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Ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MS E analysis of RNA oligonucleotides
Author(s) -
Ivleva Vera B.,
Yu YingQing,
Gilar Martin
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4683
Subject(s) - chemistry , oligonucleotide , mass spectrometry , rna , chromatography , tandem mass spectrometry , fragmentation (computing) , high performance liquid chromatography , liquid chromatography–mass spectrometry , dna , biochemistry , gene , computer science , operating system
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. Copyright © 2010 John Wiley & Sons, Ltd.