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

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.