Increasing Recovery of Phosphorylated Peptides Using ACQUITY PREMIER Technology Featuring MaxPeak High Performance Surfaces

Significance of the Topic

Phosphorylated peptides often interact with exposed metal surfaces in liquid chromatography systems, leading to sample loss, distorted peak shapes, and compromised reproducibility. These effects become critical when analyte levels are very low, such as in proteomics workflows or trace‐level pharmaceutical analyses. Mitigating metal‐induced adsorption is therefore essential to ensure accurate quantitation, reliable method performance, and high laboratory throughput.

Objectives and Overview of the Study

This application note evaluates the performance gains achieved by integrating Waters’ ACQUITY PREMIER Solution featuring MaxPeak High Performance Surfaces (HPS) into a reversed‐phase LC–MS workflow. The study compares three hardware configurations: a conventional LC system with stainless‐steel column, a conventional system equipped with an ACQUITY PREMIER column, and the full ACQUITY PREMIER Solution. The primary goal was to quantify improvements in the recovery, peak shape, and detector response of a model phosphorylated peptide at trace‐level injection amounts.

Methodology and Instrumentation

A singly phosphorylated peptide (VNQIGpTLSESIK, monoisotopic mass 1368.68 Da) was injected at 2 µL (3 pmol/µL) onto an RPLC column using a 1.85 % B/min gradient (mobile phase A: water/0.1 % formic acid; B: acetonitrile/0.1 % formic acid). Data were acquired in selected‐ion recording (SIR) mode on a single‐quadrupole mass detector. Peak integration was performed using Empower 3 software.

Used Instrumentation

• ACQUITY UPLC H-Class PLUS Bio System (conventional configuration)
• ACQUITY PREMIER System with MaxPeak HPS technology
• ACQUITY UPLC CSH C18 Column (stainless‐steel hardware)
• ACQUITY PREMIER Peptide CSH C18 Column (MaxPeak HPS treated)
• ACQUITY QDa Single-Quadrupole Mass Detector
• Empower 3 Chromatography Data System

Main Results and Discussion

The conventional setup produced a broad, tailing peak with a maximum signal of 1.1×10^6 ion counts for an 8 ng on‐column load. Substituting the column hardware with MaxPeak HPS treatment improved peak symmetry and yielded an eightfold increase in signal intensity (9.3×10^6 ion counts). Implementing the full ACQUITY PREMIER Solution further enhanced recovery, delivering a tenfold boost (1.1×10^7 ion counts) compared to the stainless‐steel configuration. Quantitative analysis of peak area as a function of surface exposure indicated a twofold increase in recovery from column surfaces alone and an additional 25 % gain when the entire flow path featured HPS treatment.

Benefits and Practical Applications

By minimizing non-specific adsorption, the ACQUITY PREMIER Solution with MaxPeak HPS technology offers:

• Enhanced sensitivity and signal intensity for metal-sensitive analytes
• Sharper peak shapes with reduced tailing
• Greater reproducibility in trace-level determinations
• Streamlined method development and faster time to results

Future Trends and Potential Applications

Advances in surface passivation technologies are poised to expand their use beyond phosphopeptide analysis to other metal-sensitive molecules, such as carboxylated metabolites and nucleotides. Integration with high-throughput proteomics platforms and automated workflows will further enhance sample preservation and data quality. Ongoing research into novel coating chemistries may enable even broader compatibility with diverse chromatographic and mass spectrometric conditions.

Conclusion

Waters’ ACQUITY PREMIER Solution featuring MaxPeak High Performance Surfaces effectively addresses metal-induced adsorption challenges in LC–MS analysis of phosphorylated peptides. By improving recovery, peak shape, and reproducibility at trace levels, this integrated hardware approach enhances data quality and laboratory productivity, facilitating robust quantitative assays in pharmaceutical development and proteomic research.