High-throughput analysis of oligonucleotides using a single quadrupole mass spectrometer for quality control

Importance of the Topic

Oligonucleotide analysis is a critical component in the development of antisense therapeutics, RNA vaccines, and molecular diagnostics. High-throughput and accurate quality control ensures integrity of custom DNA and RNA sequences in research and industrial settings.

Objectives and Study Overview

This study demonstrates a streamlined workflow using a single quadrupole mass spectrometer coupled to a binary UHPLC system and integrated software for step-by-step oligonucleotide synthesis quality control. The goal is a pass or fail report for target oligonucleotide masses across a range of chain lengths.

Methodology and Instrumentation

Sample Preparation and Chromatography

• Neat injection of 96-well plate samples directly from a DNA synthesizer
• Reversed-phase ion pairing on a 2.1×50 mm DNAPac RP column using mobile phases containing variable 0.01 to 2.0% HFIP and 0.1% TEA in water (A) or methanol (B)
• Gradient elution at 0.7 mL/min and 70 °C with UV detection at 260 nm

Mass Spectrometry and Data Analysis

• Thermo Scientific ISQ EM single quadrupole mass detector with negative HESI source
• Optimization of vaporizer and ion transfer tube temperatures, sheath and auxiliary gas pressures, and source voltage via custom injection variables in Chromeleon CDS
• Intact Protein Deconvolution engine configured for oligonucleotide mass range 2,000–20,000 Da

Instrumentation Used

• Vanquish Flex UHPLC system (Horizon/Flex base, binary pump, split sampler, column compartment, detector)
• ISQ EM single quadrupole mass spectrometer
• Chromeleon Chromatography Data System with Intact Protein Deconvolution

Main Results and Discussion

• HFIP concentration optimized at 0.1% yielded maximal signal intensity and minimal HFIP adduction, reducing reagent use by 20-fold compared to the 2% standard
• Chromatograms of 10 to 60-mer oligonucleotides showed well-resolved, symmetric peaks and removal of synthesis impurities
• Source tuning identified vaporizer and transfer tube temperatures as key factors for enhancing charge state intensity and lowering adduct formation
• Automated deconvolution accurately matched measured intact masses to theoretical values across the oligomer array

Benefits and Practical Applications

• High-throughput, minimal sample preparation workflow directly from synthesizer output
• Cost savings through reduced HFIP consumption and simplified method optimization via Chromeleon custom variables
• Rapid pass/fail reporting for quality control of large oligonucleotide arrays in research or manufacturing

Future Trends and Possibilities

Integration of longer and chemically modified oligonucleotides into the workflow could expand therapeutic and diagnostic applications. Automation and AI-driven data analysis may further accelerate method development and routine QC. Exploration of alternative ion-pair reagents and next-generation mass analyzers could enhance sensitivity and throughput.

Conclusion

The presented workflow provides an efficient and cost-effective platform for comprehensive oligonucleotide quality control using a single quadrupole MS and UHPLC. Optimized chromatographic and MS settings, combined with automated deconvolution and reporting, deliver reliable pass/fail results for target masses across diverse chain lengths.

References

1. Michelson AM, Todd AR. Nucleotides Part XXXII. J Chem Soc. 1955;2632.
2. Sinha ND et al. Nucleic Acids Res. 1984;12(11):4539.
3. Bajan S et al. Cells. 2020;9:137.
4. Rossi JJ et al. Nucleic Acid Therapeutics. 2020;30:129.
5. Scharner J, Aznarez I. Mol Ther. 2020;29(2):540.
6. Thermo Scientific TN 73670. Quality control of oligonucleotides with a single quadrupole mass spectrometer; 2020.