This document describes a novel approach to studying the production of secondary metabolites by fungi using LC/TOF-MS. Fungi grown on culture media are solvent-extracted and directly analyzed by LC/TOF-MS, allowing for easy identification of 465 secondary metabolites, mycotoxins, and other compounds of interest in a database. The methodology was validated by spiking culture media with 20 mycotoxin standards and identifying these toxins in the crude solvent extracts. Subsequently, using seven different fungi from culture collections, after culturing for 7 to 14 days in three different media, anticipated metabolites were readily identified.
The document presents a methodology for characterizing fungi by directly analyzing solvent-extracted culture media using Liquid Chromatography-Time of Flight Mass Spectrometry (LC/TOF-MS). This approach eliminates the need for time-consuming classical mycology or extensive sample clean-up. The method involves growing fungi on specific media, extracting secondary metabolites with solvents like ethyl acetate and isopropanol, and analyzing the crude extracts directly.
The application details the optimization of LC/TOF-MS operational conditions using an Agilent 6210 TOF-MS coupled to an Agilent 1200 Series HPLC. It describes the construction of a database containing exact masses for 465 mycotoxins and fungal metabolites, which is used to identify compounds based on accurate mass measurement within a tolerance of +5 ppm. The methodology was validated by spiking standards into growth media and successfully applied to identify metabolites produced by various Aspergillus and Penicillium species.
| Scan rate | 1 cycle/sec |
|---|---|
| Injected sample volume | 5 uL |
| Flow rate | 0.3 mL/min |
| Capillary voltage | 3000 V |
| Gas temperature | 300 °C |
The methodology utilizes a database containing exact masses for 465 mycotoxins and other fungal metabolites.
The plugs were extracted twice with ethyl acetate containing 1% formic acid followed by isopropanol or acetonitrile.