Rapid LC-UV-MS Characterization of Peptide Therapeutics and Impurities Using BioResolve™ Peptide Phenyl-Hexyl+ and C18 + RP Columns

Importance of the Topic

Rapid, reliable separation and molecular confirmation of peptide therapeutics and related impurities are essential across discovery, development and quality control of biopharmaceuticals. Peptides such as insulin, GLP-1 receptor agonists and amylin analogs are chemically and structurally prone to deamidation, oxidation, truncation and aggregation. Analytical approaches that deliver fast chromatographic resolution together with MS-ready conditions enable timely identification of critical quality attributes (CQAs), reduce sample consumption, and streamline workflows for regulatory and in-process testing.

Objectives and Study Overview

This application note evaluates short-format BioResolve Peptide Phenyl‑Hexyl+ and C18+ reversed-phase columns (2.1 × 50 mm, 1.6 µm, 230 Å superficially porous particles) for a single, rapid LC-UV-MS workflow capable of separating and confirming intact peptide therapeutics and a representative impurity. The study examined a mixed standard containing bovine insulin and its deamidation product, plus four peptide therapeutics (cagrilintide, semaglutide, liraglutide, tirzepatide) to demonstrate chromatographic selectivity, speed, MS-compatibility and automated intact-mass deconvolution performance.

Methodology

Sample preparation and analytes:

• Bovine insulin was prepared at 2 mg/mL in 0.01 N HCl and aged ~96 h at ~25 °C to generate a deamidation impurity; other peptide standards (cagrilintide, semaglutide acetate, liraglutide, tirzepatide) were prepared at 2 mg/mL in DPBS.
• Final mixed injections were prepared by diluting 4 µL of each 2 mg/mL stock into 200 µL of 0.1% formic acid in water:acetonitrile (98:2); injection volumes of 1–2 µL supplied ~0.45–0.91 µg total load per injection.

Chromatography and mobile phases:

• Columns evaluated: BioResolve Peptide Phenyl‑Hexyl+ and BioResolve Peptide C18+ (2.1 × 50 mm, 1.6 µm, 230 Å).
• Mobile phases: A = 0.1% formic acid in water (and a neat acetonitrile source reported); B = 0.1% formic acid in acetonitrile. Column temperature 55 °C, autosampler 5 °C, UV detection at 214 nm.
• Short gradient methods were used with analysis windows that elute all analytes within ~5–9 minutes; injection volumes 1–5 µL.

Mass spectrometry and data processing:

• MS platform: Waters BioAccord LC‑MS System operating in positive MSE mode, m/z 50–2000, scan rate 10 Hz; cone 20 V, desolvation 350 °C, capillary 1.2 kV; MSE collision energy ramp 100–150 V.
• Automated deconvolution and intact-mass analysis performed with waters_connect Intact Mass Application within waters_connect Hub to assign monoisotopic masses and charge‑state distributions.

Used Instrumentation

Key instrumentation employed in the study included:

• ACQUITY Premier UPLC System (H‑Class PLUS Bio, Premier Bio Binary Solvent Manager, column heater, FTN autosampler with active pre‑heater and TUVe low‑dispersion UV cell).
• BioResolve Peptide Phenyl‑Hexyl+ RP column (p/n: 186011725) and BioResolve Peptide C18+ RP column (p/n: 186011716), both MaxPeak Premier, SPP, 1.6 µm, 230 Å, 2.1 × 50 mm.
• BioAccord LC‑MS System for intact-mass acquisition.
• waters_connect Intact Mass Application (v1.9.0.2980) and waters_connect Hub for automated deconvolution and reporting.

Main Results and Discussion

Chromatographic performance:

• Both PH+ and C18+ columns produced comparable, high-quality separations of the peptide mixture within short run times; all analytes eluted within ~5–9 minutes.
• Baseline resolution of bovine insulin from its deamidation impurity was achieved in under six minutes on the 2.1 × 50 mm format, with retention-time difference ΔRT ≈ 0.10–0.12 minutes.
• Retention differences between the two chemistries were subtle: π–π interactions on the Phenyl‑Hexyl+ chemistry introduced small selectivity shifts, e.g., tirzepatide retained ~0.4 min longer on PH+, while some fatty‑acid‑conjugated GLP‑1 analogs were slightly more retained on C18+ by 0.08–0.50 min.
• Fatty‑acid conjugation dominated reversed-phase retention: lipidated peptides (semaglutide, liraglutide, tirzepatide, cagrilintide) eluted later than insulin despite fewer residues, emphasizing hydrophobicity driven by lipid tags.

Mass spectrometric characterization:

• Clean charge‑state distributions and low background enabled confident charge assignment and automated deconvolution for all analytes using the Intact Mass Application.
• Deconvolution revealed an approximate +1 Da mass difference between insulin and the impurity, consistent with deamidation of an asparagine residue; this assignment was consistent across both column chemistries.
• Measured monoisotopic masses showed low variability across replicates and low mass error, demonstrating reproducible mass confirmation under MS‑compatible mobile phase and acquisition settings.

Robustness and workflow advantages:

• Comparable chromatographic behavior across PH+ and C18+ chemistries indicates method transferability and consistent performance for peptide classes tested.
• Short column format reduced analysis time and solvent consumption while maintaining sufficient resolution for impurity assignment and intact-mass confirmation.

Benefits and Practical Applications

The described LC‑UV‑MS workflow offers several practical advantages for peptide analytics:

• Rapid impurity profiling and intact-mass confirmation in a single injection support high‑throughput screening during development and lot release testing.
• MS‑compatible mobile phases and clean spectra facilitate automated deconvolution, lowering manual interpretation burden and improving throughput.
• Two column chemistries provide complementary selectivity: Phenyl‑Hexyl+ can exploit π–π interactions for certain analytes while C18+ offers classic hydrophobic retention, enabling method selection based on peptide properties.
• Reduced solvent use and short runs lower per-sample cost and increase lab throughput for routine QC/characterization tasks.

Future Trends and Applications

Potential extensions and trends building on this work include:

• Broader adoption of short, high‑efficiency peptide columns for routine QC workflows and stability-indicating methods, enabling accelerated release testing.
• Integration with automated intact‑mass software, laboratory information management systems (LIMS) and cloud platforms to scale throughput and standardize reporting.
• Use of high‑resolution MS and targeted fragmentation to localize modifications (e.g., oxidation, deamidation sites) following rapid intact-mass screening.
• Method adaptation for complex matrices (formulations, serum) and development of orthogonal separations for isomeric or closely related impurities.
• Continued emphasis on green chromatography: shorter columns, reduced solvent volumes and faster gradients to minimize environmental footprint.

Conclusion

BioResolve Peptide Phenyl‑Hexyl+ and C18+ columns, in combination with a BioAccord LC‑MS system and automated intact‑mass deconvolution, provide a fast, robust and MS‑compatible platform for separation and molecular confirmation of peptide therapeutics and a key degradation impurity. The short 2.1 × 50 mm column format achieved baseline separation of insulin from a deamidation product within minutes while delivering high‑quality mass spectra and reproducible monoisotopic mass assignments. This workflow supports rapid characterization needs across development and QC, with flexibility to choose stationary‑phase chemistry based on analyte properties.

References

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