287 METHOD FOR SIMULTANEOUS QUANTITATION OF RESORCINOL (23). However, to the best of our knowledge, chemometric-assisted, stability-indicating reversed-phase HPLC methods for simultaneous determination of these compounds using minimal organic mobile phase are not found in the literature. In order to develop a new methodology, and to optimize the process parameters, several trial runs are required. Therefore, different companies use statistical and mathematical tools, such as response surface methodology (RSM), to achieve the best combination of factors and the best product or process responses. In order to optimize a response such as yield, it is necessary to fix the upper and lower limits for the goal (24–26). RSM has been applied for optimization of the process parameters in the sample preparation of cosmetic formulations (27,28). Traditional trial–error methods often become labor-, time-, and cost- intensive. The desirability function of RSM reduces the experimentation time while saving material and personnel costs. The current research focuses on optimizing the reversed- phase HPLC method for simultaneous determination of resorcinol, m-aminophenol, and p-phenylenediamine by a D-optimal combined design approach of RSM. Due to the use of a wide range of substances as dye intermediates, it is impossible to set up a universal method for their determination. However, it is of great practical interest to find a stability-indicating analytical method for the separation and simultaneous determination of the substances most frequently used in hair color products, such as resorcinol, p-phenylenediamine, and m-aminophenol the process parameters were previously set by chemometric tools. Minimal use of organic phase makes the proposed method environmentally friendly as well. EXPERIMENTS CHEMICALS AND REAGENTS The resorcinol was gifted by Swasthik Pharmaceuticals Pvt. Ltd (Vijayawada, India). The p-phenylenediamine and m-aminophenol were purchased from Loba Chemie (Mumbai, India). HPLC grades of sodium salt of hexane-1-sulfonic acid, methanol, acetonitrile (ACN), and o-phosphoric acid were purchased from Merck (Mumbai, India), and water was obtained from Cystron Laboratories (Vijayawada, India). Two commercial samples of hair dye were purchased from Garnier Color Naturals® (GR), batch No. B50031K (L’Oreal Paris, Mumbai, India) and Godrej Expert® (GG), batch No. B-745 (Godrej Consumer Products, Mumbai, India). The chromatographic separation was done on the Waters HPLC 717 Plus with ultraviolet detector, equipped with auto sampler on Luna C 8 column (250 mm × 4.6 mm i.d. 5 µm) compartment maintaining at 25oC and using Empower 2 software. OPTIMIZATION OF CHROMATOGRAPHIC CONDITIONS BY RSM For optimizing the chromatographic conditions, flow rate and ratios of mobile phase composition were considered as independent variables retention time of resorcinol, p-phenylenediamine, and m-aminophenol were considered as dependent variables and injection volume, column, and column temperature were kept as constants. Experimental design, data analysis, and generation of surface plots were performed using Design Expert Trial version 7.0 (Stat-Ease, Minnesota, United States).

288 JOURNAL OF COSMETIC SCIENCE Based on the values of desirability function and practical experimentations, the optimized chromatographic condition states the use of a mixture of acetonitrile:methanol:ion-pairing reagent maintained at 5:5:90 v/v ratios as the mobile phase flow rate was maintained at 1.5 mL min-1 with a run time of 15 minutes. Methanol was used as a diluent. The detection of analytes was carried out at 225 nm. PREPARATION OF STANDARD STOCK SOLUTION Standard stock was prepared using solutions A (50 mg of resorcinol), B (70 mg of p-phenylenediamine), and C (70 mg of m-aminophenol). Accurately weighed substances were transferred to a 100 mL volumetric flask. To this 50 mL of methanol was added, sonicated using an ultrasonic bath (UCB 30, Spectra Lab, Mumbai, India) for 5 minutes at 25oC to dissolve, and the volume was adjusted to 100 mL. A further 5 mL of these obtained solutions were diluted to 50 mL with methanol. Finally, the standard was prepared by adding 5 mL each of these three solutions (solutions A, B, and C) to a 50 mL volumetric flask and diluting with methanol. PREPARATION OF ION-PAIRING REAGENT The ion-pairing reagent was prepared by dissolving 1.68 g of hexane-1-sulfonic acid in 1 L of water. Then the pH was adjusted to 2.8 with o-phosphoric acid, and finally the solution was filtered through a 0.45 µm nylon syringe filter (Millipore®, Merck Millipore, Mumbai, India) and degassed. PREPARATION OF SAMPLE SOLUTION Two samples of commercial hair dyes were analyzed. Each of the hair color creams were weighed (0.3–0.5 g) using an analytical balance (Denver Instrument, New York, USA) and placed in a volumetric flask. Then 35 mL of methanol was added. The mixture was stirred vigorously for 15 minutes, sonicated using an ultrasonic bath (UCB 30, Spectra Lab, Mumbai, India) for 5 minutes at 25oC, and the volume was made up to 100 mL. A further 5 mL of this preparation was transferred into a 50 mL calibrated flask and diluted to volume using methanol. The final solution was filtered through a 0.45 µm nylon syringe (Millipore®, Merck Millipore, Mumbai, India). ANALYTICAL METHOD DEVELOPMENT AND VALIDATION The stock solution was diluted with methanol to give working standard solutions containing a series of 10–150 µg mL−1 concentrations. These solutions were filled into vials and placed in vial holders. The linearity was determined for resorcinol, p-phenylenediamine, and m-aminophenol by injecting nine concentrations of substances prepared in diluent, and the calibration curves were constructed by plotting peak area against the respective concentrations. The HPLC method was validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines (29). The