Beyond Liquid Chromatography: Cyclic Ion Mobility Spectrometry for Phosphorothioate Diastereomer Separation in siRNA

Phosphorothioate (PS) modifications in small interfering RNA (siRNA) moieties enhance stability and therapeutic efficacy, but introduce diastereomeric heterogeneity, complicating structural characterization. Conventional chromatographic methods, such as ion-pair reversed-phase liquid chromatography, provide limited resolution of complex stereoisomer systems, necessitating alternative analytical approaches. In this work, we systematically evaluate cyclic ion mobility spectrometry (cIMS) for the separation and identification of PS diastereomers by investigating oligonucleotide systems with varying chain length and PS linkage patterns that mimic the metabolic diversity in siRNA therapeutics. Our results demonstrate that cIMS effectively separates diastereomers in short (5-mer) to medium-length (9-mer) oligonucleotides, with separation efficiency influenced by charge state and salt adduction. Higher charge states enhance resolution by narrowing conformational distributions and enabling increased numbers of cIMS passes, while sodium and potassium adducts improve separation by promoting distinct gas-phase conformers. However, as system size increases (15-mer), the relative influence of terminal diastereomers on the overall structure diminishes, leading to reduced separation efficiency and ultimately preventing the resolution of diastereomers in long-chain siRNAs. Comparisons between experimental and computationally predicted collisional cross sections (CCSs) underscore both the potential and limitations of CCS-based diastereomer assignment, emphasizing the need for refined computational models or orthogonal validation methods. These findings highlight cIMS as a powerful and complementary tool for the structural characterization of stereochemically complex oligonucleotide therapeutics, providing valuable insights for siRNA drug development.