j, Cosmet. Sci., 53, 101-119 (March/April 2002) Optimization and validation of an analytical procedure for the determination of oxidative hair dyes in commercial cosmetic formulations URSULA VINCENT, GUY BORDIN, and ADELA R. RODRIGUEZ, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg, B-2440 Geel, Belgium. Accepted for publication December 31, 2001. Synopsis A method has been developed and validated for the analysis of commonly used intermediates of oxidative hair dyes in commercial cosmetic formulations, including both liquid and cream forms, in dark and blonde shades. The commercial formulations are submitted to extraction by an organic solvent, and the resulting aqueous phase is analyzed by reverse-phase HPLC with a gradient elution and detection with DAD and/or ESI-MS-MS. A spectra library containing 200-400 nm spectra of the target substances and their HPLC retention times has been recorded for the identification. The quantification of the target substances is also performed after spiking of the commercial formulations, using an external calibration. The recoveries obtained are very good for all selected intermediates. The whole procedure has been found to be highly suitable for the identification and quantification of dye intermediates. Also implemented has been a database containing (a) the retention times, (b) the spectral, MS, and MS/MS characteristics of the intermediates, (c) acidity constant values of some intermediates of interest experimen- tally determined and compared to the available NIST values, (d) the chromatographic conditions used, (e) the behavior towards extraction of dye intermediates, and (f) matrix compounds. INTRODUCTION A high range and variety of cosmetic formulations for permanent hair dyeing is available on the market under several forms, from cream to liquid. In this paper, the liquid form will be referred to as a shampoo. All these formulations contain matrix compounds in which hair dye intermediates at different concentrations are embedded. Oxidative hair dyes are aromatic organic compounds, e.g., amines, phenols, and derivatives, while a broad range of products can be used as matrix compounds. The different shades are obtained not only by the use of mixtures of different hair dye intermediates but also by reciprocal adjustment of the relative concentrations of the same intermediates. Two opposite shades could thus be obtained by changing the relative proportions of the same oxidative hair dyes. The choice of commercializing a formulation as a shampoo or a lol

102 JOURNAL OF COSMETIC SCIENCE cream is strongly related to the appreciation by industrial marketing of the best pro- fessional or non-professional handling. The same shades could thus be obtained either from a shampoo or a cream. The analytical challenge is thus to develop a method that allows both identification and quantification of target dye intermediates into existing commercial formulations available on the market, with the aim of implementing the European Union Cosmetic Directives 76/768/ECC and 95/17/EC (1). These directives address both targets of identifying and quantifying the authorized hair dyes and of verifying that banned hair dyes listed in Annex II are not present in the finished cosmetic products. The target hair dyes, listed in the C.T.F.A. International Cosmetic Ingredient Dictionary and Handbook (2), were selected after discussion and agreement with cosmetic industrials and scientists [COLIPA • (3) and L'Or6al data (4)], for the following reasons: ß They are representative of three major classes of oxidative hair dyes: aromatic amines, aminophenols, and phenols. ß They appear in the European directive as restricted in concentration (Annex III) or banned (Annex II) (1) for example, the choice of toluene-2,4-diamine, listed in Annex II, was deliberate in order to evaluate the detection of banned compounds as well as authorized ones. ß They appear regularly in the composition of commercial formulations. In our previous work, an analytical procedure involving a RP-HPLC separation after a three-step extraction by an organic solvent was developed that enables the separation and identification of 52 different dye-forming products according to their retention times and UV spectra, first in standard pure solutions (5,6), then in self-prepared synthetic formulations and commercial non-dyeing shampoos spiked with oxidative hair dye intermediates (7). The whole procedure was then validated to assure confidence in the final results (7). In the present contribution, the application of the methodology for the determination of thirteen oxidative hair dyes in real commercial formulations is shown as well as the validation process for this type of samples. This was carried out using commercial shampoos and creams available on the local market. Qualitative determination was clearly achieved for the twelve hair dyes initially present in the five formulations. Quantitation of each dye before and after spiking of the formulations with (i) the target dyes already present in the mixtures and (ii) additional commonly used hair dye inter- mediates was successfully achieved in the two types of formulations, and figures of merit of the method have been established. EXPERIMENTAL INSTRUMENTATION HPLC was performed using a two-piston HPLC pump with a low-pressure ternary gradient system module (System 325 from Kontron Instruments S.P.A., Milan, Italy), an Autosampler 560 with a loop of 20 lal (Kontron Instruments S.P.A), a Photodiode Array • Comit• de Liaison Europ•en de l'Industrie de la Parfumerie, de Produits Cosm•tiques et de Toilette.