Quantitative Determination of Semaglutide and Preservative in Semaglutide Injection by High Performance Liquid Chromatography

Significance of the Topic

Accurate and efficient analysis of biopharmaceutical formulations is critical for ensuring drug safety, efficacy, and regulatory compliance. Semaglutide, a long-acting GLP-1 analog used in type 2 diabetes and weight management, is formulated with phenol as a preservative. A validated HPLC method that quantifies both the active peptide and its preservative in a single injection supports streamlined quality control in pharmaceutical development and manufacturing.

Objectives and Study Overview

This work aimed to develop and validate a high-performance liquid chromatography procedure for simultaneous determination of semaglutide and phenol in multi-dose injection formulations. Key goals included achieving robust linearity over relevant concentration ranges, demonstrating method precision and accuracy, and ensuring compliance with pharmacopeial content requirements.

Methodology and Instrumentation

Sample preparation involved diluting semaglutide injection samples 50-fold with water. Mixed standard solutions were prepared by dissolving reference semaglutide and phenol in water to generate concentration levels spanning from low microgram to milligram per milliliter ranges.

Instrumentation

• HPLC system: Shimadzu Nexera LC-40D X3
• Column: C18 reverse-phase (100 mm × 2.1 mm i.d., 2 µm particle size)
• Mobile phase A: 0.1% trifluoroacetic acid in water
• Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile
• Elution: Gradient from 20% B to 90% B over 12 minutes
• Flow rate: 0.3 mL/min; column temperature: 35 °C; detection wavelength: 280 nm; injection volume: 5 µL

Main Results and Discussion

Retention times were approximately 4.6 minutes for phenol and 9.2 minutes for semaglutide, with baseline separation achieved. Calibration curves for phenol (27.5–1,100 µg/mL) and semaglutide (6.09–243.6 µg/mL) exhibited excellent linearity (r² ≥ 0.9993). Limits of detection and quantitation were low enough to meet regulatory requirements. Repeatability tests at the lowest standard concentration yielded relative standard deviations below 0.7% for peak area and below 0.1% for retention time.
Analysis of commercial semaglutide injection samples indicated semaglutide content at 104.7% of label claim and phenol at 105.0%, both within the 90–110% pharmacopeial range. Spike recovery experiments at multiple levels demonstrated recoveries between 97% and 112%, confirming method accuracy and matrix robustness.

Benefits and Practical Applications of the Method

• Simultaneous quantification of active and preservative components reduces analysis time and resource consumption.
• High precision and accuracy support regulatory compliance for batch release testing.
• The simple sample preparation and gradient program facilitate routine implementation in quality control laboratories.

Future Trends and Opportunities

Emerging advances in ultra-high-performance liquid chromatography and mass spectrometric detection may further enhance sensitivity and throughput for peptide therapeutics. Integration of automated sampling and data processing could streamline workflow, while adaptation to other peptide–preservative combinations can broaden application scope in biopharmaceutical analysis.

Conclusion

The validated HPLC method provides a reliable, accurate, and repeatable approach for simultaneous determination of semaglutide and phenol in injection formulations. Its robustness and simplicity make it well suited for routine quality control and regulatory compliance in pharmaceutical production.

Reference

Jia Zheng, Shimadzu (China) Co., LTD. Quantitative Determination of Semaglutide and Preservative in Semaglutide Injection by High Performance Liquid Chromatography. Application News, Shimadzu Corporation, First Edition: Sep. 2025.