OXIDATIVE HAIR DYES 45 Belgium). Pure water (18.2 Mll/cm quality) used for the preparation of solutions was obtained from a MilliQ Plus 185 system (Millipore, Molsheim, France). PREPARATION OF REAGENTS Solvents and chromatographic mobile phase. The solvents were mixtures of methanol (MeOH) and Soerensen buffer (40%). The Soerensen buffer (pH 8.1) was prepared as follows: 88 ml hydrochloric acid (0.1 N) and 2 g/1 L-ascorbic acid sodium salt (NaAcs) as an anti-oxidant agent were added to 112 ml sodium tetraborate solution. Mobile phase (aqueous phase B): a 0.05 M acetic acid solution was adjusted to a pH of 5.9 with a 10% ammonia solution and filtered through a 0.45-1am filter. When not in use, the eluent was stored at a temperature of 4øC to prevent microbiological growth. Preparation oft he synthetic formulations. Five synthetic formulations, each containing four hair dye intermediates (1,4-pal, 1,3-palS, 3-ap, and res) at various concentrations, were prepared according to "Les Colorants Wackherr" data (6) to reproduce the following formulations: dark brown, middle brown, light brown, dark blonde, and light blonde. They were prepared using the following substances: 1. Dark brown ß Matrix: Oxocap © (600.00 g/l), sodium acetate (10.00 g/l), TEA (30.00 g/l), AEB (3.00 g/l), MBS (4.65 g/l), ED (9.60 g/l) ß Hair dyes: 1,4-pd (12.50 g/l), 3-ap (5.00 g/l), res (36.50 g/l), 1,3-pdS (1.62 g/l) 2. Middle brown ß Matrix: Oxocap © (600.00 g/l), sodium acetate (10.00 g/l), TEA (30.00 g/l), AEB (3.00 g/l), MBS (4.65 g/l), ED (9.60 g/l) ß Hair dyes: 1,4-pd (7.90 g/l), 3-ap (2.80 g/l), res (21.00 g/l), 1,3 pdS (0.86 g/l) 3. Light brown ß Matrix: Oxocap © (600.00 g/l), sodium acetate (10.00 g/l), TEA (30.00 g/l), AEB (1.80 g/l), MBS (4.65 g/l), ED (9.60 g/l) ß Hair dyes: 1,4-pd (5.00 g/l), 3-ap (1.58 g/l), res (12.10 g/l), 1,3-pdS (0.48 g/l) 4. Dark blonde ß Matrix: Oxocap © (600.00 g/l), sodium acetate (10.00 g/l), TEA (30.00 g/l), AEB (1.80 g/l), MBS (4.65 g/l), ED (9.60 g/l) ß Hair dyes: 1,4-pd (3.16 g/l), 3-ap (0.89 g/l), res (6.95 g/l), 1,3-pdS (0.27 g/l) 5. Light blonde ß Matrix: Oxocap © (600.00 g/l), sodium acetate (10.00 g/l), TEA (30.00 g/l), AEB (1.80 g/l), MBS (4.65 g/l), ED (9.60 g/l) ß Hair dyes: 1,4-pd (2.00 g/l), 3-ap (0.50 g/l), res (4.00 g/l), 1,3-pdS (0.14 g/l) Each synthetic formulation was extracted and injected into the chromatographic system, after appropriate dilution in a mixture of MeOH and Soerensen buffer, pH 8.1 (40%). Preparation of the spiked shampoos. Three commercial shampoos available on the local market were diluted ten times w/w in a mixture of MeOH and mobile phase B, pH 5.9 (40%). These diluted matrix solutions were then spiked with 4 g/kg of each of the four hair dye intermediates (1,4-pd, 1,3-pdS, 3-ap, and res). The spiked shampoos obtained were also diluted ten times each w/w in a mixture of MeOH and mobile phase B, pH 5.9 (40%), before extraction and injection into the chromatographic system.

46 JOURNAL OF COSMETIC SCIENCE PROCEDURES Reversed-phase HPLC separations. A non-linear MeOH (A)/aqueous phase (B) gradient was used as described in our previous work (3). The total flow was 1 ml/min. The column was equilibrated by 25 ml mobile phase between injections. Each analysis was repeated three times. The column temperature was kept at 48øC. The data acquisition was carried out at the maximum UV absorbance wavelengths for each dye intermediate (Table I) in parallel with the UV spectra acquisition. Extraction of the matrix components from the final analyte solution. This was carried out by a three-step liquid-liquid extraction by n-heptane, previously shown to be 100% efficient for a large number of matrix components while not affecting the active compounds (3). Determination of concentration of dyes. The quantitation of each active compound was based on individual calibration lines in pure standard solutions [each hair dye being prepared in a mixture of Soerensen buffer (40%) and MeOH]. Each sample in a pure solution was injected in the HPLC system at concentrations ranging from 0 g/1 to 0.9 g/l (1,4-pd), 0 g/1 to 0.7 g/1 (3-ap), and 0 g/1 to 0.4 g/l (res and 1,3-pdS). The analysis was repeated three times in order to test the repeatability. Statistical evaluations and quality assurance. For the chromatography, the quality of the separations was evaluated according to different criteria classically considered in HPLC. The repeatability of the separation was calculated for each hair dye and was based on both the retention time and the peak area. Three other criteria were taken into account to evaluate the quality of the column: © the resolution R between two consecutive peaks, which characterizes the separating power of the column for two selected substances, © The peak asymmetry factor s, which is the measurement of the deviation of peak form from the theoretically expected Gaussian curve, and © the capacity factor k', i.e., the ratio of the net retention time (retention time - dead time) and the dead time. This capacity factor enables a comparison between columns of the separation of selected compounds. For the calibration, three regression types were tested (quadratic regression, linear re- gression, and trendline linear regression with intercept set at zero) in order to determine the best mathematical model applicable to the experiment (data not reported). The regression type finally chosen was that providing the most accurate results for the concentration of the maximum of selected dyes, i.e., the "trendline with intercept set at zero" regression. The pipettes were tested both by gravimetry and colorimetry, and the calculation of the uncertainties was performed according to international standards (7). Table I Maximum Absorbance Wavelengths for Four Hair Dye Intermediates Maximum UV absorbance wavelength (nm) Dye intermediate Maximum 1 Maximum 2 p-Phenylenediamine 235 290 m-Phenylenediamine sulfate 220 290 m-Aminophenol 235 280 Resorcinol 220 270