Molecular interactions between nucleotide drug isomers and cyclodextrins: Insights from ion mobility spectrometry and computational modeling

Nucleotide analogs (NAs) are widely used as antiviral and anticancer agents, but their isomers often exhibit distinct biochemical properties, posing challenges for precise analysis. Traditional separation methods are limited by low resolution and lengthy procedures. While ion mobility spectrometry-mass spectrometry (IMS-MS) offers rapid separation, its resolution is insufficient for structurally similar NA isomers. This study addresses the critical need for a high-resolution method to separate and quantify NA isomers, which is essential for drug development and quality control. We developed a host-guest chemistry approach using cyclodextrins (CDs) and metal ions to enhance IMS-MS separation of two NA isomer groups (2F-2DC/5F-5DC/5F-2DC and 2F-2DR/5F-5DR/5F-2DR). The proposed approach utilized β-CD to form binary complexes that resolved F-DC isomers (R p-p  = 1.11) through non-covalent interactions, while ternary complexes incorporating Li + ions enabled separation of F-DR isomers via metal-coordination assisted molecular recognition (R p-p  = 1.71). Theoretical calculations and non-covalent interaction analyses revealed distinct non-covalent binding sites and conformational variations, thereby elucidating the mobility separation mechanism at the molecular level. Quantitative analysis demonstrated high sensitivity LOD = 0.13–0.14 ng mL-1 for absolute quantification and LOD = 1:39–42:1 for relative quantification with recovery≥77 %, RSD ≤ 4.2 %). The method was successfully applied to drug purity testing and physiological solutions. This work introduces a novel strategy combining host-guest chemistry with IMS-MS to resolve NA isomers, overcoming limitations of traditional techniques. The molecular-level insights into CD-isomer interactions and the robust quantification framework advance the field of analytical chemistry for nucleotide-derived drugs, ensuring safer and more precise therapeutic applications.