clozapine and fipexide

A selective and sensitive approach, called extraction of product ion (XoPI) method, was developed for the detection of l-glutathione (GSH)-trapped reactive metabolites employing an Orbitrap high resolution mass spectrometer. Fragmentation of GSH conjugates in the negative ion mode leads to a product ion, deprotonated γ-glutamyl-dehydroalanyl-glycine (m/z 272.0888). As a means of utilizing this property, negative ion high resolution MS data were collected from in vitro incubations by monitoring ions from m/z 269.5 to 274.5 under in-source collision-induced dissociation. Extraction of product ions at m/z 272.0888 ± 5 ppm from this data resulted in a chromatogram exhibiting deprotonated γ-glutamyl-dehydroalanyl-glycine as the major peaks with no or very few interferences. Therefore, peaks in this extracted product ion chromatogram potentially came from GSH-trapped reactive metabolites. The GSH conjugate parent ions were then confirmed in the corresponding full scan MS data, and their structures were identified from their MS(2) fragmentation patterns. The effectiveness of the approach was assessed with four model compounds, amodiaquine, clozapine, diclofenac, and fipexide, all well-known to form GSH-trapped reactive metabolites, following incubation in human liver microsomes supplemented with β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) and GSH. The results from XoPI method were compared to two other commonly employed liquid chromatography-mass spectrometry (LC-MS) methods: precursor ion scan method and mass defect filter method. Overall, the XoPI method was more selective and sensitive in detecting the GSH conjugates. Many GSH conjugates previously not reported were detected and characterized in this study.

Topics: Amodiaquine; Chromatography, High Pressure Liquid; Clozapine; Diclofenac; Glutathione; Humans; Ions; Mass Spectrometry; Microsomes, Liver; NADP; Piperazines

Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug-induced toxicity. Reactive metabolite detection using in vitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N-(2-bromocarbobenzyloxy)-GSH (GSH-Br), for the in vitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat microsomal incubations were performed with GSH and GSH-Br for each drug with subsequent analysis by liquid chromatography/high-resolution mass spectrometry on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug-specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH-Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH-Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations.

Topics: Acetaminophen; Animals; Bromine Compounds; Chromatography, Liquid; Clozapine; Glutathione; Microsomes, Liver; Pharmaceutical Preparations; Piperazines; Rats; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Trimethoprim