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

Emilia Waraksa; Katarzyna Owczarek; Paweł Kubica; Ewa Kłodzińska; Mariusz Ozimek; Robert Wrzesień; Barbara Bobrowska-Korczak; Jacek Namieśnik

doi:10.1016/j.jchromb.2020.122197

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

J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 30:1151:122197. doi: 10.1016/j.jchromb.2020.122197. Online ahead of print.

Authors

Emilia Waraksa¹, Katarzyna Owczarek², Paweł Kubica², Ewa Kłodzińska³, Mariusz Ozimek³, Robert Wrzesień⁴, Barbara Bobrowska-Korczak⁵, Jacek Namieśnik²

Affiliations

¹ Gdańsk University of Technology, Faculty of Chemistry, Department of Analytical Chemistry, G. Narutowicza 11/12 Street, 80-233 Gdańsk, Poland; Institute of Sport - National Research Institute, Department of Analytical Chemistry and Instrumental Analysis, Trylogii 2/16 Street, 01-982 Warsaw, Poland. Electronic address: emilia.waraksa@insp.waw.pl.
² Gdańsk University of Technology, Faculty of Chemistry, Department of Analytical Chemistry, G. Narutowicza 11/12 Street, 80-233 Gdańsk, Poland.
³ Institute of Sport - National Research Institute, Department of Analytical Chemistry and Instrumental Analysis, Trylogii 2/16 Street, 01-982 Warsaw, Poland.
⁴ Medical University of Warsaw, Central Laboratory of Experimental Animal, Banacha 1 B Street, 02-097 Warsaw, Poland.
⁵ Medical University of Warsaw, Department of Bromatology, Banacha 1 B Street, 02-097 Warsaw, Poland.

PMID: 32512535
DOI: 10.1016/j.jchromb.2020.122197

Abstract

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 < 9.6%) as well as recovery (94.8 - 105.65%). The LODs were 0.0052, 0.0053, 0.0056 and 0.0043 ng/mL for BH, 2-HDMB, 3-HDMB and 4-HDMB, respectively. The developed method was applied to determine the time curses of BH and its metabolites concentrations in equine serum collected for 95.25 h following a single oral administration of BH to two healthy mares (in dose of 0.8 mg/kg). The parent drug was found at higher concentration levels than 3-HDMB (major metabolite) and 4-HDMB (minor metabolite), however, both BH metabolites lasted for a longer period of time in equine serum than the parent drug. Thus, both metabolites of BH can be considered as BH abuse markers.

Keywords: Bromhexine; Doping; Elimination study; Equestrian sports; Liquid chromatography – tandem mass spectrometry; Metabolites of bromhexine.