Single Quadrupole Mass Spectrometry Quick Workflows Specified to Oligonucleotides Screening

Importance of the Topic

Chemically synthesized oligonucleotides are critical in therapeutic, diagnostic, and research applications. Rapid and reliable verification of their molecular weight and purity is essential for ensuring product quality and regulatory compliance. Single quadrupole mass spectrometry is a cost-effective platform widely available in many laboratories. The development of automated workflows that streamline deconvolution and mass assignment without requiring a predefined full-length product (FLP) sequence accelerates analysis and reduces manual effort.

Objectives and Study Overview

This work introduces the Quick Picking algorithm within the Insight Biologics software to support automated deconvolution of multiply charged ions in oligonucleotide samples. The study aims to:

• Demonstrate the algorithm’s ability to assign molecular weights without an input FLP sequence.
• Validate performance on a 20-mer phosphorothioate antisense oligonucleotide and an 80-mer PolyT sample.
• Compare results with conventional processing that uses a defined FLP sequence.

Methodology and Used Instrumentation

Samples and Chromatography:

• 20-mer phosphorothioate-modified antisense oligonucleotide (average mass 7168.05 Da) spiked with shortmer impurities.
• 80-mer PolyT oligonucleotide (average mass 24273.62 Da).
• Ion-pair reversed-phase LC using HFIP and 10 mM TEA on a Shimadzu Nexera XSTM inert LC with Shim-pack Scepter Claris bioinert column.

Mass Spectrometry:

• Shimadzu LCMS-2050 single quadrupole in negative ion mode, m/z 600–2000, 1 s scan speed.
• Data streams: TIC, base peak chromatogram (BPC) or LC absorbance (PDA) at 260 nm.

Main Results and Discussion

• The Quick Picking algorithm automatically detected and deconvoluted multiply charged species in both 20-mer and 80-mer samples.
• Mass assignments were within 1 Da of theoretical values (e.g., 24272.68 Da vs. 24273.62 Da for 80-mer PolyT).
• Comparison with conventional FLP-based processing showed highly consistent molecular weight information for noncomplex samples.
• In complex mixtures with closely eluting components, the algorithm selects the predominant species, simplifying interpretation.

Benefits and Practical Applications of the Method

The Quick Picking workflow offers:

• Elimination of the requirement to input a known sequence, reducing setup time.
• Automated peak picking and mass deconvolution in a few clicks.
• Compatibility with existing single quadrupole LC-MS systems in quality control and research labs.
• Cost-effective and rapid screening tool for oligonucleotide synthesis confirmation.

Future Trends and Applications

Anticipated developments include:

• Extension to larger and more diverse oligonucleotides and modified nucleic acids.
• Integration with higher-resolution mass spectrometers for enhanced impurity profiling.
• Incorporation of machine learning to improve deconvolution of overlapping charge states.
• Automated reporting and database integration for proactive quality assurance.

Conclusion

The Quick Picking algorithm simplifies single quadrupole LC-MS analysis of oligonucleotides by automating charge state deconvolution without requiring a predefined sequence. Validated on 20-mer and 80-mer samples, it delivers accurate molecular weight assignments within 1 Da, streamlining workflows in QC and research environments.

References

Uchiyama K., Kato N., Wang Q., Ashton S., Loftus N., Suzuki R., Fujito Y. Single Quadrupole Mass Spectrometry Quick Workflows Specified to Oligonucleotides Screening. Shimadzu Corporation; 2024.