The Savior of Diabetes: Human Insulin and Its HPLC Analysis Method
Table of contents
Introduction
Records of diabetes date back to ancient Egypt (~1500 BC). Until just over a century ago, diabetes was often fatal. Insulin is a standard clinical therapy for diabetes, and insulin injection remains one of the most effective glucose-lowering measures.
The advent of animal-derived insulin (first-generation insulin) saved many patients, but insulin extracted from animals contained impurities, had limited purity, and was costly with low yield—all were insufficient to meet clinical demand.
In the 1980s, recombinant-DNA technology produced human insulin. Highly purified human insulin enables more precise blood-glucose control and quickly replaced animal insulin. As a second-generation product, human insulin initiated a new era of high-purity synthetic insulin.
Analytical method
Human insulin is a water-soluble macromolecular protein; its quantification is performed by size-exclusion chromatography (SEC). SEC can be prone to peak tailing, limited sensitivity, and poor reproducibility.
In this case, the Welch Xtimate® SEC-120 column demonstrated good performance for analysis of a human insulin injection.
Experimental Conditions
- Column: Xtimate® SEC-120 (7.8×300 mm, 5 µm)
- Mobile phase: A — KH2PO4 buffer (pH=3.0); B — acetonitrile (A:B=80:20, v/v)
- Flow rate: 0.8 mL/min
- Injection volume: 20 μL/50 μL
- Column temperature: 40 °C
- Elution time: 20 mins
- Detector: UV, 214 nm
| Analyte | Ret. Time | Peak Area | Area % | Plates | Tailing. |
| Human insulin | 10.83 | 10145.0928 | 100 | 19001.55 | 1.59 |
| Analyte | Ret. Time | Peak Area | Area % | Plates | Tailing. |
| Human insulin | 10.88 | 26374.3652 | 100 | 11186.34 | 2.88 |
Conclusion
Using the Welch Xtimate® SEC-120 (7.8 × 300 mm, 5 µm) column under the specified conditions above, the target compound (human insulin) eluted at a retention time of 10.9 min. Repeated injections showed stable retention times; the main peak in the injection samples was free from interference, with acceptable peak shape, satisfying the analytical requirements.