Overcoming Non-Specific Adsorption in Oligonucleotide HILIC Analysis: A Comparison of Passivation and MaxPeak™ High-Performance Surfaces Technology

Significance of the Topic

Oligonucleotides are increasingly central to research and therapeutic development but pose analytical challenges due to strong non-specific adsorption (NSA) to metal oxide sites throughout LC flow paths. NSA reduces recovery, distorts peak shape, increases variability and carryover, and compromises quantitative confidence—issues that are especially acute for HILIC analyses performed without ion-pairing reagents. Durable control of NSA is therefore critical for reliable characterization and quantification of native and chemically modified oligonucleotides in both R&D and regulated environments.

Objectives and Study Overview

This application note compares conventional stainless-steel UPLC hardware and columns with Waters ACQUITY Premier hardware and ACQUITY Premier Columns that employ MaxPeak High-Performance Surfaces (HPS). The study evaluated out-of-the-box performance, the effect of a defined passivation protocol, and the durability of adsorption mitigation across multiple injections. Performance was quantified primarily by percent recovery and reproducibility for a homopolymeric oligodeoxythymidine standard and further challenged with a mixed-base oligonucleotide representative of real-world sequences.

Methodology

Key experimental design elements:

• Separation mode: HILIC without ion-pairing additives to avoid masking NSA effects.
• Test matrix: four system/column configurations — ACQUITY Premier System or ACQUITY UPLC H‑Class PLUS System each paired with either a stainless‑steel column or an ACQUITY Premier Column of identical stationary phase chemistry.
• Sample standards: Waters MassPREP OST (oligodeoxythymidine series dT15–dT35) prepared in 20:80 water:acetonitrile; additional custom mixed‑base oligonucleotide tested on Premier platform.
• Conditioning and passivation workflow: 24-hour column equilibration, six pre‑passivation standard injections, 20 passivation injections at 2× concentration (20 µL, 80 pmol/µL), and six post‑passivation standard injections. The first post‑passivation injection served as the reference for percent recovery calculations.
• Performance metrics: percent recovery of target oligos, total peak area, and relative standard deviation (%RSD) across injections to assess stability and reproducibility.

Instrumentation Used

Instruments and principal method parameters reported in the study:

• LC systems: ACQUITY Premier UPLC System and ACQUITY UPLC H‑Class PLUS System.
• Columns: ACQUITY Premier BEH Amide Column, 1.7 µm, 2.1 × 100 mm (Premier column) and ACQUITY UPLC BEH Amide Column, 1.7 µm, 2.1 × 100 mm (stainless‑steel column equivalent chemistry).
• Detectors: Tunable UV (TUV) and PDA at 260 nm, 10 Hz sampling.
• Temperatures: column at 60 °C; sample at 10 °C.
• Flow and injection: 0.5 mL/min; standard injection 2 µL (40 pmol/µL); passivation injection 20 µL (80 pmol/µL).
• Mobile phases: A = water; B = acetonitrile; C = 50 mM ammonium acetate, pH 6.7. Sample manager washes and purges used 20:80 water:acetonitrile.
• Consumables and software: Total Recovery Vials; Empower 3.8.1 CDS for data acquisition and analysis.

Main Results and Discussion

Out‑of‑the‑box behavior:

• Stainless‑steel column configurations retained essentially all homopolymer analytes pre‑passivation, demonstrating extreme adsorption to unmodified metal surfaces.
• Replacing the stainless‑steel column with an ACQUITY Premier Column (same stationary phase) produced a substantial immediate improvement: all five oligomers were detected regardless of the system chassis, indicating column surface chemistry is a dominant factor.

Quantitative recovery and reproducibility findings (dT25 as representative species):

• ACQUITY Premier System + Premier Column: pre‑passivation recovery started at ≈88% (first injection) and reached ≈95% by the sixth pre‑passivation injection, indicating minimal initial NSA and rapid stabilization.
• ACQUITY UPLC H‑Class PLUS System + Premier Column: lower pre‑passivation recovery (≈67% to 77%) demonstrating system‑level surfaces further contribute to NSA even when the column is engineered.

Effects of passivation and durability:

• Passivation improved stainless‑steel column performance to allow peak detection, but peak shape, recovery and stability remained inferior to Premier column-based configurations.
• On both systems the first post‑passivation injection with a Premier Column produced similar recovery; however, on the ACQUITY Premier System recovery stayed effectively constant across five subsequent injections (≈100% through five injections, 99% on the sixth) with excellent reproducibility (0.3% RSD for dT25).
• On the ACQUITY UPLC H‑Class PLUS System the Premier Column’s recovery declined after the first post‑passivation injection (91% second, 86% by sixth), with higher variability (5.7% RSD), illustrating transient passivation when stainless‑steel flow‑path components remain present.
• Stainless‑steel column configurations showed rapid loss of passivation and very high variability: %RSD values of 37.3% (Premier System) and 118% (H‑Class PLUS System) for dT25, confirming unstable quantitation when unmodified metal surfaces are present.

Performance with a mixed‑base oligonucleotide:

• A custom 30‑mer containing A, C, G, and T analyzed on the ACQUITY Premier System with Premier Column produced a symmetric, well‑resolved main peak and three adjacent impurities with excellent stability: retention time %RSD ≤ 0.34% and relative area %RSD ≤ 0.05% across 12 injections.
• This demonstrates that the Premier platform maintains chromatographic integrity for structurally diverse, application‑relevant oligonucleotides beyond simple homopolymers.

Benefits and Practical Applications

Key practical outcomes reported:

• MaxPeak HPS on ACQUITY Premier System and columns minimizes analyte loss due to NSA, enabling accurate quantification for adsorption-prone oligonucleotides without reliance on mobile‑phase masking agents.
• The Premier platform sustains high recovery and low variability after passivation, providing a durable and reproducible workflow suited for routine analysis, method development, and transfer.
• The system supports analysis of mixed‑base and chemically diverse oligonucleotides representative of therapeutic development pipelines and QC testing.

Future Trends and Potential Applications

Implications and forward-looking considerations:

• Surface engineering of LC flow paths (e.g., HPS coatings, biocompatible alloys) will become increasingly critical as oligonucleotide and conjugated oligo modalities proliferate in therapeutics and diagnostics.
• Combination strategies — inert bulk materials (MP35N®, titanium, PEEK) plus targeted surface modifications — are likely to be standard practice for robust bioanalytical platforms, reducing the need for mobile‑phase additives that can complicate downstream MS or method transfer.
• Further work could extend to coupling HPS‑treated systems with LC–MS workflows, systematic evaluation of chemically modified oligonucleotides (e.g., phosphorothioates, ligand conjugates), and standardized passivation/qualification protocols for regulated laboratories.

Conclusion

This study demonstrates that NSA remains a major impediment to reliable oligonucleotide HILIC analysis when stainless‑steel components are present in the flow path. MaxPeak HPS combined with biocompatible ACQUITY Premier hardware and Premier Columns delivers substantially higher analyte recovery, superior peak shape, and markedly improved reproducibility relative to stainless‑steel hardware, both before and after passivation. Effective NSA control requires a holistic approach addressing all analyte‑contacting surfaces; surface‑engineered hardware provides a durable and practical solution for high‑quality oligonucleotide characterization and quantitation.

References

1. Eckstein F. Phosphorothioates, Essential Components of Therapeutic Oligonucleotides. Nucleic Acid Ther. 2014;24(6):374–387. doi:10.1089/nat.2014.0506.
2. Khvorova A, Watts JK. The Chemical Evolution of Oligonucleotide Therapies of Clinical Utility. Nat Biotechnol. 2017;35(3):238–248. doi:10.1038/nbt.3765.
3. Gilar M, Stoll DR. Challenges and Solutions in Oligonucleotide Analysis, Part I: An Overview of Liquid Chromatography Methods and Applications. LCGC Int. 2025;2(7).
4. Apffel A, Chakel JA, Fischer S, Lichtenwalter K, Hancock WS. Analysis of Oligonucleotides by HPLC−Electrospray Ionization Mass Spectrometry. Anal Chem. 1997;69(7):1320–1325. doi:10.1021/ac960916h.
5. Crooke ST, Witztum JL, Bennett CF, Baker BF. RNA‑Targeted Therapeutics. Cell Metab. 2018;27(4):714–739. doi:10.1016/j.cmet.2018.03.004.
6. Goodchild J. Conjugates of Oligonucleotides and Modified Oligonucleotides: A Review of Their Synthesis and Properties. Bioconjug Chem. 1990;1(3):165–187. doi:10.1021/bc00003a001.