Determination of the enaminone DM5, an anti-epileptic agent, in mouse plasma and brain tissue by high-performance liquid chromatography with ultraviolet detection

Enaminone derivatives of the 4-carbomethoxy-5-methylcyclohexane-1,3-dione series represent a new and potentially active series of compounds for the treatment of Epilepsy. Enaminone esters have been previously evaluated as compounds with potent oral anticonvulsant activity similar to class I anticonvulsants phenytoin, carbamazepine, and lamotrigine. DM5, a member of this class with -Cl in the para-substituted position, has been assessed to have the most potent pharmacological activity (ED50) in both the mouse and rat. A selective and specific high-performance liquid chromatography method was developed to quantitate DM5 in plasma and brain tissue in mice. Reverse phase chromatography with ultraviolet (lambda =307 nm) detection was utilized to quantitate eluate. A C-18 analytical column was used and the mobile phase consisted of acetonitrile and 0.05 M NaH2PO4 buffer (60:40; v/v). Liquid-liquid extraction with ether was used to extract the DM5 from plasma or brain homogenates. DM5 and carbamazepine (internal standard) eluted at similar to6.0 and 9.0 min without any interfering peaks. The calibration curves were found to be linear (r greater than or equal to0.9999) in the range of 0.1-5.0 mug/ml or mug/g. Intra-run precision's were in all in the range of 90%. The absolute recovery of the analyte in brain and plasma samples was less than or equal to 90%. The valid method accurately quantified DM5 in plasma and brain tissue samples collected from a pharmacokinetic study consisting of an intravenous bolus in the tail vein of wild type and genetically altered mice. (C) 2000 Elsevier Science B.V. All rights reserved.