HPLC-DAD Analysis of Nucleotides Using a Fully Inert Flowpath - Agilent 1260 Infinity II Bio-Inert LC System and a PEEK-lined Agilent InfinityLab Poroshell 120 HILIC-Z Column

Importance of the Topic

Phosphorylated compounds such as nucleotides frequently interact with stainless steel components in HPLC flow paths, causing adsorption, peak tailing, and quantitative losses. Reliable analysis of these molecules is critical in pharmaceutical, biopharmaceutical, and biochemical research.

Objectives and Study Overview

This application note evaluates the performance of a fully inert chromatographic system—Agilent 1260 Infinity II Bio-Inert LC—combined with a PEEK-lined InfinityLab Poroshell 120 HILIC-Z column for robust HILIC-DAD analysis of nucleosides and nucleotides. The goal is to compare recovery, peak shape, and reproducibility against a conventional stainless steel flow path.

Methodology and Instrumentation

• Instrumentation: Agilent 1260 Infinity II Bio-Inert LC System (quaternary pump, multisampler, thermostat, diode array detector) and PEEK-lined InfinityLab Poroshell 120 HILIC-Z column (2.1×100 mm, 2.7 μm).
• Reagents: Acetonitrile, water, ammonium acetate buffer at pH 9; nucleosides and nucleotides at 100 ppm.
• Chromatography: Gradient from 90% to 48% B (acetonitrile/100 mM buffer) over 12 min; flow rate 0.4 mL/min; column temperature 35 °C; detection at 260 nm.

Main Results and Discussion

Transitioning from a stainless steel flow path to a fully inert PEEK flow path markedly improved chromatographic performance:

• Stainless steel configuration exhibited significant peak tailing and reduced area, worsening with each additional phosphate group.
• The bio-inert system with PEEK-lined column delivered sharp Gaussian peaks and complete recovery (>80–110%) for all nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UTP).
• Reproducibility improved, with RSD values for retention time, area, and height generally below 1% on the inert system, compared to up to 21% on the steel system.

Benefits and Practical Applications

Employing a fully inert LC flow path eliminates metal-phosphate interactions, enabling accurate quantitation, reliable method transfer, and high-throughput analysis in pharmaceutical QC, metabolomics, and enzymatic studies.

Future Trends and Potential Applications

Further developments may include integrating inert hardware into LC-MS workflows, extending the approach to other metal-sensitive analytes (e.g., phosphopeptides, phospholipids), and adoption in multi-omics platforms for personalized medicine.

Conclusion

This study demonstrates that complete removal of metal components from both the instrument and column flow path is essential for precise, reproducible HILIC analysis of nucleotides. The Agilent bio-inert LC system with PEEK-lined HILIC column represents a robust solution for challenging phosphorylated analytes.

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