Ion-pair reversed-phase liquid chromatography-mass spectrometry analysis of oligonucleotide therapeutics using fluoroalcohol-free mobile phases

<jats:p>Ion-pair reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) is widely employed for the characterisation of oligonucleotide (OGN) therapeutics. Mobile phases utilising alkylamines and hexafluoro-2-propanol (HFIP) provide high sensitivity but are limited by high cost, low chemical stability, and environmental toxicity. This study evaluates HFIP-free mobile phases utilising low concentrations of dibutylamine (DBA) with propan-2-ol (IPA) and 2,4-dimethylpentan-3-ol (2,4-DMP) as organic modifiers. Due to the high pH of HFIP-free eluents, separation was performed on alkali-stable polymeric and hybrid silica-based stationary phases. Chromatographic resolution and electrospray ionisation (ESI)-MS sensitivity were evaluated against a benchmark DBA/HFIP system using a variety of OGN standards, antisense oligonucleotides (ASOs), and small interfering RNA (siRNA). The HFIP-free systems demonstrated comparable resolution for ssDNA standards (8–40 mer) and only marginal peak broadening for ASOs relative to DBA/HFIP. The high pH and reduced ion-pair reagent concentration resulted in a shift toward higher charge states and a significant reduction in alkylamine and metal ion adduction. Impurity profiling revealed no significant difference in the percentage fractional abundance of OGN impurities between mobile phase systems. These HFIP-free mobile phases offer a sustainable alternative for the LC-MS analysis of OGNs, enhancing signal intensity and reducing adduction while eliminating hazardous fluorinated solvents.</jats:p>