Cross-Reactivity in Urine of 53 Cannabinoid Analogs and Metabolites Using a Carboxylic Acid Enzyme-Linked Immunosorbent Assay (ELISA) and Homogenous Enzyme Immunoassay (HEIA) Kit and Immunalysis Synthetic Cannabinoid HEIA Kits

Abstract

Advancing knowledge of endocannabinoid receptor agonists and the federal legalization of hemp has created a cannabinoid market including a wide array of semi-synthetic and synthetic cannabinoid analogs. Public safety and toxicological concerns exist from a lack of regulation, limited pharmacological and metabolomic data, and minimal knowledge of detection ability. Structural similarities of the cannabinoid analogs may allow detection on immunoassays including enzyme linked immunosorbent assays (ELISA) and homogenous enzyme immunoassays (HEIA), screening platforms in forensic toxicology laboratories for rapid presumptive testing. The cross-reactivity of 27 cannabinoid analogs and 26 commercially available metabolites were evaluated on the Medica EasyRA Enzymatic Immunoassay analyzer with the Immunalysis Cannabinoids (THC) and Synthetic Cannabinoids 1-3 kits and the OraSure Technologies Cannabinoids Intercept Microplate on the Dynex DSX Automated ELISA system. The cannabinoid kits target 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (∆9-THCCOOH) at a 50 ng/mL cutoff, and the synthetic cannabinoid kits target the N-pentanoic acid metabolite of JWH-018, UR-144, and AB-PINACA at a 10 ng/mL cutoff. Cross-reactivity was evaluated at concentrations of 20, 50, 100 and 1,000 ng/mL in urine in triplicate. Absence of cross-reactivity at 1,000 ng/mL was considered undetectable. No cross-reactivity was detected on the synthetic cannabinoid kits. Cross-reactivity to ∆9-THCCOOH kits was variable with ∆8-THCCOOH and R-HHCCOOH cross-reacting to the cutoff on the ELISA, with several additional phase I metabolites cross-reacting at 100 ng/mL on both platforms. Analogs lacking the ∆9-THC tricyclic structure and pyran ring cyclization including cannabidiol were undetectable. Alicyclic bond location and alkyl chain length variably affected cross-reactivity, with alkyl lengths 2-4 having increased cross-reactivity comparatively. Compound chirality was seen to effect instrumental response, with the ELISA having increased cross-reactivity and instrumental response to R-isomers. As knowledge and prevalence of analogs increases, it is crucial to understand the impact on utilized testing platforms