Current trends in liquid chromatography-mass spectrometry analysis of small interfering RNAs: A short review

Small interfering RNA (siRNA) therapeutics represent one of the most rapidly advancing pharmaceutical modalities. Since the approval of the first siRNA-based drug in 2018, seven siRNA therapeutics have been approved to date. During manufacturing, "non-optimal duplexes" and "single-stranded impurities" can arise from synthetic byproducts such as shortmers and longmers, or from minor imbalances in strand quantification and mixing. Furthermore, phosphorothioate linkages introduce chirality, generating complex diastereomeric mixtures. The use of delivery systems, including lipid nanoparticles or N-acetylgalactosamine (GalNAc) conjugates, further increases the structural complexity and heterogeneity of the final product. Compared to single-stranded oligonucleotides, siRNA-being double-stranded-yields more complex chromatographic and mass spectral profiles, making analytical characterization particularly challenging. Therefore, the development of robust and reliable analytical methods is essential to ensure the quality of siRNA therapeutics. This review summarizes key analytical techniques for the quality evaluation of siRNA therapeutics. Liquid chromatography (LC) is one of the most widely used approaches, with separation modes such as ion-pair reversed-phase (IP-RP), anion exchange (AEX), size exclusion chromatography (SEC), and hydrophilic interaction chromatography (HILIC) being actively explored. Mass spectrometry (MS) is another powerful tool that provides molecular mass-based structural information not accessible via UV detection alone. The combination of LC and MS offers a highly effective platform for characterizing siRNA therapeutics, enabling both qualitative and quantitative assessment of impurities and isomers. Furthermore, tandem MS (MS/MS) can provide sequence-specific information for the identification of active pharmaceutical ingredients. In addition to quality assessment, the application of LC and MS techniques in the pharmacokinetic analysis of siRNA therapeutics is also briefly discussed.