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The roles of glutathione (GSH) and cysteine (Cys) in the detoxification of Microcystin-LR (MC-LR) have recently become a popular area of research. However, lacking analysis methods for MC-LR-GSH and MC-LR-Cys (two main GSH pathway metabolites) in mammals, elucidation of the detoxification mechanism and metabolic pathway of MC-LR in mammals is difficult. In this study, a novel method for the simultaneous quantitative analysis of MC-LR, MC-LR-GSH and MC-LR-Cys in rat liver was developed and validated. The analytes were simultaneously extracted from rat liver using 3 M sodium chloride solution containing 0.01 M EDTA-Na-2-5% acetic acid, followed by solid-phase extraction (SPE) on Oasis HLB and silica cartridges and determination by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS). Under the optimized pretreatment conditions and instrument parameters, good recoveries of MC-LR, MC-LR-GSH and MC-LR-Cys were obtained at three concentrations (0.2, 1.0 and 2.5 mu g g(-1) dry weight (DW)) with values ranging from 97.7 +/- 4.2 to 98.7 +/- 5.1%, 70.1 +/- 4.8 to 71.1 +/- 4.1% and 79.8 +/- 3.5 to 81.4 +/- 4.0%, respectively. The relative standard deviations (RSDs) of these compounds at 0.2, 1.0 and 2.5 mu g g(-1) DW were between 4.3% and 6.9%. The limits of detection (LODs) were 0.005, 0.007 and 0.006 mu g g(-1) DW and the limits of quantification (LOQs) were 0,017, 0.023 and 0.020 mu g g(-1) DW for MC-LR, MC-LR-GSH and MC-LR-Cys, respectively. Furthermore, this method was successfully applied to both time- and dosage-effect studies of MC-LR, MC-LR-GSH and MC-LR-Cys in vivo. (C) 2014 Elsevier B.V. All rights reserved.