286 JOURNAL OF COSMETIC SCIENCE range and variety of cosmetic products are available in the market to satisfy consumer demands (1). Due to the worldwide rapid growth of the cosmetic industry, development of low-cost, robust, analytical methodologies for routine assays of cosmetic products to ensure quality products is the need of the hour in both the cosmetic and pharmaceutical industries. Use of hair dyes for the purpose of hair coloration and beautification has increased markedly. Dyes are mostly applied to human hair in the form of aqueous solutions. Typical hair dye formulations include antioxidants, wetting agents, mordants, dye modifiers, and fragrances in variable amounts to not only impart color, but also a softening, lustrous texture (1,2). The determination of polar aromatic compounds resorcinol, m-aminophenol, and p-phenylenediamine in various environmental and industrial samples is of growing interest in analytical chemistry. These are components of cosmetics such as sun creams, skin-toning creams, and hair-coloring dyes. A commercial permanent dye usually contains a mixture of dye intermediates that involve 1,2-(pyrocatechol), 1,3-(resorcinol), and 1,4-(hydroxyquinone) dihydroxybenzenes hydroxyaminobenzenes (aminophenols) and diaminobenzenes (phenylenediamines). Some of these ingredients are of great health concern. For example, p-phenylenediamine causes allergic dermatitis, and aminophenols have a nephrotoxic effect. It was reported that all these substances have mutagenic activity, and some of them are even carcinogenic. The toxicity was found in the order of p-2 and o-2 phenylenediamines m-2 phenylenediamine phenol derivatives. The content and composition of dye intermediates in a hair dye define the resulting hair color. The use of synthetic dyes in cosmetics is regulated by legislation in many countries, allowing a maximum concentration of aminophenols of 2% and for phenylenediamines of less than 4% (3–6). Resorcinol, International Union of Pure and Applied Chemistry (IUPAC) name benzene- 1,3-diol, is a reddish-orange–colored crystalline substance with molecular formula C 6 H 6 O 2 (Figure 1A). It is used as an antiseptic and disinfectant in cosmetics, especially in hair dyes (4). p-Phenylenediamine, IUPAC name 1,4-diaminobenzene, is a white to light purple powder with molecular formula C 6 H 8 N 2 (Figure 1B). It is mainly used as a component of engineering polymers and composites. It helps darken hair color due to oxidization by air or H 2 O 2 in hair dyes, and it should be used in a concentration of not more than 1% (5). m-Aminophenol, IUPAC name 3-aminophenol, is white to beige flakes with molecular formula C 6 H 7 NO (Figure 1C). It is used as synthetic dye in hair dye preparations in a concentration of not more than 2% (6). All three compounds are soluble in water, methanol, and ethanol. Literature reports the determination of several dye intermediates in different samples by high-performance liquid chromatography-ultraviolet (7−12), high-performance liquid chromatography (HPLC) with photodiode-array detection (13), HPLC-electrochemical detection (14), differential pulse voltammetry (15,16), HPLC–mass spectrometry (17,18), Gas chromatography–mass spectrometry (19,20), desorption electrospray ionization–mass spectrometry (21), capillary electrophoresis (22), and surface-enhanced Raman spectroscopy Figure 1. Structures of (A) resorcinol, (B) p-phenylenediamine, and (C) m-aminophenol.

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).