Enhanced Metabolite Identification using Orbitrap Tribrid Mass Spectrometer

Importance of the Topic

Drug metabolism governs the conversion of lipophilic compounds into water-soluble products for excretion, impacting drug clearance, efficacy and safety. Identifying both phase I and phase II metabolites is essential to characterize metabolic soft spots, active or toxic species and clearance pathways in drug development.

Objectives and Study Overview

This work presents the Orbitrap™ ID-X Tribrid mass spectrometer coupled with the AcquireX data-dependent acquisition algorithm for comprehensive, non-targeted metabolite identification. Case studies include five small molecule drugs incubated in human liver microsomes, demonstrating the workflow’s ability to detect low-level metabolites in complex biological matrices.

Methodology and Instrumentation

• Sample Preparation: Five drugs (amprenavir, bosentan, lopinavir, ritonavir, tipranavir) at 5 µM incubated with human liver microsomes, glutathione, UDPGA and alamethicin at 37 °C for 1 h; control without drug.
• Chromatography: Thermo Vanquish Flex UHPLC with Hypersil GOLD 100 × 2.1 mm, 1.9 µm column; mobile phase A (water/0.1% formic acid), B (acetonitrile); 0–95% B gradient over 16 min; 400 µL/min flow.
• Mass Spectrometry: Thermo Orbitrap ID-X Tribrid, positive ESI; AcquireX DDA with dynamic exclusion and inclusion list generation; full MS at 120 000 resolution, MSn at 30 000; stepped HCD at 20/40/60%.

Main Results and Discussion

• AcquireX automatically builds exclusion lists from blank or control runs, then targets only sample-specific ions across multiple injections, eliminating manual list curation.
• In a bile matrix spiked at low micromolar levels, AcquireX successfully triggered MSn on the parent amprenavir ion and enabled sequential MS2/MS3 for structural elucidation.
• Compared to conventional DDA (8 triggered metabolites), AcquireX identified 21 distinct amprenavir biotransformations, including hydrolysis, oxidation, methylation and conjugation products.
• High-resolution data and isotopic pattern filtering (e.g., sulfur-containing species) enhanced selectivity and confidence in metabolite discovery.
• Data processing with Compound Discoverer 3.0 provided automatic transformation prediction, FISh scoring for fragment annotation and pattern scoring; Mass Frontier enabled mechanistic interpretation of fragmentation pathways.

Benefits and Practical Applications

The integrated AcquireX workflow streamlines metabolite profiling by removing manual list inputs, delivering high-quality MSn data in a single acquisition campaign. This approach accelerates metabolite discovery, increases coverage of low-abundance species and enhances confidence in structure assignments, supporting lead optimization, safety assessment and regulatory submissions.

Future Trends and Opportunities

Advances may include further automation and machine learning-driven real-time acquisition decisions, deeper integration of chromatographic and ion mobility separations, expanded metabolite annotation databases and application of this platform to endogenous metabolomics and systems biology studies.

Conclusion

The combination of Orbitrap ID-X Tribrid MS with AcquireX acquisition and advanced software tools constitutes a powerful platform for comprehensive, high-confidence metabolite identification, offering significant gains in throughput and data quality for drug discovery and development.

Reference

• Williams, J. et al. Drug Metab. Dispos. 2004, 32, 1201–1208.
• Pähler, G.; Brink, A. Drug Discov. Today Technol. 2013, 10, e207–e213.
• Zhang, H.; et al. J. Mass Spectrom. 2008, 43, 1181–1190.
• Zhu, P.; et al. Rapid Commun. Mass Spectrom. 2009, 23, 1563–1572.