HPLC–DAD Method Tracks Semaglutide and Tirzepatide Stability

Demand for glucagon-like peptide-1 (GLP-1) receptor agonists has surged, and with it the need for fast, reliable quality control methods for semaglutide and tirzepatide. Previously reported stability-indicating liquid chromatography (LC) methods either produced degradation below the 10–20% range expected under International Council for Harmonisation (ICH) guidelines or relied on gradient runs of up to 30 min.^1,2

Researchers at Alexandria University (Alexandria, Egypt) have developed an isocratic high performance liquid chromatography method with diode array detection (HPLC–DAD) that quantifies both peptides in bulk drug, injectable dosage forms, and spiked rat plasma, including in the presence of their degradation products. A paper based on this work was published in BMC Chemistry.¹

How Was the Chromatographic Separation Optimized?

The team began with 0.1% formic acid and methanol, but broad peaks led them to switch to acetonitrile. Raising the acetonitrile proportion above 70% caused poor peak symmetry and inadequate capacity factors. Column choice proved equally important: a C8 phase eluted the analytes in under 1 min, while a 3-µm C18 column (150 × 4.6 mm) gave poor peak shapes.

The final method used a 250 × 4.6 mm, 5-µm C18 column with 0.1% formic acid (pH 2.5)–acetonitrile (30:70, v/v) at 1 mL/min and 25 °C, a 5-µL injection, and detection at 220 nm. Semaglutide eluted at 1.42 min and tirzepatide at 1.68 min, with tailing factors of 1.07 and 1.10, respectively. The authors favored isocratic elution for its simplicity, stable UV baseline, and reproducibility compared with gradient elution.^1,3

How Did the Method Perform Under Stress Conditions and in Real Samples?

Both peptides were exposed to 0.1 M hydrochloric acid, 0.1 M sodium hydroxide, 10% hydrogen peroxide, direct light, and reflux at 60 °C. Degradation ranged from 11.84 to 18.97% for tirzepatide and 12.00 to 14.80% for semaglutide. Acid hydrolysis generated one degradant peak for each drug and base hydrolysis generated two, eluting between 2.40 and 3.30 min. Resolution between each parent peak and its nearest neighbor ranged from 3.2 to 7.6, and DAD peak purity testing confirmed the parent peaks were homogeneous.¹

The method was linear from 1 to 500 µg/mL for both analytes (r ≥ 0.9998), with limits of detection of 16 ng/mL for tirzepatide and 10 ng/mL for semaglutide. Small changes in flow rate (±0.2 mL/min), mobile phase composition (±2%), and wavelength (±2 nm) produced peak area relative standard deviations below 1%. Recoveries from commercial injections were 99.5–100.9%, and recoveries from rat plasma, prepared by acetonitrile protein precipitation, were 99.45–104.10%.¹

The authors acknowledge one limitation: the short retention times may cause overlap with poorly retained substances. They also scored the method against ten sustainability and practicality metrics, including an AGREE score of 0.75 and a Blue Applicability Grade Index of 90.¹ The authors view the method as “an invaluable broad spectrum analytical tool for laboratory quality control purposes.”¹

The study reflects a broader shift in GLP-1 analysis. As these peptides move from specialist biopharmaceutical laboratories into high-volume manufacturing and routine quality control, demand is growing for methods that are fast, inexpensive, and transferable to laboratories without mass spectrometry. Simple UV-based assays such as this one address that need for potency and stability testing.

They do not, however, replace the higher-resolution techniques required for impurity profiling. GLP-1 analogues are large, lipidated peptides whose related impurities often elute on the front or tail of the main peak, and approaches such as two-dimensional liquid chromatography–high-resolution mass spectrometry (2D-LC–HRMS) are increasingly used to resolve and identify them.⁴ With next-generation multi-agonist peptides in development and sustainability metrics becoming a routine part of method reporting, analysts are likely to need both: rapid screening methods for routine control, and orthogonal separations for full characterization.

References

1. Khalil, H. A.; Hassanein, N. A.; El-Yazbi, A. F.; Mahgoub, H. A Multimodal HPLC Stability Indicating Approach for the Estimation of Semaglutide and Tirzepatide in Bulk, Pharmaceutical Dosage Forms, and Rat Plasma: A Six-Edged Sustainability Appraisal. BMC Chem. 2026, 20, 31. DOI: 10.1186/s13065-025-01716-7
2. ICH. Stability Testing of New Drug Substances and Products Q1A(R2); International Conference on Harmonisation, 2003.
3. Schellinger, A. P.; Carr, P. W. Isocratic and Gradient Elution Chromatography: A Comparison in Terms of Speed, Retention Reproducibility and Quantitation. J. Chromatogr. A 2006, 1109 (2), 253–266.
4. Sharma, N.; Jones, K. Analytical Challenges and Emerging Strategies for GLP-1 Analysis. LCGC International www.chromatographyonline.com/view/analytical-challenges-and-emerging-strategies-for-glp-1-analysis (accessed 2026-10-09).