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_<i>P-P</i>) 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 <i>R</i>² 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.