Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin,\ntwo major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-\ndicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and\nthiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin.\nQuantification of these five markers by HPLC provides useful information on the quantity and\nstructural diversity of melanins in various biological samples. HPLC analysis of these markers\nusing the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for\nPTeCA) on a reversed-phase column had some problems, including the short lifetime of the column\nand, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by\ninterfering peaks in samples containing only trace levels of these markers. These problems can be\novercome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide\n(1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was\nincreased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers\nwith this improved method is good to excellent. This improved HPLC method was compared to the\noriginal method using synthetic melanins, mouse hair, human hair, and human epidermal samples.\nIn addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between\nboth HPLC methods. The other markers showed an attenuation of the interfering peaks with the\nimproved method. We recommend this improved HPLC method for the quantitative analysis of\nmelanin markers following AHPO because of its simplicity, accuracy, and reproducibility.
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