Stability-indicating HPLC-UV method development for GLP-1 receptor analogue semaglutide

Significance of the topic

Semaglutide is a 32–amino-acid GLP‑1 receptor agonist widely used for type 2 diabetes and chronic weight management. Reliable, stability‑indicating analytical methods are essential for quality control, regulatory submissions (including ANDAs), and to detect peptide-related impurities that can arise during synthesis, storage or stress conditions and may affect safety and efficacy.

Goals and study overview

This work aimed to develop and optimize a stability‑indicating reversed‑phase HPLC‑UV method for quantifying semaglutide and resolving closely related peptide impurities. The study compared mobile phase additives (formic acid vs trifluoroacetic acid), evaluated the influence of column temperature and gradient shape, generated thermally and oxidatively stressed samples to reveal potential degradation products, and assessed column lot‑to‑lot reproducibility for long‑term robustness.

Methodology and instrumentation

The method development combined deliberate stress generation, systematic parameter screening and analytical evaluation.

• Stress conditions: thermal degradation (60 °C for 8 days, producing ~10.1% degradation) and oxidative degradation (0.01% H2O2 at room temperature for 14 h, ~4.9% degradation).
• Sample preparation: semaglutide API at 1.0 mg/mL (5 mg in 5 mL, 20% acetonitrile/water) with triplicate stressed aliquots.
• Chromatographic variables explored: column temperature (35, 45, 55 °C), gradient starting/ending % organic (tested 25–35% start and 50–60% end B), and mobile phase additives (0.1% formic acid or 0.1% TFA in water/acetonitrile).

Instrumentation used

• Column: Thermo Scientific Hypersil GOLD Peptide, 150 × 2.1 mm, 1.9 µm.
• UHPLC system: Thermo Scientific Vanquish Flex (System Base Vanquish Horizon/Flex, Binary Pump F, Split Sampler FT, Column Compartment H, Variable Wavelength Detector F) with 11 µL bio flow cell.
• Software: Chromeleon CDS 7.3.2 for acquisition and processing.
• Detection: UV at 280 nm, sampling 2.0 Hz, 2.0 s response.

Main results and discussion

Key findings from systematic optimization are summarized below.

• Column temperature: increasing temperature from 35 °C to 55 °C improved resolution for multiple critical peak pairs; notably the resolution of the impurity immediately preceding semaglutide increased from 0.45 (35 °C) to 0.72 (55 °C). Active column pre‑heating and forced‑air mode improved retention stability and peak shape.
• Gradient optimization: a 40‑minute gradient from 30% to 50% organic (eluent B) provided the best balance between impurity separation and sensitivity. Shallow gradients improved separation but can broaden peaks and reduce sensitivity; the selected gradient minimized these trade‑offs.
• Mobile phase additive: TFA (0.1% in both aqueous and acetonitrile phases) delivered superior chromatographic resolution and sharper peaks versus FA under the tested conditions. Example: semaglutide peak width at 50% height decreased substantially (from ~0.83 to ~0.32 min) with optimized gradient/additive, improving detectability of low‑level degradants. However, TFA is known to cause ion suppression in LC‑MS, so FA remains preferable when MS detection is required.
• Stressed sample separation: several coeluting peaks observed with FA were resolved under TFA conditions for both thermal and oxidative stress samples, demonstrating the method’s ability to reveal degradation products.
• Robustness: column lot‑to‑lot reproducibility was confirmed using three manufacturing lots of the Hypersil GOLD Peptide column. Six consecutive injections per lot of the thermally stressed sample produced %RSD ≤ 1.3% for retention time, relative retention time, peak area, peak width (50%) and peak height, indicating excellent method precision and column consistency.

Benefits and practical applications

• The developed HPLC‑UV method is stability‑indicating and suitable for routine quality control of semaglutide drug substance and formulations, enabling detection and quantification of related impurities formed under common stress conditions.
• Method parameters (55 °C, active pre‑heater, 30→50% B in 40 min, 0.1% TFA) provide reproducible separations with narrow peaks that support sensitive impurity profiling by UV.
• Demonstrated column lot reproducibility supports method transferability across labs and long‑term deployment in QC environments.
• Flexibility in additive choice allows adaptation: TFA for optimal UV separations; FA if downstream LC‑MS characterization is required.

Future trends and opportunities

• Integration with MS workflows: combining the optimized chromatographic separation with MS‑compatible additives or post‑column strategies (e.g., TFA suppression or ion‑pair scavenging) could enable orthogonal impurity identification without sacrificing chromatographic performance.
• High‑throughput adaptation: shorter columns or higher flow/temperature designs, coupled with careful gradient re‑optimization, could reduce run time for routine screening while retaining critical resolution.
• Automated forced‑degradation and impurity mapping: linking stress studies with advanced data processing and peak deconvolution will improve impurity identification and support regulatory filings.
• Column chemistry evolution: further development of peptide‑focused stationary phases may improve selectivity and MS compatibility simultaneously, reducing the tradeoff between chromatographic resolution and ionization efficiency.

Conclusion

An HPLC‑UV stability‑indicating method for semaglutide was developed and validated through targeted stress studies and systematic optimization. Final recommended conditions employ a Hypersil GOLD Peptide column at 55 °C with an active pre‑heater, a 30→50% acetonitrile gradient over 40 minutes, and 0.1% TFA as mobile phase additive for best UV separation. The method demonstrated strong reproducibility across column lots and is suitable for routine impurity monitoring and QC of semaglutide, with consideration of additive trade‑offs when coupling to MS.

References

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4. Heidorn M. The Role of Temperature and Column Thermostatting in Liquid Chromatography. Thermo Fisher Scientific White Paper 71499.