APPLICATION SOLUTIONS FOR OLIGONUCLEOTIDES - APPLICATION NOTEBOOK

High-Performance UPLC and Mass Spectrometric Analysis of Oligonucleotides

• Significance

  The expanding role of synthetic oligonucleotides in diagnostics and therapeutics demands rapid, high-resolution analytical methods that deliver both qualitative and quantitative information. Conventional techniques often lack the sensitivity, throughput or structural insight required for regulatory compliance and product development.



• Instrumentation

  Waters offers an integrated platform combining ACQUITY UPLC Systems with sub-2 µm Oligonucleotide Separation Technology (OST) Columns, orthogonal ultraviolet detection (TUV/PDA) and a range of mass spectrometers: the single-quadrupole ACQUITY QDa, high-resolution Xevo G2-XS QTof and SYNAPT HDMS with Triwave ion mobility.



• Analytical Workflows

  • Ion-pair reversed-phase UPLC with TEA/HFIP or hexylammonium acetate (HAA) delivers baseline separation of 15–60 nt oligonucleotides and phosphorothioates in under 10 minutes.
  • Mass detection on QDa quantifies oligonucleotides at < fmol levels, confirming identity and purity within existing UV-based assays.
  • High-resolution UPLC–QTof provides sub-ppm mass accuracy, linear quantitation down to tens of femtomoles and rapid MS/MS ladder sequencing for full-length siRNA verification.
  • UPLC–HDMS (SYNAPT) uses ion mobility to resolve isobaric species, PEG distributions and low-level impurities beyond conventional MS capabilities.



• Automated Data Processing

  ProMass HR integrated with MassLynx streamlines high-throughput deconvolution, sequence matching and impurity profiling, presenting results in interactive HTML or plate-based formats for rapid decision-making.



• Applications

  These workflows support QC of synthetic oligonucleotides, siRNA duplex formation via on-column annealing, PS oligonucleotide impurity monitoring, PEG quality assessment and MS/MS-based sequence confirmation.



• Future Trends

  Advances in UPLC-MS™ integration, high-definition IMS, real-time process analytics and artificial intelligence–driven data interpretation will accelerate therapeutic oligonucleotide discovery and regulatory compliance.



• Conclusion

  The fusion of sub-2 µm UPLC separations, optimized ion-pair chemistries and high-definition mass spectrometry provides a comprehensive, high-throughput platform for oligonucleotide R&D and QC, enhancing speed, sensitivity and structural confidence.