Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 2: Use for selective comprehensive two-dimensional liquid chromatography

With the increasing numbers of nucleic acid-based pharmaceuticals like antisense oligonucleotides (ASO), small interfering ribonucleic acid (siRNA) entering the market, research facilities, pharmaceutical indus-tries and also regulatory authorities have been looking for efficient analytical methods for these synthetic oligonucleotides (ON). Besides of conventional one-dimensional (1D) reversed-phase liquid chromatogra-phy with or without ion-pairing (IP-RP-LC, RP-LC), hydrophilic liquid chromatography (HILIC) and mixed -mode chromatography (MMC), two-dimensional (2D) approaches combining two orthogonal chromato-graphic techniques also become more relevant due to the high structural complexity of oligonucleotides. Recently, we tested a polybutylene terephthalate(PBT)-based stationary phase under ion-pairing free RP mode for the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis of siRNA (Patisiran). In this study, retention profile and chromatographic orthogonality, respectively, were compared to other LC-modes like HILIC, IP-RPLC, another ion-pair free cholesterol-bonded RPLC and MMC considering their normalized retention times. Finally, because of higher orthogonality, the ion-pairing free PBT-bonded RPLC as first dimension ( 1 D) was hyphenated with HILIC in the second dimension ( 2 D) in a selective comprehensive 2D-LC setup leading to an enhanced resolution for peak purity evaluation of the main ON entities. (c) 2023 Elsevier B.V. All rights reserved.