Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS.

A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.