Accurate Mass Spectral Deconvolution of Multiply-charged Oligonucleotides using Unit Resolution Single Quadrupole LC/MS

Importance of the Topic

The rapid growth of nucleic acid therapeutics such as CRISPR-Cas systems, mRNA vaccines and synthetic oligonucleotide drugs creates a critical demand for reliable mass spectrometric characterization. Accurate molecular weight determination and spectral deconvolution of multiply charged oligonucleotides are essential for quality control, sequence confirmation and impurity profiling in pharmaceutical development.

Objectives and Study Overview

This study demonstrates a cost-effective workflow for precise mass analysis of a 40-mer synthetic oligodeoxythymidine using a single quadrupole LC/MS system. The main aims are:

• To achieve accurate mass assignments from unit resolution spectra.
• To compare nominal mass and calibrated mass molecular weight calculations.
• To detect and quantify adduct species via automated deconvolution.

Methodology

An oligonucleotide standard (40-mer poly-deoxythymidine) was separated on an Agilent Infinity II 1290 HPLC using a generic gradient. The eluent was analyzed by an Agilent 6135C LC/MSD XT operating in full-scan mode (m/z 700–3000). Raw profile data with zero thresholding were acquired and subsequently processed in Cerno MassWorks software. External calibration was performed by infusing an ESI-L tune mix to correct mass axis and peak shape. The Spectrally Accurate Modeling of Multiply charged Ions (SAMMI) algorithm facilitated deconvolution of overlapping charge states and identification of adducts.

Instrumentation

• Liquid Chromatography: Agilent Infinity II 1290 HPLC
• Mass Spectrometry: Agilent 6135C LC/MSD XT single quadrupole
• Software: Cerno MassWorks for external calibration and SAMMI spectral deconvolution

Main Results and Discussion

• Nominal mass spectra provided average MW 12,104.97 ± 5.99 Da (RSD 4.9%).
• Calibrated accurate mass spectra yielded MW 12,106.13 ± 0.22 Da (RSD 0.18%), closely matching theoretical value 12,106.6 Da.
• Automated “Unknown Analysis” detected three species: native oligonucleotide (80.64%), sodium adduct (+22.26 Da) and potassium adduct (+38.65 Da).
• SAMMI modeling explained 86.3% of spectral intensity by these three ion forms, confirming high spectral accuracy.

Benefits and Practical Applications

• Cost-effective alternative to high-resolution MS for routine oligonucleotide analysis.
• Improved confidence in molecular weight determinations for QA/QC and regulatory submissions.
• Automated detection and quantitation of adducts and modifications enhances impurity profiling.

Future Trends and Opportunities for Use

Integration of software-based calibration with widely available quadrupole instruments can extend accurate mass analysis to:

1. Other biopolymers such as peptides and small proteins.
2. High-throughput screening in drug discovery and process development.
3. On-line monitoring of oligonucleotide synthesis and purification workflows.

Conclusion

This work establishes that unit resolution single quadrupole LC/MS, when combined with in-spectrum calibration software, achieves accurate mass measurement and reliable deconvolution of multiply charged oligonucleotides. The approach offers a practical, affordable solution for pharmaceutical and research laboratories requiring robust oligonucleotide characterization.

Reference

Batoon PM, Bertram L, Wang Y. Accurate Mass Spectral Deconvolution of Multiply-charged Oligonucleotides using Unit Resolution Single Quadrupole LC/MS. ASMS 2024, Poster WP480.