Determination of Molecular Mass and Quantification of Oligonucleotide Therapeutics Using Quadrupole Time-of-Flight Mass Spectrometer LCMS™-9030

Significance of the Topic

Oligonucleotide therapeutics represent a growing class of precision medicines that rely on specific base sequences to modulate gene expression or protein activity. Accurate determination of their molecular mass and reliable quantification in biological matrices are essential for drug development, quality control, and regulatory compliance.

Objectives and Study Overview

This study demonstrates the use of a quadrupole time-of-flight mass spectrometer (LCMS-9030) coupled with liquid chromatography to (1) determine the exact molecular mass of a 2'-MOE modified 20-mer oligonucleotide therapeutic with high mass accuracy, and (2) establish a sensitive and linear quantification method across a broad concentration range (1–1000 ng/mL).

Methodology

Reversed-phase ion-pair HPLC was performed on a Shim-pack Scepter C18 column (2.0×75 mm, 1.9 μm) using 50 mmol/L HFIP and 10 mmol/L DIPEA (mobile phase A) and acetonitrile (mobile phase B) with a gradient from 5% to 15% B over 6 min at 0.2 mL/min and 50 °C. Electrospray ionization in negative mode was used for MS detection. The system acquired full-scan spectra (m/z 500–3000) and MRM transitions (803.4626>94.9358). Multicharged ion spectra were deconvoluted with the ReSpect algorithm in LabSolutions Insight Explore software.

Used Instrumentation

• Shimadzu LCMS-9030 quadrupole time-of-flight mass spectrometer
• Nexera HPLC system
• Shim-pack Scepter C18 column

Main Results and Discussion

Full-scan deconvolution yielded a monoisotopic mass of 6431.7239 Da with an error of 3 mDa (0.05 ppm). MRM quantification exhibited excellent sensitivity, and the calibration curve demonstrated linearity over 1–1000 ng/mL (R2 = 0.996). Representative MRM chromatograms confirmed consistent detection across the tested concentration range.

Benefits and Practical Applications

• Simultaneous structural confirmation and quantification of oligonucleotide therapeutics in a single LC-MS workflow
• High mass accuracy supports verification of modification and sequence integrity
• Wide dynamic range and sensitivity suitable for pharmacokinetic and bioanalysis studies

Future Trends and Applications

Advancements may include higher-throughput platforms, new ion-pairing chemistries to improve peak shape, integration with automated sample preparation, and expanded use of QTOF analyzers for larger and more complex nucleic acid constructs. Coupling with high-resolution separations and informatics tools will further enhance characterization and quantitation.

Conclusion

The combination of UHPLC and QTOF-MS (LCMS-9030) enables ultra-precise mass determination and robust quantification of oligonucleotide therapeutics. This approach streamlines analytical workflows for drug development and offers reliable data for regulatory submissions.