OXIDATIVE HAIR DYES 105 Time in minutes % MeOH (A) % Aqueous phase (B) 0 0 100 1 0 100 2O 19 81 30 80 20 35 8O 2O 40 95 5 5O 95 5 53 0 100 74 0 100 The total flow was also 1 ml/min. Each analysis was repeated ten times. In both cases the data acquisition was carried out at the maximum UV absorbance wavelengths for each dye intermediate in parallel with the UV spectra acquisition. The UV maxima for the dyes are p-phenylenediamine (235,90 nm), hydroquinone (220, 290 nm), m-aminophenol (230, 280 nm), toluene-2,5-diamine sulfate (235,290), resorcinol (220, 270 nm), 2,4-diaminophenoxyethanol (235,295 nm), 0-aminophenol (235,290), p-aminophenol (220, 297), toluene-2,4-diamine (235, 290), 2-methyl-5-hydroxyethyl- aminophenol (240 nm), hydroxypropyl-bis-(N-hydroxyethyl-p-phenylenediamine) HC1 (256 nm), 6-hydroxyindole (220, 290 nm), and o•-naphtol (235, 290 nm). HPLC-DAD-ESI/MS conditions. An HPLC coupled with an electrospray mass spectrom- eter was used for the identification of coeluted compounds in some commercial formu- lations. The HPLC flow (1 ml/min) was split between the photodiode array detector and the electrospray ionization source, with a flow rate ratio of 9/1. The voltage of the capillary and the source cone voltage were set at 3.2 kV and 15 kV, respectively. The source block and desolvation temperatures were 130 ø and 400øC, while the desolvation and nebulizer gas (NQ pressures were 548 1/h and 53 l/h, respectively. The acquisition was performed in the multiple reaction monitoring (MRM) mode, with a dwell time of 0.1 s, at the mass unit resolution (10% valley). The MS characteristics of the selected hair dye intermediates are given in Table I. Extraction of the matrix components from the final analyte solution. This is carried out by a three-step liquid-liquid extraction by n-heptane, previously shown to be 100% efficient Table I MS Characteristics for Five Selected Hair Dye Intermediates Cone voltage Collision energy Compounds Parent ions (volts) (eV) Daughter ions m-Aminophenol 110.06 30 20 93.11 2O 65.13 Toluene-2,5-diamine sulfate 123.09 30 20 108.08 30 77.01 2,4-Diaminophenoxyethanol 169.11 25 20 124.12 20 108.13 Resorcinol 111.04 30 15 93.17 2O 65.19 Hydroxypropyl-bis-(N- 361.31 25 25 165.22 hydroxyethyl-p- 40 121.15 phenylenediamine) HC1

106 JOURNAL OF COSMETIC SCIENCE for a large number of matrix components while not affecting the active compounds (6) except ot-naphtol. Identification of oxidative hair dye intermediates. A spectra library containing 200•400 nm spectra of the target substances and their HPLC retention times has been recorded for the identification using the acquisition software. The active compounds contained in the different formulation samples can thus be identified by means of comparison of both retention time and UV spectra. Determination of concentration of dyes. The quantitation was performed for nine of the thirteen identified oxidative hair dyes, namely p-phenylenediamine, toluene-2,4- diamine, toluene-2,5-diamine sulfate, m-aminophenol, resorcinol, hydroquinone, ot-naphtol, 2,4-diaminophenoxyethanol, and hydroxypropyl-bis-(N-hydroxyethyl-p- phenylenediamine) HC1. It was based on calibration lines separately established for each of them in pure standard solutions prepared in a mixture of mobile phase pH 5.9 (40%) and MeOH. Each sample in a pure solution was injected in the HPLC system at concentrations ranging from 0 g/1 to 0.8 g/1 (p-phenylenediamine), 0 g/1 to 2 g/1 (toluene-2,4-diamine and toluene-2,5-diamine sulfate), 0 g/1 to 0.3 g/1 (m-aminophenol), 0 g/1 to 0.8 g/1 (resorcinol), 0 g/1 to 0.3 g/1 (hydroquinone), 0 g/1 to 0.08 g/1 (ot-naphtol and hydroxypropyl-bis-(N-hydroxyethyl-p-phenylenediamine) HC1, and 0 g/1 to 0.1 g/1 (2,4-diaminophenoxyethanol). The analysis was repeated 15 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, namely the resolution R, the peak asymmetry factor s, and the capacity factor k' (7). This capacity factor enables a comparison between columns of the separation of selected compounds. For the calibration, the regression type used was as described previously (7), i.e., the "trendline with intercept set at zero" regression. The pipettes were tested both by gravimetry and colorimetry, and the cal- culation of the uncertainties was performed according to international standards (8). RESULTS AND DISCUSSION An analytical procedure was optimized and validated for the analysis of commonly used oxidative hair dye intermediates. Five commercial formulations were tested using this procedure, each of them containing initially four to six active hair dyes according to the ingredient labeling. For two of these formulations [one brown shade (DBRS) and one blonde shade (DBLS), shampoos], quantification was performed for target hair dyes before or after spiking. CHROMATOGRAPHIC SEPARATION AND IDENTIFICATION OF THE PEAKS IN NON-SPIKED FORMULATIONS Each sample prepared in mixtures of MeOH and Soerensen buffer (pH 8.1) was sub- mitted to chromatography using the gradient 1 after extraction. Each analysis was repeated three times in order to test the repeatability of both the extraction and the