Demonstrating Improved Sensitivity and Dynamic Range with MaxPeak High Performance Surface (HPS) Technology: A Case Study on the Detection of Nucleotides

Importance of the Topic

Analysis of nucleotides by LC-MS is essential for advancing our understanding of cellular metabolism, genetic functions, and the development of antiviral therapies. The strong affinity of phosphorylated nucleotides for metal surfaces in conventional LC-MS systems leads to poor recoveries and compromised sensitivity, creating a major barrier to reliable quantitation.

Objectives and Overview of the Study

This case study evaluates the performance of the Waters ACQUITY PREMIER LC System equipped with MaxPeak High Performance Surface technology. Using adenosine and its phosphorylated derivatives (AMP, ADP, ATP) as model analytes, the work demonstrates how inert chromatographic surfaces enhance detection, peak shape, and dynamic range compared to standard stainless-steel hardware.

Methodology and Instrumentation

A reversed-phase UPLC-MS method was established using a 50 mm HSS T3 column (1.8 µm) on both standard and PREMIER systems. Mobile phases consisted of 10 mM ammonium acetate (pH 6.8) and acetonitrile with a three-minute gradient. Sample injection was 1 µL of an equimolar nucleotide mixture. Detection employed a Xevo TQ-XS triple quadrupole mass spectrometer in negative electrospray mode with SRM acquisition. TruView vials minimized sample adsorption.

Main Results and Discussion

Standard stainless-steel LC systems showed complete loss of ATP and ADP and severe peak tailing for AMP. Introducing only the PREMIER column partially restored detection, but full mitigation of metal-analyte interactions required both the PREMIER system and column. Under these conditions, all four analytes exhibited sharp peaks, large areas, and linear calibration over more than three orders of magnitude with sub-pg/µL detection limits. Adenosine without phosphate groups remained unaffected across all setups, confirming that performance gains arose from surface inertness.

Benefits and Practical Applications

• Enhanced sensitivity for metal-sensitive analytes
• Improved peak shape and quantitation over wide dynamic ranges
• Compatibility with high-efficiency sub-2 µm UPLC separations
• Potential to streamline bioanalytical workflows for nucleotides and related compounds

Future Trends and Potential Applications

Further method development may explore hydrophilic interaction chromatography (HILIC) and mixed-mode columns with MaxPeak surfaces to address polar analytes in complex matrices. Incorporation of ion-pairing reagents could also expand retention and selectivity for highly polar nucleotides.

Conclusion

MaxPeak High Performance Surface technology on the ACQUITY PREMIER LC System effectively suppresses detrimental metal-analyte interactions, enabling robust and sensitive quantitation of nucleotides. This advancement opens new opportunities in metabolic profiling, drug development, and quality control analyses.

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