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 1 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 (λ=307 nm) detection was utilized to quantitate eluate. A C18 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 ∼6.0 and 9.0 min without any interfering peaks. The calibration curves were found to be linear (r≥0.9999) in the range of 0.1–5.0 μg/ml or μg/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 ≤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.