Journal
INTERNATIONAL JOURNAL OF MASS SPECTROMETRY
Volume 304, Issue 2-3, Pages 98-104Publisher
ELSEVIER
DOI: 10.1016/j.ijms.2010.06.001
Keywords
Synthetic oligonucleotides; Phosphorothioatediesters; High resolution mass spectrometry; LC-FTMS; Accurate mass measurement
Funding
- Girindus America Inc.
- The National Institutes of Health [RR019900]
- University of Cincinnati
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Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3'-terminal phosphate monoester and 3'-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N-3 (2-cyanoethyl) adducts of the full-length product. When compared with low resolution LC-MS, similar to 60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling. (C) 2010 Elsevier B.V. All rights reserved.
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