title: A liquid chromatography/tandem mass spectrometry method for detecting UGT-mediated bioactivation of drugs as their N-acetylcysteine adducts in human liver microsomes.
abstract: The detection of the reactive metabolites of drugs has recently been gaining increasing importance. In vitro trapping studies using trapping agents such as glutathione are usually conducted for the detection of reactive metabolites, especially those of cytochrome P450-mediated metabolism. In order to detect the UDP-glucuronosyltransferase (UGT)-mediated bioactivation of drugs, an in vitro trapping method using N-acetylcysteine (NAC) as a trapping agent followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed in this study. After the test compounds (diclofenac and ketoprofen) had been incubated in human liver microsomes with uridine diphosphoglucuronic acid (UDPGA) and NAC, the NAC adducts formed through their acyl glucuronides were analyzed using LC/MS/MS with electrospray ionization (ESI). The NAC adduct showed a mass shift of 145 units as compared to its parent, and the characteristic ion fragmentations reflected the parent. This is a concise and high-throughput method for evaluating reactive metabolites by UGT-mediated bioactivation.
authors.affiliation: Pharmacokinetics Research, Kissei Pharmaceutical Co., Ltd., 19-48 Yoshino Matsumoto City, Nagano 399-8710, Japan. hiroshi_harada1@pharm.kissei.co.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Harada
authors.forename: Takuro
authors.initials: T
authors.lastname: Endo
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Momose
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kusama
mesh_terms: D000111:Acetylcysteine / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016861:Cyclooxygenase Inhibitors / Q000032:analysis; D004008:Diclofenac / Q000032:analysis*; D014453:Glucuronosyltransferase / Q000032:analysis*; D006801:Humans; D007660:Ketoprofen / Q000032:analysis*; D008862:Microsomes, Liver / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D016861:Cyclooxygenase Inhibitors; D004008:Diclofenac; D007660:Ketoprofen; D014453:Glucuronosyltransferase; D000111:Acetylcysteine