Impurity Profiling Using Orbitrap Exploris 120 Mass Spectrometer and Vanquish UHPLC Coupled with Compound Discoverer Software

Significance of Topic

The precise characterization of small molecule drug impurities is essential for ensuring drug safety, quality, and regulatory compliance. High-resolution mass spectrometry combined with advanced data processing offers a streamlined approach to identify, quantify, and elucidate the structures of trace-level contaminants in pharmaceutical development.

Study Objectives and Overview

This case study focuses on profiling impurities in Mycophenolate Mofetil using a Thermo Scientific Orbitrap Exploris 120 mass spectrometer and Vanquish UHPLC system. The objectives were to demonstrate rapid full‐scan and data‐dependent MS2 acquisition with polarity switching, and to apply Compound Discoverer 3.2 software for automated impurity detection and structure elucidation.

Methodology and Instrumentation

• Sample Preparation: A 0.25 mg/mL working solution of Mycophenolate Mofetil in water with 25% acetonitrile was analyzed.
• Liquid Chromatography: Vanquish UHPLC with a Hypersil GOLD C18 column (2.1 × 100 mm, 1.9 µm) at 50 °C, 0.4 mL/min flow, gradient from 10% to 95% B (acetonitrile/0.1% formic acid).
• Mass Spectrometry: Orbitrap Exploris 120 with OptaMax NG ESI source; rapid polarity switching full MS at 60,000 resolution followed by Top‐4 DDA MS2 at 15,000 resolution, duty cycle ~1 s, m/z range 125–1500, EASY‐IC calibration.
• Data Processing: Compound Discoverer 3.2 node‐based workflow employing accurate mass, isotope patterns, FISh Scoring, database search, and user‐defined filters for targeted and untargeted impurity identification.

Key Results and Discussion

• High‐Quality Data Acquisition: Single‐run polarity switching provided comprehensive positive and negative ion spectra, capturing both polarities in one analysis.
• Impurity Identification: Multiple expected and unexpected degradants were detected, with accurate mass and isotope pattern confirming elemental formulas.
• Structure Elucidation: FISh Coverage scores supported known structure verification and unknown proposals through fragment ion matching and annotation.
• Chromatographic Separation: Retention times between 4 and 12 min allowed baseline resolution of key impurities alongside the parent API.

Benefits and Practical Applications

• Increased Throughput: Fast duty cycle and automated data mining reduce analysis time and manual interpretation.
• Enhanced Sensitivity and Confidence: High mass accuracy and dynamic range enabled detection of low‐abundance impurities.
• Comprehensive Profiling: Combined targeted, untargeted, and structure‐proposal features facilitate thorough impurity assessments for R&D and QC labs.

Future Trends and Potential Uses

• Machine Learning Integration: Leveraging AI for predictive impurity profiling and automated structure annotation.
• Broader API Applications: Extending the workflow to biologics, natural products, and complex formulation impurities.
• Next‐Generation Instrumentation: Adoption of even higher resolution and scan-speed mass spectrometers to further enhance trace-level detection.

Conclusion

This study demonstrates that coupling Orbitrap Exploris 120 MS and Vanquish UHPLC with Compound Discoverer 3.2 yields a robust, high‐throughput workflow for impurity profiling. The approach delivers confident structural assignments, efficient data processing, and supports rigorous pharmaceutical development and regulatory requirements.