CHROMtalks - Advanced IP-RPLC-MS for High-Throughput High-Resolution Oligo Impurity Profiling

Therapeutic oligonucleotides (ONs) present analytical challenges due to their structural complexity and closely related impurities. Ion-pairing reversed phase liquid chromatography (IP-RPLC) with mass spectrometry (MS) remains the gold standard for impurity profiling. A comparative method development approach was used to evaluate mobile phase additives and stationary phase properties - such as particle type, size, and pore size - for their effects on separation efficiency and MS sensitivity. Optimal conditions at 60 °C with a preheated column reduced peak distortion. Acetonitrile outperformed methanol for peak capacity and pressure. Hexafluoromethylisopropanol gave better resolution, while hexafluoroisopropanol improved MS sensitivity. Core-shell particles (1.7 µm, 100 Å) offered high resolving power for 15-35mer ONs. Fast separations were possible with short columns, while longer columns enabled high-resolution analysis of complex RNA. The method was also applied in 2D-LC-MS with anion exchange for siRNA diastereomer characterization.