High-performance liquid chromatography estimation of cross-linking of dihydroxyindole moiety in eumelanin

Eumelanin pigments consist of various ratios of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). On alkaline hydrogen peroxide oxidation, these indole moieties give rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), respectively. In a recent study, we detected considerable amounts of other pyrrole acids, pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) and pyrrole-2,3,4-tricarboxylic acid (isoPTCA), among the oxidation products of fossil ink sacs more than 160 million years old. PTeCA and isoPTCA arise from the cross-linking of the DHI moiety of eumelanin at the C2 and/or C3 positions. We mimicked the process of cross-linking by heating synthetic eumelanins prepared from various ratios of DHICA and DHI at 100 °C for 18 days (or at 40 °C for 180 days). The heated eumelanins were analyzed after alkaline peroxide oxidation as PTCA, PDCA, PTeCA, and isoPTCA by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. On heating, PTCA decreased rapidly due to decarboxylation, whereas PDCA decreased gradually. Concurrently, PTeCA increased gradually to levels close to PTCA. IsoPTCA also increased gradually at lower levels. Similar changes were observed at 40 °C at a much slower rate. These findings suggest that the PTeCA/PTCA ratio may serve as a good indicator of aging (cross-linking) of eumelanin.