Hydrophilic interaction in solid-phase extraction of antisense oligonucleotides

Abstract

The presented studies aimed to develop a new and simple extraction method based on hydrophilic interaction for antisense oligonucleotides with different modifications. For this purpose, solid-phase extraction cartridges with unmodified silica were used. All extraction steps were performed by utilizing water, acetonitrile, acetone or their mixtures. The results obtained show that a high content (95%) of organic solvent, used during sample loading, is critical to achieve a successful extraction, while elution with pure water allows effective oligonucleotides desorption. The recovery values were greater than 90% in the case of unmodified DNA, phosphorothioate, 2'-O-(2-methoxyethyl) and 2'-O-methyl oligonucleotides. For the mixture of phosphorothioate oligonucleotide and its two synthetic metabolites, the recovery values for the standard solutions were in the range of 70-75%, while for spiked human plasma, 45-50%. The developed method is simple, may be performed in a short time and requires simple solvents like water or acetonitrile/acetone, thus showing promise as an alternative to chaotropic salt-based or ion pair-based SPE methods.

Keywords: antisense oligonucleotides; hydrophilic interaction; silica cartridges; solid phase extraction.