Investigation of the stereoselective metabolism of praziquantel after incubation with rat liver microsomes by capillary electrophoresis and liquid chromatography–mass spectrometry

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Abstract

Two different separation methods for the antischistosomal drug praziquantel and its metabolites by capillary electrophoresis are described. Achiral separation was obtained by micellar electrokinetic capillary chromatography using sodium dodecyl sulfate as micelle-forming surfactant. On the other hand, the negatively charged sulfobutylether-β-cyclodextrin as a chiral selector enabled the separation of the drug and its metabolites as well as their enantioseparation. Based on this separation, the enantioselectivity of the metabolism of praziquantel was studied by incubation of the drug with rat liver microsomes. Whereas trans- and cis-4-hydroxypraziquantel were mainly formed from the R-(−)-enantiomer, another, different monohydroxylated metabolite was only formed from the S-(+)-enantiomer. Information about the structure of these metabolites was obtained, using LC–MS.

Introduction

Praziquantel (Fig. 1), an anthelminthic drug, exhibits broad activity against trematodes and cestodes. It is used in therapy as the racemate although the anthelminthic activity is mainly associated with the R-(−)-enantiomer 1, 2.

The drug undergoes extensive metabolism. After in vitro incubation with rat liver microsomes several monohydroxylated metabolites are formed, with cis-4-hydroxypraziquantel being the main metabolite 3, 4. By incubation of each praziquantel enantiomer separately with rat liver microsomes, followed by achiral high-performance liquid chromatography (HPLC) analysis of the resulting metabolites, metabolism was shown to be highly stereoselective. However, chiral separation and analyzing the enantiomeric ratio directly could only be examined for praziquantel and its metabolite trans-4-hydroxypraziquantel [5].

Capillary electrophoresis (CE) has become a powerful separation technique for the analysis of the biotransformation of drugs [6]. The development of micellar electrokinetic capillary electrophoresis (MECC) allows the separation of neutral compounds [7]. In addition to achiral analysis, CE is also suitable for the separation of enantiomers using cyclodextrins (CDs) as chiral selectors because of the high separation efficiency [8]. Charged CDs allow the analysis of even neutral components [9].

For information about the structure of metabolites liquid chromatography–mass spectrometry (LC–MS) is a powerful method. The use of HPLC in combination with a mass spectrometer in the MSn scan mode enables the analysis of different metabolites in biological samples in one step.

This paper describes a method for the separation of praziquantel and its main metabolites, cis- and trans-4-hydroxypraziquantel, by MECC and the simultaneous enantioselective separation of praziquantel and its metabolites after incubation with rat liver microsomes by using sulfobutylether-β-CD (SBE-β-CD) as a chiral selector. For the identification of these metabolites a LC–MS method was developed.

Section snippets

Chemicals

Racemic praziquantel was obtained from Merck (Darmstadt, Germany). Trans- and cis-4-hydroxypraziquantel were synthesized and characterized in our laboratory [11]. The enantiomers were separated by low-pressure LC on a cellulose triacetate (CTA) column. The optical purity of the enantiomers was determined on a Chiracel-OD column and amounts to 99.9% for praziquantel and >99% for trans-4-hydroxypraziquantel, respectively [12].

Sodium dodecyl sulfate (SDS) was purchased from Fluka (Buchs,

Achiral separation using SDS as surfactant in MECC

Praziquantel and its two metabolites cis- and trans-4-hydroxypraziquantel, as uncharged compounds, were separated by MECC using 20 mM SDS as anionic surfactant. A typical electropherogram is shown in Fig. 2. However, the migration times were very long. Finally, this method proved to be unsuitable for the analysis of the in vitro metabolism of praziquantel because of interfering components from the microsomes.

Separation of praziquantel and its metabolites with simultaneous enantiomeric separation

The separation of praziquantel and its neutral metabolites, including enantiomeric

Conclusions

An achiral MECC method using SDS was developed for the separation of the uncharged drug praziquantel and its metabolites cis- and trans-4-hydroxypraziquantel.

With the negatively charged SBE-β-CD used as a chiral additive, a simultaneous separation and enantioselective resolution of praziquantel and its metabolites was possible.

The use of a HPLC method allowed the study of the stereoselective metabolism of praziquantel only after incubation of each enantiomer separately [10], whereas with this

Acknowledgements

The authors thank the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support.

References (16)

  • P. Andrews

    Pharmacol. Ther.

    (1985)
  • F. Westhoff et al.

    J. Chromatogr.

    (1992)
  • D.K. Lloyd

    J. Chromatogr. A

    (1996)
  • S. Terabe et al.

    J. Chromatogr. A

    (1994)
  • C. Weinz et al.

    J. Chromatogr. B

    (1995)
  • P. Dayer et al.

    Biochem. Biophys. Res. Commun.

    (1984)
  • M. Bradford

    Anal. Biochem.

    (1976)
  • U. Staudt et al.

    Parasitol. Res.

    (1992)
There are more references available in the full text version of this article.

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