Development and Validation of Stability indicating HPLC method for determination of related substances and assay of Monobenzone drug substance

Monobenzone, a mono benzyl ether of hydroquinone, is used as a topical drug for skin depigmentation. It increases the excretion of melanin from melanocytes. A novel stability-indicating high performance liquid chromatographic approach has been established in the current study to identify related compounds and assay of Monobenzone. The Agilent 1260 system was used for the HPLC analysis, and a gradient method using 0.1% orthophosphoric acid in water and acetonitrile as the eluent was used to separate Monobenzone from known contaminants and degradation products on a Zorbax SB-phenyl (250 mm x 4.6 mm) 5 μm column. The flow rate was set at 0.8 mL/min, the column oven temperature was 30 °C, and the detector wavelength was maintained at 215 nm. The relative retention times for Hydroquinone and Monobenzone dimer were around 0.35 and 1.86 respectively, while the Monobenzone retention time was approximately 10.4 min. The total run time was 30 min. For all known analytes spanning the concentration range from LOQ to 200% of the specification level, the calibration plot revealed a linear relationship (minimum r=0.999). All known analytes' LOD and LOQ were found to be within 0.06–0.10 μg/mL and 0.15–0.24 μg/mL, respectively. A recovery study determined the accuracy of the proposed method. For Hydroquinone and Monobenzone dimer, mean recovery was found to be in the range of 95.86%–99.89% and 93.02%–99.84% respectively. The repeatability study showed that the method is precise enough within acceptable limits. Studies on solution stability and robustness produced results that fell within acceptable bounds. The proposed method shows excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ and system suitability findings. Additionally, the forced degradation study showed that the approach was stability indicating in nature.