Separation of phosphodiester impurities from fully phosphorothioate-modified oligonucleotides by ion-pair reversed-phase liquid chromatography.

Phosphorothioate (PS)-modified oligonucleotides are widely used in therapeutic applications; however, the separation of phosphodiester (PO)-substituted impurities remains a significant analytical challenge because these species differ only by the replacement of sulfur with oxygen within the phosphate backbone. Although ion-pair reversed-phase liquid chromatography (IP-RPLC) is widely employed for oligonucleotide analysis, the factors governing PS/PO selectivity have not been fully elucidated. In this study, we systematically investigated the effects of alkylamine structure, hexafluoroisopropanol (HFIP) concentration, and mobile-phase pH on the separation of PO-substituted impurities from a fully PS-modified oligonucleotide. Evaluation of a series of alkylamine ion-pairing reagents revealed that PS/PO selectivity was strongly influenced by alkylamine structure. Primary alkylamines afforded substantially lower resolution than secondary and tertiary alkylamines, whereas dibutylamine (DBA) provided one of the highest resolutions among the reagents examined. Investigation of HFIP concentration demonstrated that the apparent improvement in resolution observed at lower HFIP concentrations was largely attributable to concomitant changes in mobile-phase pH. Independent control of pH revealed that PS/PO selectivity increased with increasing pH and reached a maximum at approximately pH 9.5. Based on these observations, we propose that PS/PO selectivity is governed by the ion-pairing state along the oligonucleotide backbone, which is influenced by both alkylamine structure and mobile-phase pH. Bulkier secondary and tertiary alkylamines, as well as elevated pH, may reduce the extent or uniformity of ion-pair coverage, thereby allowing intrinsic differences between PS and PO linkages to contribute more effectively to chromatographic discrimination. Under optimized conditions, baseline or near-baseline separation was achieved for PO-substituted impurities containing substitutions at both terminal and internal positions. These findings provide mechanistic insights into PS/PO separation in IP-RPLC and offer practical guidance for impurity profiling of therapeutic oligonucleotides.