## 4 Discussion  
Pyrolysis-gas chromatography-mass spectrometry has been extensively used for investigating the chemical structure (and building blocks) of macromolecular materials such as lignins [26], cutting [27], soil organic matter [28; 29; 30; 31; 32; 33; 34; 35], etc.  
Many studies on the structure of fungal melanins have been published [11; 12; 17; 18; 36], however, analytical pyrolysis of melanins has attracted less attention and only a few fungal melanins (e.g., _Aspergillus niger_, _Epicoccum nigrum_, _Eurotium echinulatum_, _Hendersonula toruloida_, _Penicillium funcitolsum_, _Stachyotrys atra_) have been studied [37; 38].  
There are different approaches for analytical pyrolysis (Curie-point pyrolysis in the presence or absence of TMAH, Pyroprobe flash pyrolysis, double-shot pyrolysis-gas chromatography-mass spectrometry, stepwise pyrolysis, etc.). The pros and cons of these techniques have been widely discussed [39; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41]. In this work we adopted a double-shot pyrolysis-gas chromatography-mass spectrometry, which includes a first step at 300 \({}^{\circ}\)C for thermal desorption and a second step at 500 \({}^{\circ}\)C for pyrolysis. In this way it was possible to remove volatile, loosely held products and polysaccharides at 300 \({}^{\circ}\)C. The TMAH reagent has been proven to produce some artifacts such as benzecarboxylic acids [33] and nitrogen-compounds (e.g., triazines) among others [32; 42], but conventional pyrolysis has also limitations [24; 25; 32; 43; 44].  
Melanin is associated to the fungal cell wall. The cell wall is a multilayered structure primarily composed of polysaccharides including \(\beta\)-linked glucan, chitin, mannan and galactofuran. Proteins are also present either covalently linked or more loosely associated [16]. In addition, chitin cross-links to other cell wall polysaccharides and proteins and may form up to 40% of the fungal cell wall, while lipids may reach up to 3% [16; 45]. Extraction of fungal melanins with alkaline solvents results in a complex mixture of different materials from the cell walls and the cell itself (fatty acids, sterols, etc.).  
In the extraction process with NaOH, different materials present in the fungal mycelia can be associated by covalent bonds or co-extracted and aggregated to the real melanin moiety by hydrogen bonding, van der Waals forces, etc. Thus, the _Ochroconis_ melanins have different contributions of polysaccharides, proteins, lipids, and aromatic compounds, whose pyrolysis products are easily identified. These materials have previously been evidenced in a \({}^{13}\)C NMR and \({}^{15}\)N NMR spectroscopic study of these melanins [19].  
A review on the type of compounds evolved from the approach used in this work indicates that the pyrolysis products identified in the _Ochroconis_ melanins derived from common compounds present in the fungal cells. For instance, homologous series of alkanes, fatty acids and fatty acid methyl esters are found in the mycelia of fungi [46; 47], as well as polysaccharides [48]. Ergosterol is the major fungal membrane sterol [49]. The different series of compounds found in the pyrolyzates are discussed: