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

Significance of the topic

Phosphoramidite chemistry underpins solid-phase synthesis of oligonucleotides, which are key in therapeutic development. Impurities in phosphoramidite raw materials can compromise yield, purity, and downstream biological activity. Regulatory guidance requires detection and characterization of related impurities at levels down to 0.1% or below relative to the parent compound, making sensitive analytical workflows essential.

Objectives and study overview

This work demonstrates how the Thermo Scientific Vanquish Horizon UHPLC system coupled with an Orbitrap Exploris 120 high-resolution accurate-mass (HRAM) mass spectrometer, in combination with Thermo Scientific Compound Discoverer software, enables:

• Sensitive detection of phosphoramidite raw materials and trace impurities using LC/UV/HRAM-MS.
• Confident structure elucidation of low-abundance impurities to classify them as critical or non-critical based on high-quality MS² data.

Methodology and Instrumentation

Reagents comprised 2′-modified RNA phosphoramidites (5′-DMT-2′-F-A(bz)-CEP) from four vendors, plus a synthetic impurity spike. Sample preparation involved dissolving at 1 mg/mL in anhydrous acetonitrile. Separation and detection were performed as follows:

• UHPLC system: Vanquish Horizon UHPLC with Accucore C18 column (2.1×100 mm, 2.6 μm), gradient of 10 mM ammonium acetate to acetonitrile, flow rate 0.4 mL/min, column at 45 °C.
• Detector: Diode array at 254 nm for UV quantitation.
• Mass spectrometer: Orbitrap Exploris 120 with OptaMax NG H-ESI source, polarity-switching Top2 data-dependent MS² (MS¹ at 60 000 resolution, MS² at 15 000), mass range m/z 200–1200, normalized HCD collision energies of 10, 20, 40%.
• Data processing: Xcalibur 4.5 and FreeStyle 1.8 SP2 for acquisition and review; Chromeleon 7.2.10 CDS for UV peak detection and quantitation; Compound Discoverer 3.3 SP1 with “Impurity ID Related and Unknown with Molecular Networks” workflow for chromatographic deconvolution, elemental composition, transformation prediction, and fragment ion matching (FISh algorithm).

Main results and discussion

Sensitivity testing with a spiked impurity (5′-DMT-2′-F-A(bz)) demonstrated UV detection down to 0.01% and MS detection to 0.001% relative concentration. Comparative profiling of vendor materials revealed 25 distinct impurities at levels below 0.15% UV area, while main product purity exceeded 99.3% across all lots. Compound Discoverer’s targeted and untargeted workflows enabled automated detection and annotation, with fragment matching localizing modifications (e.g., methylation on DMT group, chlorination on DMT or benzoyl) to classify impurities as reactive/non-reactive and critical/non-critical. Differences in chlorinated isomers and oxidation, dealkylation, and substitution products were resolved among suppliers.

Benefits and practical applications

This integrated LC/UV/HRAM-MS approach allows pharmaceutical and QC laboratories to:

• Achieve trace-level impurity profiling critical for oligonucleotide drug quality control.
• Differentiate isomeric impurities and pinpoint transformation sites rapidly.
• Support regulatory compliance by characterizing impurities below 0.1%.

Future trends and opportunities

As oligonucleotide therapeutics evolve, increasing structural complexity will demand deeper impurity mapping. Emerging trends include coupling ion-mobility separation for isomer resolution, machine-learning–driven spectral annotation, and integrated online data analysis pipelines to accelerate decision-making in process development.

Conclusion

The Vanquish Horizon UHPLC system paired with the Orbitrap Exploris 120 MS and Compound Discoverer software delivers robust sensitivity, high-resolution data, and automated workflows for comprehensive impurity profiling and structure elucidation of phosphoramidite raw materials, ensuring reliable quality control in oligonucleotide manufacturing.

References

1. Roy S.; Caruthers M.S. Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries. Molecules 2013, 18, 14268–14284.
2. Kiesman W.F. et al. Perspectives on the Designation of Oligonucleotide Starting Materials. Nucleic Acid Therapeutics 2021, 31, 93–113.
3. ICH. Q3A(R) Impurities in New Drug Substances, 2003.
4. Comstock K.; Du M. Impurity Profiling of Mycophenolate Mofetil Using an Orbitrap Exploris 120 Mass Spectrometer and Vanquish Horizon UHPLC Combined with Compound Discoverer Software. Thermo Fisher Scientific Application Note 000531, 2022.