Development of multiple heartcutting two-dimensional liquid chromatography with ion-pairing reversed-phase separations in both dimensions for analysis of impurities in therapeutic oligonucleotides

•2D-LC separation of oligonucleotides using IPRP separations in both dimensions.•Elution conditions are systematically discovered through iterative retention modeling.•2D-LC separations provide both the selectivity and sensitivity needed for impurity profiling.•Application to therapeutic siRNA single strands from solid phase oligonucleotide synthesis. Oligonucleotides constitute an emerging and highly complex bioanalytical challenge and it is becoming increasingly clear that 1D methodologies are unable to fully resolve all possible impurities present in these samples. 2D-LC therefore constitutes a perfect solution wherein critical pairs can be sampled from a steep gradient 1D and separated in a shallower 2D gradient. Herein, we provide a facile 2D-LC method development approach to quickly generate high selectivity gradients utilizing ion pairing reverse phase (IPRP-IPRP). In particular we demonstrate how to iteratively generate a 12 % gradient from two training runs and then to utilize that data to predict retentions of analytes with a 2 % gradient with retention prediction errors as low as 3 and 11 %, respectively. This iterative method development workflow was applied to impurity profiling down to 1:1000 for the full-length product and phosphorothioate modified impurities. Additionally, we demonstrated the elucidation of critical pairs in complex crude pharmaceutical oligonucleotide samples by applying tailored high selectivity gradients in the second dimension. It was found that the iterative retention modeling approach allows fast and facile 2D-LC method development for complex oligonucleotide separations.