Investigating the total breakthrough behavior of oligonucleotides in ion-pairing chromatography and comprehensive HILIC × IP-RPLC

Oligonucleotides (ONs) have gained significant attention as therapeutic agents, due to their ability to selectively target genetic sequences. As ON-based drug development expands, analytical methods are essential to ensure quality and purity. While ion pairing reversed phase liquid chromatography (IP-RPLC) is the gold standard for ON impurities characterization, alternative chromatographic modes are increasingly used. However, fully resolving all impurities with a single one-dimensional method remains challenging. Comprehensive two-dimensional liquid chromatography offers improved impurity profiling by combining orthogonal separations. However, differences in elution strength between the fraction collected from the first dimension (¹D) and the mobile phase used in the second dimension (²D) can cause peak distortion and breakthrough phenomena. Interestingly, under certain conditions, these distortion effects can disappear, yielding a breakthrough peak accompanied by a symmetrical retained peak. This phenomenon, referred to as "total breakthrough," was recently described in 2D-LC separations of small molecules and peptides. Although the underlying mechanisms are not yet fully elucidated, the total breakthrough (TB) strategy provides an advantage in 2D-LC, by enabling the use of large injection volumes, effectively overcoming injection-related issues commonly caused by solvent incompatibility between dimensions. In this study, we first demonstrated that ONs exhibit TB behavior in IP-RPLC. Upon confirming this behavior, we selected a 2D HILIC × IP-RPLC configuration (a combination often considered challenging due to limited solvent compatibility) and performed an in-depth investigation of both ¹D HILIC collected fraction and ²D IP-RPLC conditions affecting breakthrough behavior. We found that understanding TB of ONs in IP-RPLC is more complex than with classical small molecules, primarily due to the interplay between ion-pairing agents in the ²D -mobile phase and residual salts originating from ¹D Through careful optimization, we successfully established a comprehensive HILIC × IP-RPLC method that enabled analysis of ONs mixture, taking advantage of the TB behavior of ONs, while preserving sufficient sensitivity.