Development of mixed-mode dispersive solid phase extraction of antisense oligonucleotides using commercial adsorbents

Abstract

Sample preparation remains a challenge in the bioanalysis of antisense oligonucleotides due to their polyanionic character, structural complexity, and interactions with proteins. In the current study, a novel mixed-mode extraction methodology for oligonucleotides was developed utilizing commercially available sorbents and subsequently applied to serum matrices. Notably, this work constitutes the first evidence that broadly available adsorbents, not specifically designed for oligonucleotide isolation, can nevertheless be effectively employed for their extraction. Five sorbents with various functional groups were systematically evaluated to elucidate their interaction with antisense oligonucleotides. As a result, dispersive solid phase extraction procedures were developed, with adsorption and desorption controlled by electrostatic, hydrogen-bonding, π-π, and hydrophobic interactions. Elution conditions were optimized using a central composite design, demonstrating that efficient desorption requires simultaneous modulation of salt pH and concentration, and organic solvent content. The results showed that extraction proceeds via a mixed mode mechanism. Two silica-based sorbents modified with phenylboronic acid and 2 (2 pyridyl)ethyl groups exhibited the most favorable balance of recovery, reproducibility, and sorption capacity, achieving recoveries up to 93%. Kinetic studies revealed rapid adsorption for both sorbents, while differences in desorption influenced method transferability from dispersive solid phase extraction to solid phase extraction. Finally, the extraction procedure for phenylboronic acid-based material enabled one step isolation of antisense oligonucleotides from serum without prior protein removal. Although substantial (1:5 v/v) serum dilution was required and recoveries were moderate (59-71%), the approach represents an alternative to currently used methods. Moreover, it expands the bioanalytical tools for mixed mode oligonucleotide extraction using widely available commercial sorbents.