Table 1.

Potential issues that can be observed for different steps in the procedures.

Step	Problem	Possible Reason	Solution
3	The fungus does not sporulate.	-	Use 1–3 mycelium disks of a solid culture. Standardize the solid media, period, and temperature of growth.
10	Low-complexity chemical profile observed in the HPLC-UV-MS analysis.	The volume of culture medium is not enough to detect the compounds, due to low yields.	Increase the amount of culture medium produced for each growth experiment. Volumes such as 100 mL, 250 mL, and 500 mL should be tested.
11	Expected chemical profile was not observed.	-	Different extraction procedures must be tested before starting this protocol.
11a	Dried filtrate is not soluble in H2O.	-	Solubilize the sample in MeOH and adsorb it in celite (1:1 m/m is recommended). Subject the celite + sample to careful evaporation (with a gentle flow of nitrogen gas, or in a Speedvac apparatus) until dryness. After drying, place the celite + sample at the top of the cartridge using a spatula.
14	Different number of variables.	-	FFED levels table using different number of variables (n) can be used. Column levels must be alternated accordingly to 2x−1, with x = column number up to x = n − 1. Last column level (x = n) is the product of preceding columns.
18	It is difficult to measure the chromatographic area and count the peaks.	Not enough resolution to separate the chromatographic peaks.	Test a different elution gradient, and/or different HPLC column.