Method Robustness Testing Using Empower™ Method Validation Manager Software Aided by the Empower Sample Set Generator to Automate Method Creation

Importance of the Topic

In pharmaceutical and analytical environments, demonstrating method robustness is essential to ensure consistent performance under slight variations of chromatographic conditions. Robustness testing provides confidence in method reliability during routine quality control and supports regulatory compliance by defining acceptable operating ranges.

Goals and Overview of the Study

This study aimed to evaluate the robustness of a reversed-phase HPLC method for the simultaneous analysis of naphazoline hydrochloride, pheniramine maleate, and related impurities. By applying a multivariable design of experiments (DoE) within Empower Method Validation Manager (MVM) software and automating method creation with the Empower Sample Set Generator (SSG), the work sought to identify critical chromatographic parameters and their interactions, then establish a control strategy.

Methodology and Instrumentation Used

The robustness investigation employed a full factorial DoE with four factors at two levels:

• Column temperature (low/high)
• Flow rate (low/high)
• % Organic solvent at gradient start (low/high)
• % Organic solvent at gradient end (low/high)

Sixteen experimental runs were prepared automatically using Empower SSG, which generated instrument methods, method sets, and a ready-to-run injection sequence. Chromatographic analysis was performed on a Waters Arc™ Premier system with XSelect™ Premier CSH C18 column and UV detection at 260 nm. Standard solutions were prepared in methanol and diluted to 0.1 mg/mL for APIs and 10 µg/mL for impurities.

Main Results and Discussion

All runs met the USP resolution criterion (≥1.5) except experiments 9, 10, and 12, where the naphazoline API peak fell below the threshold. Effect plots generated by Empower MVM revealed:

• Column temperature exerted a negative effect on resolution, with higher temperatures reducing separation.
• Flow rate had a positive effect, improving resolution as flow increased.
• Organic composition at gradient start and end influenced impurity resolution to a lesser extent, with minimal multi-factor interactions.

These insights guided the specification of critical method parameters and their acceptable ranges.

Benefits and Practical Applications of the Method

The combined use of Empower MVM and SSG offers:

• Automated generation of injection sequences and instrument methods, reducing manual workload and transcription errors.
• Enhanced understanding of parameter effects through visual effect plots.
• A clear basis for defining a control strategy to maintain performance criteria during routine operation.

Future Trends and Potential Applications

Advancements in software-driven DoE and automation are expected to further streamline method development and validation. Integration with machine learning and real-time system suitability monitoring may enable adaptive control strategies. Expansion of multivariate robustness testing to include additional variables (e.g., mobile phase pH, column age) can deepen method understanding and resilience.

Conclusion

This work demonstrates that a multivariable DoE approach, supported by Empower MVM and SSG, effectively assesses chromatographic method robustness. The generated effect plots pinpoint critical parameters, enabling definition of tight control limits to ensure reliable separation of naphazoline HCl, pheniramine, and related substances during routine use.

References

1. U.S. FDA. Analytical Procedures and Methods Validation for Drugs and Biologics Guidance for Industry. FDA, July 2015.
2. ICH Q14. Analytical Procedure Development. ICH, March 2022.
3. Swartz ME, Krull IS. Method Validation and Robustness. LCGC North America, May 2006;24(5).
4. Maziarz M, Rainville P. Efficient Method Development Using Systematic Screening Protocol. Waters Application Note 720007850, 2023.
5. Maziarz M, Wrona M, McCarthy SM. Increasing Efficiency of Method Validation for Metoclopramide HCl and Related Substances with Empower 3 MVM Software. Waters Application Note 720005111, 2018.
6. Waters Corporation. Empower Sample Set Generator (SSG) User Guide.