High sensitivity method validated to quantify estradiol in human plasma by LC-MS/MS

•A high sensitive method to quantify 17β-Estradiol (E2) were validated by LC-MS/MS.•Cleaner chromatography enabled a precise measurement in human plasma.•We proved a long stability in several conditions for E2.•Our validated method was applied in two bioequivalence studies in healthy human.

17β-Estradiol (E2) is an endogenous steroid in the human body. Its measurement is important for health and human biology understanding. However, E2 concentration in human plasma is in the range of pg/mL, which makes it difficult to detect. In this way, LC-MS/MS has been shown the most sensitive tool, although E2 is a weakly ionizable molecule. In this work, we validated a more sensitive and accurate method for E2 quantification in human plasma. Our extraction step ensured a cleaner chromatography, resulting in a precise measurement and highly reproducible method in the range of 2-150 pg/mL. Moreover, we proved a long stability for E2 in several conditions. All results indicate that our developed method is robust and sensitive enough to apply in bioequivalence studies for E2 measurement in human plasma, even at very low concentrations.