Miniaturized one-step extraction for the analysis of tetrahydrocannabinol ∆9- and ∆8- isomers, phytocannabinoids, and hexahydrocannabinol in whole blood using liquid chromatography tandem mass spectrometry

∆9-tetrahydrocannabinol (∆9-THC) plays a major role in diving under the influence of drugs investigations and workplace drug testing. Additionally, cannabidiol (CBD), as well as semi-synthetic cannabinoids, like hexahydrocannabinol (HHC) and ∆8-tetrahydrocannabinol (∆8-THC), are increasingly found on regulated and unregulated drug markets. A straightforward bioanalytical method was developed, covering 14 analytes, including ∆9-THC, CBD, ∆8-THC, and their respective metabolites, as well as HHC and a subset of minor phytocannabinoids. Sample cleanup consisted of one precipitation step conducted in a 96-well plate format and requiring only 50 µL of whole blood. For analysis an ultra high-performance liquid chromatography system coupled to a triple quadrupole mass spectrometer was used. The method was validated in accordance with ICH M10 and guidelines in forensic toxicology. The method proved to be precise and accurate with a maximal inter assay imprecision of 9.4 % and a maximal accuracy bias of 12.3 %. The validation revealed good recoveries (>86 %), highly consistent matrix effects (relative standard deviation; RSD ≤7 %) and suitable limits of quantification (LOQs), i.e. 0.5 µg/L for ∆8- and ∆9-THC. In addition, baseline separation of ∆8-THC-COOH and ∆8-THC from the corresponding ∆9-THC isomers were achieved. Method validation demonstrated a fit for purpose analytical method covering legal thresholds for ∆9-THC for driving. Samples from 4 individuals after inhalation of CBD-rich cannabis were analyzed, whereby 8 out the 14 analytes could be quantified, demonstrating the utility and suitability of the method. The method reduces sample volume, solvent use, and workload, making it well-suited for automation and clinical or forensic applications.