title: The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent.
abstract: An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry (microLC-microESI-MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH+ to [MH - 129]+ transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH+ to [MH - 129]+ transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen-GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).
authors.affiliation: Department of Exploratory Medicinal Sciences, Candidate Enhancement Group, Pfizer Central Research, Eastern Point Road, Mail Stop 8118W-114, Groton, CT 06340, USA. john_soglia@groton.pfizer.com
authors.forename: John R
authors.initials: JR
authors.lastname: Soglia
authors.forename: Shawn P
authors.initials: SP
authors.lastname: Harriman
authors.forename: Sabrina
authors.initials: S
authors.lastname: Zhao
authors.forename: John
authors.initials: J
authors.lastname: Barberia
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Cole
authors.forename: James G
authors.initials: JG
authors.lastname: Boyd
authors.forename: Leonard G
authors.initials: LG
authors.lastname: Contillo
mesh_terms: D001681:Biological Assay; D001711:Biotransformation; D002853:Chromatography, Liquid; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000032:analysis* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D004364:Pharmaceutical Preparations; C042431:S-ethyl glutathione; D005978:Glutathione