Ion-pair reversed-phase and hydrophilic interaction chromatography methods for analysis of phosphorothioate oligonucleotides

In this study we investigated the separation of a 25 mer fully phosphorothioated oligonucleotide from its truncated n-1 (24 mer) species and selected phosphodiester 25 mer impurities using ion-pair reversed-phase chromatography. The hydrophobicity of ion-pairing agents (alkylamines) impacts n-1 separation selectivity. 25 mer impurities with single and double phosphodiester bonds eluted prior to the parent phosphorothioate oligonucleotide in the same region as 24 mer impurities, which complicated the chromatographic separation. An alternative technique, hydrophilic interaction chromatography, provided a different retention pattern; 24 mer n-1 impurities eluted prior to the 25 mer, while the phosphodiester impurities eluted after the full-length oligonucleotide. This enabled an improved chromatographic separation of the truncated and phosphodiester impurities from the phosphorothioate oligonucleotide of interest.