The reversed-phase ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry method was developed during the study and successfully applied to separation and analysis of unmodified and antisense oligonucleotides. It was shown that oligonucleotide separation strongly depends on the stationary phase type. The type, pH and salt content in the mobile phase influence also their chromatographic behavior and mass spectrometry sensitivity: increasing the apparent pH causes retention decrease and increase of sensitivity, while increasing the salt concentration produces greater oligonucleotide retention and lower sensitivity. Utilization of octadecyl stationary phase with aryl rings in reversed-phase mode allows successful separation of various modified and unmodified oligonucleotides, differing in length or sequence. The developed method has numerous advantages over current liquid chromatography methods: resolution is comparable to ion pair chromatography; oligonucleotides are analyzed with salts, which allow easy purification and separation without losing column performance, whereas columns run in the presence of ion pair reagents degrade more rapidly. Moreover, the differences in mass spectrometry sensitivity between reversed-phase liquid chromatography and ion pair chromatography were not significant for the studied compounds. The developed method is an effective analytical tool for the determination of oligonucleotide impurities.

中文翻译：

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超高效反相液相色谱与 ESI-Q-TOF-MS 联用，用于分析未修饰和反义寡核苷酸

在研究过程中开发了反相超高效液相色谱四极杆飞行时间质谱法，并成功应用于未修饰和反义寡核苷酸的分离和分析。结果表明，寡核苷酸分离在很大程度上取决于固定相类型。流动相中的类型、pH 和盐含量也会影响它们的色谱行为和质谱灵敏度：增加表观 pH 会导致保留降低和灵敏度增加，而增加盐浓度会产生更大的寡核苷酸保留和更低的灵敏度。在反相模式下使用带有芳环的十八烷基固定相可以成功分离长度或序列不同的各种修饰和未修饰的寡核苷酸。与目前的液相色谱方法相比，所开发的方法具有许多优点：分辨率可与离子对色谱相媲美；寡核苷酸用盐进行分析，这样可以在不损失柱性能的情况下轻松纯化和分离，而在离子对试剂存在的情况下运行的色谱柱降解速度更快。此外，反相液相色谱和离子对色谱之间的质谱灵敏度差异对于所研究的化合物并不显着。所开发的方法是测定寡核苷酸杂质的有效分析工具。这允许在不损失柱性能的情况下轻松纯化和分离，而在离子对试剂存在的情况下运行的色谱柱降解速度更快。此外，反相液相色谱和离子对色谱之间的质谱灵敏度差异对于所研究的化合物并不显着。所开发的方法是测定寡核苷酸杂质的有效分析工具。这允许在不损失柱性能的情况下轻松纯化和分离，而在离子对试剂存在的情况下运行的色谱柱降解速度更快。此外，反相液相色谱和离子对色谱之间的质谱灵敏度差异对于所研究的化合物并不显着。所开发的方法是测定寡核苷酸杂质的有效分析工具。