240 JOURNAL OF COSMETIC SCIENCE to 0.5% and that of the corrected peak areas stays below the limit of 5%. For DMDM, this RSD value is of the order of what was obtained when DMDM was analyzed separately (see Table III). Nevertheless, DMDM has a retention time greater than 5 minutes and could (potentially) be confused with a dye intermediate (such as p- phenylenediamine, for instance). However, DMDM, which shows two chrornatographic peaks at 220 nm with exactly the same UV spectrum, does not show any absorbance signal at any wavelength greater than 250 nm. Thus, a selective detection of the dye intermediates can be cacried out at a wavelength greater than 250 nm. For EOP, Tables III and IV show that EOP has a retention time much greater than 30 minutes, and thus cannot be confused with any dye intermediate. Furthermore, the peak retention times and the peak areas of the dye intermediates are not significantly changed by the matrix compound regarding either the mean values or the RSD. Nevertheless, since EOP does not interfere with the hair dye separation in these con- ditions, an additional parameter, i.e., the concentration, has been checked. A broad range of concentrations of EOP were tested around the concentration used in formulations. Five RP-HPLC analyses were carried out for each concentration of EOP in a three-dye intermediate solution containing NaAsc as an antioxidant. Figure 2 shows the evolution of the mean corrected area of the peaks obtained for the three dye intermediates with increasing concentration of EOP in the sample. The graphs clearly show that the corrected areas do not vary significantly (p -- 0.05) when EOP has been added to the sample solution over a concentration range of 2 g/100 g-10 g/100 g. The statistical analysis of the results (data not reported) shows good repeatability of the analysis for each selected concentration of EOP. The RSD is less than 5% for the retention time and the corrected peak area. Concerning BHT, the results show that the presence of this matrix compound does not 25t • *T 4-ap • -, T • 2-n-l,4-pd ! ,,a. ß :0 't -- .. . 10 0 2 4 6 8 10 Concentration of EOP (g/100g) Figure 2. Influence of the concentration of EOP on the corrected peak areas of 4-ap, res, and 2-n-l,4-pd. (See text for experimental data and abbreviations.)

MATRIX COMPOUNDS AND OXIDATIVE HAIR DYES 241 affect either the retention times of the dyes or their RSD, nor the mean values of the corrected peak area. Nevertheless, it is important to note that the repeatability of the measurements for one component (2-n-l,4-pd) seems to be strongly affected by the presence of BHT, with the RSD increasing from 6.4% to 21%. BHT itself has a high RSD for its peak corrected area. Regarding these results, it appears, quite clearly, that although the matrix compound EOP does not interfere with the dye intermediates or with the column in a way that would affect the chromatographic behavior of the dye intermediates, it would be of advantage to remove BHT and DMDM from the sample solution before proceeding with the separation in order to obtain an accurate separation of the dye intermediates. OPTIMIZATION OF THE LIQUID-LIQUID EXTRACTION METHOD Since several matrix compounds are present in a real formulation, it is of immense interest to determine the effect of the isolation method on each of them. The optimi- zation of the extraction protocol leads to a one-step (LDA, TDS, NNO, ORA), two-step (OA, BHT, DMDM, NaAsc), or three-step (CC, DC) extraction by n-heptane, while EOP, NOL, LS, SS, TEA, PQ, MP, and PVP are not extracted. Two different approaches were considered for testing the extraction method. BHT and EOP were submitted to extraction in a mixture containing the dye intermediates as well, whereas in a second approach, the extraction was performed on single solutions of matrix compounds. Extraction of sample solutions containing a matrix compound and three dye intermediates. The extraction protocol has to be efficient to extract the matrix compounds, of course, but the target dyes should not be extracted. A one- to three-step extraction protocol was thus applied to sample solutions containing three selected dye intermediates (4-ap, res, and 2-n-l,4-pd), the antioxidant NaAsc, and a major matrix product (BHT or EOP). The aqueous phases obtained were submitted to HPLC, and the extraction yield was calcu- lated according to the following equation: ( corrected area after extraction ) yield (%) = 100 - k, correcte-------• re--•efor-•' extractio---•-• X 100 (1) Table V gives the extraction yield for each component in the solution, and the successive chromatograms obtained for the solution containing BHT before (a) and after (b) the extraction procedure are shown in Figure 3. First of all, it is clearly seen that the extraction procedure affects neither the chromatographic characteristics of the dye in- Table V Statistical Extraction Yield of the Components of a Dye-EOP and a Dye-BHT Mixture Compound One-step extraction yield (%) Two-step extraction yield (%) 4-ap 0 0 Resorcinol 0 0 2-n- 1,4-pd 0 0 NaAsc 19 50 EOP -- 0 BHT 89 100 (n.p.) n.p.: no peak.