HPLC Analysis of Fungal Melanin Intermediates and Related Metabolites

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Abstract
A convenient method to concentrate and separate intermediates and related secondary products of the pentaketide pathway leading to melanin biosynthes is has been developed. Twelve of the fourteen known compounds were separated using reverse phase high performance liquid chromatography with a 12 to 42% linear gradient of acetonitrile in 2% acetic acid. The remaining two, highly water soluble isomers, were isocraticly separated on the same column with 5% acetonitrile in 2% acetic acid. Solid phase extraction and concentration of representitive compounds in acidified aqueous brine was accomplished with reverse phase Sep-Pak cartridges. The effectiveness of the method was evaluated by determining the pentaketides produced by cell-free homogenates of Verticillium dahliae supplied with scytalone as the substrate and by cultures of two V. dahliae mutants. The method has the potential for quantifying all known pentaketide melanin metabolites and thus will allow for more comprehensive studies of fungal melanin biosynthesis.