Corynantheidine, a minor alkaloid found in Mitragyna speciosa (Korth.) Havil, has been shown to bind to opioid receptors and act as a functional opioid antagonist, but its unique contribution to the overall properties of kratom remains relatively unexplored. The first validated bioanalytical method for the quantification of corynantheidine in rat plasma is described. The method was linear in the dynamic range from 1-500鈥痭g/mL, requires a small plasma sample volume (25鈥痬uL), and a simple protein precipitation method for extraction of the analyte. The separation was achieved with Waters BEH C18 2.1鈥痻鈥?0鈥痬m column and the 3-minute gradient of 10鈥痬M ammonium acetate buffer (pH鈥?鈥?.5) and acetonitrile as mobile phase. The method was validated in terms of accuracy, precision, selectivity, sensitivity, recovery, stability, and dilution integrity. It was applied to the analysis of the male Sprague Dawley rat plasma samples obtained during pharmacokinetic studies of corynantheidine administered both intravenously (I.V.) and orally (P.O.) (2.5鈥痬g/kg and 20鈥痬g/kg, respectively). The non-compartmental analysis performed in Certara Phoenix(R) yielded the following parameters: clearance 884.1鈥?/-鈥?2.3鈥痬L/h, apparent volume of distribution 8.0鈥?/-鈥?.2鈥疞, exposure up to the last measured time point 640.3鈥?/-鈥?4.0鈥痟*ng/mL, and a mean residence time of 3.0鈥?/-鈥?.2鈥痟 with I.V. dose. The maximum observed concentration after a P.O. dose of 213.4鈥?/-鈥?0.4鈥痭g/mL was detected at 4.1鈥?/-鈥?.3鈥痟 with a mean residence time of 8.8鈥?/-鈥?.8鈥痟. Absolute oral bioavailability was 49.9鈥?/-鈥?6.4 %. Corynantheidine demonstrated adequate oral bioavailability, prolonged absorption and exposure, and an extensive extravascular distribution. In addition, imaging mass spectrometry analysis of the brain tissue was performed to evaluate the distribution of the compound in the brain. Corynantheidine was detected in the corpus callosum and some regions of the hippocampus. CI - Copyright (c) 2019 Elsevier B.V. All rights reserved.