UV-Spectrophotometric Analysis of Diazepam Using Calibration Curve and Reference Standard Methods

This study aimed to evaluate the method of the calibration curve (MCC) and the external reference standard method (MRS) to assess the accuracy and precision to validate the UV-spectrophotometric technique for quantifying diazepam at λ_max = 328 nm in 95% ethanol. Selectivity was confirmed by the absence of absorbance at λ_max = 328 nm in the UV spectrum of the blank solution, whereas this wavelength was distinctly observed for diazepam. The experimental data were analyzed using linear regression, yielding the equation y = 14387.x + 0.05. The linearity assessment included the calculation of the linear regression coefficient, which resulted in R² > 0.998. The limit of detection (LOD) and limit of quantification (LOQ) were determined as 1.84 × 10⁻⁵ g/ml and 1.04 × 10⁻⁵ g/ml, respectively. The accuracy was established by the recovery rate R (%) ± RSD (%) according to ICH guidelines: MCC yielded 100.49 ± 1.35%, while MRS resulted in 105.56 ± 1.37%. The standard deviation (SD) and relative standard deviation (RSD) values remained below 1.5, indicating a strong agreement between the measured results and the actual values. The precision evaluation showed that all values of diazepam content obtained using MCC and MRS at a confidence probability of P = 98% fell within the respective confidence intervals: MCC ranged from 4.88 mg to 4.96 mg, while MRS varied between 5.12 mg and 5.20 mg. Overall, these findings confirmed that the validated method is both accurate and reliable for the determination of diazepam concentrations in pharmaceutical formulations.