AUTOMATED LC/MS/MS HIGH THROUGHPUT MULTI-MODE IONIZATION QUANTIFICATION PROTOCOL APPLIED FOR MICROSOME STABILITY TEST IN DRUG DISCOVERY AND DEVELOPMENT

Importance of the topic

This summary describes an automated high-throughput LC/MS/MS workflow that leverages ESCi multi-mode ionization for rapid metabolic stability screening of drug candidates in microsomal assays. The approach addresses key bottlenecks in drug discovery by reducing instrument setup time and increasing sample throughput.

Goals and overview

The main objective of the study was to develop and validate a fully automated analytical protocol from method development through data reporting for in vitro microsome stability tests. The protocol integrates method optimization, MRM method generation, data acquisition, and quantification into a seamless pipeline using proprietary software tools.

Methodology and instrumentation

The workflow combines UPLC separation with a triple quadrupole mass spectrometer equipped with an ESCi source for alternating electrospray and APCI without hardware changes. Key elements include:

• Waters ACQUITY UPLC C18 BEH column (1.7 um, 2.1 x 50 mm) with binary solvent manager
• Mobile phase A: 10 mM ammonium acetate in 10/90 ACN/water; mobile phase B: 10 mM ammonium acetate in 80/20 ACN/methanol
• Flow rate of 0.6 mL/min at 40 C
• Waters Micromass Quattro Premier triple quadrupole MS with ESCi multi-mode ionization
• QuanOptimize software in MassLynx for automated MS and MS/MS scans, MRM setup, and batch quantification
• 96 well plate format with rat liver microsomal incubations at 1 uM analyte concentration, 15 min incubation time

Main results and discussion

Applying the protocol to a panel of 46 test compounds, the study demonstrated:

• Successful MS optimization and MRM method generation in a single pass, alternating among four ionization modes
• High throughput analysis of 48 compounds per plate with one compound per well
• Robust quantification of percent compound remaining and calculation of metabolic half-lives
• Typical run time under 2 minutes per injection enabling rapid screening

Benefits and practical applications

This automated protocol offers:

• Minimized analyst time through automated method creation and data processing
• Broader compound coverage by combining ESI and APCI in one injection
• Enhanced throughput supporting drug discovery timelines
• Flexible qualitative or semi-quantitative screening of ADME properties

Future trends and opportunities

Emerging advances may include integration of additional ionization modes, real-time data feedback for adaptive sampling, and broader adoption of automation tools across analytical laboratories. Further improvements in software algorithms could enable predictive method development and deeper integration with laboratory information management systems.

Conclusion

The study establishes a fully automated ESCi-LC/MS/MS workflow that significantly accelerates in vitro microsome stability testing without sacrificing data quality. The combination of multi-mode ionization and software-driven method development streamlines high-throughput ADME screening.

Reference

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