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Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides
Ultra high performance liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry was used to determine the products of the in vitro metabolism of phosphorothioate oligonucleotides. These compounds may be used during antisense therapy as synthetic fragments of genes. For this...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737794/ https://www.ncbi.nlm.nih.gov/pubmed/26758600 http://dx.doi.org/10.1007/s00216-015-9266-1 |
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author | Studzińska, Sylwia Rola, Rafał Buszewski, Bogusław |
author_facet | Studzińska, Sylwia Rola, Rafał Buszewski, Bogusław |
author_sort | Studzińska, Sylwia |
collection | PubMed |
description | Ultra high performance liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry was used to determine the products of the in vitro metabolism of phosphorothioate oligonucleotides. These compounds may be used during antisense therapy as synthetic fragments of genes. For this reason, both a sample preparation method and a qualification method were developed during this study. Liquid–liquid extraction, protein or oligonucleotide precipitation, and solid-phase extraction were tested and compared in order to select the method that yielded the highest recoveries. Ion pair chromatography was used for separation while mass spectrometry was applied for metabolite identification. The influence of the type of ion pair reagent used on the resolution and sensitivity was investigated. Results indicated that a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol, N,N-dimethylbutylamine, and methanol was the best mobile phase for maximizing both of these parameters. The developed method was applied to investigate the compounds that form during the incubation of phosphorothioate oligonucleotides with human liver microsomes. Metabolites with short sequences were created after 8 hours, while oligonucleotides constructed from a large number of nucleotide units were obtained after 12 hours of incubation. Moreover, regardless of the length of the polynucleotide chain, metabolites were produced by the same mechanism: enzymatic cleavage at the 3′ end of the sequence. |
format | Online Article Text |
id | pubmed-4737794 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-47377942016-02-09 Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides Studzińska, Sylwia Rola, Rafał Buszewski, Bogusław Anal Bioanal Chem Research Paper Ultra high performance liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry was used to determine the products of the in vitro metabolism of phosphorothioate oligonucleotides. These compounds may be used during antisense therapy as synthetic fragments of genes. For this reason, both a sample preparation method and a qualification method were developed during this study. Liquid–liquid extraction, protein or oligonucleotide precipitation, and solid-phase extraction were tested and compared in order to select the method that yielded the highest recoveries. Ion pair chromatography was used for separation while mass spectrometry was applied for metabolite identification. The influence of the type of ion pair reagent used on the resolution and sensitivity was investigated. Results indicated that a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol, N,N-dimethylbutylamine, and methanol was the best mobile phase for maximizing both of these parameters. The developed method was applied to investigate the compounds that form during the incubation of phosphorothioate oligonucleotides with human liver microsomes. Metabolites with short sequences were created after 8 hours, while oligonucleotides constructed from a large number of nucleotide units were obtained after 12 hours of incubation. Moreover, regardless of the length of the polynucleotide chain, metabolites were produced by the same mechanism: enzymatic cleavage at the 3′ end of the sequence. Springer Berlin Heidelberg 2016-01-12 2016 /pmc/articles/PMC4737794/ /pubmed/26758600 http://dx.doi.org/10.1007/s00216-015-9266-1 Text en © The Author(s) 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Research Paper Studzińska, Sylwia Rola, Rafał Buszewski, Bogusław Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides |
title | Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides |
title_full | Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides |
title_fullStr | Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides |
title_full_unstemmed | Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides |
title_short | Development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides |
title_sort | development of a method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for studying the in vitro metabolism of phosphorothioate oligonucleotides |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737794/ https://www.ncbi.nlm.nih.gov/pubmed/26758600 http://dx.doi.org/10.1007/s00216-015-9266-1 |
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