Separation of Diastereomers of Short 3-mer Phosphorothioated Oligonucleotides in Reversed-Phase Liquid Chromatography

Reversed-phase liquid chromatography is routinely used for oligonucleotide analysis, often requiring the use of ion-pairing reagents for resolving closely related oligonucleotides. However, ion-pairing reagents have their fair share of obstacles such as instrument contamination or prolonged column equilibration times. In this work, we demonstrate that their use in reversed-phase liquid chromatography is redundant when separating short 3-mer phosphorothioate oligonucleotides and their diastereomers. These short 3-mer sequences used as model compounds mimic possible degradation products or synthetic impurities. A separation of these sequences, which differ in the number and placement of phosphorothioates, was achieved by further method optimization. The BEH C18 and phenyl-based columns were utilized individually and in series, which improved the resolution of closely related sequences. A mobile phase consisting of 25 mM ammonium acetate (pH 6.8) yielded sufficient retention with acetonitrile as the organic modifier. A shallow gradient starting with a high aqueous phase (>98%) was essential to separate a fully modified 3-mer from its partially modified counterparts.