高速液体クロマトグラフィーによる血しょう中クロナゼパム定量法

Abstract

A simple, sensitive and specific method of high-performance liquid chromatography (HPLC) was developed for the determination of clonazepam in plasma. Clonazepam in plasma adjusted to the pH range of 8.0-9.0 was extracted with acetone-chloroform mixture (2: 3). The organic layer was evaporated to dryness in vacuo. The residue was dissolved in methanol containing 1-naphthol as internal standard, and a part of the solution was injected into the HPLC system. For the stationary phase, a reversed phase column maintained at 30°C was used. The flow rate of the mobile phase consisted of 0.1 M acetic acid-methanol (65: 35, pH 5.0) was 0.8 ml/min, and the elutes were detected by UV monitoring at 306 nm. In this condition, the peak of clonazepam was excellently separated from that of carbamazepine, and other anticonvulsants which were often administered with clonazepam at the same time did not affect the determination of clonazepam. The calibration curve from peak height ratio was linear and a coefficient of variation was 3.5%(10ng/ml, n=5). The recovery of clonazepam from plasma sample containing 20ng/ml was 99.9±4.6%(mean±S.D., n=5). The limit of detectability for quantitation was 5 ng/ml plasma when 1.0ml of plasma was used.