Molecular Confirmation of Oligonucleotides Using Agilent LC/MSD XT and OpenLab CDS

Significance of the Topic

Accurate molecular weight confirmation of synthetic oligonucleotides is vital for quality control in research, therapeutic development, and diagnostic applications. Rapid and reliable workflows using liquid chromatography–mass spectrometry (LC–MS) enable manufacturers and laboratories to verify sequence integrity, detect impurities, and ensure product consistency, particularly when working with modified backbones, bases, or labels.

Objectives and Study Overview

This study demonstrates a streamlined approach for molecular weight confirmation and preliminary characterization of synthetic oligonucleotides using an Agilent LC/MSD XT single quadrupole system coupled with OpenLab CDS 2.8 spectral deconvolution. The workflow is evaluated with a DNA ladder standard (15–40mer) and modified antisense oligonucleotides (ASOs) to illustrate method applicability across various lengths and chemistries.

Methodology

• Sample Preparation: DNA ladder and ASO standards dissolved in deionized water; final concentrations of 4 nmol/µL (ladder) or 50 µg/mL.
• Chromatography: Agilent AdvanceBio oligonucleotide column (2.1×50 mm, 2.7 µm); mobile phases of 100 mM HFIP/15 mM TEA (A) and methanol (B); gradient from 15% to 95% B over 11 min; 0.5 mL/min flow; 65 °C column temperature; 2 µL injection.
• Spectral Deconvolution: OpenLab CDS 2.8 with optimized settings for m/z range (1,000–3,000), charge state envelope, noise thresholds, and curve fit algorithm.

Used Instrumentation

1. Agilent 1290 Infinity II BioLC or passivated 1290 Infinity II LC system for chromatographic separation.
2. Agilent LC/MSD XT (G6135C) single quadrupole mass spectrometer with AJS ESI source.
3. OpenLab CDS 2.8 software for instrument control and MS spectral deconvolution.

Main Results and Discussion

Analysis of a 21mer DNA oligonucleotide revealed broad charge state distributions (z = 8–9 dominant) with overlapping m/z signals, underscoring the need for empirical method optimization. The DNA ladder (15–40mer) produced distinct charge envelopes, and deconvoluted masses matched theoretical values within ±1 Da (9–84 ppm). Deconvolution of 18mer and 20mer ASOs, using a narrowed mass range (6,000–8,000 Da), successfully identified full-length products and n–1 impurities, with mass errors under 1 Da. Detection of low-level impurities (0.12% of main peak) demonstrated the sensitivity of optimized processing parameters.

Practical Benefits and Applications

These workflows allow rapid molecular weight verification and preliminary purity assessment of synthetic oligonucleotides in medium- to high-throughput settings. The integration of user-friendly software and robust hardware supports routine quality control in pharmaceutical research, oligo manufacturing, and analytical laboratories.

Future Trends and Applications

Advances may include integration of high-resolution accurate mass systems for detailed impurity profiling, automated method optimization using machine learning, expanded libraries of modified oligonucleotides, and deeper structural characterization via tandem MS or ion mobility. Continued software enhancements will further streamline data processing and reporting.

Conclusion

The combination of Agilent’s 1290 Infinity II LC, LC/MSD XT, and OpenLab CDS 2.8 spectral deconvolution offers a rapid, reliable solution for molecular weight confirmation and basic characterization of synthetic oligonucleotides. This platform meets the demands of modern analytical workflows where speed, accuracy, and ease of use are paramount.

References

• Chen B.; Mason S. F.; Bartlett M. G. The Effect of Organic Modifiers on Electrospray Ionization Charge-State Distribution and Desorption Efficiency for Oligonucleotides. Journal of the American Society for Mass Spectrometry 2013, 24(2), 257–264. doi:10.1007/s13361-012-0509-5
• Basiri B.; Murph M. M.; Bartlett M. G. Assessing the Interplay Between the Physicochemical Parameters of Ion-pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides. Journal of the American Society for Mass Spectrometry 2017, 28(8), 1647–1656. doi:10.1007/s13361-017-1671-6