LC-UV analysis of large RNA using an inert UHPLC system

Significance of the topic

The accurate and sensitive analysis of oligonucleotides and large RNA (including mRNA surrogates) is critical for pharmaceutical and biopharmaceutical development, QC, and regulatory submissions. These analytes are highly anionic and prone to adsorption to metal surfaces in conventional LC systems, which degrades sensitivity, peak shape, and quantitative precision—especially at low concentrations. Minimizing metal–analyte interactions in the fluidic path therefore improves analytical reliability and reduces time and sample consumption during method development and routine testing.

Objectives and overview of the study

This technical evaluation assessed the analytical performance and system inertness of the Thermo Scientific Vanquish Amplify UHPLC system for metal-sensitive analytes. Goals included:

• Quantifying the reduction in metal–analyte interactions versus an MP35N-based Vanquish system and a third‑party UPLC (Waters Acquity Premier with MaxPeak HPS).
• Evaluating sensitivity, peak area/height, and reproducibility using a metal-binding probe (AMPcP) co-injected with adenosine and using large RNA components (500 nt and 5,000 nt).
• Demonstrating whether Amplify’s fully inert sample flow path yields stable results without pre-conditioning injections.

Methodology

Key experimental design elements:

• Probe analyte test: equimolar AMPcP (metal-binding) and adenosine (non-binding reference) at three concentrations (1,176 µM, 117.6 µM, and 11.76 µM) and three mobile phase pH values (5.2, 6.7, 8.7) using 10 mM ammonium acetate isocratic method (200 µL/min, 1 µL injection).
• Large RNA test: 500 nt and 5,000 nt RNA components from an RNA ladder, combined to 12 ng/µL each; 1 µL injections (24 ng total) analyzed by ion-pair reversed‑phase LC with TEAA mobile phases and a SurePac Oligo RP MDi column (2.1 × 50 mm, 2.5 µm).
• Injection schedules included repeated injections (multiple sets of five injections with interspersed washes) across three days to evaluate first-injection performance, saturation/conditioning effects, and inter-day reproducibility.
• Detection was by downstream diode array detectors (DAD) at 260 nm; Chromeleon CDS was used for data acquisition and analysis.

Used instrumentation

The study compared three systems and used matched columns and detectors to isolate fluidic effects:

• Thermo Scientific Vanquish Amplify UHPLC system (fully inert sample flow path: inert needle, needle seat, 25 µL loop, inert capillaries, active preheater, inert flow cell).
• Thermo Scientific Vanquish UHPLC system with MP35N biocompatible fluidics (MP35N is iron-free, corrosion‑resistant alloy but still permits some metal–analyte interactions).
• Waters Acquity Premier UPLC with MaxPeak HPS fluidics (third‑party comparison).
• Columns: Thermo Scientific SurePac Oligo RP MDi (inert column hardware) for all tests.
• Auxiliary: Vanquish DADs, Chromeleon CDS, ultrapure water, LC‑MS grade solvents, TEAA and ammonium acetate mobile phases.

Results and discussion

Summary of main findings:

• AMPcP probe results: Under the most challenging conditions (pH 5.2, lowest analyte concentration 11.76 µM), the Vanquish Amplify system delivered roughly a twofold increase in sensitivity (AMPcP:adenosine peak height ratio) compared to the Vanquish/MP35N system. The benefit was most pronounced at low concentration and acidic pH, reflecting the increased propensity for metal adsorption under those conditions.
• pH dependence: Acidic mobile phases (pH 5.2) markedly increased nonspecific surface interactions for metal-binding analytes; more basic pH and higher analyte concentrations reduced the relative benefit of the inert flow path.
• Large RNA performance: For the first injection, Amplify produced 1.35× higher peak area for the 500 nt RNA and 1.25× higher peak area for the 5,000 nt RNA compared to the Vanquish/MP35N system. Peak heights showed similar improvement factors (≈1.19–1.24×).
• Conditioning/saturation behavior: Systems with MP35N fluidics exhibited saturation behavior (requiring multiple injections to stabilize peak area). The Amplify system provided reliable and higher signal from the first injection, eliminating the need for sacrificial conditioning injections in many cases.
• Cross-vendor comparison: Amplify and the Waters Acquity Premier produced comparable performance for repeated injections (fifth injection and beyond) when matched detectors and columns were used. Amplify showed slightly better inertness for the smaller RNA fragment (500 nt) in the tested sequence.
• Wash protocols: Aggressive washes with 0.1% TFA in organic or 50 mM ammonium acetate (pH 9.2) were used to reset systems between sequences; however, the Amplify system reduced dependency on extensive conditioning washes for consistent quantitative results.

Benefits and practical applications

Practical advantages demonstrated by the Vanquish Amplify UHPLC system include:

• Improved sensitivity for metal-sensitive oligonucleotides and nucleotide analogs (up to ~2× for extreme cases), enabling lower limits of quantitation or lower sample consumption.
• Better first-injection reliability and reduced need for sacrificial injections or prolonged conditioning, saving time and scarce sample material during method development and routine assays.
• Enhanced reproducibility of peak area and height, particularly important for low-concentration samples and stability/QC testing.
• Compatibility with standard ion-pair reversed-phase methods and common columns used for oligonucleotide separations, facilitating method transfer.

Future trends and potential applications

Opportunities and expected developments:

• Broader adoption of fully inert fluidics in LC systems to improve quantitation of metal‑coordinating biomolecules, including modified oligonucleotides, siRNA, and mRNA constructs.
• Integration of inert UHPLC platforms with LC–MS workflows to combine reduced adsorption artifacts with high-sensitivity mass analysis for impurity profiling and identity confirmation.
• Refinement of mobile-phase and surface-coating chemistries to further minimize secondary interactions between ion-pair reagents, analytes, and stationary phases.
• Standardization of conditioning and wash protocols for oligonucleotide assays to improve method robustness across instrument platforms and labs.

Conclusion

The study demonstrates that replacing wetted metal components with a fully inert sample flow path, as implemented in the Vanquish Amplify UHPLC system, substantially reduces metal–analyte interactions for highly polar, anionic molecules. This yields improved sensitivity (up to ~2× for the probe analyte under worst-case conditions), higher first-injection signal for large RNA (1.25–1.35×), and more consistent quantitative performance without extensive conditioning. These benefits are particularly valuable in pharmaceutical and biopharmaceutical analyses where sample is limited and accurate quantitation of metal-sensitive oligonucleotides is required.

References

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3. Sinha ND, Jung KE. Analysis and Purification of Synthetic Nucleic Acids Using HPLC. Current Protocols in Nucleic Acid Chemistry. 2015;61(1):10.5.1. doi:10.1002/0471142700.nc1005s61