Quality control of oligonucleotides using HPLC coupled to UV and MS detection

Importance of the topic

Oligonucleotides have emerged as crucial tools in molecular biology and as a new class of therapeutics due to their high specificity and favorable toxicology profiles. Ensuring their purity and correct mass is essential in research, pharmaceutical development and quality assurance. Combining liquid chromatography with UV and mass spectrometric detection addresses both quantification and structural confirmation, meeting stringent regulatory and manufacturing demands.

Objectives and study overview

This case study illustrates a combined HPLC-UV and MS workflow for the quality control of a 26-mer oligonucleotide API. The goals were to confirm the molecular mass, quantify the main product by UV absorption at 260 nm and identify low-level impurities, including aborted sequences (N-1) and degradation products such as depurination (NDEP) and sulfur-to-oxygen back-substitution (NPO).

Methodology

Sample preparation involved dissolving a reference standard (10 mg/mL), followed by serial dilution to 25 nmol/mL, yielding a 50 pmol injection per analysis. Chromatographic separation used ion-pair reversed-phase conditions with 10 mM HFIP/7 mM TEA in water (eluent A) and methanol (eluent B). A gradient from 100 % A to 100 % B over 30 min at 0.5 mL/min and 60 °C on a C18 oligonucleotide column provided baseline separation of the main peak.

Instrumentation

• UHPLC pump: AZURA P 8.1L UHPLC
• Autosampler: AZURA AS 6.1L (cooled/heated)
• Column thermostat: AZURA CT2.1
• DAD detector: AZURA DAD 6.1L at 260 nm
• Mass spectrometer: Sciex Triple Quad™ 5500+ with ESI in negative mode
• Software: ClarityChrom 9 and Sciex OS 3.2
• Column: Waters XBridge Oligonucleotide BEH C18, 50×4.6 mm, 2.5 µm

Key results and discussion

The UV trace at 260 nm delivered precise peak areas and retention times for quantification. MS full-scan (m/z 500–1250) revealed charge states z = 7–15, with the most intense at m/z 642.94 (z = 13). Measured m/z values matched theoretical masses within ±0.1 Da, confirming the 8369.9 Da oligonucleotide. Extracted ion chromatograms (Q1MI) at z = 13 permitted selective quantification of NDEP and NPO impurities despite co-elution. The NDEP impurity was quantified at ~6 % and NPO at ~2 % relative to the main peak.

Benefits and practical applications

• Simultaneous quantification and mass confirmation accelerate QC workflows.
• High sensitivity to low-level impurities supports regulatory compliance.
• Ion-pair RP-HPLC ensures reproducible retention and compatibility with MS detection.
• Adaptable to GMP environments for therapeutic oligonucleotide production.

Future trends and applications

Advances may include integration of MS/MS or MRM for sub-percent impurity quantification, automation of sample prep and data analysis, and exploration of novel stationary phases to improve separation of closely related sequences. Emerging high-resolution MS platforms will further enhance structural characterization and real-time process monitoring.

Conclusion

The combined HPLC-UV/MS approach provides robust confirmation of oligonucleotide identity, precise quantification of the main product and sensitive detection of impurities. This workflow meets the analytical demands of research, QA/QC and drug development for synthetic oligonucleotides.

References

• Roussis S.G., Rodriguez A.A., Rentel C. Determination of individual oligonucleotide impurities by small amine ion pair-RP HPLC MS and MS/MS: n-1 impurities. Journal of Chromatography B. 1169 (2021) 122611.