Achieving Improved Sensitivity and Reliable Analytical Performances in Nucleotides Analysis

Significance of the Topic

The accurate and reproducible analysis of nucleotides is essential in biochemical research, clinical diagnostics and pharmaceutical development. Phosphate groups can interact strongly with metal surfaces in conventional UHPLC systems, causing peak tailing, variable signal intensity and poor quantitation. A fully inert flow path and metal-free column promise to suppress adsorption, improve sensitivity and ensure reliable data.

Objectives and Study Overview

This study compares two UHPLC configurations for separation and quantitation of adenosine monophosphate AMP, diphosphate ADP and triphosphate ATP at 50 µg/mL. A standard Nexera XS system with a metal-based flow path and column was evaluated against the Nexera XS inert system paired with a metal-free Shim-pack Scepter C18 column. Key performance metrics include peak shape, symmetry, injection-to-injection reproducibility, linearity of calibration curves and quantification accuracy.

Methodology

All analytes were dissolved in water and injected at 2 µL volume. Chromatographic conditions were identical for both setups: mobile phase acetonitrile/10 mmol/L ammonium formate 0.5/99.5 v/v, flow rate 0.2 mL/min, column temperature 40 °C and UV detection at 254 nm. Ten consecutive injections assessed the impact of metal adsorption on peak symmetry and area stability. Calibration curves for ATP were constructed from 1 to 50 µg/mL with six replicates per level. QC samples at 2, 20 and 45 µg/mL were used to evaluate accuracy and precision.

Used Instrumentation

• Nexera XS UHPLC system (metal-based and inert models)
• Shim-pack Scepter C18-120 column 100 mm × 2.1 mm I.D., 3 µm (metal-based and metal-free variants)
• SPD-M40 UV detector with inert flow cell
• TORAST-H Bio Vial for sample management

Main Results and Discussion

The inert configuration delivered sharp, symmetric peaks (symmetry factor ~1) and consistent peak areas over ten injections. In contrast, the metal-based system showed pronounced tailing, increasing symmetry factors and growing peak areas as surface adsorption progressed. The ATP calibration curve improved from r2=0.9918 (metal-based) to r2=0.9999 (inert). The metal-based setup failed to detect ATP at 1 µg/mL and showed large deviations at low levels, whereas the inert system quantified all levels with >96% accuracy and <1% precision. QC samples in the inert system returned accuracy between 98.2% and 100% and precision below 0.6%, compared to poor reproducibility in the metal-based configuration.

Practical Benefits and Applications

• Improved sensitivity and peak shape for phosphate-containing compounds
• Elimination of conditioning steps and mobile phase additives to suppress metal adsorption
• High linearity, accuracy and precision across a wide concentration range
• Enhanced throughput and robustness in nucleotide assays for research and QC laboratories

Future Trends and Applications

Adoption of fully inert UHPLC systems is expected to expand to metabolomics, nucleotide therapeutics and phosphorylation studies. Integration with high-resolution mass spectrometry will further enhance selectivity and detection limits. Development of more diverse metal-free stationary phases and green solvent methods will support sustainable, high-throughput analytical workflows.

Conclusion

The Nexera XS inert UHPLC coupled with a metal-free C18 column effectively suppresses metal adsorption, yielding superior peak shapes, stable signals and outstanding quantitation for nucleotides. This approach simplifies method development and enhances confidence in analytical results for phosphate-containing analytes.

Reference

• D. Fujimura, E. Ando, R. Suzuki Achieving Improved Sensitivity and Reliable Analytical Performances in Nucleotides Analysis Shimadzu Application News First Edition Feb. 2022