Reversed-Phase Ion-Pair LC/MS Analysis of siRNA under Denaturing and Non-Denaturing Conditions

Significance of the Topic

siRNA therapeutics are emerging oligonucleotide drugs that require precise quality control to distinguish between their double-stranded and single-stranded forms. Reversed-phase ion-pair LC/MS combines chromatographic separation with mass spectrometric identification, offering detailed insights into siRNA structure and purity under both denaturing and native conditions.

Objectives and Study Overview

This study demonstrates an LC/MS method using the LCMS-9050 quadrupole time-of-flight system to characterize siRNA under denaturing (single-stranded) and non-denaturing (double-stranded) conditions. It investigates the influence of column temperature on elution behavior and showcases simultaneous analysis of multiple sequences using dedicated software.

Methodology and Instrumentation

A reversed-phase ion-pair LC/MS method was established with the following setup:

• UHPLC system: Nexera XS inert
• Column: Shim-pack Scepter Claris C18-300 (100 mm × 2.1 mm, 1.9 μm)
• Mobile phase A: 100 mM HFIP, 10 mM TEA in water
• Mobile phase B: 100 mM HFIP, 10 mM TEA in methanol
• Gradient: 5 % B at 0 min → 50 % B at 10 min → 90 % B at 10.01–12 min → 5 % B at 12.1–25 min
• Flow rate: 0.3 mL/min; injection volume: 1 μL
• Column temperatures: 60 °C (denaturing) and 25 °C (non-denaturing)

Mass spectrometry parameters:

• Instrument: LCMS-9050 QTOF
• Ionization: ESI negative mode
• m/z range: 550–2500
• Nebulizing gas: 2.0 L/min; drying gas: 10.0 L/min; heating gas: 10.0 L/min
• Interface temp: 350 °C; DL temp: 250 °C; block heater temp: 400 °C

Data analysis was performed with LabSolutions Insight Biologics, enabling sequence configuration, monoisotopic mass calculation, structural formula display, and automated component identification.

Key Results and Discussion

UV chromatograms at 60 °C showed dissociation of double-stranded siRNA into separate sense and antisense peaks, indicating denaturation. At 25 °C, the duplex eluted as a single peak with a longer retention time than individual strands. LC/MS component chromatograms confirmed sequence identities under both temperatures. LabSolutions Insight Biologics accurately matched MS1 signals to sense (21 nt) and antisense (21 nt) strands, demonstrating reliable deconvolution and multi-sequence analysis.

Benefits and Practical Applications

This method allows:

• Clear differentiation between dsRNA and ssRNA forms by temperature-controlled elution.
• Simultaneous characterization of multiple oligonucleotide sequences in a single run.
• High confidence in sequence confirmation and impurity profiling via QTOF MS.
• Streamlined workflow for quality control of siRNA therapeutics.

Future Trends and Applications

Advances may include higher-throughput automation, integration with native MS for larger RNA complexes, and enhanced software for detecting chemical modifications. These developments will further improve oligonucleotide therapeutic characterization and regulatory compliance.

Conclusion

The described reversed-phase ion-pair LC/MS method using LCMS-9050 and LabSolutions Insight Biologics effectively characterizes siRNA under denaturing and non-denaturing conditions. Column temperature controls duplex stability, and the software supports efficient multi-sequence analysis, making it ideal for QC in siRNA drug development.

References

1. Nakazono J. Reversed-Phase Ion-Pair LC/MS Analysis of siRNA under Denaturing and Non-Denaturing Conditions. Shimadzu Application News No. 01-00915-EN; July 2025.
2. Application News No. 01-00595A-EN. An Oligonucleotide Impurity Analysis Workflow Using LabSolutions Insight Biologics Software.
3. Application News No. 01-00656A-EN. Simple Analysis of Impurities in Oligonucleotide Therapeutics Using a Single Quadrupole Mass Spectrometer.
4. Application News No. 01-00898-EN. Efficient Method Development for Separation of Capped mRNA Fragments.