Evaluation of Hydrophilic Interaction Liquid Chromatography, Capillary Zone Electrophoresis and Drift Tube Ion-Mobility Quadrupole Time Of Flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides
Optimization; high resolution mass spectrometry; nucleic acids; collision cross section; quality control
Abstract :
[en] Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. In this work, we studied poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS). Several analytical techniques, including ion-pair reverse phase liquid chromatography, are described in the literature to assess their quality but most of them present significant drawbacks.
In the present study, dT and PS mixtures were analyzed by hydrophilic interaction liquid chromatography (HILIC) and capillary zone electrophoresis (CZE) coupled to ultraviolet detection. In HILIC, the selectivities of three types of stationary phases (dihydroxypropane, phosphorylcholine and amide) were compared. Optimal conditions were determined and consisted of an amide stationary phase with a mobile phase made up of water, acetonitrile and 15 mM ammonium acetate (pH 5.5). In those conditions, high resolving power and good repeatability were achieved. In CZE, the effect of the background electrolyte (BGE), its pH and concentration were evaluated. A BGE made up of 300 mM ammonium acetate adjusted to pH 6.0 was selected. Finally, the two techniques were compared in terms of selectivity, repeatability and peak efficiency.
In the second part of the study, HILIC and CZE were both coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides.
Research center :
CIRM - Centre Interdisciplinaire de Recherche sur le Médicament - ULiège
Disciplines :
Pharmacy, pharmacology & toxicology
Author, co-author :
Demelenne, Alice ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Gou, Marie-Jia ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Nys, Gwenaël ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Parulski, Chloé ; Université de Liège - ULiège > Département de pharmacie > Pharmacie galénique
Crommen, Jacques ; Université de Liège - ULiège > Département de pharmacie > Département de pharmacie
Servais, Anne-Catherine ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Fillet, Marianne ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Language :
English
Title :
Evaluation of Hydrophilic Interaction Liquid Chromatography, Capillary Zone Electrophoresis and Drift Tube Ion-Mobility Quadrupole Time Of Flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides
Publication date :
2020
Journal title :
Journal of Chromatography. A
ISSN :
0021-9673
eISSN :
1873-3778
Publisher :
Elsevier, Amsterdam, Netherlands
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
FEDER - Fonds Européen de Développement Régional [BE]
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