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.
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 (R 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.
Copyright © 2020 Elsevier B.V. All rights reserved.
Publication Date: 2020-05-30 PubMed ID: 32512535DOI: 10.1016/j.jchromb.2020.122197Google Scholar: Lookup
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Summary
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This research explores a new method for detecting the use of the drug bromhexine in horses. The method uses liquid chromatography and tandem mass spectrometry to detect the presence of the drug and its metabolites in horse serum samples. The results have applications for detecting drug abuse in competitive horse racing.
Methodology of Determining Bromhexine and its Metabolites in Equine Serum
- The researchers used liquid chromatography and tandem mass spectrometry (LC-MS/MS), a popular analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry, to simultaneously determine bromhexine (BH) and its molecules that result from the process of metabolism (metabolites) in equine serum samples.
- The metabolites that were specifically monitored in this study included 4-(2-amino-3,5-dibromobenzylamino)cyclohexanol (4-HDMB), 3-(2-amino-3,5-dibromobenzylamino)cyclohexanol (3-HDMB), and 2-(2-amino-3,5-dibromobenzylamino)cyclohexanol (2-HDMB).
- The efficacy of the assay was validated in terms of linearity, accuracy, precision, recovery, and the limits of detection (LODs) for BH and the three metabolites.
Findings and Application of the Study
- The developed LC-MS/MS-based method was used to analyze the change over time in the concentration of BH and its metabolites in equine serum after a single oral dose of BH was administered to two healthy mares.
- The results showed that higher concentrations of BH were found compared to 3-HDMB and 4-HDMB, however, the BH metabolites were present in the serum samples for a longer duration than the parent drug.
- These findings suggest that the metabolites of BH can effectively serve as markers for the use of the drug, and highlight the potential use of this detection method in the field of anti-doping, particularly in competitive horse racing where the use of BH is often prohibited. Preventing the use of this drug will help ensure an equal and fair competition between horses and keep the animals’ health and welfare in check.
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Cite This Article
APA
Waraksa E, Owczarek K, Kubica P, Kłodzińska E, Ozimek M, Wrzesień R, Bobrowska-Korczak B, Namieśnik J.
(2020).
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, 1151, 122197.
https://doi.org/10.1016/j.jchromb.2020.122197 Publication
Researcher Affiliations
- 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.
- Gdańsk University of Technology, Faculty of Chemistry, Department of Analytical Chemistry, G. Narutowicza 11/12 Street, 80-233 Gdańsk, Poland.
- 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.
- Institute of Sport - National Research Institute, Department of Analytical Chemistry and Instrumental Analysis, Trylogii 2/16 Street, 01-982 Warsaw, Poland.
- Medical University of Warsaw, Central Laboratory of Experimental Animal, Banacha 1 B Street, 02-097 Warsaw, Poland.
- Medical University of Warsaw, Department of Bromatology, Banacha 1 B Street, 02-097 Warsaw, Poland.
- Gdańsk University of Technology, Faculty of Chemistry, Department of Analytical Chemistry, G. Narutowicza 11/12 Street, 80-233 Gdańsk, Poland.
Citations
This article has been cited 1 times.- Sharkawi MMZ, Safwat MT, Abdelwahab NS. Analysis of binary mixture of oxytetracycline and bromohexine in their combined veterinary formulation by four simple spectrophotometric methods with greenness assessment. BMC Chem 2024 Oct 3;18(1):192.
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