Comprehensive LC–MS-MS analysis of THC isomers, analogs, homologs, and metabolites in blood and urine

Abstract The legalization of hemp and the commercialization of hemp-based extracts have resulted in numerous cannabinoids appearing in consumer products. Although human pharmacological data are lacking for many of these isomers, analogs, and homologs of delta-9-tetrahydrocannabinol (Δ9-THC), these cannabinoids may be capable of inducing cannabimimetic effects. Structural similarities also pose unique analytical challenges due to overlapping retention times and ion transitions used to distinguish between various parent drugs and metabolites. Therefore, traditional cannabinoid assays containing Δ9-THC, 11-hydroxy-Δ9-THC (11-OH-Δ9-THC), and 11-nor-9-carboxy-Δ9-THC (Δ9-THCCOOH) are no longer sufficient to confront this new threat to public safety. A new method has been developed and validated to quantitatively confirm Δ8- and Δ9-THC, their hydroxylated and carboxylated metabolites, and 9(R)- and 9(S)-hexahydrocannabinol (HHC) stereoisomers in blood and qualitatively identify these analytes in urine. This method is also capable of qualitatively confirming Δ9,11-THC (exo-THC), HHCCOOH, Δ6a,10a-THCCOOH/Δ10-THCCOOH, and Δ8 and Δ9 THC homologs including tetrahydrocannabivarin (THCV), tetrahydrocannabutol (THCB), tetrahydrocannabihexol (THCH; Δ8-THCH in urine only), tetrahydrocannabiphorol (THCP), THC-C8, as well as 9(R)- and 9(S)-hexahydrocannabiphorol (HHCP) in blood and urine. Limits of detection were 1 ng/mL for non-carboxylated analytes and 5 ng/mL for carboxylated analytes. Calibration curves for parent and hydroxylated THC isomers and HHC stereoisomers were 1 to 50 ng/mL, whereas calibration curves for the carboxylated THC isomers were 5–250 ng/mL. This method separates all analytes of interest from potential synthesis byproducts such as Δ8-iso-THC, Δ4(8)-iso-THC, and exo-THC. Unambiguous identification of these cannabinoids will increase forensic toxicology reporting accuracy while navigating the changing landscape of cannabis regulation and product formulation.