Oligonucleotide Characterization by Bio LC and Q-TOF

Importance of the Topic

The growing adoption of synthetic oligonucleotides in therapeutics and diagnostics highlights the need for reliable analytical methods to verify their mass, purity, sequence integrity, and impurity profile. High-resolution liquid chromatography coupled with mass spectrometry (LC/Q-TOF) provides essential data for quality control, regulatory compliance, and research in biopharmaceutical development.

Study Objectives and Overview

This work evaluates two complementary workflows—Target Plus Impurities (TPI) and Sequence Confirmation (SC)—implemented on the Agilent 1290 Infinity II Bio LC system paired with the Agilent 6545XT AdvanceBio LC/Q-TOF. The goal is to demonstrate streamlined analysis of oligonucleotide standards, an 18-mer antisense oligonucleotide (ASO), and a 28-mer aptamer, covering system suitability, impurity quantification, and full sequence validation.

Methodology and Instrumentation

Sample Preparation:

• RNA Resolution Standard (Agilent)
• 18-mer ASO with 2-methoxyethoxy and phosphorothioate modifications (IDT)
• 28-mer aptamer containing fluoro U/T and methylated bases (IDT)

Chromatographic Conditions:

• Agilent 1290 Infinity II Bio LC with biocompatible flow path
• AdvanceBio Oligonucleotide column
• Mobile phases: 100 mM triethylamine and 400 mM hexafluoroisopropanol in water (A) and methanol (B)
• Negative-ion electrospray, gradient elution (2.5–7.5 min for TPI; 5 min for SC)

Mass Spectrometry:

• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Agilent Jet Stream source
• TPI workflow in MS-only mode for targeted impurity quantification
• SC workflow in MS/MS mode, optimized collision energies (15 eV for ASO, 18 eV for aptamer) and charge states (–7, –10)

Instrumentation Used

• Agilent 1290 Infinity II Bio LC System
• Agilent AdvanceBio Oligonucleotide Column
• Agilent 6545XT AdvanceBio LC/Q-TOF
• Agilent MassHunter BioConfirm 12.0 software

Main Results and Discussion

System Suitability and Separation:
High chromatographic resolution was observed for four RNA standards (14-, 17-, 20-, 21-mers), confirming the LC system’s capability to resolve closely related oligonucleotides.

Impurity Profiling (TPI Workflow):
Five-prime truncated ASO impurities were baseline separated and quantified using a targeted Find-by-Formula algorithm. Measured monoisotopic masses matched theoretical values within ±1 ppm, and relative quantitation across six replicates showed RSDs below 5%.

Sequence Confirmation (SC Workflow):
Single-injection MS/MS experiments yielded full sequence coverage for both the 18-mer ASO and the 28-mer aptamer. Optimized precursor selection and collision energy settings produced rich fragment ion spectra that support unambiguous base-by-base confirmation.

Benefits and Practical Applications

• Rapid system suitability check and standardization using commercial resolution standards
• Accurate quantification of main product and truncated impurities for QA/QC
• Confident identity and sequence verification in one-run workflow
• Enhanced data processing throughput via MassHunter BioConfirm TPI and SC modules

Future Trends and Potential Applications

The integration of advanced LC/Q-TOF workflows into automated laboratory platforms will facilitate high-throughput screening of oligonucleotide libraries. Future developments may include deeper profiling of chemical modifications, coupling with ion mobility for structural insights, and expanded compatibility with emerging nucleic acid analogs.

Conclusion

The combined TPI and SC workflows on the Agilent 1290 Infinity II Bio LC–6545XT AdvanceBio LC/Q-TOF platform deliver robust separation, precise impurity quantification, and comprehensive sequence confirmation in streamlined, reproducible methods. This approach addresses critical analytical needs in synthetic oligonucleotide development and quality control.

References

1. Wong D.; Rye P. An Integrated Workflow for the Analysis of Oligonucleotides and Their Impurities by Agilent High-Resolution LC/(Q-)TOF Mass Spectrometry. Agilent Technologies Application Note 5994-4817EN (2022).
2. Wong D.; Rye P. Comprehensive Workflow for Oligonucleotide Sequence Confirmation by Agilent High-Resolution LC/Q-TOF. Agilent Technologies Application Note 5994-5071EN (2022).