Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, Prague 12800, Czech Republic.
Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
J Chromatogr A. 2024 Aug 16;1730:465074. doi: 10.1016/j.chroma.2024.465074. Epub 2024 Jun 10.
Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioated oligonucleotides differing in the length (2-6mers and 21mer) and the number and position of phosphorothioate modifications. We investigated the influence of counterion (acetate vs. hexafluoroisopropanol) on the adsorption of eleven alkylamines on the stationary phases. A stronger adsorption of charged alkylamines on octadecyl- and phenyl-based stationary phases led to greater retention of oligonucleotides, and the adsorption of alkylamines was promoted with greater concentration of hexafluoroisopropanol in the mobile phase. Selected amines (triethylamine, dipropylamine, hexylamine) were used to study the resolution of n and n-x mers (main peak and its impurities shortened at 5´end), and diastereomeric separation of phosphorothioated oligonucleotides. The results confirmed a crucial role of alkylamine and counterion choice on the diastereomeric separation. The increasing hydrophobicity of alkylamine led to diminished diastereomeric selectivity which produced narrower phosphorothioated oligonucleotides peaks and led to improved n/n-x separation. Using hexafluoroisopropanol instead of acetate as counterion further enhances this effect (except for 100 mM concentration of hexafluoroisopropanol in combination with highly hydrophobic hexylamine). The elevated column temperature led to suppression of the diastereomeric resolution and improved resolution of n and n-x mers oligonucleotides. Baseline separation of oligonucleotides with different number of phosphorothioate linkages was achieved; this may be useful for therapeutic oligonucleotide analysis.
离子对反相液相色谱法用于分析不同长度（2-6 mers 和 21mer）和不同数量及位置硫代磷酸化修饰的天然和硫代磷酸化寡核苷酸。我们研究了反离子（乙酸盐与六氟异丙醇）对十一烷基胺在固定相上吸附的影响。带电荷的烷基胺在十八烷基和苯基固定相上的吸附更强，导致寡核苷酸的保留更强，并且随着流动相中六氟异丙醇浓度的增加，烷基胺的吸附得到促进。选择了几种胺（三乙胺、二丙胺、己胺）来研究 n 和 n-x 聚体（主峰及其 5´端缩短的杂质峰）的分辨率，以及硫代磷酸化寡核苷酸的对映体分离。结果证实了烷基胺和反离子选择对非对映异构体分离的重要作用。烷基胺疏水性的增加导致非对映异构体选择性降低，从而产生更窄的硫代磷酸化寡核苷酸峰，并改善 n/n-x 分离。使用六氟异丙醇代替乙酸盐作为反离子，进一步增强了这种效果（仅在 100 mM 浓度的六氟异丙醇与高度疏水性己胺组合时除外）。升高柱温会抑制非对映异构体的分辨率，并改善 n 和 n-x 聚体寡核苷酸的分辨率。实现了不同数量硫代磷酸化键连接的寡核苷酸的基线分离；这可能对治疗性寡核苷酸分析有用。
