Estrogens are neurosteroids, especially Estradiol (17β-E2) which is considered to be the most biologicallyactive form. 17β-E2 could have positive effects on some age-associated anomalies (memory and cognitive impairment,Alzheimer’s disease). For elucidating and better understanding the molecular and cellular mechanisms that underliethe effects within the brain, it is necessary to quantitate 17β-E2 and its metabolites (estrone and estriol) in brain andplasma.First, the RP-HPLC-ESI(-)-MS/MS method without derivatization was developed. The selectivity of the separationand the sensitivity of detection of the estrogens has been improved after optimization of MRM and chromatographicparameters.Secondly, the various derivatization agents were evaluated after their synthesis in order to improve the sensitivity,selectivity and signal enhancement. After studying the eleven synthesized derivatives of 17β-E2 in ESI-MS andMS/MS, promising results were obtained with the 17β-E2-Q8S derivative.A simple purification method using liquid-liquid extraction followed by C18 solid phase extraction has been optimizedin order to minimize assay interferences. The two assays (with and without derivatization) were then compared interms of efficiency, detection and quantification limits (LOD/LOQ), calibration linearity and reproducibility. Then,both methods were validated on biological samples (brain, hippocampus and plasma) collected from animals treatedwith known amounts of 17β-E2. Finally, the more robust method was the method without derivatization with a LODof 0.5 fmol.ÎĽL-1.

Les oestrogènes font partie de la famille des neurostĂ©roĂŻdes. Notamment, l’Estradiol (17β-E2) pourrait avoirdes effets bĂ©nĂ©fiques sur certaines anomalies liĂ©es Ă  l’âge (dĂ©clin mnĂ©sique et cognitif, maladie d’Alzheimer). Le dosagede très faibles teneurs du 17β-E2 et de ces mĂ©tabolites (Estrone, Estriol), dans les tissus cĂ©rĂ©braux et le plasma, constitueun outil indispensable pour estimer la variation de leurs niveaux avec l’âge et dans diffĂ©rentes pathologies.Tout d’abord, nous avons Ă©laborĂ© une mĂ©thode en RP-HPLC-ESI(-)-MS/MS de quantification des oestrogènes sansdĂ©rivation chimique. La sĂ©lectivitĂ© de la sĂ©paration et la sensibilitĂ© de la dĂ©tection de ces molĂ©cules ont Ă©tĂ© amĂ©liorĂ©esaprès optimisation des paramètres MRM et chromatographiques.Puis, diffĂ©rents agents de dĂ©rivation ont Ă©tĂ© synthĂ©tisĂ©s et Ă©valuĂ©s afin d’augmenter le taux d’ionisation pour amĂ©liorerla sensibilitĂ© de dĂ©tection. Après l’analyse ESI-MS et MS/MS de onze dĂ©rivĂ©s du 17β-E2, nous nous sommes intĂ©ressĂ©splus particulièrement au dĂ©rivĂ© 17β-E2-Q8S.Afin de rĂ©duire les interfĂ©rences, une mĂ©thode de prĂ©paration de l’échantillon biologique (tissus cĂ©rĂ©bral, plasma desouris) a Ă©tĂ© dĂ©veloppĂ©e. Les deux dosages (sans et avec dĂ©rivation) ont Ă©tĂ© comparĂ©es en termes de sensibilitĂ©, limitesde dĂ©tection et de quantification (LD et LQ), linĂ©aritĂ© et reproductibilitĂ©. Puis, elles ont Ă©tĂ© appliquĂ©es Ă  l’analyse desĂ©chantillons de cerveau, d’hippocampe et de plasma prĂ©levĂ©s sur des animaux jeunes et âgĂ©s traitĂ©s avec des quantitĂ©sconnues de 17β-E2. La mĂ©thode qui nous est apparue la plus robuste est la mĂ©thode sans dĂ©rivation avec une LD de0,5 fmole.ÎĽL-1.