Adding Mass Detection to Synthetic Oligonucleotide Analyses with the ACQUITY QDa Detector

Importance of the Topic

Synthetic oligonucleotides are emerging as key therapeutic agents for gene regulation and RNA interference. Reliable characterization of these molecules requires not only optical detection but also mass confirmation to verify sequence length and identify base modifications. Integrating low-cost mass detection into established liquid chromatography workflows enhances confidence in purity assessments and accelerates process development.

Study Objectives and Overview

This application study aimed to demonstrate that the ACQUITY QDa Detector can be seamlessly added to existing ion-pair reversed-phase liquid chromatography (IP-RPLC) methods for oligonucleotide analysis. A series of polyT standards (15 to 35 nucleotides) was used to evaluate mass accuracy, compatibility with triethylamine (TEA)/hexafluoro-2-propanol (HFIP) buffers, and the ease of integration into UV-based assays.

Methodology

The study employed an ACQUITY UPLC H-Class system with inline UV detection followed by the QDa mass detector. Mobile phases consisted of 15 mM TEA and 400 mM HFIP in water (A) or methanol (B), pH 8.0. A BEH C18 oligonucleotide column (2.1 × 50 mm, 1.7 µm, 130 Å) was operated at 60 °C with a 0.2 mL/min flow. PolyT standards at 10 pmol/µL were injected (5 µL, 50 pmol on column). The QDa ran in negative mode over 410–1,250 Da, 2 points/sec data rate, cone voltage 20 V, capillary 0.8 kV, probe 600 °C. Data were processed using MassLynx SCN 9.25 and MaxEnt1 deconvolution.

Used Instrumentation

• ACQUITY UPLC H-Class System
• ACQUITY UPLC TUV Detector with titanium flow cell
• ACQUITY QDa Detector
• ACQUITY UPLC BEH C18 Oligonucleotide Column (2.1 × 50 mm, 1.7 µm, 130 Å)
• MassLynx SCN 9.25 software with MaxEnt1 algorithm

Results and Discussion

The inline mass chromatograms closely matched UV traces, confirming compatibility with TEA/HFIP IP-RPLC. Multiple charge states were observed for each oligonucleotide, rising from five states for the 15 nt standard to nine for the 35 nt standard. Mass accuracy for individual charge states was within ±0.2 Da and showed RSD values below 0.02%. MaxEnt1 deconvolution of summed spectra produced zero-charge masses within +0.7 Da of theoretical values, with consistent reproducibility.

Benefits and Practical Applications

• Cost-effective addition of orthogonal mass data to routine UV assays
• Improved identification of base modifications and sequence variants
• Streamlined workflows with minimal hardware changes
• Enhanced confidence in purity and identity during manufacturing QC

Future Trends and Opportunities

Expansion of low-cost mass detection to other biotherapeutic classes such as small interfering RNAs and peptide–oligonucleotide conjugates is anticipated. Integration with automated data evaluation, real-time process monitoring and advanced deconvolution algorithms will further boost throughput and analytical depth. Coupling with high-resolution MS platforms may extend detection range for larger oligonucleotides and complex modifications.

Conclusion

The ACQUITY QDa Detector delivers accurate mass information for synthetic oligonucleotides across a broad molecular weight range using conventional IP-RPLC buffers. Its simple integration into existing UPLC-UV workflows offers a practical, cost-effective solution for routine identity and purity testing in oligonucleotide manufacturing.

References

1. Birdsall R., McCarthy S. Increasing Specificity and Sensitivity in Routine Peptide Analyses Using Mass Detection with the ACQUITY QDa Detector. Waters Application Note 2015; 720005377en.
2. Cosgrave E., Birdsall R., McCarthy S. Rapidly Monitoring Released N-Glycan Profiles During Process Development Using RapiFluor-MS and the ACQUITY QDa Detector. Waters Application Note 2015; 720005438en.
3. Birdsall R., McCarthy S. Adding Mass Detection to Routine Peptide-Level Biotherapeutic Analyses with the ACQUITY QDa Detector. Waters Application Note 2015; 720005266en.
4. Agrawal S., Zhao Q. Antisense Therapeutics. Current Opinion in Chemical Biology 1998; 2:519–528.
5. McGinnis A. C., Chen B., Bartlett M. G. Chromatographic Methods for the Determination of Therapeutic Oligonucleotides. Journal of Chromatography B 2012; 883–884:76–94.
6. Apffel A., Chakel J. A., Fischer S., Lichtenwalter K., Hancock W. S. Analysis of Oligonucleotides by HPLC–Electrospray Ionization Mass Spectrometry. Analytical Chemistry 1997; 69:1320–1325.
7. Apffel A., Chakel J. A., Fischer S., Lichtenwalter K., Hancock W. S. New Procedure for the Use of High-Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry for the Analysis of Nucleotides and Oligonucleotides. Journal of Chromatography A 1997; 777:3–21.