Variable individual responses and relationships between administered dose and CBD blood concentrations suggest therapeutic drug monitoring (TDM) as a valuable support for patient management. Currently, there is no published method for the simultaneous quantification of CBD, THC and their main active and inactive metabolites. The aim of this study was the development of an analytical method for simultaneous quantification of cannabidiol (CBD), 7-hydroxy-cannabidiol (7-OH-CBD), cannabidiol-7-oic acid (7-COOH-CBD), 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD), and 6-beta-hydroxycannabidiol (6-β-OH-CBD), Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in blood using volumetric absorptive microsampling (VAMS) and LC-MS/MS.

Glucuronide hydrolysis was conducted by adding 2μL IS solution (with 11-OH-THC-d3, THC-COOH-d3, CBD-d3, THC-d3 at 100ng/mL) and 50μL beta-glucuronidase to 30μL blood samples in a VAMS tip, diluting in 800μL water and heating at 37°C overnight. After hydrolysis, acidic and basic extractions were performed to increase the probability of isolating all metabolites. The basic extraction was performed with 4mL hexane: ethyl acetate (9:1) and 100μL ammonium hydroxide and the acidic extraction with 4mL hexane: ethyl acetate (9:1) and 15μL formic acid (≥ 99.9%). Tubes were capped, vortexed for 10sec, mixed for 10min and centrifuged at 5000g for 5min. The supernatants from both extractions were combined into a glass tube and dried under nitrogen. Samples were reconstituted with 100μL water:methanol (1:1) and transferred into autosampler glass vials, before injection of 10μL onto the chromatographic system. Chromatographic separation was performed on a C18 column, with multiple reaction monitoring (MRM) in both positive and negative ionization modes (ESI). Proof of concept of the method included analysis of blood from seven epileptic patients receiving controlled dosing of different CBD formulations (20mg/kg/day Epidiolex®, 18 drops-3.86mg/kg/day CBD oil, 3.86mg/kg/day CBD oil Enecta and 100–250mg/kg/day CBD crystals). Accurate small volume blood collection was achieved with VAMS, and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry to achieve low limits of quantification for CBD, THC, and their respective metabolites.

Method validation followed international guidelines in toxicology, with achieved linear ranges of 0.5–25ng/mL (6-α-OH-CBD, 6-β-OH-CBD, THC, 11-OH-THC and THC-COOH), 10–500ng/mL (CBD and 7-OH-CBD), and 20–5000ng/mL for 7-COOH-CBD. Limits of quantification ranged from 0.02-3.4ng/mL. Accuracy and precision were within ±20% at LOQ for all matrices and within ±15% for all quality control samples across the linear range. Analytical recoveries ranged from 61–98.6%, and matrix effects from 88.6–97.8%. Seven patients (4 males, age 3–12 years, weight 11–28.4kg; 3 females, age 8–20 years, weight 23.6–40kg), with blood samples collected at trough concentration just prior to the next administration. 7-COOH-CBD was present in the highest concentrations (211–1537ng/mL), followed by 7-OH-CBD (53.4–498ng/mL) and CBD (19.1–2501ng/mL). The isomers 6-α-OH-CBD and 6-β-OH-CBD were detected but in lower concentrations (0.9–28.9 and 1.0–38.7ng/mL, respectively), in accordance with previous studies. There were no traces of THC or its metabolites in the blood samples, as expected since the prepared CBD formulations did not contain THC.

This simultaneous cannabinoids analytical method is valuable for further investigation of CBD, THC and their metabolites in individuals treated with medical cannabis, THC or CBD pharmacotherapies and/or recreational cannabis use.