A Case Study on the HPLC Analysis Method of Human Insulin
Until a little over 100 years ago, diabetes was still a fatal disease. Insulin is a commonly used clinical drug for diabetes treatment, and injecting insulin is one of the most effective measures to lower blood sugar.
The advent of animal insulin (the first-generation insulin) saved patients who once faced a terminal diagnosis. However, the insulin extracted from animals had low purity and was accompanied by impurities. The extraction process was costly and yielded low quantities, making it difficult to meet the demand of patients.
In the 1980s, human insulin, based on recombinant DNA technology, emerged. Highly purified human insulin allowed for more precise blood sugar control and quickly replaced the first-generation animal insulin. As the second-generation insulin, human insulin ushered in a new era of high-purity synthesis.
As a water-soluble macromolecular protein, the detection of human insulin content requires the use of size-exclusion chromatography (SEC). However, SEC often encounters issues such as peak tailing, low sensitivity, and poor reproducibility.
Welch Materials' Xtimate® SEC-120 has demonstrated excellent performance in the analysis of human insulin injection solutions. Let's take a closer look at its performance in this case study.
Chromatography conditions
| Column | Xtimate SEC-120, 5μm, 7.8×300mm |
|
| Mobile Phase | Potassium dihydrogen phosphate buffer (pH=3.0): Acetonitrile = 80:20 | |
| Flow Rate | 0.8 mL/min | |
| Injection Volume | 20 μL/50 μL | |
| Column Temperature | 40℃ | |
| Detector | UV | |
| Detection Wavelength | 214 nm | |
| Elution Program | Time(min) | Potassium dihydrogen phosphate buffer (pH=3.0): Acetonitrile = 80:20 |
| 0 | 100% | |
| 20 | 100% | |
Chromatogram of Human Insulin Reference Solution Analysis
Chromatogram of Human Insulin Injection Solution Analysis
Overlay Chromatogram of Multiple Analyses of Human Insulin Standard Solution
Conclusion
Using WelchXtimate® SEC-120 chromatography column (7.8×300mm, 5μm) under these chromatographic conditions to analyze the relevant solutions, the retention time of the target compound, human insulin, is 10.9 minutes. The retention time remains stable over multiple injections. In the injection solution samples, the main peak is not interfered with, and the peak shape is good, meeting the analysis requirements.