Simple and specific high performance liquid chromatographic method for the routine monitoring of clonazepam in plasma

A simple, specific, and sensitive high performance liquid chromatographic (HPLC) method for the determination of clonazepam in plasma is described. The drug was extracted with chloroform at an alkaline pH using flunitrazepam as internal standard. The HPLC analysis was performed using a column packed with Spherisorb C6 reverse phase, the mobile phase being a solution of acetonitrile sodium acetate 0.1 M (40/60, v/v); the ultraviolet detector was set at 306 nm. The calibration curve was linear for plasma concentrations between 2 and 200 ng/ml-1 when 1 ml of sample was analyzed. Diazepam, its metabolites, and the most common antiepileptics did not interfere with the analysis. The coefficient of variation at the lowest concentration was 14%. With this method, clonazepam can also be used as internal standard for the determination of flunitrazepam. The performance of the column was still satisfactory after the analysis of more than 500 plasma samples.