Melanin is a complex polymer that provides pigmentation in a variety of biological matrices, including hair follicles and the epidermis, and occurs naturally in two distinct forms – eumelanin and pheomelanin. Current analytical techniques to quantify eumelanin and pheomelanin begin with hydrogen peroxide degradation to produce chemical markers unique to each natural form of melanin. These degradation markers are then separated and quantified by high performance liquid chromatography with ultraviolet detection (HPLC-UV) in a potassium phosphate buffer. While this characterization is compatible with simple and pure samples, identification and quantification of the chemical markers of melanin in complex biological matrices is limited by interfering signals and incompatibility of nonvolatile phosphate buffers with mass spectrometry. This study introduces two new methods for purifying and identifying the chemical markers of melanin: (1) sample purification with solid-phase extraction (SPE) and (2) selective identification of degradation markers with mass spectrometry (HPLC-MS). These adaptations enable enhanced selectivity and accuracy in the identification and quantification of melanin in complex biological matrices and may ultimately contribute to the standardization of techniques in pigment cell research. This presentation will demonstrate enhanced clarity in HPLC-UV chromatograms provided by sample preparation with SPE and unambiguous assignment of individual peaks in HPLC-MS chromatograms to specific degradation markers of melanin. Separation, identification, and quantification of the chemical markers of melanin is a critical step in elucidating the chemical structure and properties of melanin in healthy and diseased states.

Synthesis and quantification of the chemical markers of melanin in complex biological matrices

253rd National Meeting (2017)

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