This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/rcm.8173 This article is protected by copyright. All rights reserved. A New Glutathione Conjugate of the Pyrrolizidine Alkaloids Produced by Human Cytosolic Enzyme Dependent Reactions in vitro Fashe Muluneh 1,2 , Merja R. Häkkinen 1 , Rami El-Dairi 1 , Markku Pasanen 1 , Risto O. Juvonen 1 1 School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Box 1627, 70211 Kuopio, Finland; 2 Reproductive & Developmental Biology Laboratory/Pharmacogenetics Group, National Institute of Environmental Health Sciences, NIH; Research Triangle Park, NC 27709, U.S.A. ABSTRAC T Rationale. The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450 mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. Methods. The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100 mM phosphate buffer pH 7.4 containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed by using liquid chromatography connected to Finnigan LTQ ion-trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole- orbitrap mass spectrometers.