Bioanalysis of Oligonucleotide by LC–MS: Effects of Ion Pairing Regents and Recent Advances in Ion-Pairing-Free Analytical Strategies

Oligonucleotides (OGNs) are relatively new modalities that offer unique opportunities to expand the therapeutic targets. Reliable and high-throughput bioanalytical methods are pivotal for preclinical and clinical investigations of therapeutic OGNs. Liquid chromatography–mass spectrometry (LC–MS) is now evolving into being the method of choice for the bioanalysis of OGNs. Ion paring reversed-phase liquid chromatography (IP-RPLC) has been widely used in sample preparation and LC–MS analysis of OGNs; however, there are technical issues associated with these methods. IP-free methods, such as hydrophilic interaction liquid chromatography (HILIC) and anion-exchange techniques, have emerged as promising approaches for the bioanalysis of OGNs. In this review, the state-of-the-art IP-RPLC–MS bioanalytical methods of OGNs and their metabolites published in the past 10 years (2012–2022) are critically reviewed. Recent advances in IP-reagent-free LC–MS bioanalysis methods are discussed. Finally, we describe future opportunities for developing new methods that can be used for the comprehensive bioanalysis of OGNs.