Distinction between melanins derived from different precursors using pyrolysis/gas chromatography/mass spectrometry and the NIST mass spectral search algorithm

A method was required to objectively determine whether various melanins were synthesised from different precursors, and whether synthesis from the same precursor was reproducible. Melanins have a complex, heterogeneous, polymeric structure, making them difficult to characterise and compare. Pyrolysis chromatography may be useful for such large molecules that are not amenable to analysis by other methods, but the resulting chromatograms are usually complex and difficult to compare. Techniques used to objectively differentiate between such chromatograms often employ statistical methods that are difficult to use and interpret without specialised knowledge. Melanins were analysed by pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS). Software was developed to automate the conversion of the resulting total ion current (TIC) chromatograms to pseudo-mass spectra (PMS), consisting of a row vector of chromatographic peak areas (analogous to ion abundances) ordered along a retention time axis (analogous to m/z ratio). The National Institute of Standards and Technology (NIST, USA) mass spectral search program, which is used widely as an objective measure of the degree of similarity between mass spectra, was then used to generate match factors for comparisons of the generated PMS. Match factors between melanins synthesised from the same precursors were not significantly different, while match factors between melanins synthesised from different precursors were significantly smaller. The reproducibility of the pyrolysis technique was reasonable, with the majority of relative standard deviation (RSD) values of match factors from melanins synthesised from the same precursor, being below 10% (n=5 for each melanin type). While the method does not allow the unequivocal identification of individual melanin types in isolation, it can be used to compare melanins from different sources and objectively estimate the degree of similarity between them on the basis of significant differences between their pyrograms.