Validation of a novel LC–MS-MS method for the separation and differentiation of Δ⁸- and Δ⁹-tetrahydrocannabinol isomers and their major metabolites in antemortem whole blood.

This article focuses on the development and validation of a novel liquid chromatography–tandem mass spectrometry (LC–MS-MS) method to separate and quantify ∆8- and ∆9-tetrahydrocannabinol (THC) isomers and their metabolites in blood samples. Following the 2018 Farm Bill’s legalization of hemp, which contains low ∆9-THC but can be chemically converted to psychoactive ∆8-THC, forensic laboratories faced challenges distinguishing these structurally similar isomers due to coelution. The validated method achieved clear separation of ∆8- and ∆9-THC and their carboxy metabolites (THC-COOH), partial separation of their hydroxy metabolites (THC-OH), and improved limits of detection and quantification, enabling reliable identification and quantitation in forensic toxicology. The method was developed using existing laboratory reagents and equipment, followed ANSI/ASB Standard 036 guidelines, and demonstrated robustness and applicability for routine analysis of antemortem blood samples in driving under the influence cases.