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

The 2018 Farm Bill legalized hemp and defined it as cannabis plant material having not more than 0.3% ∆⁹-tetrahydrocannabinol (∆⁹-THC) by dry weight. This has opened the door for the sale of hemp-derived ∆⁸-tetrahydrocannabinol (∆⁸-THC), a psychoactive isomer of ∆⁹-THC. Hemp has minimal amounts of naturally occurring ∆⁸-THC; however, the cannabidiol found in hemp can be chemically converted into ∆⁸-THC. Unfortunately, depending on the method of conversion, the amount of ∆⁸-THC, ∆⁹-THC, and other by-products can vary widely. For many laboratories, the emergence of ∆⁸-THC products resulted in analytical challenges because of the structural similarity of the isomers resulting in coelution. In response, a novel liquid chromatography–tandem mass spectrometry method was developed to separate the two isomers, with an improved limit of detection (LOD) and lower limit of quantification (LLOQ). With this method, clear separation was achieved between ∆⁹-THC and ∆⁸-THC and 11-nor-9-carboxy-∆⁹-tetrahydrocannabinol (∆⁹-THC-COOH) and 11-nor-9-carboxy-∆⁸-tetrahydrocannabinol (∆⁸-THC-COOH) and a partial separation of 11-hydroxy-∆⁹-tetrahydrocannabinol (∆⁹-THC-OH) and 11-hydroxy-∆⁸-tetrahydrocannabinol (∆⁸-THC-OH). While ∆⁹-THC-OH and ∆⁸-THC-OH did not achieve baseline separation, sufficient separation was achieved to confidently identify and differentiate the two compounds. LOD and LLOQ were the same for quantitative compounds. A quantitative range of 0.5–100 ng/mL was achieved for ∆⁹-THC, ∆⁸-THC, and ∆⁹-THC-OH and 2.5–250 ng/mL for ∆⁹-THC-COOH. Qualitative analysis with an LOD of 0.5 ng/mL was achieved for ∆⁸-THC-OH and 2.5 ng/mL for ∆⁸-THC-COOH. To achieve the desired LODs and LLOQs, alternate multiple reaction monitoring transitions were also explored in addition to those utilized in the laboratory’s prior method and other published methods. The method was validated following the American National Standards Institute/Academy Standards Board Standard 036, Standard Practices for Method Validation in Forensic Toxicology with minor exceptions, and was proven to be reliable and robust.