title: Generic method for the absolute quantification of glutathione S-conjugates: Application to the conjugates of acetaminophen, clozapine and diclofenac.
abstract: Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although1H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with1H NMR, but with a more than 100-fold lower detection limit for absolute quantification.
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
authors.forename: Michiel W
authors.initials: MW
authors.lastname: den Braver
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
authors.forename: Nico P E
authors.initials: NPE
authors.lastname: Vermeulen
authors.affiliation: AIMMS-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands. Electronic address: j.n.m.commandeur@vu.nl.
authors.forename: Jan N M
authors.initials: JNM
authors.lastname: Commandeur
mesh_terms: D000082:Acetaminophen / Q000097:blood* / Q000737:chemistry; D000818:Animals; D002853:Chromatography, Liquid; D003024:Clozapine / Q000097:blood* / Q000737:chemistry; D004008:Diclofenac / Q000097:blood* / Q000737:chemistry; D005978:Glutathione / Q000097:blood* / Q000737:chemistry; D008297:Male; D013058:Mass Spectrometry; D055432:Metabolomics / Q000379:methods; D025341:Principal Component Analysis; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY
chemical_list: D004008:Diclofenac; D000082:Acetaminophen; D005978:Glutathione; D003024:Clozapine
keywords: Acetaminophen; Alkaline hydrolysis; Clozapine; Diclofenac; Glutathione S-conjugates; O-phthaldialdehyde