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

ABSTRACT

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.