Accelerating Oligonucleotide Research Using Novel Bioinert LC, QTOF and TQ LC-MS

Significance of the Topic

Oligonucleotide therapeutics represent a cutting-edge class of drugs that act at the genetic level, offering targeted treatments for a range of diseases. However, chemical synthesis often yields impurities and is prone to metal ion adsorption, challenging accurate characterization and quantification. Advanced analytical methods combining bioinert ultra-high performance liquid chromatography (UHPLC) with high-resolution and triple-quadrupole mass spectrometry (MS) are essential to ensure the safety, efficacy, and quality control of these molecules.

Objectives and Overview of the Study

This study aimed to develop and validate a robust, metal-free analytical workflow using Shimadzu’s Nexera XS inert UHPLC system coupled with Q-TOF LCMS-9030 and TQ LCMS-8060 instruments. Key goals included efficient separation of synthetic oligonucleotides from impurities, achieving mass accuracy below 0.05 ppm, and establishing a linear quantification range from 1 to 1000 ng/mL.

Methodology and Instrumentation

• Sample Preparation: Purified single-stranded DNA and a modified oligonucleotide ladder (10–60 bases) diluted to 5 μg/mL for LC-MS.
• Chromatography: Ion-exchange on Shim-pack Bio IEX and reversed-phase IP-RP on Shim-pack Scepter C18 metal-free columns, using 2 mM ammonium formate/0.002% formic acid buffers and filtered caps to maintain pH stability.
• Mass Spectrometry: Accurate mass detection with LCMS-9030 (Q-TOF) and quantitative MRM analysis on LCMS-8060 (TQ), monitoring the octavalent precursor at m/z 803.4626 and product ion at m/z 94.9358.

Main Results and Discussion

• Metal-free flow paths prevented metal adsorption, yielding calibration curve linearity (r² = 0.9999) and reproducible retention times.
• Accurate mass measurements achieved ≤0.05 ppm error, enabling precise molecular weight confirmation of a 20-base 2′-MOE modified oligonucleotide.
• MRM quantification demonstrated sensitivity across a 1–1000 ng/mL range with robust reproducibility.
• Ion-exchange and IP-RP LC strategies effectively separated target sequences from impurities; pH control was critical for stable analysis.

Benefits and Practical Applications

The bioinert UHPLC-MS workflow provides high sensitivity, exceptional mass accuracy, and reliable quantification, accelerating oligonucleotide drug development and quality control. It supports regulatory compliance and streamlines impurity profiling in research and industrial environments.

Future Trends and Opportunities

• Integration with high-throughput automated platforms and advanced data analytics.
• Expansion to novel oligonucleotide chemistries and delivery conjugates (e.g., lipid nanoparticles, peptide tags).
• Coupling with orthogonal techniques (ion mobility, UV absorbance) for multi-dimensional characterization.
• Development of real-time process analytical technology (PAT) for on-line QC monitoring.

Conclusion

Implementing a metal-free UHPLC-QTOF/TQ MS workflow significantly enhances the analysis of synthetic oligonucleotides by providing accurate mass determination, sensitive quantification, and robust separation of impurities. This approach underpins efficient development and rigorous quality assurance of next-generation nucleic acid therapeutics.