Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
Rapid Commun Mass Spectrom. 2010 Sep 15;24(17):2631-40. doi: 10.1002/rcm.4683.
Fast and efficient ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence-related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS(E) (alternating low and elevated collision energy scanning modes) methods. Based on the resulting fragmentation of native and chemically modified oligonucleotides, it was found that the MS(E) technique is as efficient as the traditional MS/MS method, yet MS(E) is more general, faster, and capable of producing higher signal intensities of fragment ions. Fragmentation patterns of modified oligonucleotides were investigated using RNA 2'-ribose substitutions, phosphorothioate RNA, and LNA modifications. The developed sequence confirmation method that uses the MS(E) approach was applied to the analysis of in vitro hydrolyzed RNA oligonucleotide. The target RNA and metabolites, including the structural isomers, were resolved by UPLC, and their identity was confirmed by MS(E). Simultaneous RNA truncations from both termini were observed. The UPLC quadrupole time-of-flight (QTOF) MS/MS and MS(E) methods were shown to be an effective tool for the analysis and sequence confirmation of complex oligonucleotide mixtures.
采用快速高效的超高效液相色谱/串联质谱法（UPLC/MS/MS）对短干扰 RNA 寡核苷酸进行分析，以确认目标序列相关杂质的身份。基于精确质量，鉴定出多种截断寡核苷酸和代谢物，并通过 MS/MS 和 MS(E)（交替低和高碰撞能扫描模式）方法证实其假定序列。基于天然和化学修饰寡核苷酸的产生的碎片，发现 MS(E)技术与传统的 MS/MS 方法一样高效，但 MS(E)更通用、更快，并且能够产生更高的碎片离子信号强度。使用 RNA 2'-核糖取代、硫代磷酸酯 RNA 和 LNA 修饰研究了修饰寡核苷酸的片段化模式。使用 MS(E)方法开发的序列确认方法应用于体外水解 RNA 寡核苷酸的分析。通过 UPLC 分离目标 RNA 和代谢物，包括结构异构体，并通过 MS(E)确认其身份。同时观察到从两端的 RNA 截断。UPLC 四极杆飞行时间（QTOF）MS/MS 和 MS(E)方法被证明是分析和确认复杂寡核苷酸混合物的有效工具。
