Verification of Quantitativity and Confirmation of Molecular Weight of Oligonucleotide therapeutics Using LCMS™-8060

Significance of the Topic

Oligonucleotide therapeutics are emerging as a powerful class of drugs that bind specifically to disease-related genes or proteins. Reliable quantitation and molecular weight confirmation are critical for drug development, quality control, and regulatory compliance. Mass spectrometry coupled with liquid chromatography offers high sensitivity and specificity for these analyses.

Study Objectives and Overview

This study illustrates the quantification and molecular weight confirmation of a 20-base 2'-O-(2-methoxyethyl) (2'-MOE) modified oligonucleotide using a triple quadrupole LCMS-8060 system. A calibration curve was developed in multiple reaction monitoring (MRM) mode over 1–300 ng/mL. Molecular weight was verified via deconvolution of charge-state spectra acquired in full-scan mode.

Methodology and Instrumentation

• Liquid Chromatography:
  • Column: C18 ODS, 50 mm × 2.1 mm, 2.5 µm.
  • Mobile Phase A: 50 mmol/L HFIP + 10 mmol/L DIPEA.
  • Mobile Phase B: Acetonitrile.
  • Gradient: 5%–15% B over 0.5–3 min.
  • Flow Rate: 0.2 mL/min; Column Temp.: 60 °C; Injection Volume: 2 µL.
• Mass Spectrometry:
  • Instrument: Shimadzu LCMS-8060 triple quadrupole.
  • Ionization: ESI in negative mode at –4 kV.
  • Acquisition Modes: Full scan (m/z 500–2000) and MRM (803.5→95.0).
  • Gas Flows: Nebulizing 3.0 L/min; Drying 8.0 L/min; Heating 12.0 L/min; CID gas 330 kPa.
  • Temperatures: DL 300 °C; Heat Block 450 °C; Interface 250 °C.
• Data Processing:
  • Calibration and quantitation performed in MRM mode.
  • Charge-state deconvolution executed with LabSolutions LCMS software to confirm molecular weight.

Main Results and Discussion

The MRM chromatogram displayed a sharp peak at approximately 3.3 minutes. A calibration curve across 1–300 ng/mL demonstrated linearity with R² = 0.998. In scan mode, hexavalent, heptavalent, and octavalent ions were observed and selected for analysis. Deconvolution of the multivalent spectra yielded an experimental molecular weight of 6436.37 Da, closely matching the theoretical 6436.39 Da, indicating high mass accuracy.

Benefits and Practical Applications

The described workflow enables:

• High-sensitivity quantitation of modified oligonucleotides over a wide concentration range.
• Reliable confirmation of molecular weight with minimal error.
• Streamlined analysis using standard reversed-phase conditions without ion-pair reagents that may suppress sensitivity.

Future Trends and Applications

Advances in oligonucleotide analysis will focus on higher throughput, improved software algorithms for deconvolution, and expansion to various chemical modifications. Integration with automated sample preparation and coupling to high-resolution mass spectrometers will enhance drug discovery, development pipelines, and routine QC in pharmaceutical labs.

Conclusion

The LCMS-8060 triple quadrupole system, combined with optimized reversed-phase chromatography and LabSolutions deconvolution software, provides a robust platform for both quantitative and qualitative analysis of 2'-MOE modified oligonucleotide therapeutics. The method delivers sensitive detection, excellent linearity, and accurate molecular weight confirmation.