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. 2017-07-20; 409(22): 5279-5289
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1. Anal Bioanal Chem. 2017 Sep;409(22):5279-5289. doi: 10.1007/s00216-017-0473-9.
Epub 2017 Jul 20.
Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain
highlights a local metabolic regulation after oral versus subcutaneous
Lozan E(1)(2), Shinkaruk S(2)(3)(4), Al Abed SA(3)(4), Lamothe V(3)(4), Potier
M(3)(4), Marighetto A(3)(4), Schmitter JM(1), Bennetau-Pelissero C(3)(4), Buré
(1)University of Bordeaux, CBMN, UMR 5248, CNRS, 33600, Pessac, France.
(2)University of Bordeaux, ISM, UMR 5255, CNRS, 33000, Bordeaux, France.
(3)University of Bordeaux, Neurocentre Magendie U1215 INSERM, 33077, Bordeaux,
(4)INSERM, U1215, Neurocentre Magendie, 33000, Bordeaux, France.
(5)University of Bordeaux, CBMN, UMR 5248, CNRS, 33600, Pessac, France.
(6)University of Bordeaux, CBMN, UMR 5248, CGFB, BP 68, 146 rue Leo Saignat,
33076, Bordeaux Cedex, France. .
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. Graphical abstract 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.
PMID: 28730313 [Indexed for MEDLINE]