Automating Metabolic Stability Assays and Analyses using a Robotic Autosampler and LC/MS/MS Platform

Significance of the Topic

Assessing metabolic stability in liver microsomes is essential for predicting how quickly drug candidates are cleared in vivo. Early high-throughput screening of metabolic half-lives helps prioritize leads, reduce attrition, and guide medicinal chemistry optimization.

Objectives and Study Overview

This study aimed to fully automate an in vitro metabolic stability assay and its LC-MS/MS analysis for multiple model compounds using a robotic autosampler coupled to a triple quadrupole mass spectrometer. The goal was to demonstrate throughput, reproducibility, and method performance for drug discovery applications.

Methodology and Instrumentation

The automated workflow comprised:

• Reagent preparation: working internal standards, NADPH regenerating system, buffer, and microsomes.
• Incubation: 10 µM substrate with mouse or rat liver microsomes, cofactors, at 37 °C with shaking.
• Time-course sampling: aliquots taken at 0, 5, 10, 20, 30, 40, 50, and 60 min, quenched with cold acetonitrile containing internal standard, then centrifuged.
• LC-MS/MS analysis: 2 µL injections on a C18 Poroshell column (3.0×50 mm, 2.7 µm) at 55 °C; gradient from 5 % to 95 % acetonitrile with 0.1 % formic acid over 8 min; positive-mode electrospray detection.

Instrumentation Used

• GERSTEL MPS roboticPRO autosampler with CF-200 centrifuge and heated agitator.
• Agilent 1260 HPLC system and Poroshell 120 EC-C18 column.
• Agilent Ultivo Triple Quadrupole MS with Jet Stream ESI source.

Main Results and Discussion

Calibration and QC performance:

• Linear calibration for dextromethorphan, diazepam, imipramine, phenacetin, midazolam, and verapamil with R² ≥ 0.99.
• Accuracy across QC levels ranged 73.8 %–113 % (average 95.6 %).
• Precision ≤ 5.3 % RSD (average 2.8 %).

Automated time-course studies in mouse and rat microsomes produced reproducible depletion profiles consistent with manual assays, demonstrating effective automated control of incubation temperature, timing, and sample handling.

Benefits and Practical Applications

• Significant increase in sample throughput and reproducibility compared to manual workflows.
• Reduced hands-on time and error risk through full automation of sample prep and analysis.
• Flexible platform adaptable to various drug chemotypes and microsomal systems.

Future Trends and Opportunities

Advances may include integration with compound libraries for screening, coupling to microfluidic reactors, incorporation of additional biotransformation pathways, and data-driven optimization using machine learning to further accelerate DMPK screening.

Conclusion

The fully automated microsomal stability assay on the GERSTEL MPS platform, coupled with Agilent LC-MS/MS, delivers robust, high-throughput metabolic profiling of drug candidates. Calibration and QC metrics meet industry standards, and time-course results confirm reliable performance, supporting its application in DMPK workflows.

Reference

[1] Corning Discovery Labware Inc., Mammalian Liver Microsomes, Guidelines for Use, TF000017 Rev. 2.0, April 2019.