Rapid LC-UV/MS Workflow for Routine Oligonucleotide Impurity Monitoring Using the SQ Detector 2 Mass Detector and Empower™ Chromatography Data System (CDS)

Importance of the Topic

The analysis and control of impurities in antisense oligonucleotides (ASOs) is critical for product safety, efficacy and regulatory compliance. ASOs are produced by stepwise solid-phase synthesis, which commonly produces closely related truncated, capped or chemically modified by-products that may differ by only a single nucleotide or small mass shifts. Robust, high-throughput analytical workflows that combine reliable quantitation with identity confirmation are therefore essential for routine QC release testing and lifecycle impurity control.

Objectives and Overview of the Study

This application note evaluates a rapid ion-pair reversed-phase UPLC workflow (6.5 min) combining tunable UV detection and single-quadrupole mass detection (SQ Detector 2) under Empower CDS control for routine monitoring of a lipid-conjugated ASO and related impurities. Key aims were to demonstrate: reduction of runtime and sample consumption; retention of quantitative robustness via UV; use of nominal-mass MS to confirm identity and apportion coeluting species; and implementation of an audit-ready, standardised processing and reporting workflow suitable for regulated QC environments.

Methodology

The study used a 16-residue palmitate-conjugated gapmer ASO standard (monoisotopic mass ~6046.08 Da) and two forced-degradation preparations (oxidative and thermal) to generate impurity profiles.

• Chromatography: short ACQUITY Premier Oligonucleotide C18 column (130 Å, 1.7 µm, 2.1 × 50 mm) operated at 50 °C, flow 0.52 mL/min, injection volumes 1.6–4.8 µL, total run time 6.5 min.
• Mobile phases: ion-pairing with 5 mM tributylammonium acetate (TBuAA) and 1 µM EDTA in ACN/H2O mixtures; gradient optimized for fast separation.
• Detection: in-line Tunable UV (TUV) at 260 nm as primary quantitation; SQ Detector 2 (negative ESI) to acquire nominal-mass full scans and extracted-ion chromatograms (XICs).
• Sample handling: QuanRecovery vials with MaxPeak HPS surfaces; sample temp 5 °C; use of MaxPeak Premier HPS on system and column to minimise nonspecific adsorption.
• Data system: Empower 3.8.1 for controlled acquisition, blank subtraction, processing, custom calculations and reporting.

Used Instrumentation

• ACQUITY UPLC I-Class / ACQUITY Premier LC hardware with MaxPeak Premier HPS surfaces.
• ACQUITY Premier Oligonucleotide C18 Column, 130 Å, 1.7 µm, 2.1 × 50 mm.
• ACQUITY Premier Tunable UV (TUV) Detector.
• SQ Detector 2 single-quadrupole mass detector (negative ESI).
• Empower 3.8.1 Chromatography Data System for acquisition, processing and reporting.
• QuanRecovery vials with MaxPeak HPS.

Key Data Treatment and Calibration Strategy

The workflow preserves UV as the robust primary quantifier and applies MS-derived corrections for coeluting species using the following steps:

1. Blank subtraction: TUV chromatograms are blank-subtracted in Empower to remove mobile-phase background prior to integration.
2. MS charge-state selection: the dominant -4 charge state (m/z ~1511.52) was targeted; XICs were generated for FLP and known product-related masses (e.g., FLP (P = O), ~16 Da lower).
3. Adduct discrimination: spectra acquired under standard and harsher ionisation to distinguish labile adducts (which decrease under harsh conditions) from covalent product-related species (persistent signals).
4. Calibration: linear UV calibration (blank-subtracted UV area vs on-column amount) for concentration/purity; quadratic MS calibration for summed MS response (accounts for non-linearity in MS response across load range). Quadratic coefficients are applied in Empower custom fields.
5. Apportionment: summed MS response of FLP + FLP (P = O) is apportioned between species by relative XIC areas, and the MS-derived proportions are used to correct the UV main-peak purity when species co-elute.
6. Automated processing and reporting: predefined acquisition, processing and report templates in Empower standardise calculations and produce audit-ready reports.

Main Results and Discussion

• Chromatographic performance: the 2.1 × 50 mm column and optimized gradient achieved the required separation within 6.5 minutes, substantially reducing runtime versus conventional ~40 min methods while maintaining impurity resolution for the ASO studied.
• Reduced sample and solvent use: the fast method used over fivefold less sample per injection and consumed less mobile phase and ion-pair reagent, improving throughput and reducing environmental impact.
• MS-assisted peak correction: MS XICs successfully resolved the FLP and FLP (P = O) despite partial co-elution, enabling correction of the UV main-peak to report corrected assay purity and impurities. Use of the -4 charge state provided sufficient sensitivity within the SQ mass range.
• Adduct assessment: comparison of spectra under standard and harsh ionisation conditions enabled identification of adducts versus true product-related species, improving confidence in apportionment decisions.
• Processing efficiency: dividing the sequence into subsets for processing and using Empower custom fields and templates reduced analyst time and standardised review steps. The method supports traceable review and 21 CFR Part 11–style controls (access, audit trails, e-signatures).

Benefits and Practical Applications

• Rapid throughput suitable for routine QC release testing and impurity monitoring across many samples or batches.
• Retention of UV quantitation robustness combined with MS identity confirmation reduces false quantitation from co-eluting species and provides mass-based confidence for assigned peaks.
• Lower sample and solvent consumption preserves valuable ASO standards and reduces operational costs and environmental footprint.
• Use of MaxPeak HPS surfaces (column, LC hardware, vials) reduces nonspecific adsorption of oligonucleotides, improving recovery and sensitivity for trace impurities.
• Empower CDS integration supports standardisation, reproducibility, audit readiness and reduced analyst-dependent variability.

Limitations and Considerations

• Single-quadrupole nominal-mass detection requires prior characterisation (e.g., HRMS) to define expected masses and to establish which species to monitor; HRMS remains important for initial structural elucidation of unknowns.
• MS response non-linearity requires a quadratic calibration model; careful validation of calibration and dynamic range is necessary for regulatory QC use.
• Method performance may be sequence-dependent; method transfer and validation should be performed for different ASOs and conjugation chemistries.

Future Trends and Potential Applications

• Further integration of fast chromatographic methods with automated sample handling and CDS-driven workflows will accelerate QC batch release and reduce manual review tasks.
• Advances in single‑quadrupole sensitivity and software-based deconvolution could expand the range of impurities detectable without HRMS for routine monitoring.
• Continued development of low-adsorption materials and column chemistries tailored for oligonucleotides will further improve recovery and lower detection limits for trace impurities.
• Regulatory acceptance of combined UV+nominal-MS workflows for impurity apportionment is likely to increase as standardised processing and validated methods become more widespread.

Conclusion

The presented 6.5-minute IP-RP-UPLC workflow combining TUV quantitation, SQ Detector 2 nominal-mass confirmation and Empower CDS processing provides a practical, high-throughput approach for routine ASO impurity monitoring. By maintaining UV as the primary quantitative measurement and using MS XICs to confirm identity and apportion co-eluting species, the method balances robustness and selectivity while reducing runtime, sample consumption and analyst-dependent variability. When preceded by HRMS characterisation to define targets, this approach is well suited for regulated QC environments that require traceable, standardised and efficient impurity assessment.

References

1. Capaldi D, et al. Impurities in oligonucleotide drug substances and drug products. Nucleic Acid Therapeutics. 2017;27(6):309–322. doi:10.1089/nat.2017.0691.
2. Obexer R, et al. Modern approaches to therapeutic oligonucleotide manufacturing. Science. 2024;385:eadl4015. doi:10.1126/science.adl4015.
3. Rentel C, et al. Method referenced in Agilent Technologies Application Note: Determination of Purity, Assay, and Impurity Profile for a Ligand-Conjugated Antisense Oligonucleotide Using Agilent Oligo Analysis Accelerator for OpenLab CDS. Agilent; 2026.
4. Elmekawy A, et al. At-line analysis of antisense oligonucleotide purified fractions using fast chromatographic methods. J Chromatogr A. 2025;1764:466516. doi:10.1016/j.chroma.2025.466516.