Raman spectroscopy quantification of eumelanin subunits in natural unaltered pigments

Eumelanins are large polymers composed of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Reactive oxygen species generation is higher during the synthesis of DHI than during the synthesis of DHICA; thus, the production of eumelanins with high relative DHI contents may compromise the viability of cells and organisms. Here, we show that dispersive Raman spectroscopy allows a noninvasive direct detection and quantification of DHI and DHICA in unaltered eumelanins that can be applied to the analysis of natural pigments in biological tissues. Using synthetic eumelanins purely composed of DHI or DHICA, we develop a model to determine the differential contribution of these subunits to the Raman spectra of mixed eumelanins, and exemplify the predictive capacity of the model with natural eumelanins deposited in black and gray feathers of fowl. We found that the wavenumber ranges of 550-1,200 and 1,650-2,300 cm^-1 are significant to predict the DHI:DHICA ratio in unknown samples.