Evaluation of alkylamines and stationary phases to improve LC–MS of oligonucleotides

Abstract

This study evaluated four bridged‐ethylene hybrid (BEH) columns containing C18 (130 Å), peptide C18 (300 Å), phenyl, or a mixed‐mode charged surface hybrid (CSH C18) using a wide range of antisense oligonucleotide therapeutics. The BEH C18, peptide, and phenyl columns were all capable of providing significant retention of oligonucleotide samples across multiple ion‐pairing systems using alkylamines and 1,1,1,3,3,3,‐hexafluoroisopropanol (HFIP). The retention of the oligonucleotides varied depending on the choice of alkylamine, with the order of retention being dimethylcyclohexylamine > diisopropylethylamine > triethylamine. The selectivity of these columns for several closely eluting impurities was similar. Although overall the C18, peptide, and phenyl columns were all found to be capable of analyzing oligonucleotide therapeutics, the phenyl column was found to be the most retentive and the C18 column provided the best peak shape. The CSH C18 column was found to be degraded by the alkylamine‐HFIP mobile phase despite the mobile phase being within the pH stability range of the column.

Physicochemical property evaluation of modified oligonucleotides by traveling‐wave ion mobility mass spectrometry

“…This observation corroborates with the HPLC and capillary electrophoresis (CE) results. In the HPLC and CE analyses, a broader peak was observed for the PS‐modified oligonucleotides, attributed to the many diastereomers 31,32 . Hansen's group recently reported proof of this peak widening using chiral pure PS‐modified oligonucleotides 33 …”