A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent...

Area of Science:

• * Pharmaceutical Sciences
• * Analytical Chemistry
• * Mass Spectrometry

Background:

• * The detection and structural elucidation of glutathione (GSH) conjugates are crucial for understanding drug metabolism and toxicity.
• * Existing liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods often lack the ability to broadly detect and characterize these conjugates simultaneously.
• * A need exists for a more comprehensive analytical approach to identify a wide range of GSH conjugates and their structures.

Purpose of the Study:

• * To develop and validate a novel LC/MS/MS method for the simultaneous detection and structural elucidation of diverse glutathione (GSH) conjugates.
• * To leverage high-resolution accurate mass analysis and isotope pattern triggered data-dependent product ion scans for enhanced sensitivity and specificity.
• * To demonstrate the method's capability in identifying GSH conjugates of various drug compounds and reactive intermediates.

Main Methods:

• * Development of a multiplexed high-resolution accurate mass analysis method using a LTQ/Orbitrap mass spectrometer.
• * Employing isotope pattern triggered data-dependent product ion scans for simultaneous detection and structural characterization.
• * Utilizing isotope search of mass defect filtered and control subtracted full scan accurate MS data with MetWorks software for GSH conjugate identification.

Main Results:

• * Successful detection and structural characterization of GSH conjugates for acetaminophen, tienilic acid, clozapine, ticlopidine, and mifepristone.
• * Achieved mass accuracies for precursor and product ions below 2 ppm, ensuring high data quality.
• * Demonstrated broader detection capability, independent of collision-induced dissociation behavior, and identified novel cyano-N-methylene iminium ion conjugates of verapamil.

Conclusions:

• * The developed LC/MS/MS method effectively enables simultaneous detection and structural elucidation of a broad range of GSH conjugates.
• * The method offers high mass accuracy and broad applicability for reactive intermediate trapping agents, advancing drug metabolism studies.
• * This technique provides valuable insights into both chemically tagged reactive intermediates and stable metabolites, enhancing the understanding of xenobiotic metabolism.