Increasing Chromatographic Performance of Acidic Peptides in RPLC-MS-based Assays with ACQUITY PREMIER featuring MaxPeak HPS Technology

Significance of the Topic

Metal‐ion mediated adsorption of acidic peptides during reversed‐phase liquid chromatography with mass spectrometry (RPLC‐MS) can cause peak tailing, reduced recovery and compromised assay reproducibility. These artifacts are especially critical in peptide mapping assays used for characterization and quality control of biopharmaceutical products, where detection of post-translational modifications and trace impurities is essential.

Objectives and Overview

This application note evaluates ACQUITY PREMIER columns featuring MaxPeak HPS Technology as a solution to minimize analyte–surface interactions. The study compares conventional stainless‐steel columns to MaxPeak HPS columns for RPLC‐MS peptide mapping of a NIST monoclonal antibody (mAb) tryptic digest, focusing on sensitivity, tailing, recovery, reproducibility and method compatibility.

Methodology and Instrumentation

Samples: Waters NIST mAb tryptic digest standard, 0.2 mg/mL in water with 0.1 % FA.
Chromatography: Waters ACQUITY UPLC H-Class system with binary pump, ACQUITY PREMIER Peptide CSH C18 column (2.1 × 100 mm, 1.7 µm) versus standard CSH C18 column. Mobile phases: water/0.1 % formic acid (A), acetonitrile/0.1 % formic acid (B). Flow rate 0.2 mL/min, column temp 60 °C, sample temp 6 °C.
Detection: Waters SYNAPT XS high-resolution MS (ESI+; 50–2000 m/z) and ACQUITY QDa single-quadrupole MS (ESI+; 250–1250 m/z). Data processed in Empower 3 and UNIFI.

Main Results and Discussion

Peak Tailing Reduction: The PENNYK peptide (GFYPSDIAVEWESNGQPENNYK) tailing factor decreased from 5.53 on a conventional column to 1.15 on MaxPeak HPS, enabling clear resolution of native and deamidated species.
Increased Recovery: The T14 acidic peptide (VDNALQSGNSQESVTEQDSK) at 0.2 µg load was undetected on a standard column (90 % sequence coverage) but recovered on MaxPeak HPS with 94 % coverage.
Enhanced Response and Reproducibility: The T37 peptide showed a ten‐fold increase in peak height and four‐fold in area, with MS response variability (height RSD) reduced from 11.6 % to 1.1 % over three injections.
Improved Data Quality: Fragmentation spectra in MSE mode enabled unambiguous assignment of deamidation sites on resolved T37 species, increasing confidence in peptide identification.
Operational Robustness: Over 48 hr and repeated injections on a cleaned system, tailing factor RSD for the PENNYK peptide was 8.9 % on a conventional column versus 1.0 % on MaxPeak HPS, demonstrating stable performance without lengthy passivation.
Legacy Method Compatibility: Retention times and selectivity were conserved (slope = 1.00; intercept = -0.005) when comparing MaxPeak HPS to standard columns, facilitating method transfer with minimal revalidation.

Benefits and Practical Applications

• Higher sensitivity through improved recovery and sharper peaks.
• Enhanced assay reproducibility and robustness in peptide mapping.
• Reduced method development time and avoidance of corrosive passivation steps.
• Compatibility with existing RPLC‐MS workflows and legacy methods without additional additives.

Future Trends and Opportunities

Continued development of inert surface chemistries may further mitigate adsorption for diverse analytes. Integration of MaxPeak HPS columns into high-throughput bioanalysis pipelines and expansion to other metallic‐sensitive compounds are promising. Ongoing efforts to combine surface technologies with advanced MS acquisition modes will drive deeper characterization of complex biologics.

Conclusion

ACQUITY PREMIER columns featuring MaxPeak HPS Technology effectively minimize metal‐ion mediated adsorption of acidic peptides, delivering superior peak shape, increased recovery, greater reproducibility and retention time consistency. This enhancement supports reliable RPLC‐MS peptide mapping in biopharmaceutical development and manufacturing without compromising legacy method performance.

Reference

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4. Lauber M et al. Low Adsorption LC Technology Based on MaxPeak High Performance Surfaces. Waters White Paper 720006930EN. 2020.
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