Qualification and Quantitation of Phosphorothioate Oligonucleotides Using a Quadrupole-Time-of-Flight Mass Spectrometer

Importance of the Topic

Phosphorothioate antisense oligonucleotides represent a powerful class of therapeutic agents for gene silencing. Their chemical stability and ability to modulate specific mRNA targets make them essential tools in drug development and molecular diagnostics. Accurate characterization and quantitation are critical for quality control, impurity profiling, and regulatory compliance.

Study Objectives and Overview

This study presents a combined qualitative and quantitative workflow for a series of phosphorothioate oligonucleotides, including mipomersen and three modified analogues. The main goals were:

• To confirm molecular weights with high mass accuracy
• To develop a sensitive quantitation method using high-resolution multiple reaction monitoring (MRM)
• To demonstrate a single LC/Q-TOF platform capable of both tasks

Methodology and Instrumentation

The analytical approach integrates liquid chromatography separation, Q-TOF mass spectrometry, and advanced data processing:

• Chromatography: Binary gradient of 50 mM HFIP with 10 mM DIPEA in water versus acetonitrile on a Shim-pack Scepter C18 column (2.1 × 50 mm, 1.9 µm) using a Nexera UHPLC system
• Mass Spectrometry: Shimadzu LCMS-9030 quadrupole-time-of-flight instrument operated in negative ESI mode; full-scan MS and high-resolution MRM targeting the PSO₂⁻ fragment (m/z 94.9358)
• Data Processing: Charge-state deconvolution performed with Insight™ Explore CSD software to reconstruct zero-charge spectra for accurate mass confirmation

Key Results and Discussion

• Molecular weight confirmation for all four oligonucleotides achieved with mass errors below 1 ppm after deconvolution of charge states 3–8.
• Observed monoisotopic masses closely matched theoretical values for mipomersen and its 2'-deoxy, OMe, and LNA analogues.
• High-resolution MRM delivered linear calibration curves over 1–1000 ppb (R² > 0.996), enabling detection limits down to single-digit ppb levels.
• Focusing on the characteristic phosphorothioate fragment improved selectivity and sensitivity compared to conventional ELISA or low-resolution MS methods.

Benefits and Practical Applications

The combined LC/Q-TOF workflow streamlines oligonucleotide analysis by reducing method transfer steps and reliance on multiple instruments. Key advantages include:

• High confidence in mass assignments for impurity identification
• Enhanced sensitivity for trace quantitation in complex matrices
• Flexible platform accommodating diverse oligonucleotide chemistries

Future Trends and Opportunities

Advances may include integration of microflow UHPLC, automated data processing pipelines, and expansion to next-generation nucleotide modifications. Coupling Q-TOF with ion mobility or emerging fragmentation techniques could further enhance structural characterization. The growing demand for oligonucleotide therapeutics will drive adoption of robust, high-throughput MS-based workflows in both research and regulated environments.

Conclusion

The study demonstrates that a single high-resolution LC/Q-TOF system, combined with charge-state deconvolution and high-resolution MRM, offers precise molecular weight confirmation and highly sensitive quantitation for phosphorothioate antisense oligonucleotides. This unified approach improves analytical efficiency and supports stringent quality requirements in oligonucleotide development.

Instrumentation Used

• Shimadzu LCMS-9030 quadrupole-time-of-flight mass spectrometer
• Nexera UHPLC system
• Shim-pack Scepter C18 column (2.1 × 50 mm, 1.9 µm)
• Insight™ Explore CSD charge-state deconvolution software