Quantitative analysis of underivatized 17 β-estradiol using a high-throughput LC-MS/MS assay - Application to support...

Difference in female sex hormone, β-estradiol (E₂), levels can contribute to sex differences in biological processes that underlie target tissue functions (QT interval), vulnerability to diseases (hepatitis or HIV), and response toward therapies. Accurate quantification of plasma E₂ level is thus an important aspect in both basic science research examining hormone-regulated physiological mechanisms and in clinical settings to support patient care associated with altered E₂ levels. Due to lack of a high-throughput high-sensitivity analytical method, we developed and validated a LC-MS/MS assay for accurate low-level quantification of E₂ and demonstrated its application to a guinea pig pharmacokinetic study in which guinea pigs were treated with 10 or 40 μg/kg E₂ subcutaneously and blood samples collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post-dosing. E₂ was extracted using 90 μL ovariectomized guinea pig plasma by liquid-liquid extraction. The method was robust, sensitive with linear range from 3.9 to 1000 pg/mL, and the assay met acceptance criteria for validation parameters listed in the current FDA Guidance on Bioanalytical Method Validation. Compared to the 10 μg/kg dose, more than dose proportional increase in maximum E₂ plasma concentration (C_max) and AUC_0-∞ and correspondingly longer half-life were observed after 40 μg/kg dose. This assay is a significant improvement over existing E₂ quantification methods in bioanalytical field, with high precision and accuracy, low sample and injection volumes, no derivatization, and short assay run time of 3 min. This assay is amenable in high-throughput settings requiring low-level E₂ quantitation in basic science research and clinical settings.