Impurity profiling of mycophenolate mofetil using an Orbitrap Exploris 120 mass spectrometer and Vanquish Horizon UHPLC combined with Compound Discoverer software

Importance of the topic

Impurity profiling of pharmaceutical compounds is a fundamental step in drug development and quality control. Identifying and characterizing trace-level impurities ensures the safety and efficacy of active pharmaceutical ingredients and finished drug products. The combination of ultra high performance liquid chromatography with high resolution accurate mass spectrometry provides detailed structural information critical for regulatory compliance and safety assessment.

Objectives and study overview

This application note describes a comprehensive workflow for the rapid detection and structure elucidation of impurities in mycophenolate mofetil. The study couples a Vanquish Horizon UHPLC system to an Orbitrap Exploris 120 mass spectrometer and employs advanced data mining software to profile known and unknown impurities. The aim is to demonstrate confident identification of low level impurities and degradation products in a single analytical run.

Methodology and instrumentation

The sample was prepared by diluting mycophenolate mofetil to 0.25 mg per mL in an aqueous solution containing 25 percent acetonitrile. Chromatographic separation was carried out on a C18 Vanquish Hypersil GOLD column at 50 degrees Celsius with a gradient of aqueous formate buffer and acidified acetonitrile. A flow rate of 0.4 mL per minute and a five microliter injection volume were used.

Used instrumentation

• Vanquish Horizon UHPLC system with binary pump, split sampler, column compartment and diode array detector
• Hypersil GOLD Vanquish C18 column, 2.1 x 100 mm, 1.9 micron
• Orbitrap Exploris 120 mass spectrometer with OptaMax NG electrospray source
• Compound Discoverer 3.2 software for data processing and structure proposal

Main results and discussion

Data acquisition employed high resolution full scan at 60 000 resolving power followed by data dependent MS2 at 15 000 resolution with rapid polarity switching. This approach captured both positive and negative ions in a single run. A total of 28 peaks were detected, among them the parent compound and multiple impurities. Four isomeric impurities sharing the same elemental composition were distinguished by retention time and MS2 fragmentation. Compound Discoverer software applied both targeted workflows based on expected transformations and untargeted workflows using fragment matching and isotopic pattern scoring. The software produced mirror plots comparing impurity spectra to parent spectra, enabling confident structure proposals for known and unknown impurities.

Benefits and practical applications of the method

• The high dynamic range and sensitivity of the Orbitrap Exploris 120 allows detection of low level impurities amid high concentrations of parent drug
• Rapid polarity switching produces comprehensive ionization coverage in one analysis
• Advanced data mining and node based workflows in Compound Discoverer automate impurity detection and structure elucidation
• The integrated UHPLC HRAM MS workflow increases throughput for routine impurity profiling in pharmaceutical analysis

Future trends and opportunities

Integration of ion mobility and automated library matching can further enhance separation of isomeric impurities. Machine learning models applied to HRAM spectral libraries may improve confidence in unknown structure proposals. Expansion of workflow templates in data processing software can support broader applications such as metabolite profiling and extractables and leachables analysis in packaging studies.

Conclusion

This study demonstrates an efficient workflow for impurity profiling of mycophenolate mofetil using a Vanquish Horizon UHPLC and an Orbitrap Exploris 120 mass spectrometer combined with Compound Discoverer software. The method provides high resolution full scan and MS2 data with rapid polarity switching, enabling confident detection and structural characterization of both expected and unexpected impurities. The approach streamlines routine pharmaceutical impurity analysis and can be adapted to a wide range of small molecule applications.