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Development of separation method for analysis of oligonucleotides using LC-UV/MS
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
2018 (English)Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Introduction

Oligonucleotides are short nucleic acid chains, usually 19-27mer long. They bind to their corresponding chain, making a specific inhibition possible. In pharmaceuticals, this can be used to inhibit the expression of a gene or protein of interest. Oligonucleotides are usually analyzed based on separation using both hydrophobic and ion-exchange properties. In this project, the possibility to use a mixed-mode column to separate these oligonucleotides and their impurities were explored.

Method

Liquid chromatography is used as the separation method and the method of detection is both mass spectrometry and UV. Three different columns are evaluated; C18, DNAPac RP, and mixed-mode RP/WAX.

Results and discussion

Different compositions of mobile phases and gradients are evaluated based on a literature study. Triethylamine, triethylammonium acetate, ammonium formate, hexafluoroisopropanol is used along with both methanol and acetonitrile. Phosphate buffer is evaluated on LC-UV. The results from the C18 column displays a good separation of the oligonucleotides, whilst the DNAPac RP is not as sufficient using the same mobile phases. The mixed-mode column provides good separation and selectivity using phosphate buffer and UV detection.

Conclusion

Mixed-mode column has the potential to be used for separation of oligonucleotides and one future focus would be to make the mobile phase compatible with mass spectrometry. Phosphate buffer and UV detection seems to be the go-to mobile phase using mixed-mode column even though MS is a more powerful tool for the characterization and identification of oligonucleotides. This provides a hint about the challenge in making the mobile phase MS compatible.

Place, publisher, year, edition, pages
2018. , p. 16
Series
UPTEC K, ISSN 1650-8297 ; 18028
Keywords [en]
Oligonucleotides, UPLC, liquid chromatography, time of flight, mass spectrometry, reversed-phase/weak anion exchange, AcclaimTM Mixed Mode WAX-1, DNAPacTM RP, ACQUITY UPLC BEH C18
National Category
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-403381OAI: oai:DiVA.org:uu-403381DiVA, id: diva2:1388824
External cooperation
AstraZeneca
Educational program
Master Programme in Chemical Engineering
Supervisors
Examiners
Available from: 2020-01-31 Created: 2020-01-27 Last updated: 2020-01-31Bibliographically approved

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CiteExportLink to record
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Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
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  • de-DE
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  • Other locale
More languages
Output format
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