High-Throughput In Vitro ADME Analysis with Agilent RapidFire/MS Systems: Cytochrome P450 Inhibition

Significance of the topic

Analysis of cytochrome P450 inhibition plays a critical role in drug discovery to prevent adverse drug interactions and optimize candidate selection. High throughput and cost effective screening methods enable early elimination of unsuitable compounds and support efficient ADME profiling.

Objectives and study overview

This study evaluates the performance of the Agilent RapidFire high throughput mass spectrometry system in measuring CYP450 inhibition compared to traditional LC/MS/MS. Direct and time dependent inhibition assays were conducted using FDA recommended probe substrates and human liver microsomes to determine IC50 values for multiple CYP isoforms.

Methodology and instrumentation

Direct inhibition experiments involved seven point inhibitor dilution series with pooled human liver microsomes and stably labeled isotope internal standards. Time dependent inhibition used a two time point IC50 shift approach for CYP3A4 substrates midazolam and testosterone. Samples were processed individually on RapidFire MS and on a conventional triple quadrupole LC/MS/MS platform.

Used Instrumentation

• Agilent RapidFire high throughput sample processor
• Triple quadrupole or time of flight mass spectrometer
• Traditional LC system coupled to a triple quadrupole mass spectrometer

Main results and discussion

IC50 values obtained with RapidFire MS correlated closely with traditional LC/MS/MS across eight enzyme substrate pairs R2 ~0.986. The RapidFire workflow achieved sample cycle times of approximately 6 seconds compared to 2 to 4 minutes per sample by LC/MS/MS. Time dependent inhibition assays demonstrated equivalent potency ranking while increasing assay throughput by over 20 fold. Comprehensive IC50 curves were generated in less time than that required for a single LC/MS/MS analysis point.

Benefits and practical applications

The high throughput RapidFire MS approach significantly accelerates ADME profiling by reducing analysis time and cost. Integration into drug discovery pipelines allows earlier decision making supports larger sample sets and enhances laboratory productivity. This method is applicable to routine CYP450 inhibition screening in research and QA QC environments.

Future trends and opportunities

Advances in automated sample handling and high speed mass spectrometry will further expand throughput and data quality. Emerging platforms may integrate microplate robotics multiplexed assays and data driven machine learning to predict metabolic liability. Extending this approach to other enzyme systems and metabolite profiling will broaden its impact in pharmaceutical research and personalized medicine.

Conclusion

The Agilent RapidFire MS system provides a validated reliable and high throughput alternative to conventional LC/MS/MS for CYP450 inhibition assays. It delivers equivalent IC50 data with substantial time savings and enhanced productivity supporting efficient drug discovery and development workflows.

References

1. Perloff E et al Validation of cytochrome P450 time dependent inhibition assays a two time point IC50 shift approach facilitates kinact assay design Xenobiotica 2011 39 2 99 112
2. Perloff E et al Comparison of RapidFire Ultra High Throughput LC MS MS with Traditional LC MS MS for Cytochrome P450 Inhibition Testing Abstract 106 Presented at the 11th European ISSX Meeting May 17 20 2009 Lisbon Portugal
3. Miller V et al Evaluation of High Throughput Screening Methods for Time Dependent Inhibition of CYP3A4 Utilizing RapidFire LC MS MS Technology Presented at the 11th European ISSX Meeting May 17 20 2009 Lisbon Portugal