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Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain highlights a local metabolic regulation after oral versus subcutaneous administration

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Abstract

17β-Estradiol (17β-E2) is a steroid with pleiotropic actions. In addition to being a sexual hormone, it is also produced in the brain where it modulates the reproductive axis. It has been shown that 17β-E2 also acts on synaptic plasticity and plays a role in neurological pathways and in neurodegenerative diseases. Assaying this steroid in the brain is thus interesting to improve our knowledge of 17β-E2 effects in the brain. However, 17β-E2 concentration in the central nervous system has been reported to be of a few nanograms per gram wet weight (nanomolar range concentration); therefore, its quantification requires both an efficient extraction process and a sensitive detection method. Herein is presented a derivatization-free procedure based on solid-phase extraction followed by LC-MS/MS analysis, targeted on 17β-E2, its isomer17α-E2, and its metabolites estrone (E1) and estriol (E3). This extraction process allowed reaching 96% 17β-E2 recovery from the mouse brain. Limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 2.5 pmol mL−1, respectively, were reached for both 17α-E2 and 17β-E2. LOD values for E1 and E3 were 0.01 and 0.025 pmol mL−1, respectively. The variation coefficients for intra- and inter-assays were 6 and 14%, respectively, for both estradiol forms. The method was applied to assess estrogen levels in the mouse brain and hippocampus after 17β-E2 acute (subcutaneous injection) and chronic (drinking water) physiological administration. Total estrogen levels were determined after enzymatic deconjugation and compared to free estrogen levels. While 17α-E2 was not detected in biological samples, 17β-E2 and metabolite measurements highlight a local biotransformation of estrogens after physiological administration via drinking water.

Method workflow: After oral or subcutaneous Estradiol administration, mouse brain or hippocampus was removed. Samples were homogenized and prepared according to a liquid-liquid extraction, followed by a solid-phase extraction. Then, LC-MS/MS was optimized to quantify 17ß-E2, its isomer17α-E2, its metabolites estrone (E1) and estriol (E3) and their conjugates

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Acknowledgments

The authors would like to thank the financial support of Erasmus Program for a research grant for EL, the Fondation pour la Recherche Médicale (Paris France), the IBiSA platform network, and Aquitaine Regional Government for the financial support for the mass spectrometry equipment and for the “Nouvelle Equipe Neurocampus.” The author would like to thank Dr. Boutayna Rourrhi-Frih for her discussion on SPE with EL.

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Authors and Affiliations

  1. University of Bordeaux, CBMN, UMR 5248, CNRS, 33600, Pessac, France

    Ecaterina Lozan, Jean-Marie Schmitter & Corinne Buré

  2. University of Bordeaux, ISM, UMR 5255, CNRS, 33000, Bordeaux, France

    Ecaterina Lozan & Svitlana Shinkaruk

  3. University of Bordeaux, Neurocentre Magendie U1215 INSERM, 33077, Bordeaux, France

    Svitlana Shinkaruk, Shaam Alice Al Abed, Valérie Lamothe, Mylène Potier, Aline Marighetto & Catherine Bennetau-Pelissero

  4. INSERM, U1215, Neurocentre Magendie, 33000, Bordeaux, France

    Svitlana Shinkaruk, Shaam Alice Al Abed, Valérie Lamothe, Mylène Potier, Aline Marighetto & Catherine Bennetau-Pelissero

  5. University of Bordeaux, CBMN, UMR 5248, CGFB, BP 68, 146 rue Leo Saignat, 33076, Bordeaux Cedex, France

    Corinne Buré

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  1. Ecaterina Lozan
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Correspondence to Corinne Buré.

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Research involving human participants and/or animals

All animal care and samples withdrawn were conducted in compliance with the European Communities Council Directive 2010-63-EU and approved by the Bordeaux University Animal Care and Use Committee under the protocol number 5012041. All efforts were made to minimize animal suffering, to reduce the number of animals, and to utilize alternatives to in vivo techniques, if available.

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Lozan, E., Shinkaruk, S., Al Abed, S.A. et al. Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain highlights a local metabolic regulation after oral versus subcutaneous administration. Anal Bioanal Chem 409, 5279–5289 (2017). https://doi.org/10.1007/s00216-017-0473-9

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  • DOI: https://doi.org/10.1007/s00216-017-0473-9

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