An Integrated Workflow for the Analysis of Oligonucleotides and Their Impurities by Agilent High-Resolution LC/(Q-)TOF Mass Spectrometry

Significance of Oligonucleotide Analysis

Recent advances in oligonucleotide therapeutics, including siRNA, antisense oligos, aptamers and CRISPR guides, create a pressing need for robust analytical tools. Precise identification and quantitation of the active sequence and low-abundance impurities—such as phosphorothioate-to-phosphodiester conversions, truncations, extensions and abasic variants—is essential for drug development, quality control and regulatory compliance.

Study Objectives and Overview

This application note presents an integrated, high-throughput workflow for separation, accurate-mass detection and relative quantitation of oligonucleotides and their impurities by coupling reversed-phase ion-pair chromatography with a Quadrupole Time-of-Flight (Q-TOF) mass spectrometer. The goals are to demonstrate:

• Rapid chromatographic separation of full-length oligo standards and impurity profiles
• High-resolution mass analysis with sub-ppm accuracy
• Automated data processing for target and impurity identification

Methodology and Instrumentation

The separation employed an Agilent 1290 Infinity II LC system with an AdvanceBio Oligonucleotide column (2.1×50 mm, 2.7 µm) using a binary gradient of 15 mM triethylamine/400 mM HFIP (aqueous) versus methanol at 0.5 mL/min and column temperature of 65 °C. Mass detection utilized an Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Agilent Jet Stream source in negative mode. Key MS settings: mass range 300–3200 m/z, acquisition rate 4 spectra/s, high-resolution (4 GHz) mode, VCap 3500 V, fragmentor 175 V. Data were processed in Agilent MassHunter BioConfirm software v12.0 using a Target Plus Impurities (TPI) workflow that integrates Find-by-Formula (FBF) for targeted screening and Maximum Entropy deconvolution for untargeted profiling above a user-defined mass threshold.

Main Results and Discussion

Using short gradients (~11 min), DNA/RNA ladder standards and synthetic 21-, 40- and 100-mer oligonucleotides were baseline separated and detected with excellent sensitivity. Monoisotopic and average masses matched calculated values with mass errors typically below 1 ppm for <30-mers and under 10 ppm even for 100-mers. The TPI workflow identified up to 19 truncated species of a 21-mer with relative quantitation CV <3%. In a 40-mer sample, up to eleven 5′-truncated impurities as low as 0.65% relative abundance were accurately profiled.

Benefits and Practical Applications

This integrated LC/Q-TOF approach enables:

• Comprehensive impurity profiling of oligonucleotide drug candidates
• Rapid method development with a single 11-minute gradient
• High-throughput, automated data analysis for identity confirmation and quantitation

Future Trends and Potential Applications

Ongoing advances in high-throughput HRAM platforms and intelligent software will further streamline oligonucleotide characterization. Future directions include real-time impurity monitoring during synthesis, multivariate data mining for novel modification patterns and integration of MS/MS sequencing workflows for deeper structural confirmation.

Conclusion

The described workflow demonstrates robust chromatographic separation, high-accuracy mass measurements and reliable software-driven impurity quantitation. Agilent MassHunter BioConfirm v12.0 effectively automates both targeted and untargeted analysis, reducing data-processing times and supporting quality-by-design in the development of oligonucleotide therapeutics.

Reference

1. Capaldi D. et al. Impurities in Oligonucleotide Drug Substances and Drug Products. Nucleic Acid Ther. 2017;27:309–322.
2. Okafo G.; Elder D.; Webb M. Analysis of Oligonucleotides and Their Related Substances. ILM Publications; 2012.