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Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Authors
  • Kilanowska, Anna1
  • Nuckowski, Łukasz1
  • Studzińska, Sylwia2
  • 1 Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100, Toruń, Poland. , (Poland)
  • 2 Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100, Toruń, Poland. [email protected] , (Poland)
Type
Published Article
Journal
Analytical and Bioanalytical Chemistry
Publisher
Springer-Verlag
Publication Date
Nov 01, 2020
Volume
412
Issue
27
Pages
7453–7467
Identifiers
DOI: 10.1007/s00216-020-02878-0
PMID: 32856108
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

The aim of the present investigation was the analysis and identification of antisense oligonucleotide metabolism products after incubation with human liver microsomes regarding four different oligonucleotide modifications. Separation and detection methods based on the use of liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were developed for this purpose. Firstly, the optimization of mass spectrometer parameters was done to select those which ensure the highest possible sensitivity of oligonucleotide analysis. This step was conducted for two chromatographic modes-ion pair chromatography and hydrophilic interaction liquid chromatography-due to their common application in oligonucleotide analysis. Based on sensitivity results, ion pair chromatography coupled with mass spectrometry was selected for the separation of model oligonucleotide mixtures in order to verify its selectivity for N-deleted metabolite separation. Next, the developed method was applied in the examination of oligonucleotides in vitro metabolism. First, wide optimization of incubation parameters was conducted including the concentration of the reaction buffer components. Obtained results indicated that both 3'-exonucleases and 5'-exonucleases contributed to the biotransformation of oligonucleotides. Moreover, it may be concluded that the number of metabolites depends on oligonucleotide modification and consequently its resistance to enzymatic attack. Thus, the number of the oligonucleotide metabolites decreased with the decrease of the resultant polarity of oligonucleotide caused by chemical modification. Graphical abstract.

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