Molecular-Level Insights into TIMS-MS Separation of α/β-Estradiol Isomers: Synergistic Effects of Cyclodextrins and Metal Ions

This study presents a novel approach for the rapid and high-resolution separation of estrogen isomers (α/β-estradiol and estriol: α/β-E₂/E₃) using trapped ion mobility mass spectrometry (TIMS-MS). Systematically, evaluating cyclodextrin (α-, β-, and γ-CD) as separation reagents and exploring metal ions as coordinating ligands, we achieved enhanced isomer differentiation, in which the separation resolution (R_P–P) was increased from 0.204 to 1.533. Density functional theory (DFT) calculations and noncovalent interaction visualization analysis provided molecular-level insights into the structural and interaction differences driving these separations, highlighting the critical roles of host–guest interactions and metal coordination geometry. Additionally, quantitative analysis and method validation for the estrogen isomers were measured, with a good linearity of R² value higher than 0.99 and RSD ≤ 2.28%. Finally, the method was applied to detect and quantify α/β-E₂/E₃ in human urine samples; good recoveries of ≥79.78% were obtained with RSD ≤ 4.35% for intraday and RSD ≤ 9.56% for interday. The combined experimental and theoretical results demonstrate the potential of this TIMS-MS-based strategy for clinical diagnostics and bioanalytical applications requiring precise estrogen isomer separation.