| OCR Text |
Show 68 from the calibration curve. Analysis and quantitation was performed using a Packard model 'Cobra Auto-Gamma' gamma counter. Selective analysis of eumelanin and pheomelanin Sample hydrolysis. Eumelanin and pheomelanin were measured in hair samples according to the method of Ito and Fujita (111, 112, 138) with minor modifications. Approximately 5 mg of hair was used for analysis. Commercially available AHP was used as an analytical standard for the quantitation of AHP in pheomelanin-containing hair. Sepia melanin was used as an analytical standard for the quantitation of eumelanin. 0.5 ml of 57% HI + 20 III of 50% H3P04 was added to each hair or standard sample in a conical bottom tube. Tubes were capped and heated at Boac overnight. The next day, 2 ml of 50% EtOH were then added to the cooled tubes. Samples were vortexed and centrifuged at 3000 rpm for 15 min to pellet the insoluble eumelanin fraction. The supernatant was collected for the analysis of the pheomelanin degradation product AHP (see below). The eumelanin pellet was washed with EtOH and centrifuged. The ethanol wash/centrifuge step was repeated once more. Washed eumelanin granules were solubilized in 1 ml of 1 N NaOH and 20 III of 3% H202 at lOoac for approximately 1 hour and analyzed as described below. Analysis of pheomelanin. The supernatant from the hydrolysis was evaporated to dryness in a Buchler vacuum concentrator at 60aC. Sample residues were reconstituted in approximately 1.5 ml of HPLC mobile phase (see below) for analysis. HPLC analysis of AHP was performed using a Waters model 600 pump controller equipped with a Waters 717plus autosampler (Waters Corporation, Milford, MA). Electrochemical detection of AHP was obtained using a Waters model 464 electrochemical detector set at +400 mV verses a glassy carbon electrode. The chromatographic column was a Nova Pak® C18 4 11m (particle size), 3.9 x 150 mm HPLC column purchased from Waters Corporation. Mobile phase consisted of 0.1 M sodium citrate buffer, pH 4.0, containing |
