OXIDATIVE HAIR DYES 57 500 e 300 -- 5 8 2 • 150 -- -30 I I I 0 7.5 15.0 22.5 30.0 Retention time (min) 500 4 • 300 - E --- 2 o 150 - 3 -30 I I I 0 7.5 15.0 22.5 30.0 Retention time (min) 500 4 z• 5 • 300 -- -3O I I I 0 7.5 15.0 22.5 30.0 Retention time (rain) Figure 4. Chromatographic separations of three shampoos spiked with four hair dye intermediates, after matrix extraction. a: shampoo 1. b: shampoo 2. c: shampoo 3. Peaks: (1)p-phenylenediamine (amphoteric form), (2)p-phenylenediamine (basic form), (3) m-phenylenediamine sulfate, (4) m-aminophenol, and (5) resorcinol.

58 JOURNAL OF COSMETIC SCIENCE performed on complex synthetic formulations mimicking real commercial formulations. Five synthetic formulations, each containing four hair dyes embedded in a complex matrix, have been analyzed, as well as three commercial shampoos spiked with weighed amounts of the four chosen hair dyes. In each formulation, the four hair dyes could be clearly identified using their individual retention times and UV spectra, and the analysis also showed very good repeatability in terms of retention times and peak areas for the four hair dyes in the eight formualtions. From a quantitative point of view, calibration lines were performed for each of the four hair dyes, and in all cases excellent linearity was obtained for concentration ranges commonly found in commercial formulations with very low detection limits. The concentrations of each of the hair dyes in the eight formulations could be determined with an excellent recovery rate in each formulation, in all cases higher than 90%. As a whole, the validated procedure thus appears to be highly appropriate for the identification and the quantification of oxidative hair dyes in real commercial permanent hair dyeing formulations. As a next step, the method is currently being applied and being validated for actual commercial permanent hair dyeing formulations available on the market. ACKNOWLEDGMENTS We would like to thank "Les Colorants Wackherr" and COLIPA for providing raw materials and information. We would also like to thank F. Cordeiro Raposo for his great help in the statistical calculations. The work was carried out in the frame of support to DG "Enterprise" of the European Commission. REFERENCES (1). European Union Cosmetic Directives 76/768/ECC, 0.], L262, 169-200 (September 1976) 95/17/EC, 0.], L140, 26-29 (June 1995). (2). E. Pel, G. Bordin, and A. R. Rodr guez, HPLC candidate reference method for oxidative hair dye analysis. I. Separation and stability testing,.]. Liq. Chrom. Rel. Technol., 21,883-901 (1998). (3). U. Vincent, G. Bordin, and A. R. Rodr guez, Influence of matrix compounds on the analysis of oxidative hair dyes by HPLC,.]. Cosmet. Sci., 50, 231-248 (1999). (4). J. Wenninger and G. N. McEwan, Eds., International Cosmetic Ingredient Dictionary and Handbook, 7th ed., (Cosmetic, Toiletry and Fragrance Association, Washington, DC, 1997), Vols. 1-3. (5). COLIPA: Personal communication (1994). (6). Les Colorants Wackherr: Personal communication (1998). (7). S. L. Ellison, M. Rosslein, and A. Williams, Eds., Quantifying Uncertainty in Analytical Measurement, Eurachem/CITAC Guide, 2000, pp. 1-120. (8). L.R. Snyder, J.J. Kirkland, and J. L. Glajch, Praaical HPLC Method Development, 2nd ed. (Wiley- Interscience, New York, 1997), pp. 24, 29, 208-210. (9). U. Vincent, Practical Aspects of the Determination of Oxidative Hair Dyes by RP-HPLC--Development of a Database (Annual Report, GE/R/ACH/04/99, 1999), pp. 1-24.