Improved Oligonucleotide LC–MS Performance through Inert Column Hardware and Alternative Separation Strategies

Significance of the topic

Accurate LC–MS analysis of oligonucleotides is essential for sequence confirmation, impurity profiling, and quality control in research and biopharmaceutical development. Common ion-pairing reagents and metal contact surfaces in LC systems can degrade chromatographic performance, lower recovery, complicate mass spectra through adduct formation, and introduce environmentally harmful reagents such as HFIP (a PFAS). Evaluating alternative separation modes and inert hardware addresses analytical robustness, environmental impact, and data clarity for oligonucleotide workflows.

Objectives and study overview

This work compared three LC separation strategies for oligonucleotides—conventional ion-pair reversed phase (IP-RP), ion-pair-free reversed phase (IP-free RP), and hydrophilic interaction liquid chromatography (HILIC)—and quantified the benefits of minimizing metal interactions via Ultra Inert column hardware, an inert flow path, and polypropylene solvent bottles. The goals were to assess: chromatographic resolution and MS signal quality across methods, the impact of inert hardware on equilibration and recovery, the feasibility of lowering ion-pair reagent concentration, and the effect of reducing system sodium on adduct formation.

Methodology

Samples analyzed:

• Agilent RNA resolution standard and DNA ladder
• 25-mer and 50-mer DNA
• Fully thiolated hybrid DNA
• 2'-O-methyl (2-MeO) RNA

Instrument platform:

• Agilent 1290 Infinity II Bio HPLC
• Agilent 6545XT AdvanceBio QTOF MS

Columns and stationary phases evaluated:

• Agilent AdvanceBio Oligonucleotide, 2.1 × 50 mm, 2.7 µm
• Altura Oligo HPH-C18, 2.1 × 50 mm, 2.7 µm
• Agilent AdvanceBio Glycan Mapping, 2.1 × 150 mm, 2.7 µm
• Prototype Ultra Inert Glycan Mapping, 2.1 × 150 mm, 2.7 µm

Representative LC and MS conditions (summarized):

• IP-RP mobile phases: hexylamine with HFIP in methanol/water; columns at elevated temperatures (60–75 °C in some methods) and flow rates from 0.21 to 0.6 mL/min.
• IP-free RP mobile phase: ammonium bicarbonate with methanol; analyses performed in positive ion mode with adjusted MS gas and voltages for improved signal.
• HILIC mobile phases: ammonium acetate in ACN/water mixtures run at moderate flow and lower column temperature (≈30 °C); analyses in negative ion mode typical for HILIC.

Used instrumentation

• Liquid chromatography: Agilent 1290 Infinity II Bio HPLC.
• Mass spectrometry: Agilent 6545XT AdvanceBio QTOF with tuned source parameters for each separation mode (ionization polarity, gas temps, sheath gas, fragmentor, nozzle/skimmer voltages adjusted per method).
• Columns: commercial AdvanceBio and Altura oligo columns plus prototype Ultra Inert Glycan Mapping column implemented with Ultra Inert hardware.
• Ancillary consumables: comparison of glass vs polypropylene solvent bottles to control sodium contamination.

Main results and discussion

Equilibration and recovery:

• Ultra Inert column hardware reached reproducible performance faster than stainless steel columns, decreasing the need for conditioning injections (saving time, solvent, and sample).
• Even after equilibration, stainless steel hardware showed 13–17% lower sample recovery compared with Ultra Inert hardware.

Ion-pair reagent usage and resolution:

• Using Ultra Inert hardware allowed substantial reduction of ion-pair reagent concentrations (example reduction to 15 mM hexylamine + 25 mM HFIP from higher conventional concentrations) while preserving resolution—Ultra Inert columns resolved close species (39-mer vs 40-mer) under lower ion-pair conditions where stainless steel columns failed.
• Reducing ion-pair reagents lowers costs and reduces instrument contamination risk.

MS spectral quality and adduct formation:

• Replacing glass solvent bottles with polypropylene markedly reduced sodium adduct formation, simplifying spectra and deconvolution of oligonucleotide masses.
• IP-free RP analyzed in positive ion mode produced stronger signal and fewer adducts relative to negative-mode IP-RP, yielding comparable MS data for many analytes.

Alternate separation modes:

• IP-RP still provided the highest chromatographic resolution overall, especially for longer oligonucleotides.
• IP-free RP and HILIC offered MS-friendly alternatives: for shorter oligonucleotides both provided acceptable resolution when coupled to MS detection; HILIC revealed a more complex impurity profile for 2-MeO RNA, enabling detection and characterization of low-level variants.

Mass accuracy:

• Monoisotopic mass errors for analyzed oligonucleotides were generally low (sub-1 ppm to a few ppm) across methods. Fully thiolated oligonucleotides showed larger negative mass errors under some IP-RP conditions at higher flow rates, but these improved under optimized conditions.

Benefits and practical applications of the method

• Ultra Inert hardware increases recovery and reproducibility while reducing conditioning time, improving laboratory throughput.
• Lowering ion-pair reagent load reduces reagent cost and instrument contamination and minimizes reliance on PFAS reagents such as HFIP, aligning with environmental and regulatory goals.
• IP-free RP and HILIC provide MS-compatible workflows that reduce adduct complexity and facilitate deconvolution, beneficial for QC, impurity profiling, and method development for oligonucleotide therapeutics.
• Switching to polypropylene solvent reservoirs is a simple, high-impact measure to decrease sodium adducts and simplify spectral interpretation.

Future trends and potential applications

Adoption of inert flow paths and column hardware is likely to expand for oligonucleotide analytics to enhance sensitivity and robustness. Ion-pair-free methods and HILIC will be increasingly used where MS clarity and environmental considerations are priorities. Expect further development of stationary phases and column hardware optimized for oligonucleotides, expanded use of lower-toxicity mobile-phase chemistries, and integration with high-resolution MS workflows and automated data processing to accelerate impurity identification. Prototype Ultra Inert stationary phases tailored for HILIC/RP hybrids may offer additional selectivity for difficult separations.

Conclusion

Minimizing metal interactions via Ultra Inert column hardware and using inert solvents/containers materially improves oligonucleotide LC–MS performance: faster equilibration, higher recovery, and clearer mass spectra with fewer adducts. While IP-RP maintains the best chromatographic resolution for longer sequences, IP-free RP and HILIC are viable, MS-friendly alternatives—especially for shorter oligonucleotides and impurity profiling—offering reduced reliance on PFAS-containing reagents and simpler spectral interpretation.

Reference

1. Coffey A, et al. Eliminate Metal Ions from Your Oligonucleotide LC/MS analysis. Agilent application note 5994-9018EN. 2026.
2. Ryu C. Oligonucleotide Analysis Using the Agilent InfinityLab Pro iQ and Altura Oligo HPH-C18 Column. Agilent application note 5994-9057EN. 2026.
3. Bertram L, et al. Analysis of Oligonucleotides Using an Ion-Pairing-Free Reversed-Phase Method with TOF LC/MS. Agilent application note 5994-8013EN. 2024.
4. Hsiao J, et al. Evaluating HILIC Stationary Phases for Oligonucleotide Separation by LC/MS. Agilent application note 5994-8180EN. 2025.