Abstract
Eumelanin and pheomelanin are well known and common pigments found in nature. However, their complex polymer structure and high thermostability complicate their direct chemical identification. A widely used analytical method is indirect determination using HPLC with UV detection of both types of melanin by their most abundant oxidation products: pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), thiazole-4,5-dicarboxylic acid (TDCA), and thiazole-2,4,5-tricarboxylic acid (TTCA). An increasing interest in pigmentation in biological research led us to develop a highly sensitive and selective method to identify and quantify these melanin markers in diverse biological samples with complex matrices. By introducing solid-phase extraction (SPE, reversed-phase) following alkaline oxidation we could significantly decrease background signals while maintaining recoveries greater than 70%. Our HPLC-UV-MS method allows for confident peak identification via exact mass information in corresponding UV signals used for quantitation. In addition to synthetic melanin and Sepia officinalis ink as reference compounds eumelanin markers were detected in brown human hair and a brown bivalve shell (Mytilus edulis). Brown feathers from the common chicken (Gallus g. domesticus) yielded all four eumelanin and pheomelanin markers. The present method can be easily adapted for a wide range of future studies on biological samples with unknown melanin content.
Highlights
In the scientific literature the term ‘melanin’ has been used for any number of black, dark brown to orange and yellow pigments that are non-soluble and very thermostable [1,2,3,4,5,6,7]
We demonstrate that by including a sample clean-up step by solid-phase extraction (SPE) and adapting the chromatographic system to allow for dual detection with UV and mass spectrometric (MS), two markers each for eumelanin (PDCA and PTCA) and pheomelanin (TDCA and TTCA) can be analysed within one High-performance liquid chromatography (HPLC) run
By desalting and purifying samples via SPE we could significantly reduce the background of the diverse samples we investigated
Summary
In the scientific literature the term ‘melanin’ has been used for any number of black, dark brown to orange and yellow pigments that are non-soluble and very thermostable [1,2,3,4,5,6,7]. A more accurate definition of melanin would be that they are built through enzymatic oxidative polymerisation of DOPA (L-3,4-dihydroxyphenylalanine) subunits [4, 8,9,10,11,12]. The stability and sizes of the resulting macromolecules complicate their analysis by standard analytical methods [10, 12, 13]. A well-established method in human medical studies to identify the most common. Analysis of melanin markers in biological samples
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