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 (Cmax) and AUC0-∞ 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.
Keywords: 17 β-estradiol; High-throughput; Liquid-liquid extraction; Mass spectrometry; Pharmacokinetics.
Published by Elsevier B.V.