A novel colorimetric probe for urinary 7-aminoclonazepam analysis: Method development and optimization using factorial design

Clonazepam is a widely prescribed benzodiazepine drug used for the treatment of various neurological and psychiatric disorders. Its major metabolite, 7-aminoclonazepam, is an important biomarker for monitoring clonazepam usage and metabolism in clinical and forensic settings. This study presents a sensitive and reliable colorimetric probe for the spectrophotometric determination of 7-aminoclonazepam in urine samples. The method is based on the reaction of 7-aminoclonazepam with 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the presence of Ce (IV) as oxidizing agent, which results in the formation of a blue-colored complex with maximum absorbance at 600 nm. Factors influencing the reaction, such as reagent concentrations, pH, and reaction time, were optimized using a fractional factorial design. Under the optimized conditions, the method exhibited a linear range of 0.2–4.0 μg/mL (R² = 0.9998). Accuracy and precision were within acceptable limits, with recoveries ranging from 98.24-101.94 % and %RSD less than 1.5 %. The method was successfully applied for the determination of 7-aminoclonazepam in urine samples from patients undergoing clonazepam therapy with concentrations ranging from 0.215.-1.560 μg/mL. The results demonstrate the suitability of this colorimetric method for the routine analysis of 7-aminoclonazepam in clinical and forensic laboratories posing an accessible alternative to more complex chromatographic techniques.