Development of a Robust Method for Analysis of Aspirin and Related Substances Using a Statistical Software and Quality-by-Design Approach

Importance of the Topic

Aspirin remains one of the most widely used over-the-counter and prescription medications worldwide for pain relief, fever reduction, arthritis treatment and cardiovascular event prevention. Accurate and reliable analytical methods are essential at all phases of drug development and production to ensure product quality, safety and efficacy. Applying a systematic quality-by-design approach enhances method robustness, reduces development time and facilitates successful technology transfer across laboratories.

Objectives and Study Overview

This study aimed to develop a robust, MS-compatible high-performance liquid chromatography method for simultaneous determination of aspirin active pharmaceutical ingredient and six related substances in tablet formulations. Key objectives included:

• Establishing baseline separation under mass spectrometry conditions.
• Defining a design space through risk assessment and design of experiments (DoE).
• Verifying system suitability, assay accuracy and precision according to pharmacopeial criteria.

Methodology

The method development followed analytical Quality-by-Design principles using Fusion QbD software and Empower chromatography data system. Main steps comprised:

• Definition of method performance goals, including resolution ≥2.5, peak tailing ≤1.2, RSD requirements and assay acceptance limits.
• Risk assessment to select critical method parameters: column chemistry, solvent type, mobile phase pH and gradient time.
• Screening DoE involving four column chemistries (CSH C18, BEH C18, HSS T3, HSS PFP), two organic solvents (acetonitrile, methanol), three pH levels (2.74, 3.40, 4.97) and gradient times of 7–13 minutes.
• Optimization DoE to refine gradient time (6–8 minutes), injection volume (10–25 µL), flow rate (1.0–1.5 mL/min) and column temperature (40–44 °C).
• Sample preparation using stock solutions in 60:40 water/acetonitrile with 0.1% formic acid, tablet extraction by sonication, centrifugation and filtration.
• Data modeling in Fusion QbD to identify a robust method operable design region and verification of prediction points via Empower.

Used Instrumentation

• ACQUITY Arc System with passive pre-heater and column heater/cooler.
• ACQUITY QDa Mass Detector (ESI negative mode, 50–450 m/z).
• PDA detector (210–400 nm, derived at 237 nm).
• Empower 3 chromatography data system.
• Fusion QbD method development software (v9.9.0.650 SR2b).
• XSelect HSS T3 4.6×100 mm, 2.5 µm column as final stationary phase.

Main Results and Discussion

Screening identified the HSS T3 column with acetonitrile and 0.1% formic acid at pH 2.74 over an 8.3 minute gradient as the best initial separation. Optimization yielded final conditions: flow rate 1.3 mL/min, 7 minute gradient, 10 µL injection volume and 40 °C column temperature. The design space defined a robust operable region for consistent resolution and peak shape. A diluent study showed that a 60:40 water/acetonitrile mixture with 0.1% formic acid minimized aspirin hydrolysis. The final method delivered USP resolution above 2.5 for all analytes, tailing factors below 1.2, RSD of peak areas under 2% and retention time RSD below 1%. Assay recovery ranged from 94.2 to 97.0% with precision under 5% RSD.

Benefits and Practical Applications

• Enhanced method robustness and reduced development cycles via statistical DoE.
• MS compatibility enables confirmation of known and unknown impurities by mass spectrometry.
• Streamlined workflow from DoE design to data acquisition using integrated software.
• Defined control strategy for key parameters ensures routine consistency.
• Eases method validation, transfer and regulatory compliance in pharmaceutical laboratories.

Future Trends and Opportunities

• Integration of advanced chemometric and machine learning tools to accelerate method scouting.
• Adoption of green solvents and microflow LC to reduce environmental impact and sample consumption.
• Expansion of AQbD frameworks to other analytical modalities such as UPLC-MS/MS.
• Automated column and solvent screening platforms for high-throughput method development.
• Broader use of real-time monitoring and digital twins for continuous quality assurance.

Conclusion

A robust HPLC method for aspirin and its related substances was successfully developed under MS-compatible conditions using an AQbD-based DoE approach. The final method met stringent pharmacopeial criteria for separation, sensitivity, precision and accuracy. Leveraging Fusion QbD software together with Waters ACQUITY Arc and Empower CDS streamlined method creation, enabled design space establishment and supported a clear control strategy for routine use. This approach enhances laboratory efficiency, method transferability and data reliability.

References

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