The 23 mycotoxins and fungal metabolites (Table 1) selected for this analysis represented some of the most prevalent mycotoxins and fungal metabolites in agricultural commodities. They are produced by Aspergillus and Fusarium spp. that can be found in corn, wheat, barley, and other cereal crops.⁵ Five of the Aspergillus mycotoxins are also tested in legalized cannabis in the United States.⁴ The samples were ground into a powder with a mortar and a pestle that were washed with 90% ethanol and blow-dried with air between samples. Half a gram of ground powder was placed in a silylated glass centrifuge tube along with $5\mathrm{mL}$ 80%–20% acetonitrile–water solution, followed by centrifugation at $\mathrm{1,500}\times g$ at 3,000 rpm for 5 min and collection of the supernatant. After two additional $5\text{-mL}$ washes, the supernatant was added together and passed through the QP1100 SPE purification column (Trilogy Analytical Laboratory). Next, $2\mathrm{mL}$ of the cleaned-up extract was analyzed for 20 mycotoxins and three fungal metabolites using Sciex 5500 QTrap LC-MS/MS. The analytical column was a $100\times 3\mathrm{mm}$ Kinetex $2.6\mathrm{\mu }\mathrm{m}$ C18 column with $100\AA$ pore size. The elution solvents were liquid chromatography–tandem mass spectrometry (LC-MS/MS)–grade water and methanol, each of which was buffered with 0.1% formic acid and $4\text{\hspace{0.17em}mM}$ ammonium formate. The solvent program started with 5% methanol, which was held for one minute, and then the methanol was increased to 50% over a minute. The 50% methanol composition was held constant for 3 min, after which it was ramped to 95% methanol over 5.5 min.

The LC-MS/MS analysis of mycotoxins and fungal metabolites extracted from ground cannabis flower powder used a matrix-matched calibration with hemp flower provided by the University of Kentucky Cooperative Extension Service. In this calibration, we used the hemp flower produced in a clean greenhouse with no signs of fungal infection at harvest. The flower was first tested using high-performance liquid chromatography–ultraviolet (HPLC-UV) and showed no detectable level of mycotoxins.⁶ Next, it was fortified and extracted in a process similar to that of the cannabis flower samples and also analyzed without fortification to apply any positive results to the calibration curve for accurate calculation of results. The LC-MS/MS protocol was further tested using field hemp flower samples collected by the University of Kentucky Cooperative Extension Service with signs of Fusarium infection. Three Fusarium-infected hemp flower samples were run as blind-coded positive controls, showing 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, deoxynivalenol, T-2 toxin, HT-2 toxin, and neosolaniol as expected.