Development and validation of a new RP-HPLC method for the simultaneous determination of hydroquinone, kojic acid, octinoxate, avobenzone, BHA and BHT in skin-whitening cream

Abstract

A new and simple reverse phase high performance liquid chromatography method was developed for the simultaneous determination of the major compounds of skin-whitening pharmaceutical cream, i.e., two active pharmaceutical ingredients: hydroquinone and kojic acid, two sunscreens: octinoxate and avobenzone and two antioxidants: butylated hydroxyanisole and butylated hydroxytoluene. Separation was achieved on a Zorbax SB-Phenyl (250 × 4.6 mm; 5 μm) column, using a mobile phase consisting of water with 0.1% acetic acid and acetonitrile, in a dual-mode gradient for 14 minutes. The column was maintained at 40 °C and detection was carried out at 230 nm by using a diode-array detector. The extraction procedure was studied by a design of experiments demonstrating its suitability. The structure of an additional unknown peak eluting in all chromatograms in the same proportion was identified by HPLC/MS as the keto tautomer of avobenzone. The method developed was successfully validated in accordance with ICH guidelines with regard to selectivity, linearity, accuracy, precision and robustness. Selectivity was found to be satisfactory. The linear regression analysis data for all compounds showed a good linear relationship with r² > 0.999 in the concentration range from 50% to 120% of the label claimed for each compound. The percentage RSD for precision and accuracy of the method was found to be less than 2% for all compounds. The proposed method was applied for the determination of six major compounds in skin-whitening cream prototypes developed during the formulation development stage.