Examining hybridization-based LC–MS methodologies for the bioanalysis of siRNA analytes

Hybridization-based LC-MS is rapidly emerging as a bioanalytical platform for oligonucleotides, particularly when both high sensitivity and high specificity are needed. When used to analyze single-stranded antisense oligonucleotide (ASO) therapeutics, the workflows are relatively well established, but the analysis of double-stranded small interfering RNA (siRNA) therapeutics presents additional challenges due to competition for binding from the sense strand. In the last two years, the authors have independently published extensively on hybridization-based LC-MS bioanalysis of siRNA therapeutics, and now we take a step back to evaluate the progress we have made and offer our thoughts on the future of this platform. We touch upon aspects of the sample preparation and analytical process that can either be improved upon, made more efficient, or expanded to maximize the information that can be gained from a single sample. Additionally, we discuss how hybridization-based LC-MS compares to other common oligonucleotide bioanalytical workflows, and its potential to become a frontline assay platform for use in supporting regulatory submissions. Overall, we are excited about the potential hybridization-based LC-MS has demonstrated as a bioanalytical platform and are eager to begin the conversation on where this workflow goes next.