Comparative evaluation of protein digestion and solid-phase extraction strategies for oligonucleotide quantification...

Oligonucleotide therapeutics are an emerging class of drugs, but their development is challenged by difficulties in efficient extraction and quantification, primarily due to their polarity and susceptibility to degradation by nucleases. Solid phase extraction (SPE) is one of the methods used for oligonucleotide extraction, offering sensitivity along with selectivity. Surfactant-based buffer has traditionally been used in SPE extraction of oligonucleotides, but there is now a growing focus on exploring alternatives to this method. In this study, we investigated the use of a non-surfactant buffer pretreatment module for SPE extraction of oligonucleotides using both WAX-based and mixed-mode microplates. The methods for both mixed-mode and WAX-based SPE microplates were validated and applied in an in-vitro stability study. Our study showed good recovery and easy sample preparation when using the non-surfactant buffer pretreatment module, across both microplate types. The surfactant-based buffer (SB) used with proteinase K also gave a better recovery when applied to the WAX-based SPE microplate.