Comparability Studies for the Analysis of Nucleotides on Four Different LC Systems

Importance of the Topic

Accurate analysis of nucleotides is critical in biopharmaceutical research and quality control because these phosphorylated molecules can interact with metal surfaces in standard liquid chromatography (LC) systems, leading to peak distortion and sample loss. Minimizing analyte–surface interactions ensures reliable quantification and reproducible results for processes such as nucleotide purity assessment and metabolic profiling.

Study Objectives and Overview

This study evaluates the performance of four LC systems for separating adenosine and its phosphorylated derivatives (AMP, ADP, ATP) on a PEEK-lined HILIC column. Two low-adsorption, bio-inert configurations (Agilent 1260 Infinity II Bio-Inert LC and 1260 Infinity II Prime Bio LC) are compared with two stainless steel–based systems (Agilent 1290 Infinity II LC and 1260 Infinity II LC). The goal is to assess peak shape, reproducibility, and sample loss without extensive system conditioning.

Used Instrumentation

• Column: InfinityLab Poroshell 120 HILIC-Z, 2.1×150 mm, 2.7 µm, PEEK-lined
• Mobile Phases: 10 mM ammonium acetate pH 9 (A) and ACN/100 mM ammonium acetate pH 9 (9:1 v/v) (B)
• Gradient: 90 % B to 50 % B over 12 min, 0.4 mL/min, 35 °C
• Detector: UV at 260 nm
• Systems:
  • 1260 Infinity II Bio-Inert LC (PEEK-clad flow path)
  • 1260 Infinity II Prime Bio LC (MP35N alloy)
  • 1290 Infinity II LC (stainless steel)
  • 1260 Infinity II LC (stainless steel)

Methodology

Equimolar nucleotide solutions (80–400 µM) were prepared fresh in water. Analyses proceeded in sequence across the four systems using identical columns, solvents, and injection conditions. Adenosine served as an internal standard to normalize sample loss. Retention time (RT) and peak area relative standard deviations (RSDs) were recorded over seven consecutive injections for each system.

Key Results and Discussion

Both low-adsorption systems achieved baseline separation of all four analytes with symmetrical Gaussian peak shapes and RSDs below 1 % for RT and area. In contrast, stainless steel–based systems exhibited pronounced tailing and reduced recovery for phosphorylated nucleotides. ATP and ADP showed the highest sample loss, exceeding 80 % at 80 µM on stainless steel systems. Sample loss decreased at higher concentrations but remained substantial, demonstrating persistent analyte–metal interactions.

Benefits and Practical Applications

• Avoidance of time-consuming passivation procedures and acid or chelator flushing
• Improved reproducibility and peak symmetry for metal-sensitive analytes
• Enhanced confidence in quantitative nucleotide analysis for biopharma workflows
• Extended instrument uptime and reduced maintenance costs

Future Trends and Opportunities

Developments in inert flow-path materials and surface coatings promise further improvements. Emerging stationary phases tailored for hydrophilic interaction and mixed-mode separations may enhance analyte retention without metal interactions. Integration with mass spectrometry will benefit from bio-inert interfaces to preserve sensitivity for phosphorylated compounds.

Conclusion

Low-adsorption LC systems equipped with PEEK-clad or MP35N flow paths deliver superior performance for nucleotide separations, offering reproducible, high-quality data without extensive conditioning. Stainless steel systems suffer from analyte interaction and sample loss, especially for multi-phosphorylated species. Bio-inert configurations are recommended for reliable nucleotide analysis in research and QA/QC laboratories.

References

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