Workflows for GLP-1 Receptor Agonists and Therapeutic Peptides: Identity, Impurity, Bioanalysis, and Stability Testing

Significance of the topic

Peptide therapeutics—exemplified by GLP-1 receptor agonists such as semaglutide, liraglutide, exenatide and tirzepatide—are a rapidly growing class of medicines combining favorable safety, high target specificity and tunable pharmacokinetics. Their structural diversity (amino‑acid substitutions, lipidation, linkers) and rising global demand create analytical challenges across raw material verification, impurity profiling, bioanalysis and stability testing. Robust, orthogonal and scalable analytical workflows are therefore essential across discovery, development and QC to guarantee identity, purity, potency and safety.

Objectives and overview of the compendium

This collection of application notes and studies from Agilent and collaborators presents practical workflows and experimental examples targeting key analytical tasks for GLP‑1 analogs and related peptides: raw material verification by through‑container Raman, impurity profiling using HILIC‑MS and orthogonal detectors (DAD/ELSD), automated LC/MS bioanalysis with AssayMAP sample preparation, stability and forced‑degradation characterization (UV‑Vis second‑derivative, LC/Q‑TOF and single‑quad deconvolution), and integration of structural LC/MS data with functional SPR assays.

Methodology and used instrumentation

Overview of principal methods used across studies:

• Raw material ID: spatially offset Raman spectroscopy (SORS) for through‑container verification of Fmoc‑protected amino acids (Agilent Vaya handheld Raman).
• Purity/impurity separations: hydrophilic interaction liquid chromatography (HILIC‑Z) with MS detection (single‑quadrupole LC/MSD iQ or Pro iQ Plus) and orthogonal detectors (DAD, ELSD) on low‑adsorption/Ultra‑Inert flow paths.
• Bioanalysis: automated sample cleanup (Agilent AssayMAP Bravo with RP‑S cartridges) combined with UHPLC–triple quadrupole MRM quantitation (Agilent 1290 Infinity II Bio LC with 6495D TQ).
• Stability and structural characterization: LC/Q‑TOF (Agilent 6545XT AdvanceBio) for high‑resolution mass deconvolution and identification of oxidation, truncation and addition products; UV‑Vis second‑derivative spectroscopy (Agilent Cary 3500 Multicell) for sensitive detection of aromatic residue changes; single‑quad Pro iQ Plus for routine, sensitive impurity monitoring and spectral deconvolution in QC workflows.
• Functional correlation: digital surface plasmon resonance (Nicoya digital SPR) to measure GLP‑1 receptor binding kinetics and link structural changes to activity.

Representative instrumentation (as used in the studies):

• Agilent Vaya Raman system
• Agilent 1290 Infinity III Bio LC and 1290 Infinity II Bio LC
• Agilent Altura Poroshell / Agilent AdvanceBio Peptide Mapping / Altura Peptide Plus columns (HILIC‑Z and RP peptide columns)
• Agilent InfinityLab LC/MSD iQ, Pro iQ Plus single‑quadrupole MS
• Agilent 6495D triple quadrupole LC/TQ
• Agilent 6545XT AdvanceBio LC/Q‑TOF
• Agilent AssayMAP Bravo automated sample prep
• Agilent Cary 3500 Multicell UV‑Vis
• Nicoya Digital SPR (DSPR16)

Main results and discussion

Key outcomes demonstrated across the workflows:

• Raw‑material verification: Vaya SORS reliably identified and differentiated Fmoc‑protected amino acids noninvasively through amber and HDPE containers in <40 s, preserving material integrity and supporting compliance workflows.
• HILIC impurity profiling: HILIC‑Z provided orthogonal selectivity to RPLC, resolving many product‑related impurities (isomers, truncations, amino‑acid additions, oxidized forms). When coupled to a single‑quad MS, molecular weights and charge envelopes enabled rapid identity confirmation of main peaks and trace impurities (example: Tyr addition +163 Da in tirzepatide; truncation –222 Da in semaglutide).
• Low‑adsorption flow paths: Use of Ultra‑Inert/biocompatible hardware markedly improved peak shape, recovery and detection of metal‑sensitive analytes and minor impurities (notably phosphate and oxidized peptide species) compared with stainless‑steel flow paths.
• Forced‑degradation/stability: Oxidative stress (H2O2) produced characteristic mass increments (+16, +32, +48 Da) and often partial chromatographic separation of diastereomeric oxidation products (e.g., Met sulfoxide S/R). Stability of tirzepatide showed pH dependence with higher oxidation at pH 5 even at 5 °C.
• Bioanalysis sensitivity and automation: AssayMAP RP‑S automated cleanup (after ACN/MeOH precipitation) improved assay sensitivity ~5× versus precipitation alone. Semaglutide quantitation in human plasma achieved LLOQ 0.2 ng/mL (100 μL sample) with linearity to 1,000 ng/mL; tirzepatide assay achieved LLOQ 0.05 ng/mL and similar linearity, with intra/interday QC meeting typical ±15% acceptance.
• Routine QC detection: Pro iQ Plus single‑quad MS with OpenLab deconvolution successfully detected impurities at <2% and provided cost‑effective trace impurity surveillance suitable for routine QC/QA.
• Structure–function linkage: Integrated LC/Q‑TOF and digital SPR on liraglutide demonstrated that oxidative modifications (mono/di/trioxidation) altered mass profiles but, in the reported case, did not change GLP‑1R binding affinity (KD remained ~3.5 nM). By contrast, proteolytic digestion (chymotrypsin) abolished receptor binding—illustrating that not all structural changes impact function equally and underscoring the importance of orthogonal functional assays.

Practical benefits and applications

The presented approaches provide a coherent toolkit for peptide analytics:

• Nondestructive, through‑container raw material ID reduces contamination risk and speeds material transfer to production.
• HILIC (with DAD, ELSD, MS) serves as an orthogonal separation to RPLC for comprehensive impurity mapping, while low‑adsorption hardware improves reliability for metal‑sensitive analytes.
• Automated, cartridge‑based sample cleanup (AssayMAP) enhances sensitivity and throughput for PK/TK studies without reliance on ligand binding assays.
• Single‑quadrupole MS platforms (with deconvolution) enable cost‑effective impurity monitoring in QC labs where high‑resolution MS may not be necessary.
• Combining structural MS with SPR provides direct evidence linking chemical modifications to biological function—critical for stability, comparability and risk assessment.

Future trends and potential applications

Emerging and recommended directions for peptide analytics include:

• Broader adoption of multidimensional LC (e.g., HILIC × RPLC) and solvent modulation for enhanced impurity separation and peak capacity.
• Integration of higher‑throughput automation for sample prep coupled to sensitive LC/TQ and mid‑range MS to accelerate discovery and clinical workflows.
• Wider use of low‑adsorption/biocompatible flow paths and column hardware to reduce nonspecific adsorption and improve detection of labile or metal‑sensitive species.
• Greater routine deployment of integrated structure–function platforms (LC/MS + SPR or cellular binding assays) to de‑risk formulation and storage strategies early in development.
• Application of AI/ML tools for automated spectral deconvolution, impurity annotation and trending across stability datasets to speed decision‑making.

Conclusion

This compendium demonstrates practical, interoperable analytical workflows that address the full lifecycle of GLP‑1 peptide analytics: secure raw material ID, orthogonal impurity profiling, high‑sensitivity bioanalysis and stability/structure–function correlation. By combining noninvasive screening, orthogonal chromatographic selectivity, automated sample preparation and integrated MS/SPR readouts, laboratories can enhance sensitivity, robustness and interpretability—supporting regulatory expectations and accelerating peptide therapeutic development.

References

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7. Qiu X.; Murphy S.; Wong D. L. Sensitive Quantitation of GLP‑1 Analog Tirzepatide in Plasma. Agilent Technologies application note, 2025.
8. Suresh Babu C. V. Enhanced Peptide Characterization and Stability Assessment Using UV‑Visible Second‑Derivative Spectroscopy. Agilent application note 5994‑8551EN, 2025.
9. Miladi M. Impurity Profiling of Tirzepatide Under Stress Conditions Using Agilent Pro iQ Plus. Agilent application note, 2025.
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