Oligo Purity Analysis and Sequence Confirmation by LC/MS without Ion Pairing Reagents — Sample to reports in about 5 minutes

Importance of the topic

Oligonucleotide therapeutics and diagnostic assays demand rapid, reliable quality control workflows. Conventional LC/MS methods often rely on ion-pairing reagents that can persist in the system, complicating subsequent analyses and reducing instrument versatility. Developing a fast, robust, ion-pairing-free approach expands laboratory throughput and simplifies method transfer across diverse applications.

Objectives and Study Overview

This work describes the design and evaluation of a one-minute fast HILIC-MS method for simultaneous oligonucleotide purity assessment (MS1) and sequence confirmation (auto MS/MS). Five oligos of 18–21 nucleotides were analyzed to demonstrate sub-ppm mass accuracy, low-level impurity detection, complete sequence coverage, and a total sample-to-report time of about five minutes.

Methodology

Five synthetic oligonucleotides (21-mer DNA, 21-mer BS, complementary 21-mer, 20-mer PR2, 18-mer PR8) were separated using HILIC chromatography on an InfinityLab Poroshell 120 HILIC-Z guard column (2.1 × 5 mm, 2.7 μm). Mobile phases consisted of A: 90% acetonitrile/10% water + 15 mM ammonium acetate, and B: 10% acetonitrile/90% water + 15 mM ammonium acetate. A rapid gradient from 45% to 75% B over 0.4 minutes at 0.6 mL/min, followed by a 0.6–1.0-minute cleanup at 1.75 mL/min, delivered six-second peaks.

Instrumentation

• Agilent 1290 Infinity II Bio LC system
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI

Main Results and Discussion

• Mass Accuracy: Measured monoisotopic masses matched calculated values within ±1.1 ppm.
• Impurity Profiling: Multiple low-abundance truncations and depurination products (<0.2% of target) were observed without extended gradients.
• Sequence Coverage: Auto MS/MS produced 1–2 spectra per precursor; aggregated data yielded 100% coverage for all oligos.
• Reproducibility: Overlay of 50 backpressure traces confirmed gradient reproducibility; TIC overlays across five sequences demonstrated consistent retention behavior.

Benefits and Practical Applications

This fast HILIC-MS approach eliminates ion-pairing reagents, reducing carryover and simplifying instrument maintenance. The sub-minute separation and integrated data analysis deliver high throughput suitable for QC labs and research environments requiring rapid purity and sequence validation.

Future Trends and Opportunities

• Extension to longer or modified oligonucleotides and therapeutic conjugates.
• Integration with automated sample preparation and microfluidic platforms.
• Application of machine-learning algorithms for impurity pattern recognition and automated reporting.
• Multimodal workflows combining HILIC with orthogonal separation techniques.

Conclusion

A one-minute fast HILIC-MS workflow has been established for oligonucleotide purity and sequence confirmation without ion-pairing reagents. The method delivers sub-ppm mass accuracy, detects trace impurities, achieves full sequence coverage, and completes analysis and reporting in about five minutes, making it a powerful tool for high-throughput quality control.

Reference

1. Rye P., Yang Y. Fast LC/MS oligonucleotide analysis without ion pairing. Agilent Technologies Application Note 5994-3753EN, 2022.
2. Rye P., Schwarzer C. Agilent Technologies Application Note 5994-5631EN, 2023.
3. Li G., Rye P. Agilent Technologies Application Note 5994-5632EN, 2023.
4. Wong D., Rye P. Agilent Technologies Application Note 5994-4817EN, 2022.
5. Wong D., Rye P. Agilent Technologies Application Note 5994-5071EN, 2022.