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Peer-reviewedCC BY-NC 4.0Journal of Pharmaceutical and Biomedical Analysis

Hydrophilic interaction chromatography and tandem mass spectrometry for the characterization of methylated oligoribonucleotides

  • Khaoula Adouairi
  • Carole Farre
  • Carole Chaix
  • Sébastien Pfeffer
  • Karine Faure
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Abstract

Chemical modifications on oligonucleotides, notably N-6-methyladenosine (m6A) are known for their impact on diverse classes of RNA including micro (mi)RNAs. However, the characterization of these modifications on intact sequences remains challenging. An ion-pair free hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry (HILIC-HRMS/MS) workflow was developed for the intact analysis of miRNA analogues within synthesis batches, with length up to 22 nucleotides, including sequences differing by an increase in methylation levels and positional isomers containing the same number of methyl groups. The method combines amide-based HILIC separation to top-down collision-induced dissociation (CID) MS/MS and delivers three levels of characterization in a 20-min single run. It allows (i) the separation and identification of impurities (shortmers) from full length product (FLP), (ii) the chromatographic resolution of global methylation forms (0, 1 and 2 methyl groups) up to 22-mer sequence length and (iii) positional isomers discrimination based on the presence and/or absence of specific fragments. The application of HILIC-MS/MS workflow on complex samples, such as positional isomers with identical m6A counts and non-equimolar mixtures of various methylation levels, demonstrated robust identification of each isomer, including in mixed samples and despite differences in isomer concentration.

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Cite

BibTeXRIS
BibTeX
@article{adouairi2026hydrophilic,
  title = {{Hydrophilic interaction chromatography and tandem mass spectrometry for the characterization of methylated oligoribonucleotides}},
  author = {Khaoula Adouairi and Carole Farre and Carole Chaix and Sébastien Pfeffer and Karine Faure},
  journal = {Journal of Pharmaceutical and Biomedical Analysis},
  year = {2026},
  month = oct,
  doi = {10.1016/j.jpba.2026.117546},
  url = {https://hal.science/hal-05775755v1},
  note = {CC BY-NC 4.0},
}
RIS
TY  - JOUR
TI  - Hydrophilic interaction chromatography and tandem mass spectrometry for the characterization of methylated oligoribonucleotides
AU  - Khaoula Adouairi
AU  - Carole Farre
AU  - Carole Chaix
AU  - Sébastien Pfeffer
AU  - Karine Faure
PY  - 2026
DA  - 2026/10/02
JO  - Journal of Pharmaceutical and Biomedical Analysis
DO  - 10.1016/j.jpba.2026.117546
UR  - https://hal.science/hal-05775755v1
AB  - Chemical modifications on oligonucleotides, notably N-6-methyladenosine (m6A) are known for their impact on diverse classes of RNA including micro (mi)RNAs. However, the characterization of these modifications on intact sequences remains challenging. An ion-pair free hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry (HILIC-HRMS/MS) workflow was developed for the intact analysis of miRNA analogues within synthesis batches, with length up to 22 nucleotides, including sequences differing by an increase in methylation levels and positional isomers containing the same number of methyl groups. The method combines amide-based HILIC separation to top-down collision-induced dissociation (CID) MS/MS and delivers three levels of characterization in a 20-min single run. It allows (i) the separation and identification of impurities (shortmers) from full length product (FLP), (ii) the chromatographic resolution of global methylation forms (0, 1 and 2 methyl groups) up to 22-mer sequence length and (iii) positional isomers discrimination based on the presence and/or absence of specific fragments. The application of HILIC-MS/MS workflow on complex samples, such as positional isomers with identical m6A counts and non-equimolar mixtures of various methylation levels, demonstrated robust identification of each isomer, including in mixed samples and despite differences in isomer concentration.
SN  - 0731-7085
SN  - 1873-264X
ER  -