Analysis of antisense oligonucleotides with the use of ionic liquids as mobile phase modifiers

The main goal of this study was the investigation of the impact of several ionic liquids, commonly used as free silanol suppressors, on the retention and separation of phosphorothioate oligonucleotides. Three various stationary phases (octadecyl, octadecyl with embedded polar groups and pentafluorophenyl) as well as ionic liquids with the concentration range of 0.1–7 mM were used for this purpose. The results obtained during this study showed that the increase in concentration of ionic liquids results in increasing retention of the oligonucleotides. Such an effect was observed regardless of the stationary phase used. Moreover, elongation of the alkyl chain in the structure of ionic liquids caused an increase of antisense oligonucleotide retention factors. The results obtained during retention studies confirmed that addition of ionic liquids to the mobile phase influences antisense oligonucleotide retention in a way similar to the case of commonly used ion pair reagents such as amines. A method of oligonucleotide separation was also developed. The best selectivity was obtained for the octadecyl stationary phase since separation of mixtures of antisense oligonucleotides and their metabolites differing in sequence length was successful. It has to be pointed out that ionic liquids were used for the first time as mobile phase additives for oligonucleotide analysis.