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LC–MS–MS identification of albendazole and flubendazole metabolites formed ex vivo by Haemonchus contortus

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Abstract

Resistance of helminth parasites to common anthelminthics is a problem of increasing importance. The full mechanism of resistance development is still not thoroughly elucidated. There is also limited information about helminth enzymes involved in metabolism of anthelminthics. Identification of the metabolites formed by parasitic helminths can serve to specify which enzymes take part in biotransformation of anthelminthics and may participate in resistance development. The aim of our work was to identify the metabolic pathways of the anthelminthic drugs albendazole (ABZ) and flubendazole (FLU) in Haemonchus contortus, a world-wide distributed helminth parasite of ruminants. ABZ and FLU are benzimidazole anthelminthics commonly used in parasitoses treatment. In our ex vivo study one hundred living adults of H. contortus, obtained from the abomasum of an experimentally infected lamb, were incubated in 5 mL RPMI-1640 medium with 10 μmol L−1 benzimidazole drug (10% CO2, 38 °C) for 24 h. The parasite bodies were then removed from the medium. After homogenization of the parasites, both parasite homogenates and medium from the incubation were separately extracted using solid-phase extraction. The extracts were analyzed by liquid chromatography–mass spectrometry (LC–MS) with electrospray ionization (ESI) in positive-ion mode. The acquired data showed that H. contortus can metabolize ABZ via sulfoxidation and FLU via reduction of a carbonyl group. Albendazole sulfoxide (ABZSO) and reduced flubendazole (FLUR) were the only phase I metabolites detected. Concerning phase II of biotransformation, the formation of glucose conjugates of ABZ, FLU, and FLUR was observed. All metabolites mentioned were found in both parasite homogenates and medium from the incubation.

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Abbreviations

ABZ:

Albendazole

ABZSO:

Albendazole sulfoxide

ABZSO2 :

Albendazole sulfone

DMSO:

Dimethyl sulfoxide

ESI:

Electrospray ionization

FLU:

Flubendazole

FLUH:

Hydrolyzed flubendazole

FLUR:

Reduced flubendazole

HEPES:

N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)

HPLC:

High-performance liquid chromatography

LC–MS:

Liquid chromatography–mass spectrometry

MS–MS:

Tandem mass spectrometry

QqQ:

Triple-quadrupole mass spectrometer

RPMI:

Roswell Park Memorial Institute

SPE:

Solid-phase extraction

UDP-glucose:

Uridine-5′-diphosphoglucose

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Acknowledgements

This project was kindly supported by the Czech Science Foundation (GA ČR, grant No. 524/06/1345) and the Fund of Mobility of Charles University.

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Authors and Affiliations

  1. Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic

    Viktor Cvilink, Lenka Skálová & Barbora Szotáková

  2. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic

    Jiří Lamka

  3. Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014, Helsinki, Finland

    Risto Kostiainen & Raimo A. Ketola

  4. Drug Discovery and Development Technology Center, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014, Helsinki, Finland

    Raimo A. Ketola

Authors
  1. Lenka Skálová
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  2. Barbora Szotáková
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  3. Jiří Lamka
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  4. Risto Kostiainen
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  5. Raimo A. Ketola
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Correspondence to Raimo A. Ketola.

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Cvilink, V., Skálová, L., Szotáková, B. et al. LC–MS–MS identification of albendazole and flubendazole metabolites formed ex vivo by Haemonchus contortus . Anal Bioanal Chem 391, 337–343 (2008). https://doi.org/10.1007/s00216-008-1863-9

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  • DOI: https://doi.org/10.1007/s00216-008-1863-9

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