Successful Method Migration of USP Insulin Assay to the Alliance™ iS Bio HPLC System

Significance of the Topic

Accurate quantitation of insulin and its degradation products is essential for ensuring drug potency and safety in pharmaceutical quality control. The USP Insulin Assay is a compendial method that defines regulatory acceptance criteria for insulin drug products. Successful migration of this assay between HPLC platforms allows laboratories to modernize instrumentation without compromising method performance or regulatory compliance.

Objectives and Study Overview

This study evaluates the transfer of the USP monograph for Insulin Assay from legacy HPLC systems to the new Alliance iS Bio HPLC System. Key goals include verifying system suitability parameters—such as resolution, peak tailing, and peak area precision—and comparing measured API potency across instruments to demonstrate equivalent or improved performance.

Methodology and Instrumentation

The study used an isocratic separation on an XBridge™ Peptide C18 column (4.6 × 150 mm, 5 µm) at 40 °C. The mobile phase consisted of Solution A (aqueous sodium sulfide/phosphoric acid/ethanolamine buffer, pH 2.3) mixed 74:26 with acetonitrile. Insulin and its A-21 desamido degradant were prepared at 1.5 mg/mL; system suitability solution was aged 72 hours to generate ≥ 5 % degradant content. Instruments compared included:

• Alliance e2695 HPLC with UV/Vis detector
• Alliance iS Bio HPLC with TUV detector
• System X Bio HPLC with DAD detector
• System Y HPLC with TUV detector

Data acquisition was performed using Empower™ 3 CDS. Key instrument characteristics—dwell volume and extra-column dispersion—were measured to assess their influence on retention time reproducibility and band broadening.

Main Results and Discussion

All systems met USP criteria: resolution ≥ 2.0 between insulin peaks, tailing ≤ 1.8, and peak area RSD ≤ 1.6 %. The Alliance iS Bio System achieved the lowest RSD for peak area (0.10 %) and the tightest retention time precision. Retention time shifts observed among systems were attributed to minor differences in column heater performance and extra-column effects. Measured API potency across systems varied by no more than 0.7 %, well within the ± 2 % internal criterion.

Benefits and Practical Applications

• Demonstrates robust transfer of a regulated compendial method to modern HPLC platforms.
• Improves precision in peak area and retention time measurements.
• Supports laboratory modernization without sacrificing regulatory compliance.
• Provides workflow consistency by sharing mobile phase and sample preparations across instruments.

Future Trends and Potential Applications

Advances in low-dispersion fluidics and precise column temperature control will further enhance method ruggedness. Adoption of bio-compatible HPLC systems with automated gradient and multi-channel capabilities may allow simultaneous analysis of insulin and other peptide hormones. Integration with advanced data analytics will streamline method qualification and real-time release testing.

Conclusion

The USP Insulin Assay was successfully migrated from older HPLC instruments to the Alliance iS Bio HPLC System without loss of performance. All system suitability criteria and potency measurements were met or improved. This case study validates that laboratories can adopt newer HPLC technology while maintaining compendial compliance and analytical precision.

Reference

1. United States Pharmacopeia. Insulin Assay Monograph. 2019.
2. Jenkins T., McConville P.R. Transfer of USP Insulin HPLC Method to UPLC. Waters Application Note, 2005.
3. Dlugasch A.B., Gauthier L., Hong P. Migration of USP Quetiapine Fumarate Method. Waters Application Note, 2024.