UHPLC-MS/MS Analysis of Cannabidiol and Its Metabolites in Serum of Patients with Resistant Epilepsy Treated with CBD Formulations

Cannabidiol (CBD) is a promising therapeutic agent with analgesic, myorelaxant, and anti-epileptic actions. Recently, a purified form of CBD (Epidiolex(R)) has been approved by the European Medicines Agency (EMA) for the treatment of two highly-refractory childhood-onset epilepsies (Dravet and Lennox-Gastaut syndrome). Given the interindividual response and the relationship between the dose administered and CBD blood levels, therapeutic drug monitoring (TDM) is a valuable support in the clinical management of patients. We herein report for the first time a newly developed and validated method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) to evaluate CBD and its metabolites (i.e., cannabidiol-7-oic acid (7-COOH-CBD), 7-hydroxycannabidiol (7-OH-CBD), 6-alpha-hydroxycannabidiol (6-alpha-OH-CBD) and 6-beta-hydroxycannabidiol (6-beta-OH-CBD)) in serum samples. The method reached the sensitivity needed to detect minimal amounts of analytes under investigation with limits of quantification ranging from 0.5 to 20 ng/mL. The validation results indicated in this method were accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%), and fast (8 min run time). The method resulted to be linear in the range of 1-10,000 ng/mL for CBD-COOH, 1-500 ng/mL for 7-OH-CBD and CBD and 1-25 ng/mL for 6-alpha-OH-CBD and 6-beta-OH-CBD. Serum levels of CBD (88.20-396.31 and 13.19-170.63 ng/mL) as well as of 7-OH-CBD (27.11-313.63 and 14.01-77.52 ng/mL) and 7-COOH-CBD (380.32-10,112.23 and 300.57-2851.82 ng/mL) were significantly higher (p < 0.05) in patients treated with GW pharma CBD compared to those of patients treated with galenic preparations. 6-alpha-OH-CBD and 6-beta-OH-CBD were detected in the first group and were undetectable in the second group. 7-COOH-CBD was confirmed as the most abundant metabolite in serum (5-10 fold higher than CBD) followed by 7-OH-CBD. A significant correlation (p < 0.05) between the dose administrated and a higher bioavailability was confirmed in patients treated with a GW pharma CBD preparation.

Automated summary

Cannabidiol (CBD) is a promising therapeutic agent with analgesic, myorelaxant, and anti-epileptic actions. A newly developed method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry has been validated to evaluate CBD and its metabolites, showing that patients treated with GW pharma CBD had significantly higher serum levels of CBD and its metabolites compared to those treated with galenic preparations.