Purification, characterization and analysis of sepia melanin from commercial sepia ink (Sepia officinalis)

At the moment melanins are still enigmatic bio-pigments with structure and determination methods not clearly understood for the international scientific community. Among biopolymers, melanins are unique in many aspects. Some essential biopolymers (proteins, nucleic acids and carbohydrates) are chemically well characterized and can be determined using well established analytical methodologies. On the contrary, no available methods allow us to accurately determine melanin, mostly due to the intrinsic chemical properties of melanins. These pigments, in fact, are insoluble in a broad range of solvents and pH as well as difficult to purify as a result of the heterogeneity in their structural features. Mammalian melanins exist in two chemically distinct forms: the brown to black Eumelanin and the yellow to reddish-brown Pheomelanin. Sepia melanin obtained from Sepia officinalis consists of more than 98% of Eumelanin and is therefore used as standard material in the analysis of melanic black. Commercial sepia melanin is purified according to an undescribed procedure. In this research, extraction and purification studies were carried out on sepia melanin using a hydrochloric acid (0.5-3.0 M) treatment under mechanical or ultrasonic agitation. A high degree purity sepia melanin was obtained and further characterized using Elemental Analysis (EA), Ultraviolet-Visible (UV-VIS) and Infrared (IR) spectroscopy and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for metal ion analysis. Finally, quantitative studies of the chemical degradation of Eu polymers to Pyrrole-2,3,5-tricarboxylic acid (PTCA) and Pyrrole-2,3-dicarboxylic acid (PDCA) by High Performance Liquid Chromatography (HPLC) were performed. The procedure we developed can be used to obtain a considerably less expensive sepia melanin which could prove important as a standard in future determinations of eumelanin and pheomelanin as well as in a vast field of scientific and industrial applications in such fields as human and veterinary medicine, pharmacology and cosmetics.