Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high
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RESEARCH PAPER
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 Anna Kilanowska 1 & Łukasz Nuckowski 1 & Sylwia Studzińska 1 Received: 20 April 2020 / Revised: 17 June 2020 / Accepted: 11 August 2020 # The Author(s) 2020
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. Keywords In vitro metabolism . Human liver microsomes . Antisense oligonucleotides . Ion pair chromatography . Electrospray ionization quadrupole time-of-flight mass spectrometry
Introduction Antisense oligonucleotides (ASOs) are single-stranded DNA or RNA fragments, which are chemically modified by increasing their hydrophobicity and stability in the human body [1]. Modifications of ASO structure include the introduction of
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02878-0) contains supplementary material, which is available to authorized users. * Sylwia Studzińska [email protected] 1
Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruń, Poland
different groups in the polynucleotide chain such as sulfur, fluorine, methyl, methoxy, methoxyethyl groups, and methylene bridge between carbon and oxygen atoms in the pentose moieties [2]. Their therapeutic potential has been discovered i
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