From a pharmaceutical research point of view, it seems reasonable in the early phase of discovery to eliminate those drug candidates that are subject to bioactivation to electrophilic intermediates. Although rarely detectable per se, the structures of these intermediates often can be deduced by analysis of the corresponding glutathione or N-acetylcysteine conjugates. A practical approach in this regard, therefore, is to screen drug candidates in vitro for the formation of reactive metabolites via incubations with liver microsomes or hepatocytes prepared from preclinical species or humans. These incubations are normally carried out at 37°C for a period of time in the presence of glutathione or N-acetylcysteine, followed by solid-phase extraction of the reaction mixture. The resulting samples are subject to reversed-phase chromatography. Further on-line detection and identification of thiol conjugates by mass spectrometry are performed in a stepwise manner from neutral loss and/or parent ion scans to multiple-stage mass fragmentations. This would eventually lead to elucidating the structures of reactive intermediates, from which the thiol conjugates result. The experiments require minimal sample preparation and method development and often afford ample structural information on the analytes.
Series: Methods in Pharmacology and Toxicology | Pub. Date: Aug-11-2004 | Page Range: 369-383 | DOI: 10.1385/1-59259-800-5:369
Key Words: Glutathione - N-acetylcysteine - thiol conjugation - drug metabolism - bioactivation - reactive metabolites - HPLC - mass spectrometry