Spectrofluorimetric Determination of 7‐Aminoclonazepam, a Major Clonazepam Metabolite, in Human Urine: A Factorial Design of Experiment Approach to Optimize Method Development

Clonazepam, a high‐potency benzodiazepine widely prescribed for seizure and panic disorders, carries a risk of abuse and dependency. This study developed a sensitive and selective spectrofluorimetric method for determining 7‐aminoclonazepam, the major metabolite of clonazepam, in human urine. A 2 6‐2 factorial design was employed to screen the optimal conditions for derivatization with NBD‐Cl as the fluorescent label, considering factors such as pH, reagent volumes, temperature, and reaction time. A significant model was attained ( p < 0.0001) revealing alkaline pH (9), elevated temperature (80°C), and high reagent concentrations as crucial for maximizing fluorescence intensity. The method demonstrated excellent linearity from 10 to 500 ng/mL ( R 2 = 0.9997), with limits of detection and quantitation of 3.3 and 10 ng/mL, respectively. Intra‐ and inter‐day precision (% RSD) were less than 4%, and recoveries ranged from 97.59% to 106.12%. The method also showed no significant interference from endogenous compounds, pharmaceutical excipients, or the parent drug. Applicability of the method was validated in human subjects receiving clonazepam therapy; 7‐aminoclonazepam was first detected after 12 h, peaked at 24 h (54.61 ± 9.870 ng/mL), and remained detectable up to 72 h post‐dose, offering a simple, cost‐effective spectrofluorimetric method for monitoring clonazepam metabolism in clinical and forensic settings.