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Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies

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Abstract

The use of selective oestrogen receptor modulators has been prohibited since 2005 by the World Anti-Doping Agency regulations. As they are extensively cleared by hepatic and intestinal metabolism via oxidative and conjugating enzymes, a complete investigation of their biotransformation pathways and kinetics of excretion is essential for the anti-doping laboratories to select the right marker(s) of misuse. This work was designed to characterize the chemical reactions and the metabolizing enzymes involved in the metabolic routes of clomiphene, tamoxifen and toremifene. To determine the biotransformation pathways of the substrates under investigation, urine samples were collected from six subjects (three females and three males) after oral administration of 50 mg of clomiphene citrate or 40 mg of tamoxifen or 60 mg of toremifene, whereas the metabolizing enzymes were characterized in vitro, using expressed cytochrome P450s and uridine diphosphoglucuronosyltransferases. The separation, identification and determination of the compounds formed in the in vivo and in vitro experiments were carried out by liquid chromatography coupled with mass spectrometry techniques using different acquisition modes. Clomiphene, tamoxifen and toremifene were biotransformed to 22, 23 and 18 metabolites respectively, these phase I reactions being catalyzed mainly by CYP3A4 and CYP2D6 isoforms and, to a lesser degree, by CYP3A5, CYP2B6, CYP2C9, CYP2C19 isoforms. The phase I metabolic reactions include hydroxylation in different positions, N-oxidation, dehalogenation, carboxylation, hydrogenation, methoxylation, N-dealkylation and combinations of them. In turn, most of the phase I metabolites underwent conjugation reaction to form the corresponding glucuro-conjugated mainly by UGT1A1, UGT1A3, UGT1A4, UGT2B7, UGT2B15 and UGT2B17 isoenzymes.

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Acknowledgments

This project has been supported by a research grant by the Partnership for Clean Competition, United States (Research Grant 2011–2012). The authors are also grateful to Ms. Amelia Palermo for her valuable technical assistance.

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Authors and Affiliations

  1. Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy

    Monica Mazzarino, Xavier de la Torre, Ilaria Fiacco & Francesco Botrè

  2. Dipartimento di Chimica e Tecnologia del Farmaco, “Sapienza” Università di Roma, Piazzale Aldo Moro 5, 00185, Rome, Italy

    Mariangela Biava

  3. Dipartimento di Medicina Sperimentale, “Sapienza” Università di Roma, Viale Regina Elena 324, 00161, Rome, Italy

    Francesco Botrè

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  1. Monica Mazzarino
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  2. Mariangela Biava
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  4. Ilaria Fiacco
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  5. Francesco Botrè
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Correspondence to Francesco Botrè.

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Mazzarino, M., Biava, M., de la Torre, X. et al. Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies. Anal Bioanal Chem 405, 5467–5487 (2013). https://doi.org/10.1007/s00216-013-6961-7

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  • DOI: https://doi.org/10.1007/s00216-013-6961-7

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