Determination of Nitrazepam and its main metabolites in urine by gas-liquid chromatography: Use of electron capture and nitrogen-selective detectors

Abstract

Nitrazepam and its main urinary metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, free and conjugared, were determined from 24-h fractions of human urine after a single oral dose of 5 mg of nitrazepam. Nitrazepam and the metabolites were extracted before and after glusulase hydrolysis with benzenedichloromethane (90:10) from a 1.0 ml sample. Methylnitrazepam and methylbromazepam served as internal standards. Recoveries were better than 90%. GLC analysis of nitrazepam was performed using a 63Ni electron-capture detector. The metabolites were measured by a dual flameless nitrogen selective detector. The detection limits were about 0.2 ng/ml for nitrazepam and 50 ng/ml for the metabolities. The nitrogen/selective detector responds similarly to all three compounds. The 63Ni electron-capture detector gives very poor response to 7-aminonitrazepam but allows very sensitive detection of nitrazepam. Combined use of the two detectors gives valuable information about the metabolic profile of nitrazepam.