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In vivo rat liver and brain tissue metabolites of an anticancer drug, cisplatin ((cis-diamminedichloroplatinum(II)) (CP)), have been identified and characterized by using liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with on line hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, whole liver and brain tissues were removed after intravenous administration of CP to adult male Sprague-Dawley rats (n = 3 per group). The liver and brain tissues were homogenized and extracted using a metabolite extraction procedure that involves liquid extraction with phosphate buffer containing ethyl acetate and protein precipitation with mixed solvents of methanol-water-chloroform followed by a solid-phase clean-up procedure on Oasis (R) HLB 3cc cartridges and then subjected to LC/ESI-HRMS analysis. A total of seventeen in vivo metabolites have been identified in liver tissue homogenates including mono aqua CP, monohydroxy CP, dihydroxy CP, and hydroxy aqua CP metabolites which were also observed in brain tissue homogenates. Out of the seventeen, thirteen metabolites are new, whereas the remaining four metabolites, M1-M4 were recently reported in our previous study. The structures of the metabolites were proposed using LC-MS/MS experiments and accurate mass measurements in conjunction with online deuterated experiments. These deuterated experiments were used to further support the structural characterization of drug metabolites. The identification of new metabolites provides essential information for further pharmacological and clinical studies of CP, and may also be useful to develop effective new anticancer agents.