A sensitive and selective fluorescence-based bioanalytical method for quantifying 7-aminonitrazepam in urine samples using fluorescamine derivatization and response surface methodology

Addiction is a growing public health concern, and the reliable detection of drug metabolites in biological samples is crucial for diagnosis, treatment, and forensic applications. This study presents a comprehensive method for the determination of 7-aminonitrazepam, a benzodiazepine metabolite, in urine samples using fluorescamine as a derivatizing agent for sensitive detection by fluorescence spectroscopy. The mechanism involves the reaction of fluorescamine with the primary amine group of 7-aminonitrazepam via nucleophilic addition, yielding a fluorescent product that can be quantified at λ_em 486 nm following excitation at λ_ex 385 nm. The derivatization reaction was optimized by evaluating different parameters, such as pH, reagent concentration, and reaction time, using a central composite design. This revealed a quadratic relationship between the fluorescence intensity and the reaction conditions, particularly the pH and reagent concentration. The method was then validated according to ICH M10 guidelines, demonstrating excellent linearity in the range of 25–1000 ng/mL, precision below 6 %, accuracy between 97.51 and 102.66 %, and high selectivity. The developed method was successfully applied to the analysis of rat urine samples, providing reliable quantification of 7-aminonitrazepam levels posing a promising alternative to conventional techniques.