LC/UV/HRMS-based impurity profiling and structure elucidation of phosphoramidite raw materials used for oligonucleotide synthesis

Importance of the Topic

The quality of phosphoramidite raw materials directly impacts the synthesis and therapeutic performance of oligonucleotides. Impurities introduced during manufacturing can compromise product efficacy and safety, making their sensitive detection and structural characterization crucial for quality control.

Objectives and Study Overview

This study evaluates a comprehensive workflow combining reversed-phase UHPLC with UV detection and high-resolution accurate-mass Orbitrap MS to profile and elucidate trace-level impurities in 5’-DMT-2’-F-A(bz)-CEP raw materials from multiple vendors. The goal is to demonstrate sensitivity, vendor-to-vendor impurity differences, and confident structural assignments using fragmentation data.

Methodology and Instrumentation

Samples of 5’-DMT-2’-F-A(bz)-CEP (>98% purity) were dissolved in acetonitrile and analyzed on a Thermo Scientific Vanquish Horizon UHPLC with an Accucore C18 (2.1×100 mm, 2.6 μm) column. Mobile phases were 10 mM ammonium acetate (A) and acetonitrile (B) with a gradient from 30% to 95% B. Detection utilized UV monitoring and a Thermo Scientific Orbitrap Exploris 120 MS operating in polarity switching mode with full-scan MS1 (m/z 200–1200) and data-dependent MS2. Data processing employed Chromeleon CDS for peak detection and Compound Discoverer 3.3 with FISh algorithms for automated fragmentation annotation and transformation predictions.

Main Results and Discussion

The workflow achieved UV-based impurity detection to 0.01% and HRAM-MS detection to 0.001% relative levels. UV chromatograms revealed distinct impurity profiles across four vendors. Key trace impurities, including methylated and chlorinated species, were structurally characterized by matching isotope patterns and MS2 fragment shifts. Localization of modifications to either DMT or benzoyl protecting groups was enabled by FISh-predicted fragment ions, distinguishing reactive versus noncritical impurities based on their fate in oligonucleotide synthesis.

Benefits and Practical Application

• Enables sensitive, reproducible detection of trace impurities to support QA/QC in oligonucleotide manufacturing.
• Automates spectral annotation and transformation site localization, reducing manual interpretation effort.
• Provides actionable insights for controlling critical impurities that may affect product quality.

Future Trends and Potential Applications

Advances in AI-driven spectral interpretation and predictive transformation modeling are expected to further streamline impurity profiling. Expanded application of this workflow to diverse phosphoramidite chemistries and integration into in-process monitoring will enhance real-time quality assurance. Regulatory guidelines may evolve to incorporate high-resolution MS workflows for comprehensive impurity mapping.

Conclusion

The combined UHPLC-Orbitrap and Compound Discoverer workflow offers high sensitivity and confidence in profiling and structurally elucidating phosphoramidite impurities. This approach strengthens quality control practices for raw materials in therapeutic oligonucleotide synthesis.

Reference

1. Avila J; Cruthers MS. Synthesis of DNA/RNA Analogs via Phosphoramidite Chemistry. Molecules. 2013;18:14268–14284.
2. Kiesman WF et al. Perspectives in the Design of Oligonucleotide Starting Materials. Nucleic Acid Therapeutics. 2021;31:93–113.
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4. Hackbusch S et al. LC/UV/HRAM-MS-Based Impurity Profiling and Structure Elucidation of Phosphoramidite Raw Materials Used for Oligonucleotide Synthesis. Thermo Fisher Scientific Application Note 001949; 2023.