Analysis of oligonucleotides by ion-pair reversed-phase liquid chromatography coupled with positive mode electrospray ionization mass spectrometry

  • Guofeng Weng
  • Binwen Sun
  • Zheyi Liu
  • Fangjun WangEmail author
  • Yuanjiang PanEmail author
Research Paper
Part of the following topical collections:
  1. New Insights into Analytical Science in China


Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with negative mode electrospray ionization mass spectrometry (ESI-MS) due to their highly negative charged phosphodiester backbones. Herein, the signal suppression effect of triethylamine (TEA) adducts caused the ion-pair reagent TEA/hexafluoroisopropanol (HFIP) is greatly alleviated after improving the in-source energy in positive mode ESI-MS. This strategy is applied for different RNA sequencing through analyzing their formic acid hydrolysates via IP-RPLC MS. Comparing with negative ion mode, we demonstrate that IP-RPLC MS analysis in positive ion mode is more suitable for RNA sequencing with fewer contaminant interferences. Finally, simultaneous online separation and detection of oligonucleotides and protein digests are achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.


Oligonucleotide Ion-pair reversed-phase liquid chromatography Positive mode mass spectrometry In-source energy 


Funding information

Financial supports are gratefully acknowledged for the National Key R&D Program of China (2016YFF0200503) and the National Natural Science Foundation of China (91853101, 21675152, and 21532005).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_1819_MOESM1_ESM.pdf (861 kb)
ESM 1 (PDF 860 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistryZhejiang UniversityHangzhouChina
  2. 2.CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalianChina
  3. 3.University of Chinese Academy of SciencesBeijingChina

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