From Extraction to Advanced Analytical Methods: The Challenges of Melanin Analysis

Abstract

The generic term "melanin" describes a black pigment of biological origin, although some melanins can be brown or even yellow. The pigment is characterized as a heterogenic polymer of phenolic or indolic nature, and the classification of eu-, pheo- and allo- melanin is broadly accepted. This classification is based on the chemical composition of the monomer subunit structure of the pigment. Due to the high heterogeneity of melanins, their analytical characterization can be a challenging task. In the present work, we synthesized the current information about the analytical methods which can be applied in melanin analysis workflow, from extraction and purification to high-throughput methods, such as matrix-assisted laser desorption/ionization mass-spectrometry or pyrolysis gas chromatography. Our thorough comparative evaluation of analytical data published so far on melanin analysis has proven to be a difficult task in terms of finding equivalent results, even when the same matrix was used. Moreover, we emphasize the importance of prior knowledge of melanin types and properties in order to select a valid experimental design using analytical methods that are able to deliver reliable results and draw consistent conclusions.

Keywords: MALDI; allomelanin; analytical methods; biopolymers; eumelanin; melanin; pheomelanin; pyrolysis gas chromatography.

Figure 1

Scheme of eumelanin and pheomelanin synthesis.

Figure 2

The chemical structures of (a) eumelanin and (b) pheomelanin as presented in Ref. [4].

Figure 3

TGA (solid line) and DTG (dotted line) thermogram of melanin isolated from sepia ink (SI melanin) (Partially reproduced with the permission from Ref. [10]).

Figure 4

SEM and TEM images showing the multi-scale assembly hierarchical morphology of Sepia melanin at different magnifications; melanin nanoparticle scaled at 2nm (a); melanin nanoparticle highlighting the internal organization in a form of a block-copolymer-like structure (b); secondary self-aggregation of melanin nanospheres in polydisperse large clusters (c–e); deflated clusters with a wide size distribution form of self-aggregates (f) (Reprinted with permission from Ref. [41]).

Figure 5

AFM height (left) and deflection (right) images of a typical Sepia officinalis eumelanin aggregate (the scale bar is 970 nm) (Reprinted (adapted) with permission from Ref. [67]). Copyright 2001 American Chemical Society.

Figure 6

(a) The UV-VIS spectra of synthetic melanin (green) and melanin from Sepia officinalis (blue); (b) The linear curves obtained after plotting the logarithm of absorbance against the wavelength.

Figure 7

The FTIR spectra of Sepia officinalis (red) and synthetic (black) melanin.

Figure 8

(a) The ¹H-NMR spectrum and (b) the ¹³C-NMR spectrum of melanin extracted from Lachnum singerianum YM296 mycelium (Partially reproduced with permission from Ref. [84]).

Figure 9

The ¹H-NMR spectrum of synthetic melanin.

Figure 10

Comparative MALDI-MS profiles of melanin, reproduced with permission from Ref. [97]. (67 YO; dry AMD = 67-year-old donor; dry age-related macular degeneration sample; 19 YO = 19-year-old donor sample; 78-year-old donor sample).

Figure 11

MALDI-TOF of DHN-melanin extracted from M. fijiensis mycelium. (Reproduced with permission from Ref. [36]).

Figure 12

Total ion chromatograms of products formed during pyrolysis of (a) synthetic eumelanin standard (DA-melanin) and (b) synthetic pheomelanin standard (CDA- melanin). Peak designation: (1) benzene, (2) pyridine, (3) 1H- pyrrole, (4) methylbenzene, (5) and (6) 2-methylpyrrole or 3-meth- ylpyrrole, (7) methylpyridine, (8) ethenylbenzene, (9) phenol, (10) 4-methylphenol, (11) benzyl nitrile, (12) isoquinoline, (13) indole, (14) 4-hydroxybenzothiazole, (15) methylindole, (16) 2H-1,4-ben- zothiazin-5-one, (17) 7-methyl-2H-1,4-benzothiazin-5-one, (18) 4-hydroxy-6-ethyl-benzothiazole, (19)-(21) not identified, (22) and (23) thiazolo [4,5-f]isoquinoline or thiazolo[5,4-f]isoquinoline, (24) 2-methyl-thiazoloisoquinoline, (25) 1H[1]benzothiopyrano[3,4-d]imidazol-4-one, (26) not identified. Reproduced by permission from Springer Nature, Journal of the American Society for Mass Spectrometry, GC/MS analysis of thermally degraded neuromelanin from the human substantia nigra. Dzierzega-Lecznar A, Kurkiewicz S, Stepien K, Chodurek E, Wilczok T, Arzberger T, Riederer P, Gerlach M. License no. 4643461452547; 2004 Jun 1;15(6):920-6. [131].