Pheomelanin subunit non-destructive quantification by Raman spectroscopy and multivariate curve resolution-alternating least squares (MCR-ALS)

To date no direct methods are available for the subunit quantification of pheomelanin, the sulfur-containing melanin form whose structure increases melanoma risk. Here we apply Raman spectroscopy combined with chemometrics (MCR-ALS method) to quantify benzothiazine (BT) and benzothiazole (BZ), the constituent monomers of pheomelanin. The Raman spectra of synthetic pheomelanins only composed of BT or BZ were markedly different from those of natural pheomelanins composed of mixed BT-BZ, indicating the necessity of using a multivariate curve resolution method to extract the pure profiles of these monomers in complex samples. MCR-ALS with correlation constraint was applied to the Raman spectra of pheomelanized feathers from 26 bird species to predict BT-BZ concentrations. The methodology was able to quantify BT with R-2 = 0.91 and a relative error of 9%, for the range of 100-1000 ng/mg. However, the model did not perform well for BZ quantification. In addition, the MCR-ALS model could retrieve a pure profile associated to keratin, a major component (interference) in feathers. The deconvolution of Raman spectra, which predicts total pheomelanin content, combined with MCR-ALS, which predicts BT content, can therefore provide a complete pheomelanin determination at the monomeric level. This represents a direct and non-destructive analytical method for pheomelanin subunits, opening the door to investigate the differential cytotoxicity of these subunits in a high diversity of biological samples, including melanoma.

Automated summary

The study successfully quantified the constituent monomers of pheomelanin using Raman spectroscopy combined with chemometrics. The MCR-ALS method was effective in predicting the concentrations of BT and BZ, as well as extracting interference from keratin in feathers. This method provides a direct and non-destructive analytical approach for pheomelanin subunits at the monomeric level.