Chemical degradation and determination of pheomelanin and eumelanin markers
Kazumasa Wakamatsu
Abstract
This is protocol involving chemical oxidation and reduction methods followed by high performance liquid chromatography (HPLC) detection of markers for pheomelanin and eumelanin.
Steps
Homogenize samples in water with Ten-Broeck glass homogenizer at a concentration of 10 mg/mL (if samples were < 5 mg, use 0.5 mL of water).
100 µL aliquots are then subjected to the chemical reactions.
Oxidation: Oxidize samples with 1.5% H2O2/K2CO3. After termination of reaction, leave mixtures for 20 hours at 25ºC (inducing secondary production of PTCA, PDCA, and TTCA)
Reduction: Heat samples with 57% HI in presence of H3PO2 at 130ºC for 20 hours. Analyze products, 4-AHP and 4-AHPEA
Levels of 4-AHP, the degradative product of DOPA pheomelanin, and 4-AHPEA, the degradative product of DA pheomelanin were analyzed by HPLC-ECD.
