Application of amino acid-based adsorbents for the extraction of antisense oligonucleotides from serum samples

Abstract

Antisense oligonucleotides are synthetic, short, single-stranded nucleic acid analogues used for several years as an active ingredient of drugs for treating genetic diseases. Their effective sample preparation is a topic that still requires in-depth research. Consequently, amino acid-modified silica adsorbents were utilized for the first time in the dispersive solid-phase extraction of antisense oligonucleotides. The impact of the composition of solvents used for adsorption and desorption was tested (pH, salt type, salt concentration, organic solvent type, and content in the solution). Adsorption of oligonucleotides occurred from water or salt solutions with low pH. Increasing the pH cause increase of recovery contrary to increase salt concentration. Moreover addition of methanol or acetonitrile to elution solvent reduce recovery. The developed method is based on weak ion exchange, with pH playing a key role in the extraction procedure. Thus, the adsorption and desorption are driven solely by the change in pH of the ammonium acetate solution. All tested adsorbents allowed for the efficient recovery of oligonucleotides. Nevertheless, adsorbent with bonded methionine showed the highest efficiency. The recovery depended on the structure of the antisense oligonucleotide (the highest for short and modified ones). The recovery also increases with increasing hydrophobicity of the modification. The developed method was successfully applied to oligonucleotide extraction from human serum samples resulting in a recovery of 91±1%. Only a 10 mM ammonium acetate with pH 4 (adsorption) or 11 (desorpion) is used throughout the whole procedure. The developed procedure is simple, efficient, selective, and environmentally friendly.