title: Identification and quantification of glutathione adducts of clozapine using ultra-high-performance liquid chromatography with orthogonal acceleration time-of-flight mass spectrometry and inductively coupled plasma mass spectrometry.
abstract: The application of sulphur-specific detection via ultra-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (UPLC/ICPMS) to detect and quantify the glutathione (GSH)-adducts produced via the in vitro formation of reactive metabolites is demonstrated. The adducts were formed in human liver microsomes supplemented with unlabelled GSH for clozapine. The calculation of adduct concentration was performed via comparison of the peak areas to calibration curves constructed from omeprazole, a sulphur-containing compound over the range of 0.156 to 15.62 μM of sulphur with a detection limit of 1.02 ng of sulphur on-column. Identification of the adducts was performed using conventional UPLC/time-of-flight (TOF)-MS with the calculation of clozapine intrinsic clearance carried out by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). The use of ICPMS in this way appears to offer a novel, rapid and sensitive means of determining the quantity of GSH conjugates with the combined adducts producing 0.9 μM of reactive metabolite out of a total of 3.5 μM of metabolites. The GSH adduct therefore represents 26% of this total produced as a result of the metabolism of drug to reactive species.
authors.affiliation: Discovery DMPK, AstraZeneca R&D, Charnwood, Loughborough, Leicestershire LE11 5RH, UK.
authors.forename: Cathy
authors.initials: C
authors.lastname: MacDonald
authors.forename: Christopher
authors.initials: C
authors.lastname: Smith
authors.forename: Filippos
authors.initials: F
authors.lastname: Michopoulos
authors.forename: Richard
authors.initials: R
authors.lastname: Weaver
authors.forename: Ian D
authors.initials: ID
authors.lastname: Wilson
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003024:Clozapine / Q000032:analysis* / Q000737:chemistry; D005978:Glutathione / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D013455:Sulfur; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D013455:Sulfur; D005978:Glutathione; D003024:Clozapine