Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry.
Abstract: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial. Methods: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d(9). Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MS(n) and hydrogen/deuterium exchange. Results: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana. Conclusions: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.
Copyright © 2012 John Wiley & Sons, Ltd.
Publication Date: 2012-05-05 PubMed ID: 22555927DOI: 10.1002/rcm.6225Google Scholar: Lookup
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Summary
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This research investigates the metabolism of bupivacaine, a local anesthetic, both in horses and in fungi in order to better understand how it is processed in live organisms and to improve doping control in equine sports.
Study Overview and Methods
- This study centers on the metabolism of bupivacaine, a substance widely used as a local anesthetic. The investigation was carried out specifically on horses and two types of fungi, known as Cunninghamella elegans and Cunninghamella blakesleeana.
- As bupivacaine is prohibited in equine sports, the researchers set out to delineate the metabolite profile of bupivacaine in horse urine. They undertook an administration study, giving a horse a subcutaneous injection of Marcain® (bupivacaine hydrochloride), and collected urine samples for analysis.
- The researchers also analyzed how bupivacaine is metabolized in vitro, or outside of living organisms, by incubating it with the two types of fungi. They believed that understanding this process would prove useful in future research and applications.
- The collected samples were analyzed using liquid chromatography and mass spectrometric analysis. Some samples were analyzed directly after dilution, while others underwent liquid-liquid extraction for clean-up.
Results
- In the horse urine, seven phase I metabolites of bupivacaine were identified. These included 3′- and 4′-hydroxybupivacaine, N-desbutylbupivacaine, two hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine.
- Furthermore, sulfated hydroxybupivacaine and glucuronides were detected. These metabolites were previously unrecognized in horses, with the exception of 3′-hydroxybupivacaine.
- From the results, 3′- and 4′-hydroxybupivacaine were singled out as reliable markers for doping control.
- Interestingly, the fungal samples produced the same set of phase I metabolites as the horse, indicating they are useful models for simulating mammalian metabolism of bupivacaine.
Conclusions
- The study surfaced new insights into how bupivacaine is metabolized in the horse’s body.
- It was also established that the fungi Cunninghamella elegans and Cunninghamella blakesleeana are valuable models for the metabolic processes involving bupivacaine, potentially aiding future endeavors to create analytical reference materials.
- This work significantly informs the approach to doping control in equine sports, providing specific markers that can be tested for.
Cite This Article
APA
Rydevik A, Bondesson U, Hedeland M.
(2012).
Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry.
Rapid Commun Mass Spectrom, 26(11), 1338-1346.
https://doi.org/10.1002/rcm.6225 Publication
Researcher Affiliations
- Uppsala University, Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, P.O. Box 574, SE-75123 Uppsala, Sweden.
MeSH Terms
- Animals
- Bupivacaine / analogs & derivatives
- Bupivacaine / metabolism
- Bupivacaine / urine
- Chromatography, Liquid / methods
- Cunninghamella / metabolism
- Deuterium Exchange Measurement
- Doping in Sports
- Horses / metabolism
- Horses / urine
- Liquid-Liquid Extraction
- Male
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 1 times.- Tupertsev B, Osipenko S, Kireev A, Nikolaev E, Kostyukevich Y. Simple In Vitro (18)O Labeling for Improved Mass Spectrometry-Based Drug Metabolites Identification: Deep Drug Metabolism Study.. Int J Mol Sci 2023 Feb 26;24(5).
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