Applying a Software-Assisted Analytical Quality-by-Design Approach for the Analysis of Formoterol, Budesonide, and Related Compounds by UPLC-MS

Significance of the topic

The analytical quality-by-design approach combines systematic risk-based screening with advanced software to accelerate development of robust UPLC-MS methods for pharmaceutical compounds.
This strategy optimizes critical chromatographic factors to ensure reliable, high-performance separations of formoterol, budesonide, and related impurities.
Application of AQbD supports regulatory flexibility and lifecycle management.

Study objectives and overview

The primary goal was to develop a UPLC-MS assay for formoterol, budesonide, and key related compounds using a software-assisted AQbD framework.
The study employed Fusion QbD software integrated with Empower CDS and an ACQUITY UPLC H-Class PLUS system coupled to a QDa mass detector.
Method development followed a tiered workflow: initial sample workup, chemistry system screening, high-pH screening, method optimization, and verification.

Methodology and instrumentation used

The workflow comprised:

• Initial sample workup: preparation of a test mixture of budesonide, formoterol, and USP impurities in 70:30 water:acetonitrile.
• Chemistry system screening: statistical DOE to screen pH (2.0–4.2), five column chemistries, and gradient times (5–12 min) over 44 runs.
• High-pH screening: evaluation of BEH C18 at pH 6.7–10.7 to improve peak shape, particularly formoterol tailing.
• Optimization: fine-tuning flow rate, temperature, buffer pH (8.0–9.0), and gradient times on a 100 mm column to define the operable design region.
• Verification: replicate injections under best overall answer conditions to confirm predicted performance against real results.

Instrumentation used:

• ACQUITY UPLC H-Class PLUS System with QSM, FTN, CM, PDA detector.
• ACQUITY QDa Mass Detector (ESI+, 0.8 kV capillary voltage, 600 °C source).
• Empower 3 CDS with S-Matrix Fusion QbD software.
• Columns: ACQUITY UPLC BEH C18, BEH Shield RP18, CORTECS T3, CORTECS Phenyl, HSS PFP (2.1×50 mm, 1.6–1.7 µm); final method on 2.1×100 mm BEH C18.

Main results and discussion

Fusion QbD reduced the number of necessary experiments by over 40% compared to full factorial designs.
Screening at low pH identified initial retention and wavelength (244 nm) but revealed insufficient separation and high tailing factors (>2.0) for formoterol.
High-pH screening with BEH C18 improved formoterol symmetry (tailing <1.3) and allowed separation of budesonide epimers.
Optimization on a longer column established a robust operable design region delivering baseline resolution (>2.0) for all seven analytes, formoterol tailing <1.2, across temperature (32–39 °C) and flow (0.35–0.5 mL/min) ranges.
Verification runs confirmed model predictions within 5% of actual resolution and tailing values.

Benefits and practical application

• Automation of method setup and DOE generation accelerates development timelines.
• Statistical sampling by Fusion QbD ensures comprehensive factor coverage with fewer runs.
• Integration of MS detection streamlines peak identification across changing conditions.
• Defined operable design regions support method robustness and regulatory flexibility.
• Approach applicable to routine QC, stability studies, and impurity profiling in pharmaceutical analysis.

Future trends and potential applications

Advances in AI-driven DOE and machine learning models will further reduce experimental burden and adapt methods in real time.
Integration of inline analytics and PAT tools may enable continuous monitoring and control of chromatographic performance.
AQbD frameworks will extend to novel modalities, such as oligonucleotides, biologics, and complex mixtures in life sciences.
Cloud-based software platforms may facilitate collaborative method development and cross-site harmonization.

Conclusion

The software-assisted AQbD strategy delivered a fit-for-purpose UPLC-MS method for formoterol, budesonide, and related impurities with reduced development time and high robustness.
Fusion QbD combined with Empower CDS and ACQUITY instrumentation effectively guided screening, optimization, and verification steps.
The resulting method features baseline resolution, controlled tailing, and a validated design space that supports future method adjustments without full revalidation.

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