抄録
Many studies on the determination of clonazepam by high-performance, liquid chromatograpy (HPLC method) have been reported. In their reports, however, a large volume of plasma or serum sample was used. Therefore, we developed a high sensitivity microanalytical method of clonazepam determination by HPLC with a small volume of sample, since patients treated with clonazepam were almost pediatric epileptics. A plasma or serum sample (0.2ml), to which 0.1ml of 100ng/ml triazolam was added as an internal standard, was alkalinized with 1 ml of 0.01N sodium hydroxide and then was extracted with 5 ml of diethyl ether. The organic layer was evaporated to dryness. The residue was reconstituted with 70μl of mobile phase and the resulting solution was injected into the HPLC. The HPLC condition was as follows: the column was cosmosil 5CN-R (15cm×4.6mm I.D.), the mobile phase was 5mM phosphate buffer (pH 7.0)-acetonitrile (3/1, v/v), the flow rate was 1ml/min, the column temperature was 40±0.2°C and the wavelength was 221nm. The calibration curve showed a good correlation (n=18, r=0.992); the regression equation was Y=0.0164X+0.189 within the range of 5-100ng/ml. The detection limit was found to be 5ng/ml. The coefficient of variations of the withinday and between-day precisions were less than 5 and 10%, respectively. The recovery was approximately 100%. Analysis of clonazepam in serum by this method was not influenced by other antiepileptic drugs when they were administered simultaneously.
