Development of a sample preparation and analysis method for therapeutic monitoring of diazepam and major metabolite in alcohol withdrawal syndrome treatment

Diazepam, a widely prescribed benzodiazepine, is frequently used for the management of alcohol withdrawal syndrome, anxiety, seizures, and muscle spasms. Its monitoring is critical due to its potential for abuse and the therapeutic importance of its metabolite nordiazepam. A sustainable and environmentally friendly highperformance liquid chromatography method was developed and validated for the quantification of diazepam and its active metabolite nordiazepam in human plasma samples. The chromatographic analysis was conducted on an HPLC system equipped with a UV detector. Separation was achieved using a Kinetex F5 column (150 x4.6 mm, 5 mu m). The mobile phase consisted of acetonitrile and phosphate buffer adjusted to pH 4.3 (30:70, v/v), delivered in isocratic mode at a flow rate of 1.0 mL/min, with the pH adjusted to optimize resolution. The method demonstrated excellent sensitivity, reproducibility, and linearity for both compounds, highlighting its applicability for drug monitoring and toxicological studies. Moreover, the environmentally conscious selection of materials and conditions underscores the method's compliance with green analytical chemistry principles. Both analytes were successfully detected and quantified in all fifty-three patients under the treatment of alcohol withdrawal syndrome. The measured concentrations of diazepam (0.23-1.4 mu g/mL) and its metabolite nordiazepam (0.22-3.78 mu g/mL) displayed considerable variability among individuals.