uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
2012 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 26, no 11, 1338-1346 p.Article in journal (Refereed) Published
Abstract [en]

RATIONALE: 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-d9. 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 MSn 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.

Place, publisher, year, edition, pages
2012. Vol. 26, no 11, 1338-1346 p.
National Category
Medicinal Chemistry Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-173545DOI: 10.1002/rcm.6225ISI: 000303597100009OAI: oai:DiVA.org:uu-173545DiVA: diva2:523823
Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Drug Metabolites Formed by Cunninghamella Fungi: Mass Spectrometric Characterization and Production for use in Doping Control
Open this publication in new window or tab >>Drug Metabolites Formed by Cunninghamella Fungi: Mass Spectrometric Characterization and Production for use in Doping Control
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the in vitro production of drug metabolites using fungi of the Cunninghamella species. The metabolites were characterized with mainly liquid chromatography-mass spectrometry using ion-trap and quadrupole-time-of-flight instruments. A fungal in vitro model has several advantages e.g., it is easily up-scaled and ethical problems associated with animal-based models are avoided.

The metabolism of bupivacaine and the selective androgen receptor modulators (SARMs) S1, S4 and S24 by the fungi Cunninghamella elegans and Cunninghamella blakesleeana was investigated. The detected metabolites were compared with those formed in vitro and in vivo by human and horse and most phase I metabolites formed by mammals were also formed by the fungi. The higher levels of bupivacaine metabolites in the fungal samples allowed an extensive mass spectrometric structural characterization which shows that the fungi are relevant metabolic models.

Glucuronides are important drug metabolites but they are difficult to synthesize. The discovery that the fungus Cunninghamella elegans formed large amounts of glucosides led to the idea that they could be used to form glucuronides. A new concept was developed where a fungal incubate containing a SARM S1 glucoside was mixed with the free radical tetramethylpiperidinyl-1-oxy (TEMPO), sodium bromide and sodium hypochlorite which produced a glucuronide. Isolation and characterization by nuclear magnetic resonance spectroscopy proved that the new method could produce glucuronides for use as reference material.

An investigation of reactive metabolite formation of the drugs paracetamol, mefenamic acid and diclofenac by the fungus Cunninghamella elegans was performed. It was demonstrated for the first time that the fungus could produce glutathione, glutathione ethyl-ester, cysteine and N-acetylcysteine conjugates that are indicative of a preceding formation of reactive intermediates. A comparison with conjugates formed by human liver microsomes showed that both models formed identical metabolites.

The presented investigations prove that Cunninghamella fungi are relevant drug metabolism models. They show that the fungi to a large extent forms the same metabolites as mammals and that they can produce metabolites for use as reference material in, e.g. doping control. It was also demonstrated that the fungal model can be used in the important assessment of drug toxicity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 46 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 186
Keyword
Cunninghamella blakesleeana, Cunninghamella elegans, Doping Control, Drug Metabolites, Glucuronide Production, Mass Spectrometry, Reactive Metabolites, Reference Material, Structural Characterization
National Category
Medicinal Chemistry Pharmaceutical Sciences
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-220906 (URN)978-91-554-8906-9 (ISBN)
Public defence
2014-05-09, B:41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-04-17 Created: 2014-03-23 Last updated: 2014-04-29

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Rydevik, AxelBondesson, UlfMikael, Hedeland

Search in DiVA

By author/editor
Rydevik, AxelBondesson, UlfMikael, Hedeland
By organisation
Analytical Pharmaceutical Chemistry
In the same journal
Rapid Communications in Mass Spectrometry
Medicinal ChemistryAnalytical Chemistry

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 434 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf