PREMIER Standards to Investigate the Inertness of Chromatographic Surfaces

Importance of the Topic

Ensuring chromatographic systems exhibit inert behavior toward metal-sensitive analytes is critical for reliable LC measurements.
Many biological and chelating compounds bind to exposed metal surfaces, causing adsorption, reduced sensitivity, and poor reproducibility.
Quality control standards that probe system inertness support accurate analyses in pharmaceutical, biochemistry, and industrial settings.

Objectives and Study Overview

This study introduces novel PREMIER standards based on a non-hydrolyzable ADP analog, adenosine 5’-(α,β-methylene)diphosphate (AMPcP), to assess metal interactions in LC systems.
Two standard formulations were evaluated: AMPcP alone and an equimolar AMPcP/adenosine mixture.
Comparative tests were conducted on a standard ACQUITY UPLC H-Class PLUS Bio System versus the inert ACQUITY PREMIER System to demonstrate improved analyte recovery and reproducibility.

Methodology

Standard solutions of AMPcP and the AMPcP/adenosine mixture were prepared in water and acetonitrile.
Two test approaches were applied:

• No-column injections with UV detection to quantify flowpath inertness via peak area and height reproducibility.
• Chromatographic separation using an ACQUITY PREMIER HSS T3 column and gradient elution to compare AMPcP/adenosine peak area ratios.

Instrumentation Used

• LC Systems: ACQUITY PREMIER System and ACQUITY UPLC H-Class PLUS Bio System with Binary Solvent Management.
• Detection: Tunable UV detector at 260 nm, 40 Hz, 10 mm flow cell.
• Column: ACQUITY PREMIER HSS T3, 1.8 µm, 2.1 × 50 mm.
• Mobile Phases: 10 mM ammonium acetate (pH 6.8) and acetonitrile blends.
• Software: MassLynx v4.2 for data acquisition and analysis.

Main Results and Discussion

A forced degradation study confirmed ATP’s susceptibility to hydrolysis, whereas AMPcP remained stable after prolonged heating, validating its suitability as a probe standard.
AMPcP and ATP exhibited similar adsorption to metal frits, affirming AMPcP’s sensitivity to metal interactions.
In no-column tests, switching to the ACQUITY PREMIER System reduced peak area RSD from 6.4% to 0.4% and increased absolute signal by nearly 50%.
Chromatographic testing with the AMPcP/adenosine standard showed a 16% increase in AMPcP peak area on the inert system, while adenosine response remained unchanged, enabling ratio-based assessment of flowpath inertness.

Benefits and Practical Applications

• MaxPeak High Performance Surfaces minimize metal-analyte interactions, enhancing recovery and peak shape of metal-sensitive compounds.
• PREMIER standards serve as quality control reference materials for system suitability testing on inert LC instruments.
• Routine use of these standards increases confidence in results for historically problematic analytes in pharmaceutical, biotechnological, and environmental analyses.

Future Trends and Opportunities

Adoption of robust inert surfaces may extend to other instrument platforms, including UHPLC–MS and preparative LC.
Development of additional non-hydrolyzable probe compounds could broaden system suitability tools for diverse analyte classes.
Integration with automated QC workflows and digital laboratory information systems may streamline performance monitoring and regulatory compliance.

Conclusion

The application of non-hydrolyzable AMPcP-based PREMIER standards with the ACQUITY PREMIER System provides a straightforward, sensitive approach to evaluate and ensure chromatographic inertness.
Significant improvements in analyte recovery, reproducibility, and peak shape directly support more reliable analyses of metal-sensitive compounds.

References

1. Lauber M. et al. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces. Waters Application Note, 2020.