Eliminate Metal Ions from Your Oligonucleotide LC/MS Analysis

Significance of the topic

Liquid chromatography–mass spectrometry (LC–MS) of oligonucleotides is widely used in bio/pharma laboratories for identity, purity and impurity profiling. Metal ions—both iron from stainless-steel instrument hardware and alkali metals such as sodium and potassium—can cause adsorption, peak tailing, low recovery, oxidation and complex adduct patterns in mass spectra. These effects reduce sensitivity, complicate data interpretation and increase development and QC time. Methods and hardware that minimize metal interactions and alkali adducts therefore improve analytical robustness, accuracy and throughput.

Goals and study overview

This application note compared oligonucleotide LC–MS performance under different chromatographic and hardware conditions to identify practical approaches that reduce metal-related artifacts. Key objectives were to:

• Evaluate the impact of stainless-steel versus ultra-inert coated column hardware on recovery, sensitivity and mass accuracy.
• Characterize alkali metal adduct formation and test procedural controls to reduce adduction.
• Compare a conventional ion-pair reversed-phase method (hexylamine/HFIP/methanol) with an ion-pair–free reversed-phase method (ammonium bicarbonate/methanol) for LC–MS compatibility and sensitivity.

Used instrumentation

Instrumentation used in the study (important for method transfer) included:

• Agilent 1290 Infinity II LC platform: high-speed pump (G7132A), multisampler (G7167B), multicolumn thermostat (G7116B), and diode array detector (G7115A).
• Agilent 6545XT AdvanceBio Q-TOF LC/MS (G6545BA) for high-resolution MS detection.
• Agilent MassHunter Acquisition and Qualitative Analysis software (versions as reported).

Methodology and experimental details

Samples: custom oligonucleotides were analyzed (25‑mer DNA, 50‑mer DNA and a fully thiolated 20‑mer DNA/RNA hybrid). Samples were prepared at 1 mg/mL in ultrapure water; polypropylene (bio) solvent bottles were used to minimize alkali contamination.
Chromatographic approaches compared:

• Ion‑pair reversed‑phase: hexylamine + HFIP in methanol/water on Agilent AdvanceBio Oligonucleotide column. Negative ionization MS for this mode.
• Ion‑pair‑free reversed‑phase: 20 mM ammonium bicarbonate with methanol as organic modifier on the same stationary phase, with positive-mode MS detection. This approach avoids HFIP (a volatile fluorinated ion-pair reagent) and reduces PFAS use.

Key operating observations: higher flow rates and short gradients (0.6–0.8 mL/min) caused suboptimal peak shape and increased metal adducts; lowering the flow rate (example optimized to ≈0.21–0.4 mL/min) improved peak shape, impurity resolution and reduced observed adduction.

Results and discussion

Hardware effects on recovery and sensitivity:

• Stainless-steel column hardware showed reduced initial UV peak area and poorer recovery on repeated injections, consistent with adsorption to exposed metal ion sites.
• Agilent Altura Ultra Inert (UI) coated Altura Oligo HPH‑C18 columns substantially improved UV and MS peak area, sensitivity and reproducibility compared with stainless‑steel AdvanceBio Oligonucleotide columns.

Alkali adduct formation and mass spectral complexity:

• Both column types showed sodium (+22 Da) and potassium (+38 Da) adducts and combinations ([2 Na+] +44, [Na+ K+] +60, [2 K+] +76) in many charge states, complicating charge-state envelopes and deconvolution.
• Adduct levels were reduced by careful sample handling (use of ultrapure water, polypropylene bottles), by using lower flow rates, and by using the UI coated column.

Ion‑pair vs ion‑pair‑free approaches:

• The ion‑pair free ammonium bicarbonate/methanol method produced positive-mode ionization, a slightly different charge-state distribution and generally reduced adduct levels compared with the hexylamine/HFIP method run at higher flow rates.
• Deconvoluted average masses were consistent across methods when metal-induced biases were minimized, indicating that ion‑pair‑free conditions can deliver comparable mass accuracy while avoiding HFIP.

Quantitative mass accuracy comparisons (representative):

• 25‑mer DNA: observed masses on Altura UI were within ~0.5–0.7 ppm of theoretical under ion‑pair and ion‑pair‑free conditions.
• 50‑mer DNA: observed masses on Altura UI were within ~0.5 ppm; accurate deconvolution improved with lower flow rates.
• Fully thiolated oligonucleotide: on the stainless‑steel column a large mass error was observed (≈–63.7 ppm) under ion‑pair reversed‑phase, while the Altura UI column reduced the error to ≈–5.6 ppm (and to ≈–3.5 ppm at lower flow). Under ion‑pair‑free conditions the Altura UI gave excellent accuracy (≈0.22 ppm).

These data demonstrate that column hardware and mobile-phase choices materially affect both chromatographic performance and MS mass accuracy for oligonucleotides.

Benefits and practical applications

Practical benefits observed and recommended actions:

1. Replace or avoid exposed stainless‑steel flow paths and column hardware where possible—ultra‑inert coated columns reduce adsorption, increase sensitivity and improve mass accuracy.
2. Minimize alkali metal contamination: use ultrapure reagents and polypropylene solvent bottles, avoid glass where practical, and apply blank gradients when switching away from ion‑pair reagents.
3. Consider lower flow rates and slightly longer gradients to improve peak shape, impurity resolution and reduce adduct formation.
4. Where HFIP use is a concern (PFAS), adopt an ion‑pair‑free ammonium bicarbonate/methanol method as a viable MS‑compatible alternative; this also shifts ionization polarity and can enhance sensitivity for some analytes.
5. For heavily modified oligonucleotides (e.g., fully thiolated), expect broader chromatographic peaks due to diastereomer mixtures, but mass spectra and deconvoluted masses remain interpretable if adducts are controlled.

Future trends and potential uses

Expected directions and opportunities:

• Broader adoption of ultra‑inert hardware (coatings, low-iron alloys) and biocompatible LC flow paths to reduce metal-induced artifacts across oligonucleotide and peptide analyses.
• Further development of ion‑pair‑free stationary phases and mobile-phase chemistries to eliminate PFAS/HFIP dependence while maintaining chromatographic retention and MS sensitivity.
• Integration of routine protocols for minimizing alkali contamination (consumables, sample prep, system flushing) into QC workflows to improve data reproducibility.
• Instrument manufacturers may extend coated components (valves, tubing, frits) and offer materials like MP35N or polymeric flow paths to balance mechanical strength with low metal exposure.

Conclusion

Reducing metal interactions in oligonucleotide LC–MS requires both appropriate hardware and careful LC/MS method design. Switching from stainless‑steel to ultra‑inert coated column hardware (Agilent Altura Oligo HPH‑C18 in this study), controlling alkali metal contamination, lowering flow rates and considering ion‑pair‑free mobile phases together produce measurable improvements in recovery, peak shape, impurity resolution and mass accuracy. These steps simplify spectral interpretation and improve confidence in oligonucleotide characterization for research and regulated environments.

References

1. Vanhoenacker G, et al. Evaluation of Different Ion‑Pairing Reagents for LC/UV and LC/MS Analysis of Oligonucleotides. Agilent Technologies application note, publication 5994‑2957EN, 2024.
2. Bertram L, Hsiao J. Analysis of Oligonucleotides Using an Ion‑Pairing‑Free Reversed‑Phase Method with TOF LC/MS. Agilent Technologies application note, publication 5994‑8013EN, 2024.
3. Hsiao J, Bertram L, et al. Evaluating HILIC Stationary Phases for Oligonucleotide Separation by LC/MS. Agilent Technologies application note, publication 5994‑8180EN, 2025.