Insulins Separation

Significance of the Topic

Effective separation of human insulin variants and analogs is essential for pharmaceutical quality control, clinical monitoring, and formulation development.

Objectives and Overview

This study aims to establish a robust HPLC method to resolve native human insulin and two synthetic analogs (Humalog and Lantus) within a single chromatographic run.

Methodology

A reversed-phase gradient was employed using acetonitrile and 25 mM ammonium acetate buffer at pH 3.5. The gradient increased from 20 % to 40 % acetonitrile over 30 minutes under ambient temperature conditions.

Instrumentation

• Column: Promix™ 4.6 × 250 mm
• Flow rate: 1.0 mL/min
• Detector: UV at 270 nm
• Injection volume: 5 µL
• Sample concentration: 30 µg/mL of each insulin in water

Main Results and Discussion

The method achieved clear baseline separation of three insulin peaks: native human insulin, Lantus, and Humalog, within a 30-minute run. Peak order reflected subtle differences in peptide hydrophobicity and structure. The chromatographic profile demonstrated high reproducibility and resolution suitable for routine analysis.

Benefits and Practical Applications

This rapid and reliable HPLC protocol supports pharmaceutical QC, stability testing, and bioequivalence studies. Its straightforward gradient, minimal sample preparation, and use of common instrumentation make it ideal for routine laboratory workflows and regulatory compliance.

Future Trends and Applications

Advancements may include ultra-high-pressure liquid chromatography (UHPLC) for faster throughput, coupling with mass spectrometric detection to enhance sensitivity and specificity, and incorporation of AI-driven software for automated method optimization and data interpretation.

Conclusion

The described reversed-phase HPLC method offers a simple, efficient, and reproducible approach to separate native human insulin and its analogs. It addresses key analytical challenges in pharmaceutical development and quality assurance, providing a solid foundation for further method enhancement and application.