was monitored as well. The assay was validated in terms of linearity (R2 greater than 0.9951), intra- and inter-assay accuracy (91.6 – 109.1%) and precision (CV