Comparison of biocompatible and inert UHPLC systems for LC-UV quantitation of large RNA

Comparison of biocompatible and inert UHPLC systems for LC-UV quantitation of large RNA — concise expert summary

Significance of the topic

Accurate chromatographic quantitation of large RNA and oligonucleotide therapeutics is critical for research, development, and quality control workflows. The highly anionic phosphodiester backbone of RNA can electrostatically interact with metallic surfaces in UHPLC flow paths, producing analyte loss, peak broadening, reduced sensitivity and poor reproducibility—effects that are particularly problematic at low analyte concentrations. Evaluating and minimizing these secondary interactions by using inert flow-path materials or coatings improves method robustness and lowers sample consumption for IP-RPLC and related LC-UV assays used in oligo and mRNA analytics.

Objectives and overview of the study

The study compared the Thermo Scientific Vanquish Amplify UHPLC system (an inert-coated flow path configuration) with: 1) a standard Vanquish system using MP35N alloy (biocompatible) capillaries and 2) a commercial inert platform (Waters Acquity Premier) to assess metal-induced secondary interactions. Performance was evaluated using: a metal-sensitive probe analyte (adenosine 5′-(α,β-methylene) diphosphate, AMPcP) co-injected with adenosine, and a practical test mixture of large RNA fragments (500 nt and 5,000 nt). The aim was to quantify differences in sensitivity, peak shape, and conditioning behavior across platforms under IP-RPLC conditions.

Methodology

• Separation mode: Ion-pairing reversed-phase liquid chromatography (IP-RPLC) tailored for large RNA.
• Probe test: Equimolar AMPcP and adenosine (11.7 µM total) evaluated under three mobile phase pH values (approx. pH 5.2, 6.7, 8.7) to probe pH dependence of metal interactions.
• Large RNA test: A 1:1 mix of 500 nt and 5,000 nt RNA (final concentration 12 ng/µL per component; 12 ng on-column) to assess real-sample behavior and sensitivity.
• Mobile phases for RNA: Eluent A = 100 mM triethylammonium acetate (TEAA); Eluent B = 100 mM TEAA / acetonitrile 75/25 (v/v).
• Detection and data handling: Downstream DAD UV detectors were matched between systems to ensure consistent detection conditions. Data acquired and processed with Thermo Scientific Chromeleon CDS 7.3.2 MUc.

Used instrumentation

• Thermo Scientific Vanquish Amplify UHPLC system — inert-coated flow path components (test subject).
• Thermo Scientific Vanquish UHPLC with MP35N alloy fluidics — biocompatible comparator.
• Waters Acquity Premier UPLC with MaxPeak HPS — cross-vendor inert-platform comparator.
• Chromatographic column: Thermo Scientific SurePac Oligo RP MDi, 2.5 µm, 2.1 × 50 mm.
• Detectors: Matched diode-array UV detectors installed downstream of each system’s native UV detector to standardize detection.
• Software: Chromeleon Chromatography Data System for acquisition and analysis.

Main results and discussion

• AMPcP probe results: Under conditions that favor metal interactions (notably acidic pH), the Vanquish Amplify system delivered up to a twofold increase in sensitivity for AMPcP compared with the MP35N-equipped Vanquish system. This indicates substantially reduced metal–analyte interactions for the Amplify flow path. Adenosine (non-metal-sensitive) served as an internal reference and showed no comparable sensitivity shifts, supporting the interpretation that observed differences were due to phosphate-metal interactions.
• pH dependence: Stronger secondary interactions were observed at acidic mobile phase pH; inert flow paths mitigated these effects most effectively under those conditions.
• Large RNA performance: For 5,000 nt RNA, signal responses were similar across the Vanquish Amplify and the Acquity Premier systems. For 500 nt RNA, Vanquish Amplify produced approximately 1.25–1.35× higher signal intensity versus the MP35N flow path and nearly twofold higher initial peak height versus the standard Vanquish, indicating improved sensitivity and lower secondary interactions for smaller large-RNA fragments.
• Conditioning behavior: The Acquity Premier system exhibited a measurable conditioning effect (changes across initial injections) for the 500 nt sample; the Vanquish Amplify flow path showed minimal or no conditioning, suggesting more consistent behavior over repeated injections.
• Practical testing throughput: Each platform was evaluated across repeated injections (cycles of five injections) over three days, totaling up to ~50 injections per system to probe reproducibility and conditioning trends.
• Limitations of probe analyte: While AMPcP is a sensitive indicator of metallic surface interactions, it does not fully replicate the interaction mechanisms of long phosphodiester backbones; thus combined evaluation with real RNA fragments was necessary for a practical assessment.

Benefits and practical applications of the method and findings

• Increased sensitivity: Reduced surface interactions on inert-coated flow paths raise signal intensity (up to ~2× for probe analytes and ~1.25–1.35× for certain RNA fragments), enabling lower limits of quantitation and reduced sample consumption—valuable for precious or low-concentration RNA samples.
• Improved reproducibility: Less conditioning and fewer injection-to-injection artifacts enhance quantitative reliability for QC and method transfer in oligonucleotide analytics.
• Method robustness: Inert UHPLC systems simplify method development for IP-RPLC of RNAs by minimizing variable surface adsorption effects that depend on pH and analyte size.
• Industry relevance: Findings support adoption of inert flow paths in workflows for therapeutic oligonucleotides, mRNA vaccines, and other large-RNA products where accurate LC-UV quantitation is required.

Future trends and potential applications

• Broader adoption of inert-coated or passivated flow paths in UHPLC instruments to standardize oligonucleotide analyses across laboratories and vendors.
• Extensions to mass-spectrometry workflows: Investigate compatibility and benefits of inert flow paths for LC–MS-based oligo characterization, where surface interactions can also compromise sensitivity and reproducibility.
• Development of standardized probe panels: Combine small phosphate-containing probes (like AMPcP) with representative long oligonucleotides to create method qualification kits for inertness benchmarking.
• Optimization of mobile phase chemistry: Systematic exploration of ion-pair reagents, pH, and organic modifiers to further reduce secondary interactions while preserving chromatographic resolution for a range of oligo sizes.
• Materials innovation: New alloys, coatings and single-material flow paths that minimize adsorption without compromising pressure/chemical resistance or biocompatibility.

Conclusion

This comparative evaluation demonstrates that UHPLC systems with inert-coated flow paths (Vanquish Amplify) significantly reduce metal–analyte secondary interactions for phosphate-containing analytes, improving sensitivity and reproducibility for LC-UV quantitation of large RNA and probe compounds. The Amplify system showed the largest benefits for metal-sensitive small probes and smaller large-RNA fragments (500 nt), while performance for very large fragments (5,000 nt) was comparable across the inert systems tested. Implementing inert flow-path technology can lower sample consumption, reduce conditioning artifacts, and increase confidence in quantitative oligonucleotide analytics used in R&D and QC contexts.

References

1. Lovejoy K., Žvirblis A., Grübner M., Fehrenbach T., & Steiner F. (2026). LC-UV analysis of large RNA using an inert UHPLC system (Technical Note No. 004606). Thermo Fisher Scientific.