Purification and Quantification of the Chemical Markers of Melanin to Enhance Early Diagnosis of Melanoma

Abstract

Melanoma is characterized by overproduction of the pigment melanin, which is present in two forms in human skin – eumelanin and pheomelanin. High discordance rates among experienced pathologists in the diagnosis of questionable malignant epidermal samples demonstrate a need for more robust and objective diagnostic tools. Although optical imaging qualitatively suggests that a high ratio of eumelanin to pheomelanin corresponds to the incidence of malignant melanoma, no chemical analysis to date has successfully quantified this ratio. Reproducible techniques for quantifying the natural forms of melanin may provide insight into the molecular mechanisms underlying melanoma and may be used to determine whether a high eumelanin: pheomelanin ratio is indicative of malignant melanoma. Current protocols for analyzing melanin entail chemical degradation of the pigment into markers specific to each of the two natural forms. These chemical markers are separated and quantified by high performance liquid chromatography with ultraviolet detection (HPLC‐UV). While this characterization has proved useful, identification of the chemical markers in complex biological matrices is limited by interfering signals. This study introduces two new methods for identifying and quantifying 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). Application of these techniques to melanoma cells derived from a metastatic lung site and normal skin fibroblast cells demonstrate enhanced selectivity and reproducibility in the quantification of chemical markers of melanin in complex biological matrices. Sample preparation with SPE improves clarity in HPLC chromatograms and MS detection allows unambiguous assignment of individual peaks to specific degradation markers of melanin. Enhanced selectivity and accuracy in the quantification of melanin may ultimately contribute to standardization of techniques in pigment cell research and be used as a robust diagnostic tool for detecting early stage, thin lesion melanomas.Support or Funding InformationElon University Honors ProgramElon University Undergraduate Research ProgramElon University Lumen Prize