A new approach to preparation of antisense oligonucleotide samples with microextraction by packed sorbent

Our research focused on applying microextraction by packed sorbent to extracting antisense oligonucleotides from serum samples. The tested sorbents included poly(styrene-co-divinylbenzene), octyl, octadecyl, and unmodified silica gel. As nonpolar sorbents were used for highly-polar molecules, this required ion-pair mode. Comprehensive optimization of extraction conditions was performed for 20-mer phosphorothioate oligonucleotide. Several parametres – the number of “draw–eject” cycles during the conditioning and load step, the amine type and concentration, and the volume of elution mixture – and the influence they had on recovery were studied for nonpolar sorbents, which made it possible to obtain high (ca. 90%) recovery values. The most influential parameter turned out to be the volume of elution mixture. Similar optimization was performed for silica sorbents; however, despite optimization of various parameters, the recovery values stayed relatively low. The optimized procedures for nonpolar sorbents were applied in extraction of six different oligonucleotides of various length and with different structure modifications. The highest recoveries were obtained for octyl and octadecyl sorbents, ranging between 80-99%. The developed microextraction method was used to extract phosphorothioate and 2'-O-(2-methoxyethyl) oligonucleotides and their two synthetic metabolites from enriched human plasma, with recoveries around 70-80%.