After 72 h of incubation, the contents of each flask, including the controls, were filtered through Buchner funnel into a separating funnel and extracted with three aliquots of dichloromethane (3×50 mL). The combined organic extracts were evaporated to dryness under vacuum at 40 °C using a Buchi rotary evaporator (in vitro Technologies, Noble Park North, VIC, Australia) and placed under high vacuum to remove traces of moisture. The residue was dissolved in acetonitrile to prepare 1 mg/mL stock solution and was filtered through 0.22 µM syringe filter before analysis. Cannabinoid parent drugs and metabolites were chromatographically separated using an Agilent Zorbax Eclipse XDB-C18 analytical column (150×4.6 mm, 5 μm). Mobile phases consisted of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). The gradient used consisted of 30% B (0 to 2 min), linear gradient from 30% B to 50% B (2 to 5 min), 50% B to 90% B (5 to 30 min, hold for 5 min) and 90% B to 30% B (35 to 40 min) run at 0.4 mL/min. MS data were acquired on an Agilent 6490 Triple Quadrupole mass spectrometer with an electrospray ionization source (ESI) source (positive ion mode), interfaced with an Agilent 1290 LC system. Samples prepared were injected in 2 µL volume to obtain full scan and product ion scan spectra. Product ion scan experiments were conducted on precursor ions that were presumed to be metabolites based on the comparison of full scan spectra of the samples and controls. A fragmentor voltage of 380 V with discrete collision energy of 10, 20, 30 and 40 eV (for product ion scan) was applied. The scanning mass range was set at m/z 100–1000 (scan time=500 ms). The sheath gas temperature and flow were set to 250 °C and 11 L/min, respectively. The capillary and nozzle voltages were 3000 V and 1500 V, respectively.

High resolution quadrupole Time-of-Flight mass spectrometry (HRQToFMS) experiments were carried out on an Agilent 6510 Accurate Mass QToF Mass Spectrometer, equipped with ESI source operated in positive ion mode, in order to determine accurate masses of the metabolites. The LC system and conditions used were the same as above. The following operation parameters were used: injection volume 2 µL (full scan) and 10 µL (product ion scan); capillary voltage 3500 V; nebulizer pressure 40 psi (275790 Pa); drying gas 10.0 L/min; gas temperature 350 °C; fragmentor voltage 160 V; collision energy 10, 20 and 40 eV; skimmer voltage 60 V. HRQToFMS accurate mass spectra were recorded across the range from m/z 100 to m/z 1000. The mass axis was calibrated using the mixture provided by the manufacturer over the m/z 50–3200 range. A second orthogonal sprayer with a reference solution was used as a continuous calibration using the following reference masses: m/z 121.0509 and m/z 922.0098. The chromatographic conditions and column used were same as described above. The controls were subjected to the same analysis. Analysis of the chromatographic and mass spectrometric data was conducted using MassHunter Workstation Software Qualitative Analysis (version B.06.00, Agilent). Peaks present in the fungus sample, but not in the controls, were manually identified and their fragmentation patterns and accurate masses were examined to identify the metabolites. The signal-to-noise ratio of all the identified metabolites was greater than 5.