Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resol...

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Bibliographic Details
Published in:Analytical chemistry (Washington) Vol. 96; no. 2; p. 904
Main Authors: Abdullah, A M, Sommers, Cynthia, Rodriguez, Jason D, Zhang, Deyi, Kozak, Darby, Hawes, Jessica, Sapru, Mohan, Yang, Kui
Format: Journal Article
Language:English
Published: United States 16.01.2024
Subjects:
Chromatography, High Pressure Liquid - methods
Drug Contamination
Liquid Chromatography-Mass Spectrometry
Molecular Weight
Oligonucleotides - chemistry
Tandem Mass Spectrometry - methods
ISSN:1520-6882, 1520-6882
Online Access:Get more information
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Summary:Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.
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ISSN:1520-6882
1520-6882
DOI:10.1021/acs.analchem.3c05016