Ultrafast Analysis of Metabolic Stability Assays using an Agilent Ultivo Triple Quadrupole LC/MS

Significance of the Topic

Metabolic stability assays are a critical component of drug discovery workflows, providing early insight into how drug candidates are processed by human liver enzymes. High sample volumes and the need for rapid and reliable data pose challenges for traditional LC/MS methods. Integrating ultrafast sample processing with compact instrumentation addresses throughput bottlenecks and laboratory space constraints.

Objectives and Study Overview

This study aimed to evaluate the performance of the Agilent RapidFire 365 system coupled to an Agilent Ultivo triple quadrupole LC/MS. Key goals included maintaining the ultrafast analysis cycle time demonstrated in earlier work, comparing results to both Agilent 6460 triple quadrupole LC/MS and RapidFire with Q-TOF LC/MS, and assessing data consistency across all configurations.

Methodology and Instrumentation

The workflow combined fast solid-phase extraction via RapidFire 365 with an Agilent Ultivo LC/TQ platform. Samples containing substrates, human liver microsomes, and cofactors were incubated at 37 °C and quenched at multiple time points. Extraction and elution used reversed-phase SPE cartridges and formic acid–modified buffers. RapidFire parameters included a 10 µL injection volume and 9.5 s cycle time. The Ultivo LC/TQ operated in multiple-reaction monitoring mode with optimized source conditions. Data acquisition and processing employed MassHunter Acquisition and RapidFire Analyzer software.

Results and Discussion

Analysis of eight test compounds yielded high correlation between all three instrument configurations (R2 > 0.95). Metabolic half-lives (t1/2) were determined by linear regression of log-percent remaining versus time, and compounds were categorized as fast (<20 min), intermediate (20–60 min), or slow clearance (>60 min). Calculated t1/2 values for fast and intermediate compounds showed excellent agreement across RapidFire-Ultivo, 1260/6460 LC/TQ, and RapidFire-Q-TOF setups. The ultrafast cycle time was preserved, demonstrating that the compact Ultivo system does not compromise throughput or data quality.

Benefits and Practical Applications

Using RapidFire with Ultivo LC/TQ offers:

• High throughput with 5–10 s per sample analysis
• Compact footprint suitable for space-limited labs
• Reproducible quantitative data matching larger LC/MS platforms
• Targeted MRM mode for sensitive and accurate measurements

This approach streamlines ADME screening and supports early candidate selection.

Future Trends and Potential Applications

Advancements may include expanding high-throughput assays to other ADME endpoints, integrating artificial intelligence for real-time data analysis, and further miniaturization for higher multiplexing. Combining high-resolution and targeted workflows could enable comprehensive metabolic profiling in a single run.

Conclusion

The Agilent RapidFire-Ultivo configuration delivers ultrafast, reliable metabolic stability data comparable to traditional LC/MS systems while reducing laboratory footprint. This versatile platform supports diverse analytical needs, from high-throughput screening to sensitive quantitation.

References

1. Romm MV, et al. Ultrafast Analysis of Metabolic Stability Assays Using Agilent RapidFire High-Resolution MS. Agilent Technol Appl Note 5990-8344EN, 2019.
2. McNaney CA, et al. An Automated LC-MS Process to Determine Metabolic Stability Half-Life. Assay Drug Dev Technol. 2008;6(1):121–129.
3. Obach RS. Prediction of Human Clearance of Twenty-Nine Drugs. Drug Metab Dispos. 1999;27(11):1350–1359.