Pyrolysis Products of Synthetic Cannabinoids: Chemical Profiling and Early In Vitro Neurotoxicity Assessment

Abstract

The rapid emergence of synthetic cannabinoids (SCs) as new psychoactive substances has outpaced regulatory and toxicological understanding. In particular, the neurotoxicity of their pyrolysis byproducts remains largely unexplored, despite these substances being mainly consumed via smoking. As such, this work aimed to ascertain the chemical profiling of volatile and semi-volatile compounds produced during the pyrolysis of SC-containing herbal blends (Esfinge) and to assess the in vitro neurotoxicological study of these degradation products. For chemical analysis, the herbal mixtures were subjected to controlled smoking simulations using an in-house-built smoking simulator. Pyrolysis vapours were captured and extracted using headspace-solid-phase microextraction (HS-SPME) and solvent-based methods (methanol and dichloromethane). Gas chromatography-mass spectrometry (GC-MS) enabled the identification of over 60 unique pyrolysis products, including halogenated aromatics, nitriles, and heterocyclic derivatives not present in the unburned material. To assess the blends' potential neurotoxicity, SH-SY5Y human neuroblastoma cells were exposed to smoke-derived extracts for 24h at a concentration range of 0.0986 μM to 24.67 μM, replicating inhalation exposure. Both MTT reduction and neutral red uptake assays revealed significant cytotoxicity at the highest concentration, with smoke-generated extracts exhibiting greater toxicity than their non-smoked counterparts (~58 % vs ~0 %), implicating pyrolysis byproducts in enhanced metabolic activity, cytosolic oxidoreductase activity dependent on NAD(P)H, and lysosomal dysfunction. To determine whether the plant matrix contributed to toxicity, the assays were also performed using Artemisia vulgaris and Turnera diffusa. Notably, plant-only extracts induced up to 55 % cytotoxicity, revealing a synergistic matrix effect wherein both SC-derived pyrolysis byproducts and thermolysis of phytochemicals contribute to neuronal injury. These findings demonstrate that SC pyrolysis yields a complex mixture of neuroactive compounds with higher cytotoxicity than parent SCs, and that the herbal matrix itself represents a significant source of neurotoxic compounds. This underscores the necessity of including thermal degradation products in both forensic investigations and risk assessments. Limitations include the in vitro model's inability to replicate systemic metabolism, blood-brain barrier dynamics, and immune interactions, as well as the lack of analytical standards for absolute quantification of many byproducts. Overall, this research highlights a critical public health concern: the neurotoxicity of SC smoke extends beyond the parent compounds and demands proactive regulatory and scientific attention.