Determination of clonazepam in serum by high performance liquid chromatography

A simple, specific, and sensitive high-performance liquid chromatography (HPLC) method has been developed for the routine monitoring in serum of the benzodiazepine anticonvulsant, clonazepam. Serum spiked with internal standard, methylclonazepam, was vortex-mixed for 1 min with chloroform at an alkaline pH. The evaporated extract was dissolved in the HPLC mobile phase consisting of sodium phosphate buffer, acetonitrile, and methanol. Analytics were resolved at ambient temperature on a 5-micron Supelcosil LC-PCN column (150 X 4.6 mm) equipped with a guard column. Flow rate was 2.0 ml/min, and monitoring was at 306 nm. The calibration curve was linear from 2 to 200 ng/ml. This method provides selectivity and sensitivity with a precision of 3.5%, average recovery of 99%, and no interference from 42 commonly administered drugs.