Reversed-Phase LC-MS Analysis of Low- Abundance Impurities in a GLP-1a Therapeutic Using a Charged Surface 230Å Superficially Porous Phenyl-Hexyl Column

Significance of the Topic

Reversed-phase liquid chromatography (RPLC) coupled with high-resolution mass spectrometry (HRMS) is a cornerstone technique for impurity profiling of therapeutic peptides and lipopeptides. For GLP-1 analogs such as tirzepatide, which include a hydrophobic fatty-acid side chain, conventional RPLC separations can mask backbone-related variants and necessitate long, shallow gradients or complementary orthogonal methods. Improved stationary phases that combine tailored ligand chemistry, charged surfaces and optimized particle morphology can enhance detection and separation of low-abundance impurities, supporting development, stability testing and batch release decisions.

Objectives and Study Overview

The study evaluated reversed-phase LC–HRMS methods for characterizing trace impurities in a commercial tirzepatide drug product and several synthetic impurity standards. The principal aim was to demonstrate the chromatographic and mass-spectrometric performance of a superficially porous particle (SPP) phenyl-hexyl column with a positively charged surface and large pore diameter (230 Å, 1.6 µm particles, MaxPeak Premier hardware) versus an alternative SPP phenyl-hexyl column (90 Å, 2.7 µm). The work compared mobile-phase modifiers (0.1% formic acid versus 0.1% trifluoroacetic acid) using broad 1% MeCN/min gradients to assess selectivity, sensitivity and the ability to resolve and identify low-level variants.

Methodology

Procedures combined high-efficiency RPLC separations with high-resolution QTof mass spectrometry and deconvolution workflows to detect and assign monoisotopic masses of low-abundance species.

• Sample: Commercial tirzepatide (30 mg/mL) diluted to 3 mg/mL; synthetic standards included isoAsp variants, deamidations, a substitution (Ile12→Nva) and a racemized residue (D-Ser32).
• Chromatography: Acetonitrile gradients at 1% MeCN per minute, flow 0.30 mL/min, column temperature 60 °C, sample temperature 6 °C, injection typically 0.5 µL.
• Mobile phases: 0.10% (v/v) formic acid (FA) or 0.10% (v/v) trifluoroacetic acid (TFA) in water (A) and MeCN (B).
• MS: QTof acquisition over ~400–2800 m/z with ESI source, short scan times and low collision energy to capture intact peptide charge envelopes; deconvolution applied to assign monoisotopic masses.

Instrumentation Used

Key instrumentation and configurations used in the study included:

• LC system: ACQUITY UPLC with Binary Solvent Manager, Flow-Through Needle sample manager and tunable UV detector (A215 detection).
• Columns: Waters BioResolve Peptide Phenyl-Hexyl+ RP Column (MaxPeak Premier Technology, SPP, 1.6 µm, 230 Å, 2.1 × 150 mm) and an alternative SPP phenyl-hexyl column (2.7 µm, 90 Å, 2.1 × 150 mm) for comparison.
• Mass spectrometer: Xevo G3 QTof with electrospray ionization (ESI), source/desolvation temperatures tuned for peptide ionization; data processed using UNIFI and BayesSpray deconvolution.

Main Results and Discussion

The phenyl-hexyl SPP column with 230 Å pores demonstrated superior chromatographic detail and sensitivity for low-abundance tirzepatide variants compared with the 90 Å, 2.7 µm alternative. Key observations were:

• Selectivity differences between FA and TFA: Several impurities showed notable shifts in retention depending on modifier. L-isoAsp9 gave the most pronounced alteration—eluting after the main peak with FA but before the main peak with TFA—illustrating that modifier choice can dramatically change separation order for certain backbone variants.
• Sensitivity and detectability: The RPLC–HRMS method detected many variants at very low relative abundances; a fragment with monoisotopic mass 4486.261 was observed at ~0.004–0.005% relative abundance and had sufficient signal for confident mass assignment. Overall, FA provided better MS sensitivity while TFA yielded improved UV (A215) signal for some peaks.
• Identification of synthetic standards: Several impurity standards were resolved and tentatively assigned by matching retention behavior and accurate masses—examples include L-isoAsp15 and Gln19/Gln24 deamidations. Some impurities coeluted under FA but separated under TFA (e.g., Gln19 vs Gln24), and other minor species were observed only in one modifier.
• Column performance drivers: The larger 230 Å pore diameter was better suited for the nearly 5 kDa tirzepatide molecule, and the smaller 1.6 µm SPP particles increased column efficiency, together producing enhanced resolution and deeper impurity coverage versus the 90 Å/2.7 µm column.
• Method flexibility: Broad gradients (1% MeCN/min) produced separations in ≤46 minutes; however, the authors note that longer columns (e.g., 300 mm) and extended shallow gradients can further improve resolution when needed.

Benefits and Practical Applications

The combination of the phenyl-hexyl charged-surface SPP column and HRMS offers several practical advantages for lipopeptide impurity profiling:

• Enhanced low-level impurity detection enabling identification of trace variants relevant to stability and manufacturing.
• Alternative selectivity compared with C18 phases, useful to reveal backbone-related variants that are otherwise masked by the fatty-acyl retention dominance.
• Compatibility with both FA and TFA mobile phases — allowing users to balance MS sensitivity (FA) against optical/UV detection performance (TFA) depending on analytical priorities.
• Faster workflows suitable for pairing with orthogonal methods (HILIC, IEX) or as part of a tiered analytical strategy for development and QC release testing.

Future Trends and Potential Applications

Opportunities to build on these findings include:

• Method optimization: Employing longer columns (e.g., 300 mm), shallower gradients and targeted gradient segments to resolve challenging coelutions while maintaining adequate sensitivity.
• Orthogonal integration: Systematic pairing of RP-HRMS with HILIC and ion-exchange to comprehensively map hydrophobic and hydrophilic variant spaces for regulatory impurity profiling.
• Automation and data analytics: Advanced deconvolution, database matching and machine-learning tools to accelerate identification of low-level variants and reduce manual interpretation time.
• Regulatory and QC adoption: Translating high-sensitivity RPLC–HRMS methods into validated workflows for stability testing and batch release, including standardized criteria for modifier selection and system suitability for lipopeptides.
• Column chemistry evolution: Continued development of charged-surface and ligand chemistries tailored for large peptide/lipopeptide analytes to further enhance orthogonal selectivity and robustness.

Conclusion

The charged-surface BioResolve Peptide Phenyl-Hexyl+ RP Column (SPP, 1.6 µm, 230 Å) combined with Xevo G3 QTof HRMS provides a sensitive and selective platform to profile low-abundance impurities in tirzepatide and analogous GLP-1a lipopeptides. Modifier choice (FA vs TFA), particle/pore architecture and column hardware all materially influence selectivity, detectability and practical utility. The presented methods balance analytical depth with throughput and are readily adapted—via column length and gradient tuning—or combined with orthogonal techniques to deliver comprehensive impurity characterization for development and quality control.

Reference

1. Yang H., Shiner S. Separation of a GLP-1 Receptor Agonist and Structurally Similar Impurities Using BioResolve Peptide Phenyl‑Hexyl+ and C18+ Columns. Application Brief. Waters Corporation. June 2026.
2. Yoshida K.; et al. Impurity profiling of synthetic cyclic peptides based on orthogonality between hydrophilic-interaction and reversed-phase liquid chromatography. Journal of Chromatography A, 1745 (2025): 465748.
3. Han D.; Ippoliti S.; Birdsall R. E.; Nyholm K. Accelerating Method Development and Manufacturing of GLP-1 Analogs with LC-UV/MS. Application Note. Waters Corporation. May 2025.
4. Goyon A. Advanced chromatographic strategies for comprehensive characterization of therapeutic peptides. Keynote presentation at HPLC 2026, Indianapolis IN, USA. June 2026.
5. Hanna C. M.; Koza S. M.; Shiner S. Temperature Dependence on Reversed-Phase Separations of Fatty Acid Modified GLP-1 Receptor Agonists and Their Impurities. Application Note. Waters Corporation. October 2024.
6. Koza S. M.; Chambers E. E. Selecting a Reversed-Phase Column for the Peptide Mapping Analysis of a Biotherapeutic Protein. Application Note. Waters Corporation. 2017.