Christi anburg, VA). Following digestion, samples were heated to 70°–80°C for 1h, coole d to room temperature, and diluted to 15 mL with deionized water. Each hair sam- ple was analyzed in triplicate. DYE SOLU TIONS A soluti on of dye precursors was made in deionized water with 3% ammonium hydroxide (30% active) and a pH of 10.5. The molarity of the primary intermediate was 0.031 M (PPD, HDAP) and coupler was 0.032 M (AHT, Res). Solutions were made with 0, 0.2, 1.0, and 2.0 μg/g of copper nitrate. In a 2-mL glass vial, 1720 μL of dye solution was mixed with 180 μL of hydrogen peroxide (35% active) and either 100 μL of DI water or copper nitrate solution. Three vials were made for each dye, and copper level and images taken of the color change over time. The images were taken inside a constant illumina- tion light booth with a USB camera. Each image included a reference color chart. This chart was checked to be in-line with the initial values before each measurement. The images were captured and analyzed with National Instrument’s LabView (Austin, TX) applica- tion using Vision acquisition software. COLOR MEASUR EMENT Color measur ements were taken using a Konica Minolta CM-700d handheld spectropho- tometer (Ramsey, NJ). Settings used were as follows: D65 light, 10° observer, 3 mm aperture, and specular excluded. A total of eight color re adings were made on each tress: four on each s ide. HAIR TREATMEN TS (i) Infl uence of copper ions in hair on color uptake Hair tresses pretreated with one cycle of N ice ʹN Easy Extra Light Blonde colorant were washed with water containing copper ions (0.06 μg/g) to create four sets of 12 × 2 g, 6-cm tresses with copper levels of 73 (±7) μg/g, 52 (±7) μg/g, 35 (±7) μg/g, and 21 (±7) μg/g. Each set of 12 tresses was divided into sets of four tresses which were th en colored with a permanent colorant. The three colorant tints teste d were an intense me- dium blonde/red (77/44), a medium brown/violet (4/6), and an intensive red (66/46). Each tint was mixed with a 20-volume hydrogen peroxide developer (6%) in a 1:1 ratio and applied at a dose of 4 g of mix to each gram of hair. After 30 min, hair was rinsed for 2 min, dried, and color measured using a Minolta spectrophotometer. The hair was then washed with a shampoo containing 10.5% SLE1S, 1.5% SLS, and 1.0% cocami- dopropyl betaine surfactants for a total of 14 cycles with color fade measured using a Minolta spectrophotometer after 2, 6, 10, and 14 cycles. Each wash cycle consisted of applying 0.1 g/g shampoo to the hair switch and lathering for 30 s, followed by a 30-s rinse repeated for a tot al of two shampoo applications. Hair was then dried in a hotbox at 80°C. (ii) Infl uence of histidi ne chelant on color uptake JOURNAL OF COSMETIC SCIENCE 270

A shampoo containing 10.5% SLE1S, 1.5% SLS, and 1.0% cocamidopropyl betaine sur- factants was formulated with histidine added at the 0.1% active level. Colored hair was washed for 12 cycles in tap water containing 0.06–0.09 μg/g copper with a sh ampoo with and without histidine and then analyzed for copper uptake using the ICP-OES method. Each wash cycle consisted of applying 0.1 g/g shampoo to the hair switch and lathering for 30 s, followed by a 30-s rinse repeated for a total of two shampoo applications. Ha ir was then dried in a hotbox at 80°C. This hair was then colored with two shades: an intense medium blonde/red (77/44) and a medium brown/violet (4/6). Each tint was mixed with a 20-volume hydrogen perox- ide developer (6%) in a 1:1 ratio and applied at a dose of 4 g of mix to each gram of hair. After 30 min, the hair was rinsed for 2 min, dried, and then color measured using a Minolta spectrophotometer. The hair was then washed with a shampoo containing 10.5% SLE1S, 1.5% SLS, and 1.0% cocamidopropyl betaine surfactants with and with- out 0.1% histidine for a total of 12 cycles with color fade measured using a Minolta spectrophotometer after 2, 5, 8, and 12 cycles. Each wash cycle consisted of applying 0.1 g/g shampoo to the hair switch and lathering for 30 s, followed by a 30-s rinse re- peated for a total of two shampoo applications. Hai r was then dried in a hotbox at 80°C. HDAP–AHT DYE SYNTHESIS 0.009 M HDAP and AHT were adde d to a 500-mL fl ask. Ethanol (20 mL) and am- monium hydroxide (0.083 M) were added to completely dissolve the solid. To an- other 250-mL Erlenmeyer fl ask, K3Fe(CN)6 (0.037 M) and water (100 mL) were added. A sonicator was used to dissolve the ferrate salt and form a homogeneous solu- tion. The ferrate solution was then added to the solution of dye precursors. The mix- ture was stirred continuously at room temperature for 30 min. The precipitate was fi lte red and washed with water to give a dark red solid (Z)-5-amino-4-((2-amino-1- (2-hydroxyethyl)-1H-pyrrol-3-yl)imino)-2-methylcyc lohexa-2,5-dien-1-one. This crude product was triturated with ethyl acetate and then dried in an oven (50°C) to afford 2.15 g of pure compound (yield 91.5% 98% purity confi rmed by electro- spray ionisation mass spectrometery and proton Nuclear Magnetic Resonance). DYE EXTRACTION 300 mL of metha nol was combine d wi th 150 mL of DI water in a 500-mL solvent bottle and allowed to equilibrate to room temperature before use. 100 mg of hair (four replicates fo r each copper level) were weighed into 4-mL vials, and 4 mL of extraction solvent was added to each vial and allowed to stand for 96 h. At the end of the extraction period, extracts were transferred to fresh vials and diluted 1:5 times and transferred to a disposable cuvette immediately before UV spectro- photometric analysis. A Cary 100 UV-vis spectrophotometer (Agilent, Santa Clara, CA) was used (waveleng th range: 400–800 nm, scan rate: 600 nm/min, and data inter- val: 1.0 nm). Absorbance data were normalized to account for differences in sample weight. REDOX METALS IN COLOR FORMATION IN A HAIR COLORANT 271