Effective Workflow for Pharmaceutical API Impurity Analysis using HR- LCMS and Compound Discoverer

Importance of the Topic

Pharmaceutical impurity analysis is essential for ensuring drug safety, efficacy and regulatory compliance. High resolution mass spectrometry combined with advanced software accelerates identification of both known and unknown impurities, supporting R&D, manufacturing and post-marketing surveillance.

Objectives and Study Overview

This study demonstrates a streamlined workflow for Fexofenadine API impurity profiling using a Thermo Scientific Orbitrap Elite mass spectrometer and Compound Discoverer software. It aims to achieve rapid, accurate and confident characterization of impurities.

Methodology and Used Instrumentation

• Sample Preparation: Fexofenadine dissolved in acetonitrile/water (1:1) at 0.3 µg/mL.
• Liquid Chromatography: Thermo Scientific Accela 1250 HPLC pump, Open Accela autosampler and PDA detector; Accucore C18 (2.1×150 mm, 2.6 µm); mobile phases water (A), acetonitrile (B) and water with 0.05% ammonium hydroxide pH 9 (C); gradient from 60% A to 5% A over 19 min; flow rate 400 µL/min; injection volume 5 µL.
• Mass Spectrometry: Orbitrap Elite with HESI II source; positive ESI; full scan (160–1500 amu) at 120 000 resolution; data-dependent MS/MS at 15 000 resolution; optimized gas flows and temperature settings.
• Data Processing: Compound Discoverer node-based workflow including Expected Finder, Compound Generator, FISh Scoring, Unknown Detector and Peak Consolidator for comprehensive impurity identification and annotation.

Main Results and Discussion

The workflow rapidly generated a base peak chromatogram of Fexofenadine and detected expected API-related impurities alongside structurally unrelated unknowns. High-accuracy mass measurements and isotopic pattern fidelity confirmed elemental compositions. FISh scoring matched fragment ions to proposed structures, while automatic adduct grouping and result filters streamlined data review. Both targeted and untargeted impurities were identified with high confidence.

Benefits and Practical Applications

• Accelerated impurity profiling with high resolution and sensitivity.
• Flexible software workflows for both targeted and unknown impurity detection.
• Automated fragment matching and isotope confirmation reduce manual interpretation.
• Improved data visualization and filtering enable efficient review for QA/QC and regulatory reporting.

Future Trends and Potential Applications

Integrating artificial intelligence for enhanced structure elucidation, expanding workflows to cover metabolites and degradation products, and increasing automation for routine QC testing. The methodology can be adapted to other APIs, natural products and complex matrices.

Conclusion

Combining Orbitrap Elite high resolution LC-MS with Compound Discoverer software provides a powerful, reliable and efficient platform for comprehensive pharmaceutical impurity analysis, enabling confident identification and structural characterization of both known and unknown compounds.