An automated method to study the rapid intramolecular transacylation of drug acyl glucuronides using Cyclic Ion Mobility Spectrometry-Mass Spectrometry

Importance of the Topic

The study of acyl glucuronide transacylation is critical in drug development because these metabolites can undergo intramolecular rearrangements linked to toxicity and drug withdrawal. Rapid, reliable assessment of the rate of acyl migration helps predict safety risks and supports regulatory compliance for carboxylic acid–containing drugs.

Objectives and Overview of the Study

This work aimed to develop an automated, high-throughput approach using Cyclic Ion Mobility Spectrometry-Mass Spectrometry (cIM-MS) to monitor the rapid intramolecular transacylation of drug acyl glucuronides in real time. The focus was on 1-β-O-acyl naproxen glucuronide (sNAG) as a model compound to demonstrate method feasibility.

Methodology and Instrumentation

• Sample Preparation and Incubation

• sNAG prepared at 100 µM in MeCN/H₂O/0.1% HCOOH and 10 mM NH₄Ac (pH 7.4).
• Incubation performed at 37 °C in an LC sample manager, with aliquots drawn every ~2 min over 1.5 h.

• Flow Injection Analysis (FIA)

• Injection flow rate of 40 µL/min directly into the mass spectrometer, eliminating the need for LC separation during kinetic monitoring.

• Cyclic IMS-MS Conditions

• Waters ACQUITY UPLC I-Class PLUS and SELECT SERIES Cyclic IMS.
• Electrospray in negative mode (–1.5 kV), desolvation at 500 °C, gas flow 800 L/hr.
• Ion mobility separation time: 81 ms with multiple passes to resolve isomeric forms.
• Predicted collisional cross-sectional (CCS) values obtained via machine learning to guide mobility separation.

Key Results and Discussion

Cyclic IMS resolved the 1-β-O isomer of sNAG from its 2, 3 and 4-O isomers based on distinct arrival time distributions. Integration of mobility peak areas enabled kinetic plots showing the rapid disappearance of the 1-β-O form and concurrent rise of migrated isomers. Half-life values were calculated from these real-time data, matching literature trends. The method required no prior LC method development and captured transacylation kinetics with high temporal resolution.

Benefits and Practical Applications of the Method

• Eliminates lengthy LC method development and discrete sampling workflows.
• Provides real-time kinetic data, enabling precise half-life determination.
• Automatable in standard LC-MS autosamplers, supporting high-throughput safety screening.
• Broad applicability to other acyl glucuronides and labile metabolite classes.

Future Trends and Potential Applications

• Extending the approach to a wider range of phase II metabolites and complex biological matrices.
• Integration with predictive CCS databases and machine learning for automated isomer identification.
• Coupling to high-resolution MS and tandem IMS for structural elucidation of minor species.
• Adoption in preclinical and regulatory settings for streamlined safety assessment.

Conclusion

This study demonstrates an automated cIM-MS workflow for rapid, in-situ monitoring of acyl glucuronide transacylation. By bypassing LC separation and leveraging cyclic ion mobility, the method enables high-resolution isomer separation and real-time kinetics, significantly accelerating safety evaluation of reactive metabolites.

Reference

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