Cannabidiol metabolites identified by LC-QTOF after controlled dosing

Cannabidiol (CBD) is a non-intoxicating cannabinoid found in cannabis and often used for its purported therapeutic benefits. In the form of Epidiolex®, CBD is an FDA-approved treatment for seizure disorders in children. After the 2018 Farm Bill removed hemp (cannabis with <0.3% THC) from the Controlled Substance Act in the United States, non-pharmaceutical CBD became widely available on the retail market. With increased use of CBD, it is important to measure CBD in various biological matrices. In urine, previous studies have measured 7-hydroxy-CBD and 7-carboxy-CBD, analogous to the major metabolites of Δ9-tetrahydrocannabinol (THC). The aim of this study was to identify metabolites of CBD and verify if 7-hydroxy-CBD and 7-carboxy-CBD are the major metabolites. To identify CBD metabolites, 34 urine samples collected after controlled dosing of 100 mg CBD, representing a wide range of time points (1.5-22 hours), and formulations (Epidiolex, syrup, and vaporized administration) were analyzed by liquid-chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) with and without hydrolysis and compared to 11 samples collected after placebo dosing. Thirteen CBD metabolites were identified, including hydroxylation, carboxylic acid formation, alkyl loss, and dihydrodiol formation. The most abundant metabolites included 7-hydroxy-CBD, 6α-hydroxy-CBD, and a novel metabolite indicating hydroxylation on the pentyl sidechain. Most metabolites were >90% conjugated demonstrating that hydrolysis is required for detection in urine. After oral dosing, metabolite concentrations were higher in urine samples collected 4 and 6 h after dosing compared to 1.5 and 11-22 h. CBD concentrations were higher when CBD was administered as Epidiolex compared to synthetically derived CBD in oral syrup or vaping. In conclusion, the results support the use of 7-hydroxy-CBD as a marker of CBD exposure in hydrolyzed urine, but also identified several novel metabolites that might further our understanding of CBD pharmacokinetics.