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Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry

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Abstract

Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 μm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene.

Structures of clomiphene isomers and overlay of MRM-chromatograms of clomiphene and its metabolites

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Acknowledgments

We are grateful to Svitlana Igel and Gabriele Böhmer for supervising the clinical trial. This work was supported by grants from the Robert Bosch Foundation (Stuttgart, Germany) and from the Federal Ministry for Education and Research (BMBF, Berlin, Germany; 03IS2061C and 01ZP0502).

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Authors and Affiliations

  1. Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tübingen, Auerbachstr. 112, 70376, Stuttgart, Germany

    Boian Ganchev, Georg Heinkele, Reinhold Kerb, Matthias Schwab & Thomas E. Mürdter

  2. Department of Clinical Pharmacology, Institute of Experimental and Clinical Pharmacology and Toxicology, University Hospital, Otfried-Müller-Straße 45, 72076, Tübingen, Germany

    Matthias Schwab

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  1. Boian Ganchev
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  2. Georg Heinkele
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  3. Reinhold Kerb
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  5. Thomas E. Mürdter
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Correspondence to Thomas E. Mürdter.

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Ganchev, B., Heinkele, G., Kerb, R. et al. Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry. Anal Bioanal Chem 400, 3429–3441 (2011). https://doi.org/10.1007/s00216-011-5045-9

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  • DOI: https://doi.org/10.1007/s00216-011-5045-9

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