OXIDATIVE HAIR DYES 113 00- V• 7 RP-HPLC/DAD M .0 7.5 15.0 22.5 l•t•tio• tim, (m• '0 1 ! (Total Ionic Curre 100 $.i.: TIC 2.64e6 •[ ø'4øl tl $.t.: 169.1 124.1 It. ]l $.i.: 2.81e3 5 S.t.: 123.1 108.1 S i ....... .-e,..-m.. x-...-va. ........ Tim e .... •.=• .•_.. 2.09e6 ............................. - -' .... Time (ram) 0.0 7.5 15.0 0.0 7,• 1•.0 Figure 3. Identification of the coeluted compounds in a dark blonde shade commercial formulation using MS/MS detection. S.t.: specific transition. S.i.: signal intensity. Peaks: (4)m-aminophenol (5) toluene-2,5- diamine sulfate (6) 2,4-diaminophenoxyethanol (7) resorcinol (13) hydroxypropyl-bis-(N-hydroxyethyl- p-phenylenediamine) HC1 and (M) matrix compound. the peaks labeled 5 and 6 can be attributed clearly to the two compounds toluene-2,5- diamine sulfate and 2,4-diaminophenoxyethanol. IN-HOUSE VALIDATION OF THE ANALYTICAL PROCEDURE FOR THE DETERMINATION OF NINE INTERMEDIATES IN TWO SPIKED COMMERCIAL FORMULATIONS Chromatographic separation and identification of the peaks. In order to fully validate the procedure, including the quantitation aspect, another approach was considered. Two of the five formulations (DBRS and DBLS) were spiked with known concentrations of both initially present hair dyes and additional ones. Each sample was prepared in mixtures of MeOH and mobile phase pH 5.9 (40%) and was submitted to chromatography using gradient 2 (see Experimental) after extraction. Each analysis was repeated ten times in order to test the repeatability of both the extraction and the chromatographic separation. The chromatographic separations obtained for the dark brown shade shampoo (DBRS) and the dark blonde shade shampoo (DBLS) are depicted in Figure 4. With these conditions of pH and gradient, the closest eluted compounds in these ranges

114 JOURNAL OF COSMETIC SCIENCE 3000 2250 - 1500 - 750 - 1 4 7 10 -200 I I 0.0 7.5 15.0 22.5 13 Retention time (min) 35.0 3000 2250 - 1500 - 750 - -200 0.0 b 4 14 13 I I I 7.5 15.0 22.5 35.0 Retention time (min) Figure 4. Chromatographic separations of two commercial formulations after spiking and matrix extrac- tion. a: "dark brown" shade shampoo. b: "dark blonde" shade shampoo. Peaks: (1)p-phenylenediamine (2) hydroquinone (4) m-aminophenol (5) toluene-2,5-diamine sulfate (6) 2,4-diaminophenoxyethanol (7) resorcinol (9) toluene-2,4-diarnine (listed in Annex II, deliberately spiked) (10) N,N'-bis-(2- hydroxyethyl)-p-phenylenediamine sulfate (13) hydroxypropyl-bis-(N-hydroxyethyl-p-phenylenediamine) HC1 (14) ot-naphtol and (M) matrix compound. See text tbr experimental data and acronyms.