LC–MS-MS quantification of Δ⁸-THC, Δ⁹-THC,THCV isomers and their main metabolites in human plasma

In recent years, potential therapeutic applications of several different cannabinoids, such as Δ⁹-tetrahydrocannabinol (Δ⁹-THC), its isomer Δ⁸-THC and Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), have been investigated. Nevertheless, to establish dose–effect relationship and to gain knowledge of their pharmacokinetics and metabolism, sensitive and specific analytical assays are needed to measure these compounds in patients. For this reason, we developed and validated an online extraction high-performance liquid/liquid chromatography–tandem mass spectrometry (LC/LC–MS-MS) method for the simultaneous quantification of 13 cannabinoids and metabolites including the Δ⁸ and Δ⁹ isomers of THC, THCV and those of their major metabolites in human plasma. Plasma was fortified with cannabinoids at varying concentrations within the working range of the respective compound and 200 μL was extracted using a simple one-step protein precipitation procedure. The extracts were analyzed using online trapping LC/LC–atmospheric pressure chemical ionization–MS-MS running in the positive multiple reaction monitoring mode. The lower limit of quantification ranged from 0.5 to 2.5 ng/mL, and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 82.9% to 109% and 4.3% to 20.3% (coefficient of variance), respectively. Of 534 plasma samples following controlled oral administration of Δ⁸-THCV, 236 were positive for Δ⁸-THCV (median; interquartile ranges: 3.5 ng/mL; 1.8–11.9 ng/mL), 383 for the major metabolite (−)-11-nor-9-carboxy-Δ⁸-tetrahydrocannabivarin (Δ⁸-THCV-COOH) (95.4 ng/mL; 20.7–328 ng/mL), 260 for (−)-11-nor-9-carboxy-Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV-COOH) (5.8 ng/mL; 2.5–16.1 ng/mL), 157 for (−)-11-hydroxy-Δ⁸-tetrahydrocannabivarin (11-OH-Δ⁸-THCV) (1.7 ng/mL; 1.0–3.7 ng/mL), 49 for Δ⁸-THC-COOH (1.7 ng/mL; 1.4–2.3 ng/mL) and 42 for Δ⁹-THCV (1.3 ng/mL; 0.8–1.6 ng/mL). We developed and validated the first LC/LC–MS-MS assay for the specific quantification of Δ⁸-THC, Δ⁹-THC and THCV isomers and their respective metabolites in human plasma. Δ⁸-THCV-COOH, 11-hydroxy-Δ⁸-THCV and Δ⁹-THCV-COOH were the major Δ⁸-THCV metabolites in human plasma after oral administration of 98.6% pure Δ⁸-THCV.