Metabolic screening using on-line ultrafiltration mass spectrometry

Area of Science:

• Pharmacology
• Analytical Chemistry
• Biochemistry

Background:

• Hepatic cytochromes P450 are crucial for drug metabolism.
• Characterizing drug metabolites is essential for drug development and safety.
• Existing methods for metabolite identification can be time-consuming.

Purpose of the Study:

• To develop and validate an on-line mass spectrometric method for generating and identifying drug metabolites.
• To enable rapid screening of drugs for their metabolic profiles.
• To characterize primary metabolites formed by microsomal cytochromes P450.

Main Methods:

• Developed pulsed ultrafiltration-mass spectrometry using a 100,000 molecular weight cut-off membrane.
• Utilized rat liver microsomes, substrates (imipramine, chlorpromazine, pentoxyresorufin), and NADPH in a continuous-flow system.
• Employed electrospray mass spectrometry and tandem mass spectrometry for metabolite detection and structural confirmation.

Main Results:

• Successfully generated and identified metabolites of imipramine, including 10-hydroxyimipramine, 2-hydroxyimipramine, imipramine N-oxide, and N-desmethylimipramine.
• Demonstrated no metabolite formation in control experiments (boiled microsomes, no NADPH).
• Achieved high throughput screening with at least 20 incubations per hour, potentially up to 60.

Conclusions:

• On-line ultrafiltration electrospray mass spectrometry is a streamlined and high-throughput method.
• The technique facilitates efficient in vitro formation and characterization of microsomal drug metabolites.
• This method accelerates the screening and identification of drug metabolites in drug discovery.