Bypassing LC System Passivation Requirements Using ACQUITY PREMIER with MaxPeak HPS Technology for the Recovery of a Phosphorylated Peptide

Significance of the Topic

Accurate analysis of phosphorylated peptides is critical in proteomics research and biopharmaceutical quality control. Surface interactions in liquid chromatography systems can lead to significant analyte loss, poor peak shape, and inconsistent results. Conventional approaches often rely on aggressive chemical passivation, which adds complexity, time, and potential contamination to routine assays.

Objectives and Study Overview

This study evaluated the ability of the Waters ACQUITY PREMIER system equipped with MaxPeak High Performance Surfaces (HPS) technology to recover a model phosphorylated peptide without requiring system passivation. Performance metrics were compared against a well-conditioned stainless-steel LC system before and after phosphoric acid treatment.

Methodology and Instrumentation

A reversed-phase gradient using water and acetonitrile (both containing 0.1% formic acid) was employed to separate a four-component peptide mixture, including a doubly phosphorylated insulin receptor peptide (TRDIpYETDpYYRK). Three system configurations were tested:

• Conventional LC system and column (stainless-steel flow path) without passivation
• Same conventional system after a 30% phosphoric acid wash and equilibration
• ACQUITY PREMIER system with MaxPeak HPS technology and a Peptide CSH C18 column (130 Å, 1.7 µm, 2.1 × 100 mm), no passivation

The MaxPeak HPS hardware features corrosion-resistant flow paths with an inert surface treatment designed to minimize metal-analyte interactions.

Main Results and Discussion

• Conventional LC without passivation failed to detect the phosphorylated insulin receptor peptide due to surface adsorption.
• After phosphoric acid passivation, full recovery of the peptide was observed, illustrating the need for harsh chemical treatment in traditional systems.
• The ACQUITY PREMIER system with MaxPeak HPS technology achieved complete recovery of all four peptides off the shelf, without any passivation steps.
• Repeatability tests over 15 injections showed a peak area %RSD of 6.22% for the passivated conventional system versus 0.48% for the MaxPeak HPS setup, with an average signal increase of approximately 1.5-fold.

Benefits and Practical Applications

• Eliminates time-consuming and corrosive system passivation procedures
• Enhances analyte recovery and improves peak shape for phosphorylated peptides
• Boosts method robustness and throughput in proteomics and biopharmaceutical workflows
• Reduces potential contamination from acid treatments

Future Trends and Opportunities

Advancements in surface treatments will continue to refine LC performance for sensitive biomolecules. Integration of inert hardware with automated conditioning protocols may further streamline proteomics analyses. Expansion of MaxPeak HPS concepts to other chromatographic formats could benefit small-molecule, lipidomic, and metabolomic applications.

Conclusion

The Waters ACQUITY PREMIER system with MaxPeak HPS technology provides an off-the-shelf solution for reliable recovery of phosphorylated peptides without system passivation. It delivers superior repeatability and signal intensity compared to conventional stainless-steel LC workflows, simplifying method development and enhancing laboratory efficiency.

References

1. Gjerde DT, Hanna CP, Hornby D. Appendix 2: System Cleaning and Passivation Treatment. In: DNA Chromatography. Wiley-VCH Verlag GmbH & Co.; 2002.
2. Day R. Passivating Stainless Steel in HPLC Systems. Waters Lab Highlights. 2008; LAH 0376.