Haglöf Jakob, Nilsson Jenny M, Ludvigsson Jufang Wu
Department of Medicinal Chemistry, Uppsala Biomedical Centre, Uppsala University, Uppsala, Sweden.
Global Pharmaceutical Development, Pharmaceutical Technology & Development, Operations, AstraZeneca, Gothenburg, Sweden.
J Sep Sci. 2025 Aug;48(8):e70238. doi: 10.1002/jssc.70238.
Oligonucleotides are traditionally analyzed using ion-pair reversed-phase liquid chromatography. In this project, we explored an alternative approach utilizing a reversed phase-weak anion exchange mixed-mode column, which combines separation based on both charge and hydrophobicity. Our findings indicate that the mixed-mode column provided stronger retention and superior separation compared to the C18 column, eliminating the need for ion-pairing reagents. Additionally, selectivity can be adjusted using gradient pH and buffer concentration as well as acetonitrile content. However, the use of a phosphate buffer was necessary to ensure adequate retention and separation, despite its incompatibility with MS detection. To address this issue, a 2DLC-UV-qTOF setup with multiple loops was established, where the second-dimension LC, connected to the MS detector, required only an MS-compatible mobile phase. Using a HILIC-type LUNA Omega Sugar column in the second-dimension LC, the MS intensity of the heart-cut from the first dimension was increased 100-fold, allowing for the characterization of the main peak.
传统上，寡核苷酸是使用离子对反相液相色谱法进行分析的。在本项目中，我们探索了一种替代方法，即使用反相-弱阴离子交换混合模式柱，该柱结合了基于电荷和疏水性的分离方法。我们的研究结果表明，与C18柱相比，混合模式柱具有更强的保留能力和更好的分离效果，无需使用离子对试剂。此外，可通过梯度pH、缓冲液浓度以及乙腈含量来调节选择性。然而，尽管磷酸盐缓冲液与质谱检测不兼容，但为确保足够的保留和分离，仍有必要使用它。为解决此问题，建立了一种带有多个定量环的二维液相色谱-紫外-四极杆飞行时间质谱联用装置，其中连接到质谱检测器的二维液相色谱仅需要与质谱兼容的流动相。在二维液相色谱中使用HILIC型LUNA Omega糖柱，一维流出物中心切割峰的质谱强度提高了100倍，从而能够对主峰进行表征。
