Analytical and Bioanalytical Chemistry

, Volume 410, Issue 14, pp 3375–3384 | Cite as

Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry

  • N. M. Elzahar
  • N. Magdy
  • Amira M. El-Kosasy
  • Michael G. Bartlett
Research Paper


Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3′- and 5′-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences.

Graphical abstract

Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS


Antisense Chemical stability Ion-pair chromatography LC/MS/MS Phosphorothioate oligonucleotides 



The authors would like to thank ProQR Therapeutics and Ionis Pharmaceuticals for their support of this work. Also, the authors would like to thank the Egyptian Joint Supervision Missions Department of the Ministry of Higher Education, Cairo, Egypt.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1032_MOESM1_ESM.pdf (1.4 mb)
ESM 1 (PDF 1.34 mb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • N. M. Elzahar
    • 1
    • 2
  • N. Magdy
    • 1
  • Amira M. El-Kosasy
    • 1
  • Michael G. Bartlett
    • 2
  1. 1.Pharmaceutical Analytical Chemistry Department, Faculty of PharmacyAin Shams UniversityCairoEgypt
  2. 2.Department of Pharmaceutical and Biomedical Sciences, College of PharmacyUniversity of GeorgiaAthensUSA

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