Ion mobility high resolution mass spectrometry coupled to HILIC and CZE for the characterization of phosphodiester and phosphorothioate oligonucleotides

Abstract

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. With the emergence of oligonucleotides on the market, it is of increasing importance to develop analytical methods to study those modified oligonucleotides and their impurities. Chromatographic and electrophoretic approaches may be used for that purpose. In this work, poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS) were studied. For the chromatographic approach, Hydrophilic Interaction Liquid Chromatography (HILIC) was chosen as it represents good alternative to commonly used ion-pair reversed-phase liquid chromatography for the analysis of polar compounds. Moreover, it avoids the use of ion pairing agents, which makes it more compatible with mass spectrometric detection. For the electrophoretic approaches, a capillary zone electrophoresis (CZE) with a MS-compatible background electrolyte was employed. Both techniques were 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